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PMC006xxxxxx/PMC6644400.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145784310.1021/acsomega.7b00848ArticleMagnetic Co-Doped MoS2 Nanosheets for Efficient Catalysis of Nitroarene Reduction Nethravathi C. Prabhu Janak Lakshmipriya S. Rajamathi Michael Materials Research Group, Department of Chemistry, St. Joseph’s College, 36 Lalbagh Road, Bangalore 560027, India E-mail: mikerajamathi@rediffmail.com.18 09 2017 30 09 2017 2 9 5891 5897 23 06 2017 30 08 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Co-doped MoS2 nanosheets have been synthesized through the hydrothermal reaction of ammonium tetrathiomolybdate and hydrazine in the presence of cobalt acetate. These nanosheets exhibit a dominant metallic 1T phase with cobalt ion-activated defective basal planes and S-edges. In addition, the nanosheets are dispersible in polar solvents like water and methanol. With increased active sites, Co-doped MoS2 nanosheets exhibit exceptional catalytic activity in the reduction of nitroarenes by NaBH4 with impressive turnover frequencies of 8.4, 3.2, and 20.2 min–1 for 4-nitrophenol, 4-nitroaniline, and nitrobenzene, respectively. The catalyst is magnetic, enabling its easy separation from the reaction mixture, thus making its recycling and reusability simple and efficient. The enhanced catalytic activity of the Co-doped 1T MoS2 nanosheets in comparison to that of undoped 1T MoS2 nanosheets suggests that incorporation of cobalt ions in the MoS2 lattice is the major reason for the efficiency of the catalyst. The dopant, Co, plays a dual role. In addition to providing active sites where electron transfer is assisted through redox cycling, it renders the nanosheets magnetic, enabling their easy removal from the reaction mixture. document-id-old-9ao7b00848document-id-new-14ao-2017-00848eccc-price ==== Body 1 Introduction Two-dimensional (2D) MoS2 nanosheets1 have garnered interest as a potential noble-metal-free catalyst for the electrochemical generation of hydrogen from water2−4 and hydrodesulfurization of petroleum.5,6 Theoretical and experimental studies indicate that the catalytic activity of the thermodynamically stable 2H polymorph of MoS2 is associated with its metallic edges, whereas its semiconducting basal plane is catalytically inert.2,4 In this context, nanostructures of MoS2, amorphous7−9/crystalline,10−13 and vertically aligned structures14,15 have been explored to maximize the number of active edge sites. MoS2 is also hybridized with conducting/semiconducting/magnetic materials (graphene15−19/CoSe220/CoS21−24/CdS25,26/Fe3O427) to enhance the catalytic activity through synergetic coupling effects. Metastable, intrinsically metallic, octahedral 1T MoS2 obtained through exfoliation of trigonal prismatic 2H MoS2 has proven to be an excellent catalyst for H2 evolution reactions as the 1T phase facilitates electrode kinetics by increasing the electric conductivity and proliferation of the catalytic active sites.28−30 Introducing transition metal ions (Co, Ni, Fe) into the MoS2 matrix has been the classic route to maximize the catalytic activity of MoS2, as the doped ions alter the electronic properties at the coordinatively unsaturated catalytic S-edges.10,31,32 These strategies have been designed, largely, to either optimize the density of active edge sites by reducing the dimensions along the z direction or xy direction (nanostructures)33 or increase the conductivity by stabilizing the 1T MoS2 polytype.19,28,29 The question is, would it be possible to tune both the structural features and electronic properties simultaneously to increase the catalytic active sites? Doping 2H MoS2 with Co has been shown to increase its catalytic efficiency through increased active sites in the basal planes in addition to edges.34 It would be of interest to prepare Co-doped 1T MoS2 because in addition to all of the above effects, there would be increased conductivity. One of the standard reactions to test the electron transfer catalytic action is the reduction of nitroarenes by NaBH4. Nitroarenes, with aromatic rings associated with H-bonding −NH2 and −OH groups, enable the reduction to be carried out in water, making it a green reaction. This study demonstrates a single-step robust strategy to synthesizing 1T Co-doped MoS2 nanosheets. With increased active sites, Co-doped MoS2 nanosheets exhibit exceptional catalytic activity in the reduction of nitroarenes. The observed turnover frequency (TOF) is far superior in comparison to that of other MoS2 architectures and noble-metal-based catalysts, reported so far. 2 Results and Discussion The XRD pattern of as-prepared Co-doped MoS2 nanosheets (Figure 1A,a) exhibits a broad 002 reflection at 11.0 Å, indicating the presence of guest species in the interlayer.35,36 The guest entity could possibly be NH3/NH4+ ions released as byproducts of hydrazine used as a reductant in the hydrothermal reaction. Figure 1 (A) XRD patterns of Co-doped MoS2 nanosheets (a) as-prepared and (b) treated with 1 N HCl and of (c) MoS2 prepared in the absence of cobalt. (B) Raman spectrum of Co-doped MoS2 nanosheets in comparison with bulk MoS2. On treating Co-doped MoS2 nanosheets with 1 N HCl solution, the 002 reflection (Figure 1A,b) disappears, indicating deintercalation of the guest species. However, the low intensity of the 002 reflection or its absence (Figure 1A,a,b) suggests that Co-doped MoS2 nanosheets are poorly ordered along the stacking direction and comprise largely exfoliated layers. The asymmetric 2D reflections at 2θ = 33 and 57° reveal the presence of stacking faults37,38 within the few-layered Co-doped MoS2. The undoped MoS2 is also poorly ordered and exhibits increased basal spacing due to NH3/NH4+ intercalation (Figure 1A,c). The Raman spectrum of Co-doped MoS2 (Figure 1B) exhibits the in-plane E2g (380 cm–1) and out-of-plane A1g (406 cm–1) Mo–S vibration modes, characteristic of the MoS2 layered structure. An additional peak at 220 cm–1 in Figure S1 (Supporting Information, SI) indicates the presence of 1T polytype. Increased full width at half-maximum and the shift in the A1g and E2g modes of Co-doped MoS2 in comparison to those of bulk MoS2 clearly indicate softening of A1g and E2g modes and phonon confinement that is expected for mono- to few-layer MS2, thus indicating that the Co-doped MoS2 comprises mono to few layers.39,40 The chemical composition of the Co-doped MoS2 was further probed by X-ray photoelectron spectroscopy (XPS). Mo 3d and S 2p spectra (Figure 2a,b; Table 1) correspond to Mo4+ and S2– of the 1T polytype of MoS2. A small proportion of the 2H polytype coexists with the 1T phase.41 The N 1s spectrum (Figure 2c; Table 1) indicates the presence of NH3 and NH4+ ions, which are accommodated in the interlayer of MoS2 nanosheets, as suggested by the XRD pattern (Figure 1a).35 The core-level Co 2p spectra (Figure 2d; Table 1) confirm the presence of Co2+ species. The XRD pattern (Figure 1a) and the XPS Co 2p spectra confirm the absence of CoS2 and CoMo2S4. The binding energy of 779.2 eV is close to what has been observed for CoMo2S4, suggesting that Co2+ substitutes Mo atoms along the {002} or the S-edge planes of MoS2. The atomic percentages of Co, Mo, S, and N are 4.68, 24.66, 59.38, and 11.27, respectively, leading to a chemical composition of Co0.16Mo0.83S2(NH3)0.38. Figure 2 XPS spectra showing Mo 3d (a), S 2p (b), N 1s (c), and Co 2p (d) core-level peak regions of Co-doped MoS2. Table 1 Summary of the Binding Energies of Mo, S, Co and N in Co-doped MoS2 binding energy (eV) Mo 3d phase S 2p Co 2p N 1s Mo0.83Co0.16S2(NH3)0.38 228.6 & 231.8 1T 161.49 & 162.80 779.2 & 794.0 397.6 – NH3 229.0 & 232.5 2H 163.97 & 164.77 400.4 – NH4+ To understand the nature of the chemical environment of Co2+, the Co-doped MoS2 was treated with 1 N HCl when the intercalated or undoped Co2+ species, if any, was expected to be leached out. Cobalt estimation of the leachate showed that only about 30% of the cobalt could be leached out by acid. This was further confirmed by the atomic percentages (Co-3.36, Mo-27.11, S-69.54) in the acid-leached Co-doped MoS2 arrived at from XPS data. The composition of the acid-leached sample is Co0.1Mo0.78S2. The XPS spectra of acid-treated Co-doped MoS2 (SI, Figure S2) indicate that whereas the nitrogen-containing species, NH3 and NH4+ ions, are absent, Co2+ and 1T phase of MoS2 nanosheets are retained. These further suggest that Co2+ is present in the MoS2 lattice. Hydrothermally synthesized MoS2 has been shown to have a defective basal plane as well as unsaturated S-edges.42 Recent studies by Liu et al.34 demonstrate that Co2+ is doped at S vacancies in basal planes as well as at the unsaturated S-edges. The magnetic hysteresis loop measured on the powder sample indicates a weak ferromagnetic behavior (Figure 3). The saturation magnetization (MS) at 300 K of Co-doped MoS2 nanosheets is 0.0029 emu g–1, which is comparable to that of exfoliated 1T MoS2 reported in the literature.43 Because our control 1T MoS2 is nonmagnetic, it is fair to assume that the magnetism in Co-doped MoS2 arises as a consequence of doping. The magnetism in monolayer MoS2 and its doped analogues depends on the nature of edges, type of edge defects, lattice strain, and the dopant concentration. Theoretical calculations by Wang et al.44 reveal that low concentrations of 4 and 6% of Co2+ doping in the Mo vacant sites of the basal planes result in stable magnetic moments at room temperature. Yun et al.45 and Saab et al.46 also reported tuning of electronic and magnetic properties due to doping of metal ions in the MoS2 lattice. The very low MS observed for Co-doped MoS2 suggests that the weak ferromagnetism here originates from the strain in the layer rather than from ordering of Co2+ ions. Co-doped MoS2 (Figure 3) as well as the acid-leached product is weakly magnetic, suggesting that Co2+ ions are doped in the MoS2 layers. In addition, the presence of Co2+ in the MoS2 lattice could be the reason for the retention of metastable 1T structure even after deintercalation of the intercalants (SI, Figure S2). All of these results indicate that Co2+ is possibly doped in the basal plane and S-edge planes of MoS2 layers. Figure 3 Hysteresis loop of the Co-doped MoS2 nanosheets at 300 K. Clusters of layers are observed in the SEM image (Figure 4a) of as-synthesized Co-doped MoS2. The bright-field transmission electron microscopy (TEM) image (Figure 4b) indicates that the transparent layers are few-layer thick and few hundred nanometers in lateral dimensions. The HRTEM image (Figure 4c) shows lattice fringes with a spacing of 1.1 nm, which correlates with the basal spacing observed in the XRD pattern (Figure 1a), suggesting the presence of intercalants. The HRTEM image in Figure 4d clearly shows that the layers are crystalline, exhibiting (100) lattice planes. Except for the circled regions representing the 2H phase, the layers largely exist as the 1T polytype.43 Figure 4 (a) SEM image, (b) low-magnification bright-field TEM image, and (c, d) HRTEM images of Co-doped MoS2. Figure 5 schematically depicts the catalytic reduction of nitroarenes. Catalytic performance of Co-doped MoS2 in the reduction of 4-nitrophenol (4-NP) in water is summarized in Figure 6a,d–f. UV–visible absorption spectra (Figure 6a) of the reaction mixture indicate that 4-nitrophenol converts to 4-aminophenol within 7 min. The absorption peak at 400 nm is due to the nitro phenolate ion, and the intensity of this peak decreases with time and disappears completely at 7 min. Peaks at 235 and 308 nm emerge due to the formation of amino phenolate, and their intensities increase with time. The log (absorbance) versus time plot (Figure 6e) is linear (R2 = 0.979), indicating a pseudo-first-order kinetics47,48 with a rate constant of 1.976 × 10–3 s–1. The turnover frequency (TOF) values, defined as the number of moles of the product formed per unit time per mole of the catalyst, of the materials studied are given in Table 2. Figure 5 Schematic representation of the catalytic reduction of nitroarenes using Co-doped 1T MoS2. Figure 6 Reduction of nitroarenes was traced through UV–visible absorption spectra of the reaction mixture containing 10 mg of the Co-doped MoS2 catalyst, 400 mM NaBH4, and nitroarene. Evolution of absorption spectra with time in the case of 4-nitrophenol (a), 4-nitroaniline (b), and nitrobenzene (c). Plots of absorbance (d) and log (absorbance) (e) against time for 4-nitrophenol reduction. Efficiency of the catalyst (as TOF) in six consecutive cycles of 4-nitrophenol reduction (f). Table 2 Catalytic Activity of the Catalysts in the Reduction of Nitroarenes substrate catalyst time (min) TOF (min–1) 4-nitrophenol (37 mM) Co-doped MoS2 (4.7% doping) 7 8.41 Co-doped MoS2 (∼2% doping) 13.5 4.36 Co-doped MoS2 (∼1% doping) 18 3.27 acid-leached Co-doped MoS2 8 7.36 ammoniated MoS2 90 0.65 4-nitroaniline (14 mM) Co-doped MoS2 (4.7% doping) 7 3.15 nitrobenzene (50 mM) Co-doped MoS2 (4.7% doping) 4 20.2 Apart from exhibiting a high TOF, the catalyst also has the advantage of recyclability. As the catalyst is weakly magnetic, it is easily separated from the reaction mixture using a strong magnet, enabling easy recycling (Figure 5). The catalytic activity of Co-doped MoS2 remains nearly constant over a number of cycles (Figure 6f). The morphology and composition of the catalyst remain almost the same after six cycles of catalysis. Earlier attempts to make MoS2-based catalysts magnetic have been through hybridization of MoS2 nanosheets with magnetic nanoparticles such as Fe3O4.27 One of the shortcomings of such approaches is the increased net weight of the catalyst because the magnetic component of the hybrid does not provide sites for catalytic action. Here, the advantage is that the dopant that improves the catalytic efficiency also makes the catalyst magnetic. Treating Co-doped MoS2 with an acid leads to deintercalation of interlayer NH3/NH4+ and removal of about 30% of Co2+, which were either intercalated or in the edge planes. Catalytic reduction of 4-NP using acid-leached Co-doped MoS2 exhibits a slight decrease in catalytic activity (Table 2). In contrast, ammoniated 1T-MoS2 synthesized in the absence of a cobalt source exhibits relatively very poor catalytic activity toward 4-NP reduction (Table 2). The comparison of the catalytic activities (Table 2) of the catalysts used in 4-NP reduction suggests that incorporation of cobalt ions in the MoS2 lattice is crucial to maximizing the efficiency of the catalyst. Figure 6b,c traces the reduction of nitroaniline and nitrobenzene, respectively, in the presence of as-prepared Co-doped MoS2. The results show that the catalyst is universally effective in the reduction of nitro groups in different substrates and in at least two polar solvents. In fact, the catalyst is most efficient in the reduction of nitrobenzene, a reaction that is of importance in the removal of toxic nitrobenzene from effluents. In all of the cases, reduction of nitroarene does not occur in the absence of the catalyst. In comparison to what has so far been reported in the literature (Table 3), the enhanced TOF and recyclability make Co-doped MoS2 a superior catalyst. The TOF of Co-doped MoS2 is 1 order greater than that of the best MoS2-based catalyst and ∼20% higher than the best value reported so far. Table 3 Comparison of TOF for the Reduction of Nitroarenes by Various Catalytic Materials Reported in the Literature catalyst TOF (min–1) reference 4-nitrophenol reduction Co-doped MoS2 8.41 present work 1T chemically exfoliated MoS2 0.74 (49) 2H chemically exfoliated MoS2 0.015 MoS2-Fe3O4 4.0 × 10–2 (27) MoS2-Fe3O4/Pt 6.0 × 10–4 (50) MoS2-Pd 3.2 × 10–3 (51) MoS2-Pt MoS2 2.5 × 10–3 MoS2-Au MoS2-Ag Ni0.33Co0.66 2.0 × 10–3 (52) citrate capped Au nanoparticles 1.4 (53) Ag dendrites 0.13 (54) Pd supported on CNTs 6.3 (55) 4-nitroaniline reduction Co-doped MoS2 3.15 present work 1T chemically exfoliated MoS2 1.39 (49) Au nanowires 0.10 (56) dodecahedral Au nanoparticles 0.10 (57) In Co-doped MoS2, Co2+ takes residence at the coordinatively unsaturated sulfur vacancies on the basal plane and edge sites.10,58 This leads to a conversion of a fraction of Mo4+ to Mo3+, thus stabilizing the 1T polytype.15 Doped Co2+ distorts the close-packed sulfur layer of MoS2 and induces lattice strain.59,60 These sites would lower the reaction free energy.28 Nitroarenes are adsorbed at the strained active sites of the MoS2 surface.59−61 At these sites, the electron transfer to the substrate is facilitated by Co2+/Co3+ redox couple. The electrons generated by the hydrolysis of NaBH4 are transferred to the Co2+-accommodated basal and edge sites of 1T MoS2 and are promoted into the MoS2 conduction band.59 The Co-substituted sites not only instigate faster electron transfer for nitroarene reduction through reversible reduction–oxidation reactions but also serve as an electron reserve and aid in the retention of the 1T phase with enhanced electrical conductivity. The role of Co in improving the catalytic efficiency is very clear from the fact that the control MoS2, which has intercalated NH3/NH4+ and hence has similar access to surface as that of Co-doped MoS2, shows poor activity (TOF is 1 order lower). As Co-doped MoS2 is largely few-layer thick, the lattice expansion by intercalated species may not be very important and this is borne out from the almost similar activity of the acid-treated sample, which does not have intercalated species. This observation may be important when these catalysts are used in the hydrogen evolution reaction, the reaction medium of which is usually fairly acidic. To further ascertain the role of Co sites in catalysis, the catalytic activity of Co-doped MoS2 with varying cobalt contents was studied. The increase in TOF of 4-nitrophenol reduction with an increase in Co doping (Table 2) further confirms the importance of Co sites in the catalyst. 3 Conclusions Magnetic, 1T Co-doped MoS2 nanosheets with the cobalt ion-activated defective basal planes and S-edges are synthesized in a single-step hydrothermal reaction. Readily dispersible in polar solvents like water or methanol, Co-doped MoS2 nanosheets exhibit exceptional catalytic activity toward reduction of nitroarenes at ambient temperature. In addition to exhibiting a high turnover frequency, the catalyst can be magnetically separated from the reaction mixture, thus enabling recyclability simple and efficient. The superior catalytic activity of the Co-doped MoS2 layers may be due to a combination of (a) stabilization of the metallic 1T phase and (b) better electron capture from the hydride and electron supply to the nitroarene substrates through reversible redox reactions at the Co sites. 4 Experimental Section 4.1 Preparation of Co-Doped MoS2 Nanosheets Cobalt acetate (0.214 g) was dissolved in 45 mL of water. Ammonium tetrathiomolybdate (0.442 g) was added to the pink Co2+ solution, and the mixture was stirred for 15 min. Hydrazine hydrate (5 mL) was added to the solution, and stirring was continued for another 15 min. The black-brown solution was transferred to a teflon-lined autoclave and sealed in a stainless steel canister. The autoclave was heated in a hot-air oven at 180 °C for 24 h and cooled to room temperature under ambient conditions. The pH of the supernatant at the end of the reaction was ∼12. The black precipitate was washed with distilled water till the pH of the washings is ∼7, followed by washing with acetone. The product was dried in air at ambient temperature. The preparation was repeated using 0.107 and 0.054 g of cobalt acetate to vary the cobalt content in the product. 4.2 Acid Leaching of Co-Doped MoS2 Nanosheets To extract the intercalated and free cobalt species, 100 mg of Co-doped MoS2 was stirred in 5 mL of 1 N HCl for 24 h. The supernatant was collected. The process was repeated thrice. The cobalt content in the supernatant was estimated. The black solid was washed with water followed by acetone and dried in air at ambient temperature. 4.3 Preparation of MoS2 Nanosheets As a control experiment, the synthesis was repeated in the absence of cobalt acetate, which results in ammoniated MoS2 nanosheets. 4.4 Reduction of Nitroarenes Using Co-Doped MoS2 Nanosheets as a Catalyst 4.4.1 Reduction of 4-Nitrophenol (4-NP) The catalyst (10 mg) was dispersed in 100 mL of water by stirring for 1 h. 4-NP (512 mg, 37 mM) was dissolved in 100 mL of the catalytic dispersion. An excess of NaBH4 (1.51 g, 400 mM) was added with constant stirring [4-NP:NaBH4 molar ratio was 1:12]. The progress of the reaction was monitored by measuring the absorbance of 4-NP at 400 nm. 4.4.2 Reduction of 4-nitroaniline (4-NA) The catalyst (10 mg) was dispersed in 100 mL of water by stirring for 1 h. 4-NA (192 mg, 14 mM) was dissolved in 100 mL of the catalytic dispersion. An excess of NaBH4 (1.51 g, 400 mM) was added with constant stirring [4-NA/NaBH4 molar ratio was 1:28]. The progress of the reaction was monitored by measuring the absorbance of 4-NA at 380 nm. 4.4.3 Reduction of Nitrobenzene (NB) The catalyst (10 mg) was dispersed in 100 mL of methanol by stirring for 1 h. Nitrobenzene (0.52 mL, 50 mM) was dissolved in the dispersion. An excess of NaBH4 (1.51 g, 400 mM) was added with constant stirring [NB/NaBH4 molar ratio was 1:8]. The progress of the reaction was monitored by measuring the absorbance of nitrobenzene at 259 nm. In all of the cases, an excess amount of NaBH4 was used to ensure that its concentration could be considered constant throughout the reaction and the molar ratio in each case was optimized at the lowest NaBH4 concentration that results in the shortest reaction time. For comparison, the nitroarene reduction reactions were carried out (a) in the absence of the catalyst, (b) using control ammoniated MoS2 nanosheets as a catalyst, and (c) acid-treated Co-MoS2 nanosheets. 4.5 Characterization All of the samples were analyzed by recording powder X-ray diffraction (XRD) patterns using a PANalytical X’pert pro diffractometer (Cu Kα radiation, secondary graphite monochromator, scanning rate of 1° 2θ/min). The amount of Co2+ was estimated by a Varian AA240 atomic absorption spectrometer using a Co hallow cathode lamp in an air–acetylene flame at a wavelength of 324.4 nm. X-ray photoelectron spectroscopy (XPS) measurements were carried out with Kratos axis Ultra DLD. All spectra were calibrated to the binding energy of the C 1s peak at 284.51 eV. Scanning electron microscopy (SEM) analysis was carried out using a Zeiss, Ultra 55 field emission scanning electron microscope equipped with energy-dispersive X-ray spectroscopy (EDS). Transmission electron microscopy (TEM) images were acquired with Tecnai T20 operated at 200 kV. UV–visible spectra of the reaction mixtures were recorded on a PerkinElmer (LS 35) UV–visible spectrometer. The Raman spectra of the samples were recorded on HORIBA Jobin-Yvon LabRAM HR800 at 532 nm excitation wavelength. Isothermal magnetization [M vs H] was measured using a superconducting quantum interference device (SQUID) magnetometer. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00848.Raman spectrum of Co-doped MoS2, and XPS data of acid leached Co-doped MoS2 (PDF) Supplementary Material ao7b00848_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was funded by SERB, India (EMR/2015/001982). ==== Refs References Chhowalla M. ; Shin H. S. ; Eda G. ; Li L.-J. ; Loh K. P. ; Zhang H. 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PMC006xxxxxx/PMC6644401.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145783710.1021/acsomega.7b00699ArticleResonance Control of a Graphene Drum Resonator in a Nonlinear Regime by a Standing Wave of Light Inoue Taichi Anno Yuki Imakita Yuki Takei Kuniharu Arie Takayuki Akita Seiji Department of Physics and Electronics, Osaka Prefecture University, Sakai 599-8531, Japan E-mail: akita@pe.osakafu-u.ac.jp.14 09 2017 30 09 2017 2 9 5792 5797 29 05 2017 30 08 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. We demonstrate the control of resonance characteristics of a drum-type graphene mechanical resonator in a nonlinear oscillation regime using the photothermal effect, which is induced by a standing wave of light between graphene and a substrate. Unlike the resonance characteristics of a conventional Duffing-type nonlinearity, those of the nonlinear oscillation regime are modulated by the standing wave of light with a contribution of the scattered light of an actuation laser, despite a slight variation of amplitude. Numerical calculations conducted with a combination of equations of heat and motion with the Duffing-type nonlinearity explain this modulation: the photothermal effect delays the modulation of graphene stress or tension. document-id-old-9ao7b00699document-id-new-14ao-2017-00699hccc-price ==== Body 1 Introduction Graphene has attracted much attention as a nanoelectromechanical system (NEMS) component because of its superb mechanical and electrical properties. Especially in terms of a graphene NEMS, a graphene mechanical resonator (G-MR)1−10 is expected to be a highly sensitive mass and force sensor and a component of the computing application. To improve the G-MR sensitivity, it is important to realize a nanomechanical resonator with a high quality factor (Q-factor). The intrinsic Q-factor depends solely on the material itself, so the modulation of the intrinsic Q-factor is not easy. By contrast, the apparent Q-factor can be modulated easily by modulating the loss part of the motion of the resonator by application of the active11,12 or passive feedback13−15 to the resonating system under a linear response regime. In the case of passive feedback, the photothermal self-oscillation and laser cooling of the G-MR with a Fabry–Perot cavity between graphene and the substrate have been demonstrated by photothermal back-action induced, respectively, by positive and negative feedback under a linear oscillation regime.3 This photothermal back-action was analyzed based on the delayed response component in the resonating system.8,15 In addition to linear oscillation, nonlinear effects on mechanical resonators, which are commonly described by the Duffing effect at large oscillation amplitude, are important for the improvement of sensitivity or other applications such as mode coupling toward computing applications.6,7,16−22 Nevertheless, few reports describe resonance control of the G-MR in a nonlinear resonance regime with the delayed component using optical back-action.4,6 In addition, the delayed component using optical back-action with a steep optical field gradient should have position dependence. Therefore, we must analyze the combination of the Duffing-type nonlinear equation and the delayed-time component with position dependence. Unfortunately, because equations of this type present difficulty in obtaining accurate analytical solutions, detailed analysis of the nonlinear resonance and control of its resonance properties of the G-MR in a nonlinear regime persist as challenging tasks. This study demonstrates resonance control of the G-MR in the nonlinear resonance regime with a Fabry–Perot cavity using optical back-action induced by the standing wave of light in the Fabry–Perot cavity. In addition to the experiment, we conduct numerical calculations to investigate the relation between nonlinearity and the position-dependent delayed-time component induced by optical back-action in the nonlinear oscillation regime. 2 Results and Discussion This study investigated a drum-type G-MR similar to that presented schematically in Figure 1a. Figure 1b shows that the drum-type G-MR with a 5.4 μm diameter was fabricated successfully. Raman spectroscopy was used to investigate the graphene degradation after the fabrication process. Figure 1c shows that the D band originating from the defects on the Raman spectrum at the suspended part of the G-MR is rarely observed. This result implies that this fabrication process induces no significant degradation of graphene. Figure 1 Drum-type G-MR with a Fabry–Perot cavity. (a) Schematic illustration of the drum-type G-MR with a Fabry–Perot cavity. (b) SEM image of a drum-type G-MR with a drum diameter of 5.4 μm. The scale bar is 5 μm. (c) Raman spectrum for whole of the suspended part of the drum-type G-MR. Figure 2a shows that the resonance of the drum-type G-MR was measured using optical detection methods in vacuum at approx. 10–3 Pa. The intensity of light interference induced between the G-MR and the Si substrate was measured to ascertain the displacement of the G-MR. A laser, used as a probe, irradiated the center of the drum with wavelengths of 406 or 521 nm. To oscillate the G-MR, a 660 nm wavelength laser was irradiated on the substrate near the support edge of the G-MR with a spot diameter of approx. 1 μm, as illustrated schematically in Figure 2a, which was modulated at a certain frequency f through an objective lens with a numerical aperture (NA) of 0.7. Figure 2 Measurement setup for resonance characteristics with probe lasers. (a) Schematic illustration of the optical detection and actuation system. (b) Position dependence of the heat induced by the interference of the probe laser (406 or 521 nm) between the substrate surface and the graphene membrane obtained from finite element method analysis. (c) Resonance curve measured under various actuation laser intensities, where the wavelength and the intensity of the probe laser are 406 nm and 6.3 μW, respectively. Frequency f is normalized by the resonance frequency f0 (approx. 9.83 MHz) at linear oscillation. Figure 2b portrays the graphene position dependence of the induced heat on the G-MR by the interfered probe laser light between the graphene and the Si substrate, which were calculated using the finite element method, where the complex refractive indexes of graphene (2.6 – 1.3i)23 and Si (5.6 – 0.4i at 400 nm)24 were used. The gray area between 300 and 335 nm corresponds to the graphene height from the substrate deduced from the SiO2 and electrode thickness, as illustrated schematically in the inset of Figure 2b, where the possibility that the graphene edge partially adheres to the side of the Au electrodes exists. The graphene height was difficult to measure accurately using atomic force microscopy (AFM) because of insufficient tension of graphene, which caused unexpected perturbation on the AFM image. In the gray area, the slopes of the profiles for wavelengths λp of 406 and 521 nm were opposite. This difference causes a different response of the photothermal effect induced by the probe laser on the drum-type G-MR for respective λp. The thermal expansion coefficient, α, of graphene is negative (−8 × 10–6 K–1) at around room temperature.25 Therefore, higher heat generation at graphene induces higher stress10 or tension of the graphene membrane. Figure 2c presents an example of a frequency response curve of the drum-type G-MR taken from lower frequency (f0 ≈ 9.83 MHz at the resonance of the linear oscillation regime), where the actuation laser intensity (Pact) was changed from 296 to 736 μW with λp = 406 nm. All frequency response curves presented in this paper were taken from the lower frequency. At a higher intensity of actuation, a hardening-type nonlinear vibration is clearly observed, where the resonance curve deforms from symmetrical to unsymmetrical and where the resonance peak shifts toward a higher frequency with increasing actuation. The nonlinear vibration observed here can be excited by increasing the amplitude, which led to the appearance of the nonlinearity term in resilience. In this case, the Duffing-type equation of motion with an external force F cos ωt is applicable with amplitude x described as 1 where m stands for the mass of the G-MR, γ signifies linear damping related to the Q-factor, k0 represents the intrinsic spring constant, β is the nonlinear coefficient (the Duffing constant), and ω denotes the angular frequency. The spring constant of the drum-type G-MR is ascertained from the stress or tension acting on the graphene. At low amplitude, k0 is deduced to be constant, whereas at larger amplitude, the graphene deflection induces additional hardening like k0 + Δk, which results in nonlinear vibration. Results confirmed that the resonance properties under the linear regime showed no marked dependence of Pp (6.30–9.28 μW), as shown in Figure S2 of the Supporting Information. This result implies that no marked modulation of γ or k0 under the linear regime is induced by the photothermal effect attributable to the standing wave of the probe lasers. To modify the resonance characteristics of the G-MR in the nonlinear oscillation regime using the interference light, the probe laser intensity (Pp) dependence was investigated at Pact = 516 μW corresponding to the weak nonlinear oscillation presented in Figure 2c. Figure 3a depicts the frequency response curves obtained at various probe laser intensities from 6.30 to 9.28 μW for respective λp. The frequency responses show weak nonlinearity for both λp. For detailed analysis of the nonlinearity, we evaluated the slopes of the frequency response of the phase shift dφ/df at the resonance as the measure of its nonlinearity, where φ is the phase shift between the deflection of the G-MR and the actuation force. Briefly, in the linear oscillation regime, dφ/df is determined by 2m/γ, which is expected to be independent of λp. For the nonlinear oscillation regime, the frequency response of the phase shift shows an abrupt change from positive to negative, so-called the jumping phenomenon, so that dφ/df at the resonance is infinity. Consequently, one can expect that the weak nonlinear condition corresponding to the intermediate region gives a certain slope dφ/df depending on the nonlinearity. It is noteworthy that, in the case of the conventional nonlinear oscillation governed by eq 1, the frequency at the resonance shifts to a higher frequency with increasing amplitude. The nonlinearity increases with increasing amplitude. Figure 3 Nonlinearity control of the drum-type G-MR by the interference of light. (a) Frequency response curves with various probe laser intensities Pp measured by λp = 406 and 521 nm under Pact = 516 μW. Arrows indicate respective resonances, where the frequency f is normalized by the resonance frequency at the linear response measured under weak actuation. (b) Probe laser intensity Pp dependence of the amplitude at the resonance shown in (a). (c) Probe laser intensity Pp dependence of nonlinearity determined by dφ/df. Solid lines are a guide for the eyes. Figure 3b,c present the probe laser intensity dependence of the maximum amplitude and nonlinearity dφ/df at the resonance, respectively. For λp = 406 nm, the dφ/df at the resonance decreased despite the slight increase in amplitude that occurred with increasing Pp. Moreover, as clearly portrayed in Figure 3a, the peak position for λp = 406 nm shifts 0.4% toward lower frequency with increasing Pp. These changes induced by the probe laser are completely opposite for the conventional nonlinear phenomenon explained by eq 1. For λp = 521 nm, the amplitude at the resonance shows no significant probe laser intensity dependence. The peak position at the resonance fluctuates within 0.3% and shows no specific dependence. Only nonlinearity dφ/df increases with increasing Pp, which is the opposite dependence observed at λp = 406 nm, which might derive from the opposite slopes of the profiles of the standing wave of the probe laser presented in Figure 2b. The changes cannot be explained using the conventional Duffing equation described by eq 1. It is noteworthy that the resonance characteristics in the linear regime show no significant change for changing the probe laser intensity as described above. Note that the scattered light from the actuation laser may modify the nonlinear property because of the additional photothermal effects on the resonance. To investigate the unexpected resonance properties in the nonlinear regime, we consider the delayed time response, which was regarded as the origin of the modification of the resonance properties in the linear resonance regime.13 For the linear resonance regime, the delayed time response on the oscillating system acts on the phase component, which leads to the modulation of damping, γ.13,14 A similar delay component induced by the photothermal effect of the standing wave of light should be considered for the G-MR under a nonlinear regime. The temperature change of the G-MR, Δθ, modifies the stress or tension acting on graphene. Actually, Δθ depends not only on position x but also on time t attributable to thermal relaxation time τth determined by the heat capacity and the thermal resistance, where the heat capacity considered here is expected to include not only graphene itself but also the substrate in contact with the graphene. In the case of a nonlinear oscillator with the time-delayed component, the analytical solution is difficult to obtain. Assuming a linear relation between the spring constant and temperature, the ratio of the spring constant Δk/k is given as 2 where α denotes the temperature coefficient of the spring constant difference. In the case of an one-atom-thick resonator, the spring constant mainly comes from its tension.26 For simplicity, we assumed the graphene resonator as a linear string with length l and concentrated mass m at the center of the string, the spring constant and the resonance frequency ω0 are expressed as k = 4T0/l and , where T0 is the internal strain of the resonator. The internal strain of graphene, T0, can be obtained from the resonance frequency (9.86 MHz) at the linear response regime to be 1 × 10–7 N. Using the thermal expansion coefficient, α, and Young’s modulus, E, of graphene (−8 × 10–6/K and 1 TPa), the tension is modified by temperature with αAE ≈ 1.36 × 10–8 N/K, where A is the cross-sectional area of the graphene resonator (5 μm wide and 0.34 nm thick). As a result, one can estimate it as α0 = 0.15/K from αAE/T0. Additionally, the position dependence of the probe laser-induced heat is approximated as linear because of its small oscillation amplitude compared to the gap separating graphene and the substrate. For simplicity, the time t is scaled by ω0 as τ = ω0t, which is a nondimensional parameter. In addition, we infer that this simplified model with a lumped capacitance model for the heat equation, Δθ, is applicable as 3 where q0 is a slope of the position dependence of the induced heat normalized by the system heat capacity on graphene given by mC and ω0, which is defined from Pp and profiles of the photogenerated heat, as presented in Figure 2b, where C is the heat capacity of graphene. In addition, the photothermal effect from the scattered light of the actuation laser is also implemented as described by the third term of the right hand side, where Pscat is the light intensity of the scattered light of the actuation laser normalized by the system heat capacity on graphene and ω0. This expression is similar to the case described in an earlier report13 for linear oscillation, except for the position dependence term q0x(t) proposed here. Consequently, the additional spring constant Δk is expected to be a function with nonlinear dependence on t and x as Δk(x,t). In this case, the Duffing-type motion equation described by eq 1 using τ = ω0t is now modified to 4 where Q is the quality factor determined as Q–1 = γ/mω0 and x0 = F/mω02 = F/k. The term Δk(x,t) consists of higher order terms of x with certain phase shifts resulting from the time-delayed component, which gives rise to the modification of Q and β in addition to the spring constant itself. In the case of the one-atom-thick resonator, the Duffing term comes mainly from its tension depending on the vibration amplitude.26 On the basis of the same assumption as the string mentioned before, the Duffing term is expressed as 5 where ωE2 = 4AE/ml. Solving simultaneous differential eqs 2–5 numerically, one can qualitatively evaluate the photothermal effect induced by the probe laser, where we also scaled the position variable x by x0 for simplicity. Parameters used in this calculation are presented in Table 1. As a reference, the nonlinear behavior of this model was examined as portrayed in Figure 4a, where the driving amplitude x0 was changed without the photothermal effect induced by the probe laser. A linear response is obtainable at x0 = 1. Nonlinearity with an apparent hardening effect increases with increased driving amplitude. The jumping phenomenon on the frequency response curve is readily apparent. Figure 4 Numerical calculation of the Duffing-type nonlinear resonance with a delayed effect without the scattered light of the actuation. (a) Conventional Duffing resonance curves with various driving amplitudes. (b,c) Frequency response curves calculated with delayed time constants. (d) Frequency response curves calculated with various probe laser intensities Pp. Table 1 Parameters Used for Numerical Calculations f0 (MHz) ω0 (rad s–1) l (μm) A (m2) Q ρ (kg/m3) C (J/kg K) ε (TPa) β (K–1) 9.86 2πf0 5 1.7 × 10–15 300 2250 700 1 0.15 To evaluate the effect of the delayed time constant τth on nonlinearity, ω0τth is used as a parameter13 with the ratio of the oscillation period at linear oscillation, ω0–1, and the delayed time constant τth. At ω0τth < 1 corresponding to the faster response of temperature to the oscillation period, the nonlinearity decreases with increasing ω0τth, which indicates that the fast response, that is, no delay effect, imparts no effect on the phase component of the resonator. Consequently, the nonlinearity returns to the original state with decreasing ω0τth. In the case of ω0τth > 1 corresponding to a slow temperature response, the nonlinearity increases with increasing ω0τth as observed in Figure 4c. The temperature modulation induced by the oscillation is decreased, finally becoming zero at longer τth, which results in the elimination of the delayed effect. The delayed time response greatly influences the resonance characteristics. The condition of ω0τth ≈ 2 is most efficient to suppress the nonlinearity of the resonator as shown in Figure S3 of the Supporting Information, which closely resembles the case for laser cooling of the mechanical resonator.8 Figure 4d presents the light intensity of the probe laser dependence of the frequency response at ω0τth = 2 without the scattering of the actuation laser, that is, Pscat = 0. The nonlinearity decreases with increasing light intensity. The amplitude at the resonance is ∼0.7, which is much greater than the 0.2 obtained in the case of no delay effect portrayed in Figure 4a. Further increase of q0 has a softening effect on the resonance characteristics. These results indicate that the delay effect does not merely suppress the nonlinearity by decreasing the amplitude as the conventional nonlinear oscillator: it directly reduces the nonlinear term in eq 4. However, the effect of the slope difference of the standing wave of the probe laser cannot be explained. Figure 5 shows simulated frequency response curves for ω0τth and probe light intensity dependences with a positive or negative slope of the profiles of the probe laser, where the contribution of the scattered light from the actuation laser (1% of x0) was implemented as described in eq 3. In this case, a clear slope dependence of nonlinearity appeared. In the case of the negative slope corresponding to the 406 nm probe laser, nonlinearity is suppressed at larger ω0τth without a certain minimum as shown in Figure 5a and Figure S3c of the Supporting Information. On the contrary, the clear nonlinearity at the positive slope of the probe laser profile corresponding to the 521 nm probe laser is observed at all of ω0τth even with the larger ω0τth, which is different from the case without the scattered light effect as shown in Figure 5b (see also Figure S3d of Supporting Information). As can be observed in Figure 5c, the decrease of the suppression of the nonlinearity is enhanced at higher q0 at the negative slope. Further increase of q0 induces softening nonlinearity. In the case of the positive slope, the suppression of the nonlinearity is hardly observed. Thus, the combination of the probe laser and the scattered light of the actuation laser is required for the development of the slope dependence of the nonlinearity modification. Figure 5 Numerical calculation of the Duffing-type nonlinear resonance with a delayed effect with the scattered light of the actuation. (a,b) Frequency response curves calculated with various delayed time constants for the negative or positive slope of the probe laser profile. (c,d) Frequency response curves calculated with various q0 for the negative or positive slope of the probe laser profile. We performed numerical simulations to fit the simulated results to experimental data as shown in Figure S4, where the probe laser intensity dependences of the amplitude and nonlinearity were investigated under the condition of the presence of the probe laser and the scattered light of the actuation laser. Figure 6 represents the best fit to the experimental data. Note that the actual laser intensity dependence of q0 on the photothermal effect is unknown, so that the horizontal axis was adjusted with an arbitrary ratio of the actual probe laser intensity and q0. The thermal relaxation time ω0τth, and the scattered light intensity are the dominant fitting parameters. Apparently, both the vibration amplitude and the nonlinearity (dφ/df) obtained from the experiments agree with the results of the numerical simulation. Figure 6 Comparison between the experimental data and simulations. (a,b) Probe laser intensity dependence of vibration amplitude and nonlinearity determined by dφ/df obtained from the experiments shown in Figure 3c and the simulations, where solid lines are smooth fitting for the numerical calculations indicated by tiny dots. 3 Conclusions We demonstrated control of drum-type G-MR nonlinearity using the photothermal effect induced by the standing wave of light between graphene and the substrate, that is, a Fabry–Perot cavity. Unlike the conventional Duffing-type nonlinearity, the nonlinearity was modulated with a slight oscillation amplitude variation by changing the probe laser wavelength and intensity. This phenomenon is explainable: the delayed thermal response induced by the photothermal effect causes the modulation of the effective spring constant of the graphene membrane in the phase space. We used numerical calculations consisting of the heat equation and the Duffing-type equation of motion with the delayed-time response to investigate the modulation of resonance characteristics in a nonlinear oscillation regime. Results show that the weak amplitude dependence of nonlinearity observed in the experimentally obtained results was qualitatively consistent with the calculated results. We believe that this modification of oscillation characteristics in a nonlinear regime opens the way to realize an NEMS circuit using nonlinear effects such as up-conversion of the frequency or mode coupling. 4 Experimental Section The sample fabrication process is illustrated schematically in Figure S1 of the Supporting Information. First, metal electrodes consisting of Cr/Au (5 nm/30 nm) as a support of the graphene drum were fabricated on a heavily doped Si substrate with a 300 nm-thick SiO2 layer using conventional photolithography processing (Figure S1b). Subsequently, after the monolayer graphene was transferred onto the substrate using polymethyl methacrylate (Figure S1c), it was trimmed using oxygen plasma etching to form the drum-type G-MR (Figure S1d), where graphene was synthesized using low-pressure chemical vapor deposition at 1000 °C using a Cu foil as a catalyst.27,28 To form the drum-type G-MR suspended by metal electrodes, the SiO2 layer underneath the graphene drum was etched using buffered hydrofluoric acid (Figure S1e), where the metal electrodes are used as the metal mask for etching. Trenches at both sides of the drum were necessary for uniform etching of the SiO2 layer. They served as an evacuation channel for the air between the graphene drum and the substrate during the measurement of resonance characteristics. The samples thus fabricated were finally dried using supercritical drying to prevent sticking of the suspended graphene induced by the surface tension of water. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00699.Schematic illustration of the flow of device fabrication, resonance properties of the G-MR under a linear oscillation regime, simulated delayed time constant dependence of nonlinearity of the G-MR with or without the scattering light effect, and simulated delayed time constant dependence of amplitude and nonlinearity of the G-MR with the scattering light effect (PDF) Supplementary Material ao7b00699_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was partially supported by JSPS KAKENHI grant numbers JP15H05869, JP16K14259, JP16H00920, JP17H01040, and JP16H06504. ==== Refs References Bunch J. S. ; van der Zande A. M. ; Verbridge S. S. ; Frank I. W. ; Tanenbaum D. M. ; Parpia J. M. ; Craighead H. G. ; McEuen P. L. Science 2007 , 315 , 490 –493 . 10.1126/science.1136836 .17255506 van der Zande A. M. ; Barton R. A. ; Alden J. S. ; Ruiz-Vargas C. S. ; Whitney W. S. ; Pham P. H. Q. ; Park J. ; Parpia J. M. ; Craighead H. G. ; McEuen P. L. Nano Lett. 2010 , 10 , 4869 –4873 . 10.1021/nl102713c .21080681 Barton R. A. ; Storch I. R. ; Adiga V. P. ; Sakakibara R. ; Cipriany B. R. ; Ilic B. ; Wang S. P. ; Ong P. ; McEuen P. L. ; Parpia J. M. ; Craighead H. G. Nano Lett. 2012 , 12 , 4681 –4686 . 10.1021/nl302036x .22889415 Croy A. ; Midtvedt D. ; Isacsson A. ; Kinaret J. M. Phys. Rev. B: Condens. Matter Mater. Phys. 2012 , 86 , 235435 10.1103/physrevb.86.235435 . Jiang J.-W. ; Park H. S. ; Rabczuk T. 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PMC006xxxxxx/PMC6644402.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145887010.1021/acsomega.8b00767ArticleComparative Study of Potassium Salt-Loaded MgAl Hydrotalcites for the Knoevenagel Condensation Reaction Devi Rasna Begum Pakiza Bharali Pankaj Deka Ramesh C. Department of Chemical Sciences, Tezpur University, Napaam, Tezpur 784028, India E-mail: ramesh@tezu.ernet.in. Tel: +91 3712 275058. Fax: +91 3712 267005/6.29 06 2018 30 06 2018 3 6 7086 7095 20 04 2018 07 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A series of potassium salt-loaded MgAl hydrotalcites were synthesized by wet impregnation of KNO3, KF, KOH, K2CO3, and KHCO3 salts over calcined MgAl hydrotalcite (Mg–Al = 3:1). The samples were characterized by X-ray diffraction, Fourier transform infrared, thermogravimetry–differential thermal analysis, scanning electron microscopy, and N2 absorption–desorption techniques to investigate their structural properties. The results showed formation of well-developed hydrotalcite phase and reconstruction of layered structure after impregnation. The prepared hydrotalcites possess mesopores and micropores having pore diameters in the range of 3.3–4.0 nm and Brunauer–Emmett–Teller surface area 90–207 m2 g–1. Base strengths calculated from Hammett indicator method were found increasing after loading salts, where KOH-loaded hydrotalcite showed base strength in the range of 12.7 < H– < 15, which was found to be the preferred catalyst. Subsequently, KOH loading was increased from 10 to 40% (w/w) and catalytic activity was evaluated for the Knoevenagel condensation reaction at room temperature. Density functional theory calculations show that among all of the oxygen atoms present in the hydrotalcite, the O atom attached to the K atom has the highest basic character. In this study, 10% KOH-loaded hydrotalcite showing 99% conversion and 100% selectivity was selected as the preferred catalyst in terms of base strength, stability, and catalytic efficiency. document-id-old-9ao8b00767document-id-new-14ao-2018-00767nccc-price ==== Body 1 Introduction Design, synthesis, and application of solid base catalysts have been of major concern in recent years due to their tunable basicity, environmentally acceptable nature, and capacity to catalyze diverse reactions.1,2 Although a large number of basic materials have been reported in the literature, the study of structure–property relationship of supported solid bases and their basic properties has aroused renewed interest recently.3 Host materials like zeolites (e.g., NaY, KL, NaX), porous metal oxides (e.g., Al2O3, ZrO2), mesoporous silica (e.g., SBA-15, MCM-41), mixed metal oxides, montmorillonite clay, ALPO series, etc. are employed to prepare solid strong bases and super bases.4,5 Among various guest materials and alkali and alkaline earth metal salts, transition metals like Ag, Cu, Co, etc. are the most common.6,7 Among various alkali metal salts, potassium salt-loaded catalysts, e.g., KNO3/Al2O3, KNO3/ZrO2, KF/NaY, K2CO3/Y zeolite, KOH/NaX, KOH/Al2O3, KOH/ZrO2, KOH/MgO, etc., have been reported as excellent basic materials for a good number of organic reactions.8−10 On the other hand, oxide hosts due to their low surface area and silicon-containing hosts due to reaction with some basic guests have limited their use for many reactions.11 Hydrotalcites (HTs) and hydrotalcite-like layered materials are another class of solid hosts that are used to generate strong bases.12 This is a class of solid superbase that can catalyze a large number of organic reactions, such as transesterification, aldol condensation, Knoevenagel condensation, isomerization, and epoxidation reaction.13,14 However, selection of metal salts to suit the host material and their effect over structure and basicity of the host are yet to be explored for hydrotalcite-like compounds. Besides, conditions for a suitable catalyst in terms of its base strength, catalytic activity, stability of host material, recovery, etc. are not easily met.15 Recently, Zhao et al. have reported that potassium-loaded MgAl mixed oxides using KOH has base strength (H–) above 26.5 and shows high catalytic activity for the Knoevenagel condensation reaction at room temperature.16 On the basis of the aforementioned ideas, we aimed to investigate the structure and basicity of Mg–Al hydrotalcite after modification with a series of potassium salts. The Knoevenagel condensation is a versatile C–C bond-forming reaction between a carbonyl compound and an active hydrogen compound to form an unsaturated compound, which can lead to the formation of various chemically and biologically important intermediates, such as α-cyanocinnamates, α,β-unsaturated esters, α,β-unsaturated nitriles, cinnamic acid, etc. These intermediates are commonly used for the production of perfumes, cosmetics, drugs, antihypertensives, calcium antagonists, polymers, fine chemicals, pharmaceuticals, etc. Kantam et al. reported a modified method for activation of MgAl hydrotalcite and applied them for the quantitative formation of the Knoevenagel condensation product in liquid phase.17 In another report, Ebitani et al. showed that reconstructed hydrotalcite is more active than untreated hydrotalcite and provides a unique acid–base bifunctional surface capable of promoting the Knoevenagel and Michael reactions.18 They have also found that reconstructed hydrotalcite gives three times more yield than untreated hydrotalcites for aldol and Knoevenagel condensation reactions. Similarly, modified Mg–Al hydrotalcite with tert-butoxide anion, layered double hydroxide fluoride, and hydrotalcite in ionic liquid medium are reported as efficient catalysts for this reaction.19 Garcia et al. and Čejka et al. have recently reviewed varieties of effective catalysts, such as cation-exchanged zeolites,20 metal-organic frameworks,21−24 mesoporous molecular seives,25 etc., for the Knoevenagel condensation reaction. Other types of solids like ZnO, MgO, alumina, potassium carbonates, zeolites, modified zeolites, natural phosphates, etc. are reported as potential catalysts for this reaction.26 These are considered as more benign in comparison to traditionally used alkaline hydroxides.27 In this work, we have impregnated a series of potassium salts, i.e., KNO3, KF, KOH, K2CO3, and KHCO3, over calcined Mg–Al hydrotalcite (3:1 ratio), studied the effect of base precursors on the structure and basicity of the host, and finally employed for the Knoevenagel condensation reaction. 2 Results and Discussion Powder X-ray diffraction (XRD) patterns of parent hydrotalcite and potassium salt-loaded hydrotalcites are presented in Figure 1. Reflections at 2θ values of 11.5, 23.05, 34.7, 38.35, 45.95, 60.5, and 61.75° corresponding to (003), (006), (102), (105), (108), (110), and (113) planes indicate the formation of highly crystalline layered structure of hydrotalcite, which corroborates well with literature reports.28 Again all potassium salt-loaded samples show strong peaks for (003) and (006) planes, which clearly shows the presence of hydrotalcite phase; this confirms rehydration and reconstruction of hydrotalcite phase after loading. However, shifting of 2θ positions to slightly higher or lower values and diminished intensity of loaded samples can be attributed to the presence of potassium salts on hydrotalcite structure. It has been observed from Figure 1 that the salts KOH, KHCO3, and K2CO3 are well dispersed over the support, whereas KF- and KNO3-loaded samples showed some additional diffraction lines in the XRD pattern. Hence, preferred catalyst can be chosen among KOH, KHCO3, and K2CO3 in terms of phase stability. This observation was further investigated through crystallinity study, which did not follow the similar trend. The crystallinity study revealed that the highest crystallinity after loading was achieved for KOH/HT and the lowest was for KHCO3/HT (Figure S1, Supporting Information). It shows that although KHCO3 disperses better than KF and KNO3 onto the host, it decreases the crystallinity of the overall catalyst. Thus, crystallinity loss is dependent on the nature of the salt but not on dispersion. Thus, observing both crystallinity and phase stability, we can observe that KOH/HT is a stable and highly crystalline catalyst among KOH, KHCO3, and K2CO3. Therefore, KOH/HT is chosen as the preferred salt from XRD. We have also calculated crystallite sizes, unit cell parameters (a), and basal spacing between the layers (d), which are summarized in Table 1. Peaks for (003) and (006) reflections were considered to calculate basal spacings between the layers. Peak for (110) reflection was used to calculate unit cell parameter “a” according to the formula a = 2d, whereas the (003) plane was used to calculate “c” according to the formula c = 3d.29 Comparison of the original hydrotalcite to the loaded hydrotalcites shows that the basal spacings and unit cell parameters increase after loading. Increase of basal spacings and unit cell parameters further supports increase of Mg (+2) ions gradually in the LDH, indicating interaction of some Al (+3) ions with potassium salts and decrease of Coulombic interaction between interlayer anions and brucite-like layers.30,31 The increases of both “d” and a are larger for KOH/HT, K2CO3/HT, and KNO3/HT (d003 = 7.80–7.93; d006 = 3.88–3.91; a = 3.06–3.07) than for KF/HT and KHCO3/HT (d003 = 7.70–7.73; d006 = 3.85–3.86; a = 3.05–3.06). Figure 1 Powder X-ray diffraction pattern of potassium-loaded hydrotalcites. Table 1 Calculation of Lattice Parameter and Basal Spacings for Potassium Salt-Loaded Hydrotalcites sample (003) reflection, 2θ (deg) d003 (Å) (006) reflection, 2θ (deg) d006 (Å) (110) reflection, 2θ (deg) d110 (Å) a (Å) c (Å) crystallite size (003) HT 11.50 7.70 23.05 3.85 60.50 1.53 3.06 23.10 128.09 KF/HT 11.50 7.70 23.00 3.86 60.60 1.52 3.05 23.10 135.56 KHCO3/HT 11.45 7.73 23.10 3.85 60.55 1.52 3.05 23.19 153.65 K2CO3/HT 11.35 7.80 22.70 3.91 60.35 1.53 3.06 23.40 135.70 KNO3/HT 11.35 7.80 22.90 3.88 60.20 1.53 3.07 23.40 164.93 KOH/HT 11.15 7.93 22.85 3.89 60.35 1.53 3.06 23.79 82.31 Thus, a stability order of the salts over the support can be understood from overall results of X-ray diffraction study, which follows the trend KOH/HT ∼ K2CO3/HT > KHCO3/HT > KF/HT ∼ KNO3/HT. Hence, among the studied salts over hydrotalcite support, KOH is chosen as the preferred salt. Smaller crystallite size of KOH/HT in comparison to the others is again in favor of its selection as preferred catalyst for base-catalyzed reaction.32 Following these results, we have loaded KOH amount of 15–40% (w/w) by identical impregnation method to gain better understanding of the effect of the salt over the support. Figure 1 shows that hydrotalcite layered structure preserves up to 20% loading of KOH and collapses above it, where the reflection for the (003) plane was completely destroyed for 25–40% loaded samples. However, crystallinity loss was quite higher for 15–40% loaded samples. When 15% KOH was loaded, crystallinity loss was increased 7 times more in comparison to 10% loaded sample. Therefore, KOH loading beyond 10% is believed to be not effective over hydrotalcite support. Hence, 10% KOH/HT has been conceded as the best catalyst in this study. The thermogravimetric analysis (TGA) and differential thermal analysis (DTA) results obtained from thermal analysis of uncalcined samples in the temperature range of 20–500 °C shows four decomposition steps, which are typical for hydrotalcite-like compounds (Figure S2, Supporting Information). The first weight loss step in the temperature range 20–90 °C corresponds to the loss of physically adsorbed water on the surface, which is found to be in the range of 1–12% of the total weight loss (Table S1, Supporting Information). In the second step, 12–15% weight loss takes place in the range of 80–250 °C due to the loss of interlayer water molecules. We have observed that the weight loss steps of all loaded samples are similar to the parent hydrotalcite, except the step for interlayer water loss. For HT, KF/HT, and KNO3/HT, interlayer water loss takes place only in a single step, whereas it takes place in several steps for KOH/HT, KHCO3/HT, and K2CO3/HT. This shows that the water molecules may be linked in different environments in the presence of different guest molecules. In the third step i.e., 250–415 °C, dehydroxylation and partial loss of carbon dioxide take place, showing 13–22% weight loss. It is noteworthy that dehydroxylation becomes slower after loading of the salts and collapse of the brucite layers starts at low temperature in comparison to the parent hydrotalcite. Besides, dehydroxylation is the fastest in case of KNO3/HT and the slowest in case of KOH/HT, which in turn confirms that the HT phase is thermally less stable in the presence of KNO3 salt and more stable in the presence of KOH. In the fourth weight loss step, loss of carbon dioxide from the samples takes place in the temperature range of 395–518 °C, showing a weight loss of 2–4%. Thus, TGA analysis reveals that the hydrotalcite layer collapses earlier for KNO3/HT and KF/HT in comparison to the other three, i.e., KOH/HT, KHCO3/HT, and K2CO3/HT, which gives thermal stability order of the loaded salts as KNO3/HT < KF/HT < KHCO3/HT < K2CO3/HT < KOH/HT. Thus, TGA analysis also reveals that KOH/HT is the most thermally stable catalyst and the preferred catalyst in our study. The Fourier transform infrared (FTIR) spectra of prepared hydrotalcites are presented in Figure 2. Characteristic bands for hydrotalcite at around 3450, 1640, 1380, 860, 661, and 510 cm–1 clearly indicate the presence of interlayer water molecules, hydroxyl groups in the brucite-like layers, carbonate ions in the interlayer galleries, and Mg–O and Al–O bonds in all samples. Again, bands at around 3600–2800 cm–1 for KOH/HT, K2CO3/HT, and KHCO3/HT samples are broader compared to KNO3/HT and KF/HT, which may be due to the high water content of these samples in the surface as well as in the interlayer spaces. This again correlates well with thermogravimetric analysis of the samples, confirming high water content of these samples in the interlayer spaces. Figure 2 FTIR patterns of hydrotalcites loaded with potassium salts. Nitrogen adsorption–desorption measurements were carried out to investigate the surface areas, pore sizes, and pore volumes of the materials. Table 2 shows textural properties of the parent hydrotalcite and potassium salt-loaded hydrotalcites. The parent hydrotalcite exhibits a Brunauer–Emmett–Teller (BET) surface area of 207 m2 g–1. Introduction of potassium species decreases the BET surface area of all of the samples. The surface area of hydrotalcite loaded with K2CO3 is lost to a greater extent compared to the other four salts and decreases up to 90 m2 g–1. This is due to the blockage of pores and interlayer spaces of hydrotalcite by CO32– anions of K2CO3 salt, thus preventing N2 molecules to adsorb onto the surface. This observation has been further confirmed by the pore volume calculation of the samples, which shows that the pore volume of K2CO3/HT is the smallest and decreases from 0.23 cm3 g–1 of parent hydrotalcite to 0.19 cm3 g–1 of the loaded one. On the other hand, surface areas of KNO3/HT and KF/HT are 201 and 197 m2 g–1, respectively. This can be expected for low water content of these materials, as described in TGA and DTA analysis. We have found intermediate values of surface areas for the samples KOH/HT and KHCO3/HT. This can be attributed to the high water content in these samples. On the other hand, the KOH/HT sample shows the largest pore volume and pore diameter with comparable surface area to the parent hydrotalcite. Thus, surface areas and pore sizes of loaded hydrotalcites other than K2CO3 salt are not abruptly affected after loading the salts. The N2 adsorption–desorption measurements showed that all hydrotalcites exhibit type II isotherm and H3 hysteresis loop characteristics of both monolayer and multilayer adsorption typical for aggregated powders like clays or cements having no uniform pore structures (Figure S3, Supporting Information).33,34 Type H3 hysteresis loops are generally found for nonrigid aggregates of platelike particles or assemblages of slit-shaped pores in the sample. It has again been confirmed from the pore size distribution curves of the samples, which show broad range of pore sizes in the samples. However, narrow pore size distribution centered at a pore radius of 20 Å and large pore volume of KOH/HT has led us to select it as the optimum sample in this study. Table 2 Textural Properties of Potassium Salt-Loaded Hydrotalcites entry sample BET area (m2 g–1) pore volume (cm3 g–1) pore diameter (nm) base strengths (H–) total basicity (mmol g–1) soluble basicity (mmol g–1) 1 HT 207 0.23 3.30 9.6 < H– < 11.1 0.12 0.00a 0.00b 2 KF/HT 197 0.32 3.38 12.7 < H– < 15 0.18 0.02 3 KHCO3/HT 184 0.25 3.95 9.6 < H– < 11.1 0.18 0.03 4 K2CO3/HT 90 0.19 3.99 12.7 < H– < 15 0.23 0.05 5 KNO3/HT 201 0.32 3.66 12.7 < H– < 15 0.19 0.00 6 KOH/HT 185 0.36 4.00 12.7 < H– < 15 0.22 0.03 a Hydrotalcite. b Reconstructed hydrotalcite obtained by immersion of MgAl mixed oxide in water for 24 h. The scanning electron microscopy (SEM) images of parent hydrotalcite and KOH/HT in magnification of X5500 and X4000 are shown in Figure 3. The parent hydrotalcite has crystal sizes in nanometer ranges with homogeneous shapes of the crystals. On loading KOH onto it, particle size decreases, showing layers of agglomerated sheets in the range of 1–2 μm. Figure 3 Scanning electron micrographs of (a1, a2) HT and (b1, b2) KOH/HT at two different resolutions. The results of base strengths of all hydrotalcites, which were determined by the Hammett indicator method, are summarized in Table 2. Hydrotalcites loaded with KF, K2CO3, KNO3, and KOH after drying at 80 °C for 15 h showed color change in the presence of Tropaeolin O (H– = 11.1–12.7) indicator and could not change the color of 2,4-dinitroaniline (H– = 15). Therefore, they showed similar base strength in the range of 12.7 < H– < 15. On the other hand, the unloaded parent hydrotalcite and KHCO3/HT showed color change with phenolphthalein (H– = 8.0–9.6), whereas no change was observed with Tropaeolin O (H– = 11.1–12.7). Thus, their base strengths lie in the range of 9.6 < H– < 11.1. Total basicity measurement of the samples shows that the number of total basic sites for K2CO3/HT and KOH/HT are higher than the other samples. This is reflected in the catalytic activities shown in Table 4 that the conversions for KOH/HT and K2CO3/HT are higher than the other catalysts. On the other hand, the total number of basic sites of all of the catalysts is relatively low compared to calcined hydrotalcites reported in the literature. However, it is quite obvious from the results that although the number of total basic sites is not quite high, uncalcined hydrotalcites also can fairly catalyze the Knoevenagel condensation reaction with its moderate base strengths. Thus, loading of alkali metal compounds over hydrotalcites would be acknowledged to catalyze some mild base-catalyzed organic reactions, such as the Knoevenagel condensation reaction, nitroaldol condensation reaction, aldol condensation, etc. Soluble basicities of all of the samples are low, where no soluble base was found at all for KNO3/HT. This again indicates more interaction of KNO3 with host hydrotalcite structure, thus preventing their loss as soluble base. We again calculated the reactivity of the O atoms using density functional-based reactivity descriptor, the Fukui function. Fukui functions, fO+ and fO–, are evaluated using Hirshfeld population analysis (HPA) and Mulliken population analysis (MPA) schemes to locate the nucleophilic and electrophilic sites, respectively. Although an analytical expression for the Fukui function is not available, it is usually calculated by finite difference approximation, which is called the condensed Fukui function. The condensed Fukui function of an atom “O” in a molecule with N electrons at constant external potential v(r⃗) can be expressed as 1a 1b where ρO(No), ρO(No + ΔN), and ρO(No + ΔN) are charge densities on atom O of the system with No, No + ΔN, and No – ΔN electron systems, respectively. In conventional Fukui function computations, a value of 1.0 is used for ΔN. In the present calculation, we have used a value of 0.1 for ΔN. The values of Fukui functions are given in Table 3. We have calculated the Fukui functions (fO+ and fO–), relative electrophilicity (fO+/fO–), and relative nucleophilicity (fO–/fO+) for those oxygen atoms in the hydrotalcite system having higher values of these reactivity parameters. In general, it is observed that for a particular atom in a molecule, the increase in fO– values is the indication of high basicity. From Table 3, it has been seen that the highest fO– value is obtained for the oxygen atom number 38, which is the one attached to the potassium atom. Relative electrophilicity (fO+/fO–) and relative nucleophilicity (fO–/fO+) are better reactivity parameters to locate the preferable site for nucleophilic and electrophilic attacks, respectively, in a chemical system.35,36 The basicity of a system increases with the increase of the fO–/fO+ ratio. Thus, the oxygen attached to the potassium having the highest value of relative nucleophilicity (fO–/fO+) will be the most basic site. From this calculation, we have inferred that the O atom attached to the K atom has highest values of fO– as well as fO–/fO+. Therefore, we can conclude that O 38 is the most basic site in the hydrotalcite system. The optimized structure of the hydrotalcite is shown in Figure 4. The oxygen atoms for which the reactivity parameters have been evaluated are marked, and these are the atoms having higher basic character in the system. Figure 4 Optimized structure for the most stable geometry of Mg3Al(OH)8KOH. The green balls represent magnesium, pink balls represent aluminum, purple balls represent potassium, red balls represent oxygen, and gray balls represent hydrogen atoms in the optimized geometry. The oxygen atoms having larger values of f(−) (higher basicity) are numbered, and the bond lengths are in angstrom. Table 3 Values of Fukui Functions with Respect to Mulliken and Hirshfeld Charges of the Basic Oxygen Atoms of the Metal Oxide Fukui function (fO+) Fukui function (fO–) relative electrophilicity (fO+/fO–) relative nucleophilicity (fO–/fO+) atom MPA HPA MPA HPA MPA HPA MPA HPA O 38 0.026 0.025 0.019 0.027 1.37 0.93 0.73 1.08 O 65 –0.001 0.006 0.002 0.005 –0.50 1.20 –2.00 0.83 O 69 0.021 0.022 0.021 0.022 1.00 1.00 1.00 1.00 O 80 0.004 0.006 0.002 0.006 2.00 1.00 0.50 1.00 O 83 0.017 0.019 0.017 0.020 1.00 0.95 1.00 1.05 The catalytic activities of the parent and potassium salt-loaded hydrotalcites were evaluated for liquid-phase Knoevenagel condensation reaction (Scheme 1) at room temperature with a variety of aldehydes. The conversion of reactants to the products in an organic reaction is mainly influenced by parameters such as solvent, temperature, effect of substituted groups in the substrates, amount of catalysts, etc. Taking these points into consideration, we have optimized the reaction conditions by varying the conditions. First, the reaction was performed at room temperature without any catalyst by taking malononitrile and p-nitrobenzaldehyde as model reactants and methanol as solvent. To our expectation, no formation of the product was observed. Therefore, we have performed all other reactions in the presence of catalysts, i.e., with modified hydrotalcites and rehydrated hydrotalcite under the same reaction conditions. The results are summarized in Table 4. Reaction with rehydrated hydrotalcite is comparatively slower than with the loaded hydrotalcite (Table 4, entry 2). It has been observed that the reaction in methanol is not selective with all of the catalysts and conversions are lower than reported methods. However, KNO3/HT and KF/HT (Table 4, entries 3 and 4) catalysts show better results than the other catalysts in terms of selectivity and it can be correlated to their larger surface areas than the other loaded catalysts. This can be correlated again from TGA results described above that due to low water content in KNO3/HT and KF/HT, aprotic environment of the catalyst makes the aldol-type intermediate dehydrate easily, thus showing better selectivity of the Knoevenagel product compared to KOH/HT, KHCO3/HT, and K2CO3/HT (Table 4, entries 5–7), where more interlayer water content of these catalysts offers protic environment in the catalysts, thus stabilizing the intermediate aldol-type product before the dehydration step. However, low conversions of the product with KNO3/HT and KF/HT catalysts can be attributed to the interaction of the salts with the support forming new phases and thus reducing active sites of the catalysts (XRD). On the other hand, the preferred catalyst of this study, i.e., KOH/HT (Table 4, entry 7), showed the highest conversion, largest pore volume, longest pore diameter, and good selectivity within 30 min and therefore the reaction was further carried out with this catalyst to optimize the reaction conditions. High conversion of KOH/HT is also supported by its small crystallite sizes (Table 1), which favors the active hydroxyl groups to take part in the reaction.37 Scheme 1 Table 4 Knoevenagel Condensation Reaction with Different Potassium Salt-Loaded Hydrotalcites at Room Temperaturea entry sample time (min) % conversionb % selectivityb 1 no catalyst 30 0 0 2 HTc 30 41 90 3 KNO3/HT 30 57 91 4 KF/HT 30 42 90 5 K2CO3/HT 30 63 69 6 KOH/HT 30 66 81 7 KHCO3/HT 30 61 76 8 KOHd 30 0 0 a Conditions: p-nitrobenzaldehyde (1 mmol), malononitrile (1 mmol), methanol (3 mL), catalyst amount: (25 mg, 8 w % of potassium ion), reaction temperature: room temperature. b Obtained from 1H NMR analysis of the crude reaction mixture. c Reconstructed hydrotalcite obtained by immersion of MgAl mixed oxide in water for 24 h. d Reaction with KOH salt (8 wt % of 25 mg). Following this study, we have investigated the effect of solvents with 10% KOH/HT at room temperature by taking six different solvents of different polarities. It is observed that solvents play a significant role on both conversions and selectivities. When aprotic polar solvents were used (Table 5, entries 2, 4, and 5), 81–99% conversion was observed giving 100% selectivity of the product within 15 min. On the other hand, when protic polar solvent methanol was used, the reaction was slow and both conversion and selectivity was poor (Table 5, entry 3). Nonpolar solvents like toluene and diethyl ether (entries 1 and 6) take longer reaction time than polar solvents, giving 61–99% conversion and 100% selectivities within hours. It is noteworthy to mention that in this study dimethylformamide (DMF) is superior to the most commonly used solvent toluene for the Knoevenagel condensation reaction in the presence of hydrotalcite catalysts. It can be attributed to the fact that the reactants are miscible well in polar environment and the catalyst mixes homogeneously in the reaction mixture during vigorous stirring condition. Thus, interaction of the catalyst with reactants becomes feasible in DMF compared to nonpolar solvents toluene or diethyl ether. Therefore, it is clear from Table 5 that DMF is the best choice in terms of conversion, selectivity, and reaction time, and 10% KOH/HT can be chosen as the optimum catalyst with DMF. Table 5 Effect of Various Solvents on the Knoevenagel Condensation Reaction at Room Temperaturea entry solvent time % conversionb % selectivityb 1 toluene 40 min 99 100 2 DMF 15 min 99 100 3 MeOH 30 min 66 81 4 acetonitrile 15 min 81 100 5 DCM 15 min 99 100 6 diethyl ether 4 h 61 100 a Conditions: p-nitrobenzaldehyde (1 mmol), malononitrile (1 mmol), solvent (3 mL), catalyst (25 mg); catalyst: 10% KOH/HT. b Obtained from 1H NMR yield of the crude reaction mixture. We next used 10% KOH/HT for the Knoevenagel condensation reaction with various aldehydes bearing electron-donating and electron-withdrawing groups, active methylene compounds (malononitrile and diethyl malonate), and DMF at room temperature. The results are summarized in Table 6. The Knoevenagel condensation reaction has been reported previously with hydrotalcite-like catalysts, such as rehydrated Mg–Al hydrotalcite and metal-loaded Mg–Al hydrotalcite in different reaction media and optimized reaction conditions.38 At this time, rehydration and loading have been done in the same step and found an efficient catalyst for this reaction. Aldehydes with both electron-donating and electron-withdrawing groups reacted efficiently with malononitrile under similar reaction condition to give 99% conversions and 100% selectivity of the corresponding olefins (entries 5 and 7). Aldehydes containing electron-withdrawing groups in ortho and para positions do not have much effect over the reaction time (Table 6, entries 2 and 5) and reacted faster than aldehydes bearing electron-donating groups (entries 7 and 8). On the other hand, unloaded catalyst takes comparatively longer reaction time than loaded catalysts and gives 99% conversion and 100% selectivity within 1.5 h (Table 6, entry 3). It may be due to the presence of potassium salts and improved basicity of the loaded catalysts, thus completing the reaction within short time. Heterocyclic aldehyde, i.e., 2-furaldehyde, also reacted faster, giving 99% conversion within 15 min. However, reactions with aliphatic aldehydes and aldehydes with big molecular size are comparatively slower than simple aldehydes showing moderate yield (52–65%, entries 12–13) within 5 h. With diethyl malonate as active methylene compound, the acidity of the acidic protons decreases due to two ester groups, thereby increasing the reaction time for all substrates and giving 70–90% conversion and 100% selectivity within 4–8 h. Polycyclic aromatic aldehydes, such as 1-naphthaldehyde, also reacted efficiently, giving high conversion to the product. It is observed that uncalcined KOH/HT with base strength in the range of 12.7 < H– < 15 shows comparable results to calcined mixed oxide catalyst, i.e., 21 wt % MgO–ZrO239 and 10.3 wt % K–MgAl(O),40 having base strength 26.5 ≤ H– < 33.0 (Table 6, footnotes c and d). This can be understood as high BET surface area of KOH/HT compared to other two made the reaction comparable to each other. We have also evaluated the efficiency of the catalyst by performing the reaction between malononitrile and 4-chlorobenzaldehyde for four repetitive cycles. It is observed that the catalyst is active even at the fourth cycle of the reaction, which gives 99% conversion and 100% selectivity within 15 min (Table 6, entry 20). On the whole, potassium hydroxide-loaded MgAl hydrotalcite acts as an efficient solid base catalyst for the Knoevenagel condensation reaction at room temperature. Table 6 Knoevenagel Condensation Reaction of Different Aldehydes and Active Methylene Compounds with 10% KOH/HTa entry R X Y time % conversionb % selectivityb 1 Ph CN CN 30 min 99 100 2 4-NO2C6H4 CN CN 15 min 99 100 15 min 97c 10 min 99d 3 4-NO2C6H4 CN CN 40 min 99e 100 4 4-NO2C6H4 CN CN 90 min 99f 100 5 2-NO2C6H4 CN CN 15 min 99 100 6 4-ClC6H4 CN CN 10 min 99 100 7 4-CH3C6H4 CN CN 60 min 99 100 8 4-OHC6H4 CN CN 60 min 99 100 9 1-naphthyl CN CN 60 min 99 100 10 2-furyl CN CN 15 min 99 100 11 propionyl CN CN 90 min 99 100 12 isobutyl CN CN 5 h 52 100 13 cinnamic CN CN 5 h 65 100 14 4-NO2C6H4 COOEt COOEt 4 h 99 100 15 2-NO2C6H4 COOEt COOEt 4 h 99 100 16 4-ClC6H4 COOEt COOEt 4 h 99 100 17 4-CH3C6H4 COOEt COOEt 8 h 82 100 18 4-OHC6H4 COOEt COOEt 8 h 77 100 19 1-naphthyl COOEt COOEt 8 h 68 100 20 4-ClC6H4 CN CN 15 min 99g 100 a Conditions: aldehyde (1 mmol), active methyl compound (1 mmol), DMF (3 mL), 10% KOH/HT (25 mg). b Obtained from 1H NMR yield of the crude reaction mixture. c Aldehyde (2 mmol), active methylene compound (2 mmol), 21 wt % MgO–ZrO2 (20 mg), DMF (1 mL). d Aldehyde (2 mmol), active methylene compound (2 mmol), 10.3 wt % K–MgAl(O) (20 mg), DMF (1 mL). e Reaction with rehydrated hydrotalcite. f Reaction with as-prepared hydrotalcite after drying at 80 °C for 15 h. g Fourth run with recovered catalyst. Following this, the leaching of potassium species into solution was tested by flame photometry study. For this purpose, the reaction was performed with 2 mmol p-nitobenzaldehyde, 2 mmol malononitrile, and 100 mg of 10% KOH/HT in DCM solvent. After completion of the reaction, 10 mL of water was added to dissolve the leached out potassium and to separate organic portion. We observed that 0.355 mg of K was leached out from 100 mg of catalyst, which is quite low compared to the loading amount. Thus, leaching of a small amount of potassium does not affect the overall reaction speed and conversion. 3 Conclusions In conclusion, we have found that KOH-loaded MgAl hydrotalcite prepared through wet impregnation of potassium salts over calcined MgAl hydrotalcite is the most stable while KNO3-loaded hydrotalcite is the least stable among a variety of potassium salt-loaded MgAl hydrotalcites, i.e., KF, KOH, KNO3, KHCO3, and K2CO3. It was found that 10% (w/w) KOH/HT is the best catalyst and DMF is the best solvent in the study and catalyzes the reaction efficiently giving 99% conversion and 100% selectivity within 10 min. It is also concluded that polar aprotic solvents like DMF and acetonitrile speed up the reaction, whereas nonpolar solvents slower the reaction. 4 Experimental Section 4.1 Preparation of MgAl Hydrotalcite and MgAl(O) Mixed Oxide Magnesium aluminum carbonate (MgAl-CO3) hydrotalcite was prepared according to the procedure reported by Nyambo et al.41 In this method, solution A containing 38.46 g of Mg(NO3)2·6H2O (0.15 mol) and 18.75 g of Al(NO3)2·9H2O (0.05 mol) in 125 mL of deionized water was added dropwise over 1 h to solution B containing 14 g of NaOH (0.35 mol) and 15.9 g of Na2CO3 (0.1 mol) in 145 mL of deionized water at pH 10–12 with vigorous stirring. The solution was kept stirring vigorously for another 1 h and the white precipitate formed was aged without stirring for 24 h at 65 °C, cooled to room temperature, filtered, and washed several times with deionized water until the filtrate becomes neutral. Finally, the precipitate was dried at 80 °C for 15 h and then calcined at 450 °C for 6 h to obtain MgAl(O) support. 4.2 Preparation of Potassium Salt-Loaded MgAl Hydrotalcites The potassium salt-modified hydrotalcites (HTs) containing same amount of potassium ions (8% w/w) were prepared by a wet impregnation method reported earlier. In this method, a solution of the metal salt containing 1 mmol of the salt (except for K2CO3, where 0.5 mmol was taken) in 8 mL of deionized water was stirred with 500 mg of calcined hydrotalcite for 24 h and the slurries were dried at 80 °C for 15 h to obtain the loaded catalysts. The samples were denoted as KNO3/HT, KOH/HT, K2CO3/HT, KHCO3/HT, and KF/HT. Following the same procedure, 10–40% KOH/HT was prepared by taking appropriate quantity of KOH and the support. 4.3 Theoretical Calculations For the theoretical part, density functional calculations were performed using DMol3 program package,42 as implemented in the Materials Studio program system.43 Geometry optimization was done by treating the exchange–correlation interaction with generalized gradient approximation (GGA) using the Perdew, Burke, and Ernzerhof (PBE) functional.44 We used the double numerical with polarization1 basis set for our calculations. The PBE functional was demonstrated to be reliable for predicting structures of inorganic oxides and it is one of the most universally applied GGA functionals.45 4.4 Characterization Powder X-ray diffraction patterns were recorded on a Rigaku (MiniFlex, U.K.) X-ray diffractometer with Cu Kα radiation (1.5418 Å) at a scan speed of 7° min–1 and 2θ range of 5–70° at 30 kV and 15 mA. The percentage crystallinities of the samples were determined by integrating XRD peaks and using the formula, % crystallinity = (AS × 100)/AR, where AR is the integrated area of the reference material under the peaks between a set of 2θ limits and AS is the integrated area of the sample under the peaks between the same set of 2θ limits as that of the reference. The crystallite sizes were determined by X-ray line broadening method considering 003 reflection plane and using the Debye–Scherrer equation (t = 0.89/β cos θ, where “t” is the crystallite size, “β” is the full width at half-maximum, and “θ” is the angle of diffraction).37 FTIR spectra of various catalysts were recorded on a Nicolet Impact model-410 spectrometer with 1 cm–1 resolution and 32 scans in the mid-IR (400–4000 cm–1) region using the KBr pellet technique. The SEM measurements were carried out using a JEOL JSM-6390LV scanning electron microscope. Thermogravimetric analysis was performed using a Shimadzu thermogravimetric analyzer (TGA-50) in the temperature range of 25–600 °C at a heating rate of 10 °C min–1, and differential scanning calorimetric analysis was performed using a Shimadzu differential scanning calorimeter (DSC-60) in the temperature range of 25–300 °C at a heating rate of 10 °C min–1. Both analyses were performed under nitrogen atmosphere using 10–15 mg of sample. The specific surface areas were determined from N2 adsorption–desorption isotherms obtained by the Brunauer–Emmett–Teller method using a Quantachrome NOVA 1000e surface area and pore size analyzer at −196 °C. Pore size distributions were calculated using the Barrett–Joyner–Halenda equation from the amount desorbed at a relative pressure of about 0.05–1 using N2 desorption branches of the isotherm. Flame photometry analysis was performed in Systronics Flame Photometer 128. The strengths of basic sites were determined by the Hammett indicator method, following the procedure reported by Fraile et al.46 The following Hammett indicators were used: neutral red (H– = 6.8–8.0), phenolphthalein (H– = 8.0–9.6), Tropaeolin O (H– = 11.1–12.7), and 2,4-dinitroaniline (H– = 15). In this procedure, 25 mg of loaded catalyst was shaken with 1 mL of indicator solution (0.1% in methanol) and allowed to equilibrate for 2 h. The color of the catalyst was then noted. The base strength was reported as stronger than the weakest indicator, which exhibits a color change, and weaker than the strongest indicator, which exhibits no color change.47 Total basicities of the samples were determined by titration with benzoic acid. In this method, a suspension of the loaded hydrotalcite (0.15 g) in a toluene solution of phenolphthalein (2 mL, 0.1 mg mL–1) was stirred for 30 min and titrated with a toluene solution of benzoic acid (0.01 M). To determine the leachable basicities, 100 mg of loaded hydrotalcite was added to 10 mL water and the mixture was shaken well at room temperature for 1 h. The catalyst was filtered off, and a methanol solution of phenolphthalein (1 mL, 0.1 mg mL–1) was added to the filtrate, which was then titrated with a methanol solution of benzoic acid (0.01 M). 4.5 Catalytic Reactions The Knoevenagel condensation reaction was carried out in a 50 mL round-bottom flask at room temperature. The catalysts were oven-dried at 80 °C for 12 h prior to use. In the typical procedure, 0.025 g of catalyst was added to a mixture of 1 mmol aldehyde, 1 mmol active methylene compound, and 3 mL of DMF at room temperature. The whole mixture was stirred for the required time and monitored by thin-layer chromatography. After completion of the reaction, DMF was evaporated and the product was extracted with ethyl acetate, dried over anhydrous Na2SO4, evaporated, purified, and finally analyzed using a 1H NMR spectrometer (JEOL JNM-ECS 400) taking Me4Si as the internal standard and CDCl3 as solvent. The used catalysts were washed several times with acetone, dried, and then reused. The conversions (%) were determined from integration of 1H NMR signal of the crude reaction mixtures. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00767.Crystallinity (%) of potassium-loaded hydrotalcites (Figure S1); thermogravimetric analysis and differential thermal analysis patterns (Figure S2); nitrogen adsorption–desorption isotherms (Figure S3); and weight loss percentage of hydrotalcites (Table S1) (PDF) Supplementary Material ao8b00767_si_001.pdf The authors declare no competing financial interest. 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PMC006xxxxxx/PMC6644403.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145905110.1021/acsomega.8b01018ArticleCavitand-Decorated Silicon Columnar Nanostructures for the Surface Recognition of Volatile Nitroaromatic Compounds Tudisco Cristina †∥Motta Alessandro ‡Barboza Tahnie §Massera Chiara §Giuffrida Antonino E. †Pinalli Roberta §Dalcanale Enrico §Condorelli Guglielmo G. †† Dipartimento di Scienze Chimiche, Università di Catania, and INSTM UdR Catania, V.le A. Doria 6, 95125 Catania, Italy‡ Dipartimento di Chimica, Università degli Studi di Roma “La Sapienza” and INSTM UdR Roma, P.le A. Moro 5, 00185 Roma, Italy§ Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, Università di Parma and INSTM UdR Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy* E-mail: enrico.dalcanale@unipr.it (E.D.).* E-mail: guido.condorelli@unict.it (G.G.C.).15 08 2018 31 08 2018 3 8 9172 9181 16 05 2018 19 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. Nanocolumnar Si substrates (porous silicon (PSi)) have been functionalized with a quinoxaline-bridged (EtQxBox) cavitand in which the quinoxaline moieties are bonded to each other through four ethylendioxy bridges at the upper rim of the cavity. The receptor, which is known to selectively complex aromatic volatile organic compounds (VOCs) even in the presence of aliphatic compounds, has been covalently anchored to PSi. The larger surface area of PSi, compared to that of flat substrates, allowed one to study the recognition process of the surface-grafted receptors through different techniques: Fourier-transform infrared spectroscopy, thermal desorption, and X-ray photoelectron spectroscopy. The experiments proved that surface-grafted cavitands retain the recognition capability toward aromatic VOCs. In addition, the affinities of EtQxBox for various aromatic compounds (i.e., benzene, toluene, nitrobenzene, and p-nitrotoluene) have been studied combining density functional theory computations and thermal desorption experiments. Computational data based on the crystal structures of the complexes indicate that this cavitand possesses a higher affinity toward aromatic nitro-compounds compared to benzene and toluene, making this receptor of particular interest for the detection of explosive taggants. The results of computational studies have been validated also for the surface-grafted receptor through competitive recognition experiments. These experiments showed that EtQxBox-functionalized PSi can recognize nitrobenzene in the presence of a significant excess of aromatic vapors such as benzene (1:300) or toluene (1:100). document-id-old-9ao8b01018document-id-new-14ao-2018-01018bccc-price ==== Body Introduction The detection of volatile nitroaromatic compounds is an area of active research, particularly regarding environmental monitoring and social security.1 Among nitroaromatics, nitrobenzene (NB) and 4-nitrotoluene (NT) are molecules of particular interests because NB, which is widely used in organic industries, is considered a highly polluting substance due to its toxicity, carcinogenicity, and biological persistence,2 whereas NT is an explosive taggant.3 Previous papers reported the detection of these nitroaromatic compounds adopting different analytical techniques such as liquid chromatography/mass spectrometry,4−6 spectrophotometry,7 chromatography,8 fluorescence quenching methods,9,10 electrochemical methods,11 surface-enhanced Raman spectroscopy,12,13 and using various organic and inorganic materials such as metallic nanoparticles,2,14 metal–organic frameworks,15−17 polymers,18 carbon nanostructures,19,20 and self-assembled monolayers.21,22 General overviews of nitroaromatic detection approaches can be found in some recent reviews.23−25 Although all of these methods allow the detection of NB at low concentrations, they often require complex instruments and time-consuming sample preparations and, in some cases, suffer from low selectivity or lack of reversibility. On the contrary, detection methods based on supramolecular receptors (such as quinoxaline-bridged and iptycene-roofed cavitands for aromatic volatile organic compounds (VOCs) or higher iptycenes for nitrocompound detection26−28) have recently been explored as alternatives to classical approaches for a fast and easy recognition of the presence of aromatic contaminants.29−35 Conformationally mobile quinoxaline-based cavitand (QxCav, Chart 1)36 showed remarkable selectivity and sensitivity for the detection of benzene, toluene, ethylbenzene, and xylenes (BTEX) in air.29 The complexation properties of this receptor are due to the presence of a deep, hydrophobic cavity capable of engulfing aromatic rings, interacting with them via a set of CH···π and π-stacking interactions.33 These complexation proprieties are retained after the covalent anchoring of this receptor on different surfaces (i.e., Si and ZnO).37,38 Recently, a new conformationally rigid quinoxaline cavitand (EtQxBox) delimited by four quinoxaline walls linked via ethylendioxy bridges (Chart 1) was synthetized.39 This new receptor exhibits an enhanced trapping efficiency for BTEX compared to the parent QxCav receptor in air. Moreover, the conformational rigidity of this cavitand maximizes the binding of toluene, ethylbenzene, and xylenes (TEX) with respect to benzene by increasing the number and strength of their synergistic CH···π interactions.39 Chart 1 Chemical Structures of the Cavitands Discussed in this Study However, the use of cavitands as bulk material limits the degree of miniaturization achievable and, in addition, it can affect the receptor selectivity due to residual nonspecific adsorptions at the solid–gas interface. On the contrary, one of the limitations of the use of monolayers on flat substrates is related to the low surface area and, in turn, to the low amount of material involved in the recognition process, thus jeopardizing sensitivity. A possible solution to these limitations is the deposition of receptor monolayers on high surface area substrates.40−42 Herein, we report on the covalent anchoring of a specifically designed EtQxBox cavitand to columnar porous silicon (PSi) through hydrosilylation of the undecylenic feet decorating the lower rim of the receptor. The high surface area of PSi allowed us to study the heterogeneous recognition process of the surface-grafted receptors versus aromatic analytes through different analytical techniques: Fourier-transform infrared spectroscopy (FTIR), thermal desorption, and X-ray photoelectron spectroscopy (XPS). An unprecedented selectivity of EtQxBox@PSi toward nitroaromatic compounds compared to benzene and toluene has been assessed and rationalized combining thermal desorption experiments with a density functional theory (DFT) approach. Results and Discussion Material Synthesis and Characterization The desired cavitand was prepared following a step-by-step synthetic approach, starting from resorcinarene Res [C10H19, H] (Scheme 1) presenting four terminal double bonds at the lower rim, essential for the grafting of the cavitand onto porous silicon. Scheme 1 Synthesis of EtQxBox Resorcinarene Res [C10H19, H] was obtained from the standard condensation of resorcinol and undecylenic aldehyde in acidic conditions;43 Res [C10H19, H] was reacted in anhydrous conditions with four equivalents of 2,3-dicloro-5,8-dimethoxyquinoxaline A, in a microwave reactor, in the presence of K2CO3 as the base and dimethylformamide (DMF) as the solvent, affording the octamethoxy-quinoxaline cavitand 1 in good yields. The subsequent reaction step was the deprotection of the methoxy groups to have four couples of neighboring free OHs on the quinoxaline ring for cavity rigidification by the introduction of four ethylendioxy bridges. In a previously reported procedure,39 the deprotection of the methoxy groups of quinoxaline was performed by aluminum trichloride in anhydrous toluene. In this case, the presence of the four double bonds at the lower rim of the resorcinarene is not compatible with the use of AlCl3 as a deprotecting agent. Therefore, new deprotection conditions were elaborated to preserve the presence of the four double bonds. The deprotection of cavitand 1 was performed in two steps. The first one consisted in the oxidative elimination of the eight methoxy groups by cerium ammonium nitrate, leading to the tetraquinone-quinoxaline cavitand 2. Crude 2 was purified by column chromatography, and the deprotection was confirmed through 1H NMR by the disappearance of the methoxy signal. Owing to its low stability, cavitand 2 was immediately used in the next step, which consisted in the reduction of the quinone groups by sonication in the presence of metallic zinc (reducing agent) and acetic acid (proton donor) to give cavitand 3. The final reaction step was the rigidification of the cavitand via introduction of four ethylendioxy groups bridging the eight OH moieties at the upper rim. Cavitand 3 was reacted in dry conditions under microwave irradiation with ethylene glycol ditosylate in the presence of anhydrous Cs2CO3 as base and dry DMF as solvent, affording the desired EtQxBox cavitand in 50% yield (Figure S1). PSi functionalization was performed through thermal hydrosilylation of the EtQxBox terminal double bonds at the lower rim, following a reported procedure.44 FTIR and XPS were carried out to characterize porous silicon slides (PSi) functionalized with EtQxBox receptors (EtQxBox–PSi). Figure 1 compares the following FTIR spectral regions of bare PSi (black line) and EtQxBox–PSi (red line): (a) the C–H stretching region between 3200 and 2700 cm–1; (b) the Si–H stretching region between 2300 and 2000 cm–1; and (c) the region between 1500 and 950 cm–1, which contains SiOx and C–O stretching vibrations. The EtQxBox–PSi spectrum shows two strong bands due to the CH2 symmetric (νs(CH2)) and antisymmetric (νa(CH2)) stretches at 2852 and 2928 cm–1 and a lower band at 3050 cm–1 assigned to the aromatic C–H (ν(CH)) stretches of the cavitand.44 The presence of these bands, combined with the absence of the analogue ones in the PSi spectrum, indicates that the receptor is grafted to the PSi surface. In the spectrum of bare PSi, three distinct signals at 2085, 2107, and 2267 cm–1, due to SiH, SiH2, and SiH3 stretches, respectively, are present.45 After EtQxBox anchoring, these SiHx peaks broaden slightly and decrease, leading to a single broad band, as a result of the hydrosilylation reaction, which causes the partial replacement of Si–H terminations with Si–C bonds. In addition, the low broad band at 2251 cm–1 observed in the EtQxBox–PSi spectrum can be assigned to the OSi–Hx stretches of the partially oxidized silicon substrate.46 The 1500–950 cm–1 region of the EtQxBox–PSi spectrum shows two bands at 1172 and 1272 cm–1 attributed to the aromatic C–O bond of the ethylendioxy bridges. In addition, the EtQxBox–PSi spectrum shows also some features in the 1500–1300 cm–1 region associated with the breathing modes of the aromatic ring. All of these features are not detectable in the PSi spectrum. Figure 1 FTIR spectral regions in the 3200–2700 cm–1 (left), 2300–2000 cm–1 (middle), and 1500–950 cm–1 (right) ranges of bare PSi (black line) and EtQxBox–PSi (red line). XPS characterization gives further indication of the success of the anchoring process. XPS C 1s and N 1s spectral regions of PSi and EtQxBox–PSi samples are shown in Figure 2. The C 1s spectrum of PSi (Figure 2a) mainly consists of a component centered at 285.0 eV due to adventitious carbon and a low shoulder around 286.0 eV due to oxidized adventitious carbons. The EtQxBox–PSi spectrum (Figure 2b) consists of three main components. The first component, centered at 285.0 eV, represents aliphatic and aromatic hydrocarbons, whereas the second one, centered at 286.5 eV, can be attributed to the carbon in the cavitand phenyl ring bonded to one oxygen. Note that the possible formation of Si–O–C groups due to the reaction between Si–H and Si–OH termination with oxidized carbon species can contribute to this latter component. The third component at 287.6 eV is due to quinoxaline carbons that bond both oxygen and nitrogen atoms. The N 1s spectrum of EtQxBox–PSi (Figure 2d) consists of a main band centered at 399.9 eV due to the nitrogens of the quinoxaline rings.47 A much less intense side component at 401.1 eV could be due to protonated N atoms or forming H bonds. The presence of these bands, combined with the absence of the analogue signals on PSi (Figure 2c), is a clear evidence of the grafting of EtQxBox on the surface. Figure 2 XPS C 1s (left) and N 1s (right) spectral regions of PSi (a, c) and EtQxBox–PSi (b, d) samples. Complexation Studies Crystal Structure of NT@EtQxBox To evaluate the inclusion ability of EtQxBox toward nitroaromatic compounds, the crystal structure of complex NT@EtQxBox·10DMSO was determined by single-crystal X-ray diffraction methods (Figure S2). This complex was obtained by slow evaporation of a dimethyl sulfoxide (DMSO) solution of hexyl-footed EtQxBox39 and NT from a 1:1 stoichiometric ratio mixture. Crystallographic details for the structures are reported in the Supporting Information, Table S1. Different views of the molecular structure are shown in Figure 3, whereas geometrical details of the interactions responsible for the complex formation are given in Figure S3 and Table S2. NT enters the cavity with the methyl group, which interacts with the aromatic walls at the lower rim of the host via two C–H···π interactions, dictating the orientation of the guest into the cavity. These interactions are strengthened by the presence of the nitro substituent on the aromatic ring, which renders the methyl group more “acidic”. The guest is further stabilized by the presence of two bifurcated C–H···N weak H bonds involving the aromatic hydrogen atoms ortho to the methyl group and the nitrogen atoms of two quinoxaline rings (see Figure S3 and Table S2). The nitro group does not interact with the cavity rim. Figure 3 Side and top views of the molecular structure of NT@EtQxBox. Color code: C, gray; O, red; N, blue. Hydrogen atoms and solvent molecules have been omitted for clarity. The guest is represented in space-filling mode. Nitroaromatic Vapor Complexation by EtQxBox–PSi The affinity of the cavitand-grafted silicon surfaces toward aromatic compounds has been evaluated by exposing EtQxBox–PSi to either NB (about 30 Pa) or NT (about 15 Pa) vapors calculated according to the Antoine equation.48 The detection of the surface-complexed molecules was performed using both XPS and FTIR techniques. In addition, a control reference surface (Ref-PSi) was prepared and similarly exposed to the nitroaromatic vapors. Ref-PSi consisted of a PSi surface functionalized with an inactive organic monolayer formed by a mixture (4:1) of 1-dodecene and a linear naphtyridine, the 2,7-diamido-1,8-naphthyridine.49 The mixed alkene/naphtyridine monolayer was chosen for its elemental composition (C/N/O = 14:1:0.5) similar to that of EtQxBox (C/N/O = 13.5:1:2). Figure 4a shows the evolution of EtQxBox–PSi FTIR spectra in the 1800–1200 cm–1 range after exposure to NB or NT vapors. The spectrum of Ref-PSi after NB exposure has been reported as reference. The spectra of EtQxBox–PSi after exposure to the analytes show the characteristic bands at 1345 and 1523 cm–1 associated with the asymmetric and symmetric N–O stretching of NO2, respectively. These bands are absent in the Ref-PSi spectrum under the same conditions (Figure 4a bottom), indicating that surface-bound EtQxBox is needed for the recognition and that nonspecific interactions can be neglected. Figure 4 (a) FTIR spectra in the 1800–1200 cm–1 range of EtQxBox–PSi after exposure to NB (up) or NT (middle) vapors. The spectrum of Ref-PSi after NB exposure (bottom) has been added for reference. (b) Intensity variation of the 1523 cm–1 band of NO2 after cycles of absorption/desorption of NB with EtQxBox–PSi. The reversibility of the complexation process was then evaluated through the FTIR monitoring of the intensity variation of NO stretches during NB adsorption/desorption cycles. These cycles were obtained through sample exposure to NB vapors (adsorption), followed by a mild treatment at 80 °C under N2 flushing for 20 min (desorption). The decrease of the feature associated with the NO2 group down to a 20% of the maximum after the desorption step indicates that the process operates with good reversibility (Figure 4b). The complexation process was also monitored by XPS. Figure 5 reports the N 1s spectral regions of EtQxBox–PSi (Figure 5a) and Ref-PSi (Figure 5b) before and after the complexation process with NB. After the exposure to NB, the N 1s region of EtQxBox–PSi (Figure 5a middle trace) shows a new broad peak at ∼406.0 eV, besides the typical peak at 399.9 eV, attributable to the presence of the NO2 group of NB in the cavity. In the case of Ref-PSi, only the N 1s band centered at 400.0 eV assigned to the nitrogens of the 2,7-diamido-1,8-naphthyridine is present before and after vapor exposure (Figure 5b). Figure 5 N 1s spectral regions of (a) EtQxBox–PSi and (b) Ref-PSi before and after the NB exposure. The NB desorption induced by sample heating was also monitored by XPS analysis, confirming the process reversibility observed in the FTIR experiments (Figure 5a upper trace). Nitroaromatic vs Aromatic Complexation at the Gas–Solid Interface The complexation of benzene (104 Pa), NB (30 Pa), and NT (15 Pa) vapors on EtQxBox–PSi slides and their thermal desorption under ultra-high vacuum (UHV) conditions (total pressure = 10–8 Torr) were studied by in situ mass spectrometry. Various ions associated with the fragmentation of the desorbing molecules have been detected by the mass spectrometer during the heating of the substrate (Figure 6). In the case of benzene desorption, the molecular C6H6+ (main ion) ion and the C6H5+ fragment (<20%) start to desorb below 50 °C, and the desorption is completed at about 150 °C. Figure 6 Thermal desorption experiments of EtQxBox–PSi (left) and Ref-PSi (right) after the adsorption of benzene (up) and NB (bottom). In the case of NB, the complexation is proved by the desorption of the C6H5+ fragment, which in this case is the most intense, whereas the observed molecular C6H5NO2+ ion is about 30% of the main fragment. Note that the desorption temperature range of NB is broader than the one observed for benzene. The same trend is observed for the thermal desorption of NT (Figure S4). Similar experiments performed on the inert Ref-PSi slides showed a minimal uptake of the guests, further confirming that the presence of the cavitand on the surface is essential for the aromatic compound complexation. To evaluate the different affinities of EtQxBox–PSi toward aromatic and nitroaromatic compounds, a set of experiments with simultaneous adsorptions of vapors of NB/benzene (1:300 ratio) and of NB/toluene (1:100 ratio) was performed. The comparison between benzene and NB adsorption emphasizes the effects on the cavitand–arene complex if a nitro group is added to the aromatic ring. On the contrary, through experiments adopting toluene and NB mixtures, the role of the two substituents can be compared. After the adsorption of the benzene/NB mixture, the main ions observed during the thermal desorption were C6H5+, which is the most intense peak arising from NB fragmentation, and the C6H5NO2+ molecular ion (Figure 7). Despite the large excess of benzene in the gas phase, only a reduced amount of benzene (peak C6H6+) was released during heating. Note that the contribution of benzene to the intensity of the fragment C6H5+ is below 10% because the amount of this ion, deriving from benzene fragmentation, is less than 20% of C6H6+. This experiment has shown that NB is preferentially complexed with respect to benzene by EtQxBox–PSi. It therefore indicates that the addition of the nitro group to the aromatic ring increases the stability of the cavity–arene complex. Figure 7 Thermal desorption experiments of EtQxBox–PSi (up) and Ref-PSi (bottom) after the adsorption of (left) benzene/NB (300:1) and (right) toluene/NB (100:1) mixtures. During thermal desorption after toluene/NB exposure, the characteristic toluene ions (i.e., C7H8+ e C7H7+) and the NB C6H5NO2+ and C6H5+ ions are clearly present. Despite the excess of toluene in the gas phase, the amount of adsorbed NB is comparable to that of the adsorbed toluene, suggesting a higher affinity of EtQxBox toward NB. However, in this case, the gap is less marked when compared to that of the benzene/NB mixture. Rationalization of the Observed Selectivity via a DFT Study DFT calculations of the insertion of aromatic compounds inside the EtQxBox cavity were performed to rationalize the higher affinity of the cavitand toward nitroaromatics compared to that toward simple aromatic hydrocarbons. In particular, complex formation with benzene, toluene, NB, and NT is compared. In all cases, deep insertion of the aromatic moiety into the cavity is observed (Figure 8). Figure 8 Optimized structures for the host–guest complex between aromatic guests and the EtQxBox cavitand: (a) benzene@EtQxBox; (b) NB@EtQxBox; (c) toluene@EtQxBox; and (d) NT@EtQxBox. Benzene and toluene insert deeper than the respective nitro derivatives (see also the corresponding crystal structures in ref (39)). In the case of toluene, both orientations of the methyl group with respect to the cavity are energetically accessible, but the one with the methyl group protruding outside the cavity is preferred (Figure 8c). The nitro group is always positioned outside the cavity (Figure 8b,d). The host–guest formation is in all cases exothermic and exoergonic (Figure 9). The stabilization energies are mostly due to the CHguest···πhost and CHguest···Nhost interactions, as also evidenced by the NT@EtQxBox crystal structure. In the cases of NB and NT, a further stabilizing contribution of about 7 kcal/mol, compared to benzene and nitrobenzene, respectively, is ascribable to the dipole–dipole interaction between the nitroaromatics and EtQxBox (Figure S5). The much higher affinity of the cavity toward NB compared to benzene, observed in the adsorption/desorption experiments, is due to this difference. Smaller differences are observed in the adsorption affinity between NB and toluene, as confirmed by the calculated lower energy gap (about 3 kcal/mol) between the host–guest complexes. In the case of toluene, the lack of the dipole–dipole stabilization due to the nitro group is partially balanced by the formation of the CHguest···πhost interactions of the methyl group.39 The process is entropy opposed due to the confinement of the guest inside the cavity. In fact, we found that Gibbs free energy values are always 13–16 kcal/mol higher than the respective enthalpy values (at 298 K). The entropic loss is quantified in about 13–16 kcal/mol in the gas phase. Figure 9 Interaction energy of the four guests with EtQxBox. Conclusions In this work, we report the synthesis, characterization, and complexation properties of a new organic–inorganic hybrid material based on a porous silicon surface decorated with a conformationally rigid quinoxaline-bridged cavitand. The recognition properties of EtQxBox toward aromatic VOCs have been transferred to the silicon surface. The success of the grafting protocol has been demonstrated by combining different analytic techniques (XPS and FTIR). Reversible host–guest complexation of aromatic and nitroaromatic compounds on the functionalized porous surface has been evaluated by XPS, FTIR, and desorption experiments. We demonstrated through the combination of experimental results and DFT modeling that the affinity of the EtQxBox–PSi system toward nitroaromatic compounds is significantly higher than the one toward benzene because of the stabilizing contribution due to the dipole–dipole interaction. Experimental Section Materials Purchased chemicals were used as received unless otherwise noted. Water used in PSi preparation and functionalization was Milli-Q grade (18.2 MΩ cm) and was passed as a final step through a 0.22 μm filter. Unless stated otherwise, reactions were conducted in flame-dried glassware under an atmosphere of argon using anhydrous solvents (either freshly distilled or passed through activated alumina columns). Silica column chromatography was performed using silica gel 60 (Fluka 230–400 mesh or Merck 70–230 mesh). 1H NMR spectra were obtained using a Bruker Avance 300 (300 MHz) and a Bruker Avance 400 (400 MHz) spectrometer at 298 K. All chemical shifts (δ) were reported in ppm relative to the proton resonances resulting from incomplete deuteration of the NMR solvents. High-resolution matrix assisted laser desorption ionization-time of flight (MALDI-TOF) was performed on AB SCIEX MALDI-TOF-TOF 4800 Plus (matrix: α-cyano-4-hydroxycinnamic acid). 2,3-Dicloro-5,8-dimethoxyquinoxaline A(50) and undecylenic-footed resorcinarene Res [C10H19, H]43 (Scheme 1) were prepared according to published procedures. Synthesis of Cavitand EtQxBox Synthesis of Octametoxy-Quinoxaline Cavitand 1 First, 1 g (9.61 × 10–4 mol) of resorcinarene Res [C10H19, H] was dissolved in 50 mL of dry DMF; K2CO3 (2.12 g, 0.01 mol) and quinoxaline A (0.99 g, 3.84 × 10–3 mol) were then added. The reaction was conducted in a microwave reactor, in open-vessel modality, at 120 °C for 1.5 h. The crude was diluted in a large excess of ethyl acetate and washed with water. The organic phase was dried over sodium sulfate and evaporated. The compound was purified by flash column chromatography (SiO2, CH2Cl2/acetone 95:5–90:10), affording a yellow solid, active in fluorescence, in 69% yield. 1H NMR (CDCl3, 300 MHz): 7.43 (s, 4H, Hup), 6.93 (s, 8H, ArHQuin), 6.84 (s, 4H, Hdown), 5.79 (m, 4H, −CH=CH2), 4.96 (m, 8H, −CH=CH2), 4.02 (s, 24H, −OCH3), 3.95 (t, 4H, ArCH–R, J3 = 7.9 Hz), 2.30 (m, 8H, −ArCH–CH2), 1.33 (m, 56H, −CH2−). MALDI: m/z = 1787.89 [M + H]+. Synthesis of Tetraquinone-Quinoxaline Cavitand 2 Cavitand 1 (1.02 g, 5.72 × 10–4 mol) was dissolved in 100 mL of tetrahydrofuran, and cerium ammonium nitrate (3.76 g, 6.86 × 10–3 mol, previously diluted in a minimum amount of water) was added. The mixture was stirred at room temperature for 30 min and then quenched by the addition of water. After extraction with ethyl acetate and evaporation, the compound was purified by flash column chromatography (SiO2, CH2Cl2/acetone 85:15), affording an orange solid in 80% yield. Owing to its poor stability, the compound was characterized only through 1H NMR and immediately used for the next reaction. 1H NMR (CDCl3, 300 MHz): 7.38 (s, 4H, Hup), 7.18 (s, 8H, ArHQuin), 6.80 (s, 4H, Hdown), 5.80 (m, 4H, −CH=CH2), 4.97 (m, 8H, −CH=CH2), 3.63 (t, 4H, ArCH–R, J3 = 7.4 Hz), 2.30 (m, 8H, ArCH–CH2−), 1.33 (m, 56H, −CH2−). Synthesis of Octahydroxy Quinoxaline Cavitand 3 Tetraquinone-quinoxaline cavitand 2 (0.21 g, 1.26 × 10–4 mol) was dissolved in 6 mL of acetone. Zinc (0.92 g, 0.01 mol) and 0.5 mL of acetic acid were added in this order. The suspension was sonicated for 5 min. The crude was filtered over celite, dissolved in ethyl acetate, and washed with water, until complete removal of acetic acid. After evaporation, the pure compound was obtained as a bright yellow solid in 90% yield. 1H NMR (DMSO- d6, 400 MHz): 8.32 (s, 8H, ArOH), 8.10 (s, 4H, Hup), 7.90 (s, 4H, Hdown), 6.94 (s, 8H, ArHQuin), 5.83 (m, 4H, −CH=CH2), 4.95 (m, 8H, −CH=CH2−), 4.08 (t, 4H, ArCH–R, J3 = 7.3 Hz), 2.30 (m, 8H, −ArCH–CH2−), 1.22 (m, 56H, −CH2−). MALDI-MS: m/z =1673.71 [M + H]+. Synthesis of Cavitand EtQxBox Cavitand 3 (80 mg, 4.78 × 10–5 mol) was dissolved in 5 mL of dry DMF in a microwave vessel. Cs2CO3 (202 mg, 6.20 × 10–4 mol) and ethylene glycol ditosylate (106 mg, 2.86 × 10–4 mol) were added under nitrogen. The mixture was reacted in a microwave reactor at 120 °C for 1.5 h. The crude was extracted with CH2Cl2/H2O, dried over sodium sulfate, and evaporated. The compound was purified by preparative thin layer chromatography (SiO2, acetone 100%), affording the desired compound as a bright yellow solid, in 50% yield. 1H NMR (CDCl3, 400 MHz): 8.31 (s, 4H, Hup), 7.55 (s, 4H, Hdown), 6.74 (s, 8H, ArHQuin), 5.88 (m, 4H, −CH=CH2), 5.81 (t, 4H, ArCH–R, J3 = 7.6 Hz), 5.04 (m, 8H, −CH=CH2), 4.64–4.50 (m, 16H, ArOCH2CH2OAr), 2.11 (m, 8H, −ArCH–CH2−), 1.32 (m, 56H, −CH2−). MALDI-MS: m/z = calculated for C108H113N8O16 [M + H]+ 1777.82745, found 1777.8230, calculated for C108H112N8NaO16 [M + Na]+ 1799.8094, found 1799.8094 [M + Na]+. PSi Preparation PSi has been prepared by wet metal-assisted chemical etching according to a published method.39,ref40 In particular, Czochralski-grown, p-type Si(100) slides having a resistivity of 1.5–4 Ω cm were treated for 5 min with an hydrofluoric acid (HF) (0.14 M) and AgNO3 (5 × 10–4 M) water solution. After the deposition of Ag particles, the slides were dipped in HF, H2O2, and H2O (40% HF/30% H2O2/H2O 25:10:4 v/v) solution for 1 min and then rinsed with Milli-Q water and dried under N2 flow. Cavitand Grafting For grafting on porous substrates, the etched PSi was dipped in a EtQxBox solution in mesitylene. The solution was refluxed at 200 °C under N2 for 5 h; then functionalized PSi slides were cleaned by two washing cycles in an ultrasonic bath (5 min each) in dichloromethane. Cavitand Complexation Tests Adsorption experiments were performed by placing samples for 30 min in a closed chamber saturated with aromatic VOC vapors. The saturated chamber at 25 °C was obtained by placing one beaker (or two beakers for the mixtures) containing 3 mL of the aromatic compound (benzene, toluene, or NB) and allowing vapor saturation for 4 h. The saturation of NT vapor was obtained by placing for 2 h ∼50.0 mg of NT in a closed chamber kept at 50 °C. Sample Characterization XPS spectra were obtained with a PHI 5600 multitechnique ESCA-Auger spectrometer equipped with a monochromated Al Kα X-ray source. Analyses were carried out with a photoelectron takeoff angle of 45° (relative to the sample surface) with an acceptance angle of ±7°. The XPS binding energy scale was calibrated by centering the C 1s peak due to hydrocarbon moieties and “adventitious” carbon at 285.0 eV. FTIR spectra were obtained with JASCO FTIR 430, using 100 scans per spectrum (scan range 560–4000 cm–1, resolution 4 cm–1). Thermal desorption experiments were performed in a UHV chamber (basic pressure ∼ 10–8 Torr). For the experiments, the holder (Vacuum Science, Italy) was resistively heated with a ramp of about 10 °C/min from 30 to 200 °C. The desorbed molecules were detected with a Smart-IQ + (Thermo Electron Corporation) quadrupole mass spectrometer equipped with an electron filament as ion source and a multiplier detector (mass range 1–300). Crystal Structure of NT@EtQxBox The crystal structure of NT@EtQxBox was determined by single-crystal X-ray diffraction methods. Intensity data and cell parameters were obtained at 190 K on a Bruker APEX II equipped with a charge-coupled device area detector and a graphite monochromator (Mo Kα radiation λ = 0.71073 Å). The data reduction was carried out using the SAINT and SADABS51 programs. The structure was solved by Direct Methods using SIR9752 and refined on Fo2 by full-matrix least-squares procedures, using SHELXL-2014/753 in the WinGX suite version 2014.1.54 All of the nonhydrogen atoms were refined with anisotropic atomic displacements, with the exclusion of some atoms belonging to the disordered DMSO lattice molecules and of a terminal methyl carbon atom of the alkyl chain. The hydrogen atoms were included in the refinement at idealized geometries (C–H 0.95/0.99 Å) and refined “riding” on the corresponding parent atoms. The weighting schemes used in the last cycle of refinement was w = 1/[σ2Fo2 + (0. 0.1232P)2 + 5.6500P], where P = (Fo2 + 2Fc2)/3. Geometric calculations were performed with the PARST97 program.55 Crystallographic data (excluding structure factors) for the structure reported have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-1831062 and can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge, CB2 IEZ, U.K. (fax: +44-1223-336-033; e-mail deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). Computational Details Calculations were performed adopting the M06 hybrid meta-generalised gradient approximation functional.56 The standard all-electron 6-31G** basis57 was used for all atoms. Molecular geometry optimization of stationary points was carried out without symmetry constraints and used analytical gradient techniques.58 Frequency analysis was performed to obtain thermochemical information about the reaction pathways at 298 K using the harmonic approximation. All calculations were performed using the G16 code59 on Linux cluster systems. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01018.1H NMR spectrum of the EtQxBox cavitand; crystallographic data, refinement details, and ORTEP view of NT@EtQxBox·10DMSO; crystallographic geometrical parameters and view of the host–guest interaction for the complex NT@EtQxBox; thermal desorption experiments for NT vapors; DFT calculated values of the dipole moment of the EtQxBox host and used guests (PDF) Supplementary Material ao8b01018_si_001.pdf Author Present Address ∥ Laboratory of Adsorption and Catalysis, Department of Chemistry, University of Antwerpen (CDE), Universiteitsplein 1, 2610 Wilrijk, Antwerpen, Belgium (C.T.). The authors declare no competing financial interest. Acknowledgments This work was supported by the European Union through the DOGGIES project (Grant FP7-SEC-2011-285446) and University of Catania through the project “Piano della Ricerca di Ateneo 2016–2018”. Centro Interfacoltà di Misure “G. Casnati” and the “Laboratorio di Strutturistica Mario Nardelli” of the University of Parma are kindly acknowledged for the use of NMR and MALDI facilities and of the Diffractometer. ==== Refs References Bhatkhande D. S. ; Pangarkar V. G. ; Beenackers A. C. M. Photocatalytic degradation of nitrobenzene using titanium dioxide and concentrated solar radiation: chemical effects and scaleup . Wat. Res. 2003 , 37 , 1223 –1230 . 10.1016/S0043-1354(02)00490-6 . Rastogi P. K. ; Ganesan V. ; Krishnamoorthi S. Palladium nanoparticles incorporated polymer-silica nanocomposite based electrochemical sensing platform for nitrobenzene detection . Electrochim. Acta 2014 , 147 , 442 –450 . 10.1016/j.electacta.2014.09.128 . Singh S. Sensors-An effective approach for the detection of explosives . J. Hazard. Mater. 2007 , 144 , 15 –28 . 10.1016/j.jhazmat.2007.02.018 .17379401 Sarkar P. K. ; Prajapati P. K. ; Shukla V. J. ; Ravishankar B. ; Choudhary A. K. 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PMC006xxxxxx/PMC6644404.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145802110.1021/acsomega.8b01868ArticleRoom-Temperature, Copper-Free Sonogashira Reactions Facilitated by Air-Stable, Monoligated Precatalyst [DTBNpP] Pd(crotyl)Cl Pohida Katherine Maloney David J. ‡Mott Bryan T. †Rai Ganesha National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States* E-mail: bmott12@uab.edu (B.T.M).* E-mail: bantukallug@mail.nih.gov. Phone: 301-827-1756 (G.R).10 10 2018 31 10 2018 3 10 12985 12998 03 08 2018 26 09 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A novel application of [DTBNpP] Pd(crotyl)Cl (DTBNpP = di-tert-butylneopentylphosphine) (P2), an air-stable, commercially available palladium precatalyst that allows rapid access to a monoligated state, has been identified for room-temperature, copper-free Sonogashira couplings of challenging aryl bromides and alkynes. The mild reaction conditions with TMP in dimethyl sulfoxide afford up to 97% yields, excellent functional group tolerability, and broad reaction compatibility with access to one-pot indole formation. document-id-old-9ao8b01868document-id-new-14ao-2018-01868wccc-price ==== Body Introduction The Sonogashira reaction, catalyzing C(sp)-C(sp2) bond formation, is a vital tool in academia and industry, for its ability to increase conjugation and rigidity in natural product synthesis, drug development, molecular electronics, nanoscale scaffolds, and heterocyclic chemistry.1−4 Historically, the use of copper co-catalysts with palladium allowed for mild reaction conditions due to transmetallation, as reported by Sonogashira.5 However, undesirable homocoupling of acetylenes, air sensitivity, moisture sensitivity, and difficulties in pharmaceutical purification processes prompted the need for well-refined, copper-free systems while broadening the scope for challenging substrates. Classic addition of a ligand to an air-stable Pd-source (such as Pd(OAc)2 or Pd2(dba)3) may encounter disruption from inadvertent ligand coordination, catalyst impurity, or delayed catalyst generation due to in situ formation.6−10 Even with the variety of palladium sources and ligands currently available, arguably the most popular conditions for Sonogashira reactions are Pd(PPh3)4, a catalyst lacking air stability, and Pd(PPh3)2Cl2. These reactions frequently require high temperatures, copper salts, or additional ligands that may be pyrophoric or noncommercial, limiting feasibility.11 Among newly published protocols some call for atypical solvents, additives, or catalysts, and many requiring long reaction times and show few examples of pharmaceutically relevant heteroaromatics.12−17 Although few attractive methods are available in the literature,18,19 it is still desirable to develop a more robust protocol broadly applicable for complex substrates. Recently developed preformed palladacycles20 and palladium dimers21,22 allow for a defined ligand ratio and exhibit greater catalytic rates than their individual counterparts, exemplifying the utility of preformed palladium complexes for numerous carbon–carbon couplings. Additionally, these palladium precatalysts pose an improvement to traditional catalysts by exhibiting greater stability and feasibility in reaction set up while still providing the same active catalyst. Despite the coordinative potential of palladium, monoligated adducts (L1Pd0) frequently produce a more efficient catalyst and thus there is a desire for precatalysts capable of producing monoligated palladium in situ.23−25 The proposed mechanism for monoligated precatalysts begins by activation to the LPd0 state, utilizing bulky, electron-rich ligands to stabilize a reactive catalytic species that facilitates faster oxidative addition and efficient catalytic cycle.26,27 One of the prominent monoligated precatalyst backbones was introduced by Buchwald in 2007: air-stable palladacycles that are readily activated by deprotonation under mild reaction conditions to obtain the monoligated Pd0 complex via reductive elimination of the intramolecular amine–aryl group.28−32 Another successful, commercially available precatalysts synthesized in 2002 by Nolan and co-workers utilize an allyl-based palladium precatalyst that incorporates the NHC-carbene ligand. This monoligated precatalyst is air stable, room-temperature activated and capable of performing efficient Suzuki, Buchwald, and other cross-couplings.33,34 In 2010, Shaughnessy35 and Colacot36 found success replacing the NHC-ligand on Pd(allyl)Cl complexes with bulky phosphines or varying the π-allyl substituents in Suzuki, α-arylation, and amination reactions. These base-promoted, bench-stable precatalysts reductively eliminate a noninhibitory olefin byproduct while creating an active palladium complex proficient in a wide range of cross-coupling reactions, including Sonogashira.27,37−39 Results and Discussion The Sonogashira coupling has become a prominent intermediate step and functional group addition for medicinal chemistry projects. A quick survey of prominent medicinal chemistry journals finds 18 references already this year and 75 in 2017 employing the alkyne coupling, yet, Pd(PPh3)4, Pd(PPh3)2Cl2, and palladium salts with copper-dominated catalyst conditions. During our medicinal chemistry efforts in synthesizing novel inhibitors of human lactate dehydrogenase, we required a reliable and facile Sonogashira coupling condition for library synthesis. These classical conditions were minimally successful with our diversely functionalized substrates and risked catalyst poisoning by incorporating nitrogen and sulfur containing aryl groups, especially when attempting scale-up procedures. We sought an improved catalyst and optimized condition applicable to a wide variety of functional groups for application in our work as well other medicinal chemistry projects that experienced limitations similar to those listed above. The initial catalyst search was based off the work of Soheili et al.,40 who performed Sonogashira couplings at room temperature with allyl palladium chloride and P(t-Bu)3, without copper, and hypothesized the formation of a monoligated active L1Pd0 catalyst using these substrates. In view of the success of Buchwald and allyl monoligated palladium catalysts in numerous cross-coupling reactions,41 we envisioned that preformed Buchwald and allyl monoligated palladium precatalysts with bulky, electron-rich phosphines, have the capability to be a bench-stable, monoligated precatalyst for copper-free Sonogashira couplings. These next generation catalysts would provide a powerful synthetic solution by expanding the scope, increasing catalytic rates, and eliminating the use of pyrophoric ligands. Herein, we report the catalytic efficiencies of several Buchwald palladacycles and allyl monoligated palladium catalysts (Figure 1) applied to the Sonogashira reaction. Figure 1 Currently established palladium precatalysts: P1–P4, P6–P15. Ligand for in situ catalyst: L1. P5 was synthesized in house. Initial efforts were focused on the screening of precatalysts P1–P14 using challenging coupling partners. The 3,5-dimethoxyphenyl bromide (1), which does not give any product under classical Sonogashira conditions40 (entry 1 in Table 1) due to hindered oxidative addition step, and an electron-deficient heteroaromatic alkyne (2), a representative for difficult heteroaromatic alkynes that are frequently employed in medicinal chemistry and pharmaceutical development, were coupled with base DABCO in tetrahydrofuran (THF), conditions similar to those used by Soheli et al. For comparison, several difficult electron-rich substrates, such as p-bromophenol (entry 5m), p-bromoaniline (entry 5n), or 3-bromothiophene (entry 5r), were coupled using our protocol. The π–allyl palladium-based catalyst (P1), which incorporated P(t-Bu)3 and a crotyl ligand, successfully produced coupling product 3, without copper, in moderate yield (52%, entry 2). In changing the phosphine ligand to DTBNpP (P2) and XPhos (P4) with the same palladium precatalyst, the P2 catalyst revealed to be a more efficient catalyst with a 75% yield (entries 3 and 5).42 However, the activity diminished dramatically when P(t-Bu)3 was replaced with P(Cy)3 (P3, entry 4). Attempting to improve yields with the η3-1-t-Bu-indenyl ligand (P5) (investigated by Hazari et al.)43 led to only 53% yield (entry 6). The original π–allyl palladium complex without a bulky phosphine ligand, unsurprisingly showed no appreciable product (entries 7 and 8). Switching to Buchwald type precatalysts, both the P8 and P9 catalysts that contain the P(t-Bu)3 ligand were able to produce the desired product, but much less effectively than P2 (23–27% yield, entries 9 and 10). However, P10 and P11 incorporating the DTBNpP ligand markedly increased the yields to 63 and 56%, respectively (entries 11 and 12). Furthermore, P12 and P13 catalysts with P(t-Bu)2(Me) and P(t-Bu)2(n-Bu) ligand, respectively, almost completely diminished catalyst activity (entries 13 and 14), proving the specificity of the DTBNpP ligand for this system. The catalyst P14, a precursor to L1Pd0 and successfully used in amination reactions,10 demonstrated negligible activity within these conditions (entry 15). Additionally, a commercially available NHC precatalyst, P15, with a cinnamyl and chloride ligand similar to the phosphine-based allyl precatalysts, was tested for comparison; however, it afforded no product (entry 16). After finding P2 as the most efficient precatalyst, we then compared its reactivity with the catalyst formed in situ. The catalyst formed from 1:1 (Pd/P) ratio of P6/L1 exhibits a slightly higher yield after 18 h compared to P2, but a lower yield within the first hour, likely due to formation of the active catalyst (entry 17). Exchanging the crotyl ligand for cinnamyl to form the in situ catalyst exhibited no significant improvement and provided comparable product formation over time (entry 18). The addition of L1 to P2 (1:1) significantly retarded P2 activity with almost no conversion observed during the first 3 h (entry 19). However, the activity progressed gradually to produce a similar yield as independent P2 after 18 h (Figure 2 and entry 17 vs entry 3). This could be due to the initial formation of the coordinately saturated 14-electron L2Pd0. The remainder of catalysts in Figure 2 tapered off their rate of product formation after 6 h, except for P10, which exhibited an increase in activity after 3 h possibly due to delayed release of the aromatic amine. A comparison of the DTBNpP-containing catalysts in Figure 2 demonstrates the efficiency of P2 compared to its individual ligand/palladium sources and over the Buchwald precatalysts for the Sonogashira reaction. Figure 2 Conversion of 2 over an 18 h period using catalysts containing the DTBNpP ligand. Table 1 Catalyst Screening with 1-Bromo-3,5-dimethoxybenzene (1) and 3-Ethynylpyridine (2)a entry catalyst 3 (yield, %)b 1 Pd(PPh3)2Cl2, CuI 0c 2 P1 52 3 P2 75 4 P3 0 5 P4 57 6 P5 53 7 P6 0 8 P7 0 9 P8 27 10 P9 23 11 P10 63 12 P11 56 13 P12 0 14 P13 4 15 P14 1 16 P15 0 17 P6 with L1 (1:1) 84d 18 P7 with L1 (1:1) 78d 19 P2 with L1 (1:1) 71d 20 P2 2e a Reaction conditions: 1 (0.5 mmol), 2 (0.8 mmol), cat. (.025 mmol), DABCO (1.0 mmol), THF (2.5 mL), rt for 18 h under argon atmosphere. b Yield was determined by liquid chromatography/mass spectrometry (LC/MS) with pyrene as internal standard. c Followed standard Sonogashira reaction procedure. d Catalyst and ligand stirred for 5 min prior to reagent addition. e Without degassing using argon or nitrogen. With the finding of efficient catalyst P2, the effects of base and solvent were next examined (Table 2). By using DABCO as the base, the reaction achieved less than 50% yield with nonpolar (e.g., DCM and MTBE) or polar protic solvents (e.g., MeOH and EtOH) (entries 1–4). In contrast, using polar aprotic solvents (e.g., THF, ACN, DMF, and DMSO) provided better conversions with 62–100% yield, DMSO being the best solvent in this reaction condition (entries 5, 7, 8, and 11). Several exceptions were also observed during the solvent screening. For instance, a good yield (74%) was observed by using the nonpolar 1,4-dioxane solvent (entry 6), but the reaction performed poorly in NMP, a polar solvent (40% yield, entry 9). Moderate yields were found by using Lipshutz’s Sonogoshira protocol in water using 3% nonionic amphiphile PTS (entry 10).44 DMSO was carried through the screening of various bases (entries 12–29). As expected, the lack of base afforded no product (entry 12). A wide variety of inorganic bases produced excellent yield over the course of 18 h (entries 13–16). Among them, NaOAc was the only one that reached a high yield (86%) in 2 h (entry 13). Although KHCO3 as the base is less effective, Cs2CO3 and TBAF were found to be totally ineffective (entries 17–19). Continuously, various organic bases were screened under the same conditions. Sterically hindered amines, such as TMP, t-BuNH2, (i-Pr)2NH, and Hunig’s base, reached high yields (>84%) in 2 h (entries 20–24). However, using Et2NH and Et3N as the bases only produced product in moderate yield (56–58%) after 18 h (entries 24 and 25). Some cyclic amines, e.g., pyrrolidine and piperidine, were also effective bases, but required 18 h to reach completion (entries 26 and 27). Morpholine and DBU were found to be much less effective (entries 28 and 29). Of the bases, (i-Pr)2NH and TMP afforded product 3 (100%) within 2 h, making them both attractive bases for this reaction. TMP was used in subsequent optimization due to faster product formation within 2 h; although, (i-Pr)2NH is a valuable cost-effective substitute. Table 2 Optimization of Base and Solvent in the Coupling of 1 and 2a 3 (yield, %)b entry base solvent t = 2 h t = 18 h 1 DABCO MTBE 42 54 2 DABCO DCM 25 35 3 DABCO MeOH 27 35 4 DABCO EtOH 26 40 5 DABCO THF 46 72 6 DABCO 1,4-dioxane 40 74 7 DABCO ACN 67 82 8 DABCO DMF 42 62 9 DABCO NMP 26 40 10 DABCO 3 wt % PTS in H2O 33 50 11 DABCO DMSO 91 100 12 none DMSO 0 0 13 NaOAc DMSO 86 100 14 KOH DMSO 50 100 15 K2CO3 DMSO 50 100 16 K3PO4 DMSO 51 100 17 KHCO3 DMSO 26 43 18 Cs2CO3 DMSO 0 0 19 TBAF DMSO 0 0 20 t-BuNH2 DMSO 86 89 21 iPr2NH DMSO 100 100 22 Hunigs’ base DMSO 84 100 23 TMP DMSO 100 100 24 Et2NH DMSO 15 56 25 Et3N DMSO 53 58 26 pyrrolidine DMSO 27 100 27 piperidine DMSO 42 100 28 morpholine DMSO 20 58 29 DBU DMSO 2 10 a Reaction conditions: 1 (0.5 mmol), 2 (0.8 mmol), P2 (0.025 mmol, 5 mol %), base (1.0 mmol), solvent (2.5 mL), rt for 18 h under argon atmosphere. b Yield was determined by LC/MS with pyrene as internal standard. TMP was re-evaluated with alternative and sustainable solvents45 to broaden the applicability of these conditions for purposes, such as process chemistry (Table S1). Although the THF/TMP combination was not as productive as DMSO/TMP (Table S1, entry 1), high yields were still obtained after 18 h (70%). Sustainable solvents, such as 2-MeTHF and sulfolane, were able to provide acceptable yields after 18 h if a greener solvent is desired (Table S1, entries 2 and 3). EtOAc, a solvent recommended for environmentally friendly chemistry and a top 10 solvent used in GSK pilot operations in 2005, was able to produce a 62% yield in 18 h that could likely be increased with heating (Table S1, entry 4). ACN was the most effective solvent from this table (Table S1, entry 5) with product formation similar to DMSO as the solvent, a positive result considering its use in process chemistry and recommended use in medicinal chemistry. TMP and DMSO were chosen to evaluate catalyst loading (Table 3). The 5 mol % catalyst loading produced a 96% yield in only 0.5 h, whereas a 2.5 mol % achieved 77% (entries 1 and 2). Both conditions were highly effective, reaching 100% yield in 1.5 h. Decreasing loading to 1 or cot0.5 mol % resulted in significant product formation delays (48 and 42%, respectively), though both went to completion by 18 h (entries 3 and 4). Increasing the temperature to 60 and 100 °C allowed the catalyst load to be decreased to 0.5 mol % while still achieving 80 and 85% yield, respectively, in 0.5 h. For our purposes, a 2.5 mol % catalyst load was chosen to limit palladium usage while retaining rapid product formation at room temperature. Table 3 Effect of Catalyst Loading in the Coupling of 1 and 2a 3 (yield, %)b entry catalyst load (%) temperature (°C) t = 0.5 h t = 1.5 h t = 18 h 1 5 rt 96 100 100 2 2.5 rt 77 100 (86)c 100 3 1.0 rt 25 48 92 4 60 100 100 100 5 100 100 100 100 6 0.5 rt 15 42 88 7 60 80 97 100 8 100 85 93 100 9 0.1 60 15 33 56 10 100 25 26 39 11 0.01 60 0 0 2 12 100 4 5 10 a Reaction conditions: 1 (0.5 mmol), 2 (0.8 mmol), [DTBNpP] Pd(crotyl)Cl, TMP (1.0 mmol), DMSO (2.5 mL), rt for 18 h under argon atmosphere. b Yield was determined by LC/MS with pyrene as internal standard. c Parentheses indicate an isolated yield. The attention was then shifted to exploration of coupling partner scope using the optimized reaction condition, namely, P2 (2.5 mol %), TMP, and DMSO. Various bromides were tested, and the results are summarized in Scheme 1. In general, electron-withdrawing group-substituted aryl bromides (5a–d and 5f–i) and heteroaryl bromides (5o–r) were completed within 2–4 h with high isolated yields (65–92%). The exception was the 2-CF3 substitution (5e), requiring 18 h to reach completion. Though the aryl bromides with electron-donating substitutions (5j–n) had slightly lower yields (52–78%), the electronic substitution effect seemed minimal. These examples also demonstrated excellent tolerability of functional groups, including nitrile, nitro group, ketone, carboxylic acid, ester, amide, as well as unprotected hydroxyl (5l, 5m) and amino (5n) groups. However, some heterocycles, such as 5t–aa, required slightly elevated temperature (60 °C) to push to completion with moderate to good yields (42–87%). Despite the challenging sterics of 4-bromo-3,5-dimethylisoxazole, it achieved 61% conversion over 32 h, however, difficulty in purification likely due to instability limited isolated yields (5z). In addition, an 87% yield was attained using 4-bromo-1H-indole in 6 h (5aa). The present protocol is not applicable for aryl chlorides, as exemplified by 5i, only exhibiting bromide coupling. Scheme 1 Scope of Bromides,, Reaction conditions: 4 (0.5 mmol), 2 (0.8 mmol), P2 (2.5 mol %), TMP (1.0 mmol), DMSO (2.5 mL), rt under argon atmosphere. Isolated yield. Stir at rt for 3 h then increase temperature to 60 °C. With the broad scope of bromides obtained, various aryl- and alkyl-substituted alkynes with electron-donating bromide 1 were then evaluated (Scheme 2). The phenylacetylene and aryl acetylene with electron-donating groups typically proceeded to completion in 2 h with excellent isolated yields (7a–c). The heteroaryl acetylene, such as N-methylated imidazole (7d), and aryl acetylene with electron-withdrawing substitutions (7e–g) required prolonged reaction time (12–24 h) to secure a good yield, but were still completed at room temperature. Finally, the alkenyl- (7h) and alkyl (7i–n)-substituted alkynes were efficiently coupled with 1 under similar conditions to produce the desired product in excellent yield (75–96%). Scheme 2 Coupling of 1 with Aryl and Alkyl Acetylenes,, Reaction conditions: 4 (0.5 mmol), 6 (0.8 mmol), P2 (2.5 mol %), TMP (1.0 mmol), DMSO (2.5 mL), rt with argon atmosphere. Isolated yield. Stir at rt for 3 h then increase temperature to 60 °C. To test the feasibility in large-scale preparation, a 2 g scale reaction was performed on 1 and 2 using 2.5 mol % catalyst loading. To our gratification, the reaction reached 100% conversion in 2 h with 92% isolated yield that further confirmed the efficiency and effectiveness of this coupling protocol (Scheme 3). Scheme 3 Indole Synthesis via Sonogashira Coupling with 2-Bromoanilines, Reaction conditions: 1. 8 (0.5 mmol), 9 (0.63 mmol), P2 (5.0 mol %), TMP (1.0 mmol), ACN (2.0 mL), rt under argon atmosphere. 2. HCl (2.5 mmol), 90 °C. Isolated yield. To further test the utility of these conditions, 2-bromoanilines were coupled with various alkynes in hopes of intramolecular cyclization to form the indole. Moderate yields were achieved using an unoptimized one-pot indole synthesis utilizing the Sonogashira protocol followed by refluxing in the presence of concentrated HCl. The method accommodated both electron-withdrawing and electron-donating functionalities on the bromide (10a–d), producing isolated yields up to 78%. Additionally, the cyclization tolerated alkynes containing an alkyl or phenol substituent while still maintaining yields around 55% (10e, 10f). With further optimization, we believe this one-pot approach may provide a rapid pathway toward diverse indole library synthesis. Conclusions In summary, a robust, copper-free Sonogashira coupling reaction is described. Through extensive optimization campaign, the homogeneous precatalyst P2, [DTBNpP] Pd(crotyl)Cl, was found to be the most effective catalyst. Together with optimized base (TMP) and solvent (DMSO), this condition provides a simple, mild, scalable, and versatile alternative for the coupling of variety of aryl bromides and alkynes. Additionally, this precatalyst provides rapid access to indoles via the one-pot method, further expanding on the utility of P2. We believe the preformed, air-stable P2 provides a reliable and more effective alternative to the commonly used catalysts, such as Pd(PPh3)4 and PdCl2(PPh3)2, by retaining or surpassing coupling efficiencies, bypassing in situ catalyst formation, forgoing usage of pyrophoric agents, and decreasing the likelihood of catalyst poisoning. A broad scope of both coupling partners together with high functional group tolerability and excellent isolated yield makes it an attractive addition to the existing Sonogashira coupling conditions for chemical library generation, medicinal chemistry, and with potential use in process chemistry. Experimental Section All commercial solvents and reagents were purchased from commercial sources and used without alteration. The modified water solvent (3% PTS in H2O) was created using a 15% PTS in H2O stock from Sigma-Aldrich and water from our lab, heating to achieve uniform consistency. Precatalysts were purchased from Strem Chemicals (46-0028 (P8), 46-0365 (P13), 46-0358 (P11), 46-0385 (P9), 46-0275 (P6), and 46-0295 (P7)), Johnson Matthey (Pd-162 (P1), Pd-163 (P2), Pd-170 (P4), Pd-178 (P3), and Pd-113 (P14)), and Sigma-Aldrich (RNI00185 (P10) and 794198 (P12)). Additional P2 was synthesized with procedures published by Seechurn et al.36 NMR data comparing the commercially available and homemade catalyst is provided in spectral data. Reaction monitoring was performed on the Agilent 1200 series LC/MS containing a Luna C18 (3 mm × 75 mm, 3 μm) reversed-phase column, utilizing UV detection at λ = 220 nm. The LC/MS ran a 3 min gradient spanning 4–100% acetonitrile in H2O modified by trifluoroacetic acid (0.05%) at a flow rate of 0.8 mL/min. Flash column chromatography was carried out on Teledyne Isco CombiFlash Rf+ systems using 24G Isco Silica Gel columns (230–400 mesh) and HPLC grade solvents. Products purified on 1H NMR and 13C NMR spectra were analyzed by the Varian 400 MHz in DMSO-d or CDCl3. 1H NMR spectra were referenced to 7.26 ppm for CDCl3 and 2.50 ppm for DMSO-d. 13C NMR were referenced to 77.23 ppm for CDCl3 and 39.5 ppm for DMSO-d6. 31P NMR was referenced externally to 0.00 ppm for H3PO4. Splitting patterns are reported as: singlet (s), doublet (d), triplet (t); quartet (q), septet (s), and other combinations of these patterns. Coupling constants are reported in Hertz. High-resolution mass spectrometry (HRMS) was obtained by the Agilent 6210 Time-of-Flight (TOF) LC/MS system. Proton and sodium adducts may be observed from the salt exposure in the purification and mass spectrometry instrument. In certain cases, heating was utilized. General Procedure for Synthesis of (3), (5), and (7) To an oven-dried Biotage microwave process vial (#355630), bromide (0.50 mmol, 1.00 equiv), alkyne(0.75 mmol, 1.50 equiv), 2,2,6,6-tetramethylpiperidine (169 μL, 1.00 mmol, 2.00 equiv), and P2 (5.17 mg, 0.013 mmol, 0.025 equiv) were added to DMSO (1.5 mL). Remaining DMSO (1 mL) was added to rinse the sides. The reaction vessel was sealed and bubbled with in-house argon for 5 min. The reaction stirred at room temperature for the designated time. Work up involved ammonium chloride, EtOAc, and brine. The organic layer was concentrated and purified on Isco silica gel columns to give the resulting product using a gradient of 0–100% for ethyl acetate/hexanes with a 0.1% NH4OH modifier, 0–20% for methanol/DCM with a 0.1% NH4OH modifier, or 0–100% water/ACN with a 0.1% TFA modifier. 3-((3,5-Dimethoxyphenyl)ethynyl)pyridine (3) Tan solid. Yield: 97 mg, 85%. Time: 1.5 h. 1H NMR (400 MHz, DMSO-d6) δ 8.75 (dd, J = 2.2, 1.0 Hz, 1H), 8.59 (dd, J = 4.9, 1.6 Hz, 1H), 7.97 (dt, J = 7.9, 1.9 Hz, 1H), 7.46 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 6.75 (d, J = 2.3 Hz, 2H), 6.59 (t, J = 2.3 Hz, 1H), 3.78 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 160.41, 151.59, 149.02, 138.51, 123.57, 123.07, 119.22, 109.17, 102.26, 92.29, 85.64, 55.40. HRMS (ESI+) in m/z: Expected 240.1019 [M + H+] (C15H14NO2+). Observed 240.1015. 4-(Pyridin-3-ylethynyl)benzonitrile (5a) While solid. Yield: 92 mg, 90%. Time: 0.5 h. 1H NMR (400 MHz, DMSO-d6) δ 8.84–8.77 (m, 1H), 8.63 (dd, J = 4.9, 1.7 Hz, 1H), 8.03 (dt, J = 7.9, 1.9 Hz, 1H), 7.95–7.88 (m, 2H), 7.82–7.75 (m, 2H), 7.50 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.78, 149.60, 138.79, 132.62, 132.23, 126.51, 123.65, 118.59, 118.29, 111.39, 90.68, 89.96. HRMS (ESI+) in m/z: Expected 528.1904 [M + H+] (C14H9N2+). Observed 205.0758. 5a is a known compound.46 1-(4-(Pyridin-3-ylethynyl)phenyl)ethan-1-one (5b) White solid. Yield: 102 mg, 92%. Time: 0.5 h. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (dd, J = 2.3, 0.9 Hz, 1H), 8.62 (dd, J = 4.9, 1.6 Hz, 1H), 8.08–7.96 (m, 3H), 7.78–7.69 (m, 2H), 7.49 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 2.61 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 197.18, 151.71, 149.38, 138.68, 136.56, 131.69, 128.45, 126.17, 123.62, 118.87, 91.42, 88.97, 26.73. HRMS (ESI+) in m/z: Expected 222.0913 [M + H+] (C15H12NO+). Observed 222.0914. 5b is a known compound.47 3-(Phenylethynyl)pyridine (5c) Tan solid. Yield: 79 mg, 88%. Time: 1 h. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (dd, J = 2.2, 0.9 Hz, 1H), 8.59 (dd, J = 4.9, 1.6 Hz, 1H), 7.98 (dt, J = 7.9, 2.0 Hz, 1H), 7.59 (ddd, J = 6.7, 3.0, 1.6 Hz, 2H), 7.46 (tt, J = 6.2, 1.8 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 151.55, 148.96, 138.47, 131.43, 129.18, 128.77, 123.56, 121.64, 119.32, 92.21, 86.08. HRMS (ESI+) in m/z: Expected 202.0627 [M + Na+] (C13H9NNa+). Observed 202.0634. 5c is a known compound.48 3-((3-Nitrophenyl)ethynyl)pyridine (5d) Tan solid. Yield: 99 mg, 87%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 2.1 Hz, 1H), 8.63 (dd, J = 4.9, 1.7 Hz, 1H), 8.39 (t, J = 1.9 Hz, 1H), 8.28 (ddd, J = 8.4, 2.5, 1.1 Hz, 1H), 8.04 (ddt, J = 7.6, 5.6, 1.6 Hz, 2H), 7.75 (t, J = 8.0 Hz, 1H), 7.55–7.44 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.82, 149.53, 147.89, 138.79, 137.54, 130.49, 125.97, 123.85, 123.64, 123.25, 118.62, 89.89, 88.20. HRMS (ESI+) in m/z: Expected 225.0659 [M + H+] (C13H9N2O2+). Observed 225.0657. 3-((2-(Trifluoromethyl)phenyl)ethynyl)pyridine (5e) Yellow oil. Yield: 88 mg, 71%. Time: 18 h. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (dd, J = 2.1, 1.0 Hz, 1H), 8.64 (dd, J = 4.9, 1.7 Hz, 1H), 7.97 (dt, J = 7.9, 1.9 Hz, 1H), 7.86 (dd, J = 7.6, 1.4 Hz, 2H), 7.80–7.71 (m, 1H), 7.66 (tdd, J = 8.6, 2.2, 1.1 Hz, 1H), 7.50 (ddd, J = 7.9, 4.9, 1.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.42, 149.59, 138.53, 134.03, 129.57, 123.74, 122.13, 119.45, 118.70, 91.26, 87.89. HRMS (ESI+) in m/z: Expected 248.0682 [M + H+] (C14H9F3N+). Observed 248.0675. 5e is a known compound.49 3-(Pyridin-3-ylethynyl)benzoic Acid (5f) Tan solid. Yield: 69 mg, 65%. Time: 4 h. 1H NMR (400 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.80 (dd, J = 2.2, 0.9 Hz, 1H), 8.61 (dd, J = 4.9, 1.6 Hz, 1H), 8.10 (t, J = 1.7 Hz, 1H), 8.01 (ddt, J = 13.2, 7.8, 1.7 Hz, 2H), 7.83 (dt, J = 7.7, 1.4 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.49 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.38, 151.61, 149.09, 138.74, 135.37, 132.08, 131.42, 129.79, 129.29, 123.63, 122.08, 119.08, 91.21, 86.83. HRMS (ESI+) in m/z: Expected 224.0706 [M + H+] (C14H10NO2+). Observed 224.0706. 4-(Pyridin-3-ylethynyl)benzaldehyde (5g) Tan solid. Yield: 91 mg, 88%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.83–8.77 (m, 1H), 8.63 (dd, J = 4.9, 1.7 Hz, 1H), 8.03 (dt, J = 7.9, 2.0 Hz, 1H), 8.00–7.94 (m, 2H), 7.84–7.77 (m, 2H), 7.50 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 192.42, 151.75, 149.49, 138.74, 135.82, 132.13, 129.64, 127.44, 123.65, 118.77, 91.35, 89.54. HRMS (ESI+) in m/z: Expected 208.0757 [M + H+] (C14H10NO+). Observed 208.0755. 5g is a known compound.50 Methyl 3-(Pyridin-3-ylethynyl)benzoate (5h) Tan solid. Yield: 103 mg, 87%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (dd, J = 2.2, 1.0 Hz, 1H), 8.61 (dd, J = 4.9, 1.7 Hz, 1H), 8.14–8.08 (m, 1H), 8.05–7.97 (m, 2H), 7.86 (dt, J = 7.8, 1.4 Hz, 1H), 7.65–7.58 (m, 1H), 7.48 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 3.88 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 165.35, 151.71, 149.23, 138.65, 135.77, 131.87, 130.24, 129.59, 129.47, 123.58, 122.29, 118.97, 90.95, 87.07, 52.37. HRMS (ESI+) in m/z: Expected 238.0863 [M + H+] (C15H12NO2+). Observed 238.0851. 3-((4-Chlorophenyl)ethynyl)pyridine (5i) White solid. Yield: 95 mg, 89%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J = 2.6 Hz, 1H), 8.60 (dd, J = 4.3, 2.2 Hz, 1H), 7.99 (dd, J = 8.0, 2.3 Hz, 1H), 7.66–7.57 (m, 2H), 7.52 (dd, J = 8.7, 2.2 Hz, 2H), 7.50–7.43 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.63, 149.19, 138.58, 133.97, 133.22, 129.00, 123.63, 120.56, 119.08, 91.06, 87.17. HRMS (ESI+) in m/z: Expected 214.0418 [M + H+] (C13H9ClN+). Observed 214.0413. 5i is a known compound.51 3-([1,1′-Biphenyl]-2-ylethynyl)pyridine (5j) Yellow oil. Yield: 101 mg, 79%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.58–8.46 (m, 2H), 7.78–7.68 (m, 2H), 7.68–7.62 (m, 2H), 7.58–7.38 (m, 7H). 13C NMR (101 MHz, DMSO-d6) δ 151.20, 148.90, 143.44, 139.59, 138.05, 132.79, 129.64, 129.53, 129.07, 128.12, 127.79, 127.58, 123.62, 119.89, 119.48, 92.24, 88.64. HRMS (ESI+) in m/z: Expected 256.1121 [M + H+] (C19H14N+). Observed 256.1118. Anal. Calcd for C19H13N: C, 88.86; H, 5.39; N: 5.76. Found: C, 88.75; H, 5.14; N, 5.38. 3-(p-Tolylethynyl)pyridine (5k) Tan solid. Yield: 75 mg, 78%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (dd, J = 2.2, 1.0 Hz, 1H), 8.58 (dd, J = 4.9, 1.6 Hz, 1H), 7.96 (dt, J = 7.9, 1.9 Hz, 1H), 7.53–7.40 (m, 3H), 7.26 (d, J = 7.8 Hz, 2H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 151.48, 148.79, 139.05, 138.37, 131.35, 129.39, 123.54, 119.52, 118.63, 92.45, 85.51, 21.02. HRMS (ESI+) in m/z: Expected 194.0964 [M + H+] (C14H12N+). Observed 194.0958. 5k is known compound.52 2-Hydroxy-5-(pyridin-3-ylethynyl)benzamide (5l) White solid. Yield: 90 mg, 76%. Time: 6 h. 1H NMR (400 MHz, DMSO-d6) δ 13.42 (s, 1H), 8.75–8.69 (m, 1H), 8.61–8.50 (m, 2H), 8.17 (d, J = 2.1 Hz, 1H), 8.05 (s, 1H), 7.94 (dt, J = 7.9, 1.9 Hz, 1H), 7.62 (dd, J = 8.6, 2.0 Hz, 1H), 7.46 (ddd, J = 8.0, 4.9, 0.9 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 171.10, 161.73, 151.35, 148.73, 138.25, 136.89, 131.74, 123.60, 119.57, 118.21, 114.84, 111.58, 92.01, 84.57. HRMS (ESI+) in m/z: Expected 239.0815 [M + H+] (C14H11N2O2+). Observed 239.0808. Anal. Calcd for C14H10N2O2: C, 70.58; H, 4.23; N, 11.76. Found: C, 70.48; H, 4.31; N, 11.78. 4-(Pyridin-3-ylethynyl)phenol (5m) Tan solid. Yield: 50 mg, 52%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.70 (d, J = 2.1 Hz, 1H), 8.54 (dd, J = 4.9, 1.6 Hz, 1H), 7.91 (dt, J = 7.9, 1.9 Hz, 1H), 7.48–7.36 (m, 3H), 6.85–6.77 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.40, 151.31, 148.42, 138.13, 133.16, 123.51, 119.96, 115.78, 111.78, 93.04, 84.18. HRMS (ESI+) in m/z: Expected 196.0759 [M + H+] (C13H10NO+). Observed 196.0757. 4-(Pyridin-3-ylethynyl)aniline (5n) Tan solid. Yield: 60 mg, 62%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (d, J = 2.1 Hz, 1H), 8.50 (dd, J = 4.9, 1.7 Hz, 1H), 7.86 (dt, J = 7.9, 2.0 Hz, 1H), 7.40 (dd, J = 7.9, 4.9 Hz, 1H), 7.28–7.19 (m, 2H), 6.63–6.52 (m, 2H), 5.63 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 151.07, 149.86, 147.92, 137.78, 132.70, 123.46, 120.48, 113.57, 107.33, 94.49, 83.35. HRMS (ESI+) in m/z: Expected 195.0917 [M + H+] (C13H11N2+). Observed 195.0919. 5n is a known compound.53 1,2-Di(pyridin-3-yl)ethyne (5o) Tan solid. Yield: 83 mg, 92%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.80 (dd, J = 2.2, 1.0 Hz, 2H), 8.62 (dd, J = 4.9, 1.7 Hz, 2H), 8.02 (dt, J = 7.9, 1.9 Hz, 2H), 7.49 (ddd, J = 7.9, 4.9, 0.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 151.66, 149.37, 138.64, 123.62, 118.81, 89.05. HRMS (ESI+) in m/z: Expected 181.076 [M + H+] (C12H9N2+). Observed 181.0762. 5o is a known compound.54 5-(Pyridin-3-ylethynyl)isoquinoline (5p) Tan solid. Yield: 90 mg, 78%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 9.42 (d, J = 1.0 Hz, 1H), 8.93 (dd, J = 2.2, 0.9 Hz, 1H), 8.71–8.61 (m, 2H), 8.27–8.19 (m, 2H), 8.15 (dt, J = 7.9, 2.0 Hz, 1H), 8.10 (dd, J = 7.2, 1.1 Hz, 1H), 7.75 (dd, J = 8.2, 7.2 Hz, 1H), 7.52 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 152.91, 151.80, 149.33, 144.30, 138.73, 134.94, 134.61, 129.08, 127.90, 127.22, 123.63, 119.06, 118.32, 118.09, 91.94, 88.69. HRMS (ESI+) in m/z: Expected 231.0919 [M + H+] (C16H11N2+). Observed 231.0917. Anal. Calcd for C16H10N2: C, 83.46; H, 4.38; N: 12.17. Found: C, 83.22; H, 4.53; N, 12.07. 5-(Pyridin-3-ylethynyl)pyrimidine (5q) Tan solid. Yield: 72 mg, 90%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 9.06 (s, 2H), 8.82 (dd, J = 2.2, 1.0 Hz, 1H), 8.65 (dd, J = 4.9, 1.7 Hz, 1H), 8.04 (dt, J = 7.9, 1.9 Hz, 1H), 7.51 (ddd, J = 7.9, 4.9, 1.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 158.79, 157.14, 151.72, 149.77, 138.77, 123.69, 118.35, 118.33, 92.34, 85.79. HRMS (ESI+) in m/z: Expected 182.0713 [M + H+] (C11H10N3+). Observed 182.0713. 5q is a known compound.55 3-(Thiophen-3-ylethynyl)pyridine (5r) Tan solid. Yield: 72 mg, 78%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 8.73 (dd, J = 2.2, 1.0 Hz, 1H), 8.58 (dd, J = 4.9, 1.6 Hz, 1H), 7.98–7.91 (m, 2H), 7.68 (ddd, J = 5.0, 2.9, 1.0 Hz, 1H), 7.45 (ddd, J = 7.9, 4.9, 1.0 Hz, 1H), 7.30 (dt, J = 5.0, 1.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.42, 148.83, 138.35, 130.65, 129.53, 127.10, 123.56, 120.53, 119.42, 87.83, 85.41. HRMS (ESI+) in m/z: Expected 186.0372 [M + H+] (C11H8NS+). Observed 186.0368. 5r is a known compound.56 Methyl 4-Oxo-6-(pyridin-3-ylethynyl)-1,4-dihydroquinoline-2-carboxylate (5s) Yellow solid. Yield: 38 mg, 25%. Time: 6 h. 1H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 8.86–8.71 (m, 1H), 8.60 (dd, J = 4.8, 1.7 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.05–7.95 (m, 2H), 7.87 (dd, J = 8.7, 2.0 Hz, 1H), 7.48 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 6.69 (s, 1H), 3.97 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.69, 151.71, 149.12, 138.68, 134.78, 128.32, 126.19–124.88 (m), 123.72, 120.13 (d, J = 157.9 Hz), 117.30, 110.62, 91.87, 86.68, 53.62, 29.03. HRMS (ESI+) in m/z: Expected 327.0752 [M + Na+] (C18H12N2O3Na+). Observed 327.0755. 5-(Pyridin-3-ylethynyl)-3,4-dihydroquinolin-2(1H)-one (5t) White solid. Yield: 66 mg, 53%. Time: 4 h. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (dd, J = 2.2, 0.9 Hz, 1H), 8.59 (dd, J = 4.9, 1.6 Hz, 1H), 7.98 (dt, J = 7.9, 2.0 Hz, 1H), 7.59 (ddd, J = 6.7, 3.0, 1.6 Hz, 2H), 7.46 (tt, J = 6.2, 1.8 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 169.90, 151.53, 149.04, 138.76, 138.47, 127.26, 125.40, 125.29, 123.58, 120.49, 119.29, 116.01, 90.28, 90.01, 29.80, 23.28. HRMS (ESI+) in m/z: Expected 249.1025 [M + H+] (C16H13N2O+). Observed 249.1022. 3-(Pyridin-3-ylethynyl)imidazo[1,2-a]pyrimidine (5u) Tan solid. Yield: 46 mg, 42%. Time: 6 h, Temperature: 60 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.17 (dd, J = 6.8, 1.9 Hz, 1H), 8.90 (dd, J = 2.2, 0.9 Hz, 1H), 8.70 (dd, J = 4.1, 2.0 Hz, 1H), 8.62 (dd, J = 4.9, 1.7 Hz, 1H), 8.22 (s, 1H), 8.10 (dt, J = 7.9, 1.9 Hz, 1H), 7.51 (ddd, J = 8.0, 4.9, 1.0 Hz, 1H), 7.29 (dd, J = 6.8, 4.2 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.91, 151.34, 149.16, 148.33, 139.63, 138.15, 134.53, 123.55, 118.84, 110.17, 106.07, 95.71, 79.04. HRMS (ESI+) in m/z: Expected 221.0822 [M + H+] (C13H9N4+). Observed 221.0826. 1-Methyl-4-(pyridin-3-ylethynyl)-1H-pyrazole-3-carbaldehyde (5v) Tan solid. Yield: 69 mg, 63%. Time: 20 h, Temperature: 60 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.98–9.93 (m, 1H), 8.71 (dd, J = 2.2, 1.0 Hz, 1H), 8.58 (dd, J = 4.9, 1.6 Hz, 1H), 8.31 (s, 1H), 7.93 (dt, J = 7.9, 1.9 Hz, 1H), 7.46 (ddd, J = 7.9, 4.9, 1.0 Hz, 1H), 4.00 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 184.92, 151.33, 149.42, 148.91, 138.30, 136.69, 123.59, 119.49, 102.13, 88.69, 82.95. HRMS (ESI+) in m/z: Expected 212.0818 [M + H+] (C12H10N3O+). Observed 212.0822. 3-((1-Methyl-1H-imidazol-5-yl)ethynyl)pyridine (5w) Tan solid. Yield: 63 mg, 69%. Time: 24 h, 60 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (dd, J = 2.2, 1.0 Hz, 1H), 8.59 (dd, J = 4.9, 1.6 Hz, 1H), 7.98 (dt, J = 7.9, 1.9 Hz, 1H), 7.81 (s, 1H), 7.47 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 7.37 (d, J = 1.0 Hz, 1H), 3.73 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 151.22, 149.01, 139.85, 138.12, 134.44, 123.57, 119.03, 114.54, 92.74, 80.76, 31.73. HRMS (ESI+) in m/z: Expected 184.0869 [M + H+] (C11H10N3+). Observed 184.0868. 5w is a known compound.57 4-(Pyridin-3-ylethynyl)furan-2-carbaldehyde (5x) Brown solid. Yield: 101 mg, 83%. Time: 20 h, Temperature: 60 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.64 (d, J = 0.9 Hz, 1H), 8.75 (dd, J = 2.2, 1.0 Hz, 1H), 8.61 (dd, J = 4.9, 1.6 Hz, 1H), 8.56 (d, J = 0.9 Hz, 1H), 7.98 (dt, J = 8.0, 1.9 Hz, 1H), 7.76 (d, J = 0.9 Hz, 1H), 7.48 (ddd, J = 7.9, 4.9, 1.0 Hz, 1H), 1.64 (s, 0H). 13C NMR (101 MHz, DMSO-d6) δ 178.61, 152.39, 151.86, 151.49, 149.30, 138.52, 123.62, 123.51, 118.79, 108.88, 88.69, 82.06. HRMS (ESI+) in m/z: Expected 198.055 [M + H+] (C12H8NO2+). Observed 198.0549. 4-(Pyridin-3-ylethynyl)thiazole (5y) Tan solid. Yield: 73 mg, 78%. Time: 18 h, Temperature: 60 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J = 1.9 Hz, 1H), 8.78 (dd, J = 2.2, 1.0 Hz, 1H), 8.62 (dd, J = 4.9, 1.6 Hz, 1H), 8.21 (d, J = 1.9 Hz, 1H), 8.01 (dt, J = 7.9, 1.9 Hz, 1H), 7.53–7.43 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 154.99, 151.59, 149.31, 138.63, 136.28, 125.41, 123.63, 118.71, 86.72, 85.38. HRMS (ESI+) in m/z: Expected 187.0324 [M + H+] (C10H7N2S+). Observed 187.0324. 3,5-Dimethyl-4-(pyridin-3-ylethynyl)isoxazole (5z) Yellow oil. Yield: 30 mg, 30%. Time: 32 h, Temperature: 60 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (dd, J = 2.3, 1.0 Hz, 1H), 8.59 (dd, J = 4.9, 1.8 Hz, 1H), 7.98 (dq, J = 8.0, 1.9 Hz, 1H), 7.51–7.41 (m, 1H), 2.52 (d, J = 1.7 Hz, 4H), 2.30 (d, J = 1.7 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 172.12, 160.04, 151.54, 149.13, 138.46, 123.59, 119.08, 99.88, 90.96, 80.45, 11.72, 10.07. HRMS (ESI+) in m/z: Expected 199.0866 [M + H+] (C12H11N2O+). Observed 199.0860. Anal. Calcd for C12H10N2O: C, 72.71; H, 5.08; N, 14.13. Found: C, 72.96; H, 5.24; N, 13.85. 4-(Pyridin-3-ylethynyl)-1H-indole 9 (5aa) Tan solid. Yield: 95 mg, 87%. Time: 6 h. 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.85–8.79 (m, 1H), 8.58 (dt, J = 4.9, 1.4 Hz, 1H), 8.08–7.99 (m, 1H), 7.54–7.42 (m, 3H), 7.31–7.23 (m, 1H), 7.19–7.09 (m, 1H), 6.67 (ddt, J = 3.0, 1.9, 1.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 151.50, 148.59, 138.33, 135.50, 128.92, 126.60, 123.55, 122.88, 120.89, 119.97, 113.02, 112.42, 100.48, 91.91, 87.93. HRMS (ESI+) in m/z: Expected 219.0917 [M + H+] (C15H11N2+). Observed 219.0918. 1,2-Bis(3,5-dimethoxyphenyl)ethyne (7a) Tan solid. Yield: 136 mg, 91%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 6.71 (dd, J = 2.4, 0.8 Hz, 4H), 6.56 (t, J = 2.3 Hz, 2H), 3.77 (d, J = 0.8 Hz, 12H). 13C NMR (101 MHz, DMSO-d6) δ 160.40, 123.57, 109.07, 101.99, 88.95, 55.41. HRMS (ESI+) in m/z: Expected 299.1278 [M + H+] (C18H19O4+). Observed 299.1268. 7a is a known compound.58 3-((3,5-Dimethoxyphenyl)ethynyl)phenol (7b) Yellow solid. Yield: 116 mg, 91%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 7.21 (t, J = 7.9 Hz, 1H), 6.97 (dt, J = 7.6, 1.2 Hz, 1H), 6.91 (dd, J = 2.5, 1.5 Hz, 1H), 6.82 (ddd, J = 8.2, 2.5, 1.1 Hz, 1H), 6.70 (dd, J = 2.3, 1.1 Hz, 2H), 6.57–6.52 (m, 1H), 3.77 (d, J = 1.3 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 160.40, 157.37, 129.89, 123.74, 122.98, 122.24, 117.77, 116.38, 109.05, 101.84, 89.02, 88.79, 55.40. HRMS (ESI+) in m/z: Expected 255.1016 [M + H+] (C16H15O3+). Observed 255.1017. Anal. Calcd for C16H14O3: C, 75.57; H, 5.55; N, 0. Found: C, 75.53; H, 5.64; N, <0.02. 1,3-Dimethoxy-5-(phenylethynyl)benzene (7c) Brown oil. Yield: 113 mg, 95%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 6.54–6.46 (m, 3H), 5.41 (s, 1H), 3.74 (s, 5H), 1.83 (dd, J = 12.0, 4.8 Hz, 2H), 1.75–1.36 (m, 9H), 1.30–1.16 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 160.41, 131.40, 128.87, 128.76, 123.68, 122.12, 109.05, 101.90, 89.39, 88.87, 55.40. HRMS (ESI+) in m/z: Expected 239.1067 [M + H+] (C14H15N2O2+). Observed 239.1072. 7c is a known compound.59 5-((3,5-Dimethoxyphenyl)ethynyl)-1-methyl-1H-imidazole (7d) Yellow solid. Yield: 112 mg, 92%. Time: 12 h. 1H NMR (400 MHz, DMSO-d6) δ 7.78 (s, 1H), 7.31 (d, J = 1.1 Hz, 1H), 6.71 (d, J = 2.3 Hz, 2H), 6.56 (t, J = 2.3 Hz, 1H), 3.77 (s, 6H), 3.71 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.43, 139.61, 134.02, 123.29, 108.73, 101.91, 95.89, 77.33, 55.44, 31.74. HRMS (ESI+) in m/z: Expected 243.1128 [M + H+] (C14H15N2O2+). Observed 243.1134. Anal. Calcd for C14H14N2O2: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.34; H, 5.87; N, 11.37. Methyl 4-((3,5-Dimethoxyphenyl)ethynyl)benzoate (7e) Tan solid. Yield: 136 mg, 92%. Time: 24 h. 1H NMR (400 MHz, DMSO-d6) δ 8.02–7.97 (m, 2H), 7.74–7.65 (m, 2H), 6.75 (d, J = 2.3 Hz, 2H), 6.59 (t, J = 2.3 Hz, 1H), 3.87 (s, 4H), 3.78 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 165.60, 160.44, 131.71, 129.42, 129.36, 126.90, 123.10, 109.24, 102.36, 92.36, 87.95, 55.44, 52.34. HRMS (ESI+) in m/z: Expected 297.1135 [M + H+] (C18H17O4+). Observed 297.1135. Anal. Calcd for C18H16O4: C, 72.96; H, 5.44; N, 0. Found: C, 72.79; H, 5.48; N, <0.02. 4-((3,5-Dimethoxyphenyl)ethynyl)benzonitrile (7f) White solid. Yield: 123 mg, 93%. 1H NMR (400 MHz, DMSO-d6) δ 7.95–7.85 (m, 2H), 7.79–7.67 (m, 2H), 6.76 (d, J = 2.3 Hz, 2H), 6.60 (t, J = 2.3 Hz, 1H), 3.78 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 160.45, 132.61, 132.18, 127.03, 122.84, 118.42, 111.03, 109.31, 102.54, 93.34, 87.45, 55.47. HRMS (ESI+) in m/z: Expected 286.0838 [M + Na+] (C17H14NO2Na+). Observed 286.0834. 4-((3,5-Dimethoxyphenyl)ethynyl)benzoic Acid (7g) Yellow solid. Yield: 79 mg, 56%. 1H NMR (400 MHz, DMSO-d6) δ 13.16 (s, 1H), 7.97 (d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H), 6.75 (d, J = 2.3 Hz, 2H), 6.59 (t, J = 2.3 Hz, 1H), 3.78 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 166.67, 164.03, 160.44, 131.57, 130.62, 129.56, 126.46, 123.19, 109.22, 102.32, 92.03, 88.13, 55.45, 40.43, −1.92. HRMS (ESI+) in m/z: Expected 283.0978 [M + H+] (C17H15O4+). Observed 283.0973. Anal. Calcd for C17H14O4: C, 72.33; H, 5.00; N, 0. Found: C, 71.88; H, 4.95; N, <0.02. 1-(Cyclohex-1-en-1-ylethynyl)-3,5-dimethoxybenzene (7h) Orange oil. Yield: 101 mg, 83%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 6.55 (d, J = 2.3 Hz, 2H), 6.49 (t, J = 2.3 Hz, 1H), 6.19 (tt, J = 3.8, 1.6 Hz, 1H), 3.74 (s, 6H), 2.13 (dtdd, J = 9.6, 5.8, 4.1, 2.3 Hz, 4H), 1.69–1.49 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 160.29, 135.51, 124.25, 119.88, 108.78, 101.30, 90.81, 86.80, 55.28, 28.68, 25.18, 21.77, 20.94. HRMS (ESI+) in m/z: Expected 243.138 [M + H+] (C16H19O2+). Observed 243.1376. 4-(3,5-Dimethoxyphenyl)-2-methylbut-3-yn-2-ol (7i) Yellow oil. Yield: 100 mg, 91%. Time: 20 h. 1H NMR (400 MHz, DMSO-d6) δ 6.57–6.39 (m, 3H), 5.45 (d, J = 1.2 Hz, 1H), 3.74 (d, J = 1.3 Hz, 6H), 1.45 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 160.31, 124.09, 108.82, 101.35, 95.67, 80.37, 63.57, 55.32, 31.57. HRMS (ESI+) in m/z: Expected 221.1172 [M + H+] (C13H17O3+). Observed 221.1171. 7i is a known compound.60 1-((3,5-Dimethoxyphenyl)ethynyl)cyclohexan-1-ol (7j) White solid. Yield: 112 mg, 86%. Time: 5 h. 1H NMR (400 MHz, DMSO-d6) δ 6.50 (dt, J = 6.5, 2.3 Hz, 3H), 5.41 (s, 1H), 3.74 (s, 6H), 1.83 (dd, J = 12.5, 4.8 Hz, 2H), 1.71–1.58 (m, 2H), 1.58–1.41 (m, 5H), 1.29–1.18 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 160.32, 124.19, 108.87, 101.29, 94.60, 82.56, 66.87, 55.32, 39.63, 24.90, 22.74. HRMS (ESI+) in m/z: Expected 261.1485 [M + H+] (C16H21O3+). Observed 261.1481. Anal. Calcd for C16H20O3: C, 73.82; H, 7.74; N, 0. Found: C, 73.9; H, 7.95; N, <0.02. tert-Butyl 1-((3,5-Dimethoxyphenyl)ethynyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (7k) Clear oil. Yield: 128 mg, 75%. Time: 2 h. 1H NMR (400 MHz, DMSO-d6) δ 6.56–6.52 (m, 2H), 6.48 (dd, J = 2.7, 1.7 Hz, 1H), 3.73 (d, J = 1.1 Hz, 6H), 3.68 (dd, J = 10.1, 5.6 Hz, 0H), 3.54–3.29 (m, 3H), 2.02–1.93 (m, 1H), 1.47–1.32 (m, 9H), 1.22 (dd, J = 8.2, 4.8 Hz, 1H), 0.74 (t, J = 5.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 160.28, 153.77, 123.99, 109.05, 101.39, 78.89, 55.31, 51.13, 50.81, 47.58, 47.33, 28.05, 26.05, 25.21, 17.64. HRMS (ESI+) in m/z: Expected 366.1687 [M + Na+] (C20H25NO4Na+). Observed 366.169. Anal. Calcd for C20H25NO4: C, 69.95; H, 7.34; N, 4.08. Found: C, 70.05; H, 7.49; N, 4.04. tert-Butyl 4-((3,5-Dimethoxyphenyl)ethynyl)-4-methylpiperidine-1-carboxylate (7l) Clear oil. Yield: 174 mg, 97%. Time: 24 h. 1H NMR (400 MHz, DMSO-d6) δ 6.55 (d, J = 2.3 Hz, 2H), 6.48 (t, J = 2.3 Hz, 1H), 3.88 (d, J = 13.1 Hz, 2H), 3.73 (s, 5H), 3.04 (s, 3H), 1.72–1.64 (m, 2H), 1.40 (s, 11H), 1.28 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ 160.28, 153.78, 124.27, 109.06, 101.31, 93.85, 82.77, 78.64, 55.33, 31.43, 28.95, 28.09. HRMS (ESI+) in m/z: Expected 382.1989 [M + Na+] (C21H29NO4Na+). Observed 382.1985. Anal. Calcd for C21H29NO4: C, 70.17; H, 8.13; N, 3.90. Found: C, 70.45; H, 8.34; N, 3.93. 1-(Cyclopropylethynyl)-3,5-dimethoxybenzene (7m) Orange oil. Yield: 97 mg, 96%. Time: 10 h. 1H NMR (400 MHz, DMSO-d6) δ 6.50 (d, J = 2.3 Hz, 2H), 6.45 (t, J = 2.3 Hz, 1H), 3.72 (s, 6H), 1.52 (tt, J = 8.3, 5.0 Hz, 1H), 0.93–0.82 (m, 2H), 0.76–0.66 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.25, 124.64, 108.98, 100.93, 93.42, 75.67, 55.26, 8.34. HRMS (ESI+) in m/z: Expected 203.1067 [M + H+] (C13H15O2+). Observed 203.1060. 7m is a known compound.61 2-(4-(3,5-Dimethoxyphenyl)but-3-yn-1-yl)isoindoline-1,3-dione (7n) White solid. Yield: 141 mg, 84%. Time: 8 h. 1H NMR (400 MHz, DMSO-d6) δ 7.95–7.88 (m, 2H), 7.86 (dt, J = 5.0, 3.4 Hz, 2H), 6.45 (t, J = 2.3 Hz, 1H), 6.39 (d, J = 2.3 Hz, 2H), 3.82 (t, J = 6.9 Hz, 2H), 3.68 (s, 6H), 2.78 (t, J = 6.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 167.66, 160.23, 134.57, 131.50, 124.03, 123.13, 108.86, 101.15, 86.55, 81.91, 55.24, 36.20, 18.44. HRMS (ESI+) in m/z: Expected 336.123 [M + H+] (C20H18NO4+). Observed 336.1229. Anal. Calcd for C20H17NO4: C, 71.63; N, 5.11; H, 4.18. Found: C, 71.65; H, 5.19; N, 4.13. General Procedure for Synthesis of (10) To an oven-dried Biotage microwave process vial (#355630), 2-bromoaniline (0.50 mmol, 1.00 equiv), alkyne (0.63 mmol, 1.25 equiv), 2,2,6,6-tetramethylpiperidine (169 μL, 1.00 mmol, 2.00 equiv), and P2 (10.33 mg, 0.025 mmol, 0.05 equiv) were added to ACN (1.0 mL). Remaining ACN (1.0 mL) was added to rinse the sides. The reaction vessel was sealed and bubbled with in-house argon for 5 min. The reaction was stirred at room temperature for the designated time. Concentrated HCl (2.50 mmol, 5.0 equiv) was added via syringe directly to the reaction mixture and stirred at 90 °C for 9 h. Work up involved ammonium chloride, EtOAc, and brine. The organic layer was concentrated and purified on Isco silica gel columns to give the resulting product using a gradient of 0–100% for ethyl acetate/hexanes. 2-Phenyl-1H-indole (10a) Tan solid. Yield: 49 mg, 51%. Time: 1. 1 h; 2. 9 h. 1H NMR (400 MHz, chloroform-d) δ 8.31 (s, 1H), 7.71–7.63 (m, 3H), 7.46 (t, J = 7.7 Hz, 2H), 7.41 (d, J = 8.1 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.26–7.18 (m, 1H), 7.15 (t, J = 7.4 Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H). 13C NMR (101 MHz, chloroform-d) δ 138.02, 136.96, 132.52, 129.42, 129.16, 127.85, 125.30, 122.50, 120.81, 120.42, 111.03, 100.15. HRMS (ESI+) in m/z: Expected 194.0964 [M + H+] (C14H12N+). Observed 194.0967. 10a is a known compound.62 2,2-Difluoro-6-phenyl-5H-[1,3]dioxolo[4,5-f]indole (10b) Yellow solid. Yield: 40 mg, 29%. Time: 1. 3 h; 2. 9 h. 1H NMR (400 MHz, chloroform-d) δ 8.37 (s, 1H), 7.66–7.57 (m, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.37–7.30 (m, 1H), 7.22 (s, 1H), 7.08 (s, 1H), 6.79 (dd, J = 2.3, 1.1 Hz, 1H). 13C NMR (101 MHz, chloroform-d) δ 140.90, 139.50, 138.20, 131.89, 131.86, 129.10, 127.82, 124.86, 124.24, 100.35, 100.25, 92.61. HRMS (ESI+) in m/z: Expected 274.0686 [M + H+] (C15H10F2NO2+). Observed 274.0682. Methyl 2-(4-Methoxyphenyl)-1H-indole-7-carboxylate (10c) Tan solid. Yield: 66 mg, 47%. Time: 1. 1 h; 2. 9 h. 1H NMR (400 MHz, chloroform-d) δ 10.04 (s, 1H), 7.92–7.73 (m, 2H), 7.73–7.63 (m, 2H), 7.14 (t, J = 7.8 Hz, 1H), 7.03–6.94 (m, 2H), 6.75 (d, J = 2.3 Hz, 1H), 4.01 (d, J = 1.2 Hz, 3H), 3.86 (d, J = 1.2 Hz, 3H). 13C NMR (101 MHz, chloroform-d) δ 168.25, 159.77, 139.19, 136.93, 130.70, 126.83, 125.92, 124.78, 123.89, 119.48, 114.62, 112.20, 98.42, 55.50, 51.98. HRMS (ESI+) in m/z: Expected 282.1125 [M + H+] (C17H16NO3+). Observed 282.1133. 2-Phenyl-1H-indol-7-amine (10d) Brown solid. Yield: 81 mg, 78%. Time: 1. 8 h; 2. 8 h. 1H NMR (400 MHz, chloroform-d) δ 8.20 (s, 1H), 7.76–7.59 (m, 2H), 7.49–7.41 (m, 2H), 7.36–7.30 (m, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.97 (td, J = 7.7, 2.0 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H), 6.61 (d, J = 7.3 Hz, 1H), 3.66 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 136.25, 133.66, 132.52, 129.25, 128.86, 127.07, 126.42, 124.66, 120.56, 108.51, 105.21, 99.24. HRMS (ESI+) in m/z: Expected 209.1073 [M + H+] (C14H13N2+). Observed 209.1077. 10d is a known compound but no analytical data can be found online. 2-Cyclopropyl-5-(trifluoromethoxy)-1H-indole (10e) Brown solid. Yield: 73 mg, 61%. Time: 1. 1 h; 2. 8 h. 1H NMR (400 MHz, chloroform-d) δ 8.01 (s, 1H), 7.34 (d, J = 2.5 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.03–6.91 (m, 1H), 6.21–6.06 (m, 1H), 1.96 (tt, J = 8.4, 5.1 Hz, 1H), 1.07–0.94 (m, 2H), 0.84–0.72 (m, 2H). 13C NMR (101 MHz, chloroform-d) δ 144.00, 143.28, 134.15, 129.12, 122.26, 119.72, 114.97, 112.31, 110.72, 98.35, 9.04, 7.64. HRMS (ESI+) in m/z: Expected 242.0787 [M + H+] (C12H11F3NO+). Observed 242.0790. 3-(1H-Indol-2-yl)phenol (10f) Tan solid. Yield: 54 mg, 52%. Time: 1. 1 h; 2. 9 h. HRMS (ESI+) in m/z: 1H NMR (400 MHz, chloroform-d) δ 8.30 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.25–7.17 (m, 1H), 7.16–7.10 (m, 2H), 6.82–6.76 (m, 2H), 4.85 (s, 1H). 13C NMR (101 MHz, chloroform-d) δ 156.19, 137.59, 136.94, 134.22, 130.45, 129.31, 122.66, 120.88, 120.48, 117.89, 114.87, 112.29, 111.06, 100.48. Expected 210.0913 [M + H+] (C14H12NO+). Observed 210.0915. 10f is a known compound, but no analytical data can be found online. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01868.Catalyst and compound characterization data (PDF) Supplementary Material ao8b01868_si_001.pdf Author Present Address † UAB School of Medicine, 1670 University Blvd, Birmingham, Alabama 3523, United States (B.T.M.). Author Present Address ‡ Nexus Discovery Advisorsm 7820B Wormans Mill Road, Suite 208, Frederick, Maryland 21701, United States (D.J.M.). The authors declare no competing financial interest. Acknowledgments We thank Shyh-Ming Yang, Patrick Morris, Dan Jansen, Samarjit Patnaik, and Sara Kearney for valuable suggestions; Dingyin Tao and Yuhong Fang for analytical chemistry support. The authors gratefully acknowledge funding by the Intramural Research Program, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH). ==== Refs References Chinchilla R. ; Najera C. Recent advances in Sonogashira reactions . Chem. Soc. Rev. 2011 , 40 , 5084 –5121 . 10.1039/c1cs15071e .21655588 Chinchilla R. ; Najera C. The Sonogashira reaction: a booming methodology in synthetic organic chemistry . Chem. Rev. 2007 , 107 , 874 –922 . 10.1021/cr050992x .17305399 Jenny N. M. ; Mayor M. ; Eaton T. R. Phenyl-Acetylene Bond Assembly: A Powerful Tool for the Construction of Nanoscale Architectures . Eur. J. Org. Chem. 2011 , 2011 , 4965 –4983 . 10.1002/ejoc.201100176 . Wang D. ; Gao S. 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PMC006xxxxxx/PMC6644405.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145884910.1021/acsomega.8b00835ArticleVersatile Approach for Reducing Propagation Loss in Wet-Electrospun Polymer Fiber Waveguides Ishii Yuya †Omori Keisho ‡Sakai Heisuke §Arakawa Yuki ∥Fukuda Mitsuo ‡† Faculty of Fiber Science and Engineering, Kyoto Institute of Technology, Kyoto, Kyoto 606-8585, Japan‡Department of Electrical and Electronic Information Engineering and ∥Department of Environmental and Life Sciences, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan§ School of Materials Science, Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan E-mail: yishii@kit.ac.jp.22 06 2018 30 06 2018 3 6 6787 6793 27 04 2018 08 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Wet-electrospun (WES) polymer micron and submicron fibers are promising building blocks for small, flexible optical fiber devices, such as waveguides, sensors, and lasers. WES polymer fibers have an inherent cylindrical geometry similar to that of optical fibers and a relatively large aspect ratio. Furthermore, WES fibers can be produced using low-cost and low-energy manufacturing techniques with large-area fabrication and a large variety of materials. However, the high propagation loss in the fibers, which is normally on the order of tens or thousands of decibels per centimeter in the visible light region, has impeded the use of these fibers in optical fiber devices. Here, the origin of propagation losses is examined to develop a comprehensive and versatile approach to reduce these losses. The excess light scattering that occurs in fibers due to their inhomogeneous density is one of the primary factors in the propagation loss. To reduce this loss, the light transmission characteristics were investigated for single WES polymer fibers heated at different temperatures. The propagation loss was significantly reduced from 17.0 to 8.1 dB cm–1 at 533 nm wavelength, by heating the fibers above their glass transition temperature, 49.8 °C. In addition, systematic verification of the possible loss factors in the fibers confirmed that the propagation loss reduction could be attributed to the reduction of extrinsic excess scattering loss. Heating WES polymer fibers above their glass transition temperature is a versatile approach for reducing the propagation loss and should be applicable to a variety of WES fibers. This finding paves the way for low-loss WES fiber waveguides and their subsequent application in small, flexible optical fiber devices, including waveguides, sensors, and lasers. document-id-old-9ao8b00835document-id-new-14ao-2018-00835accc-price ==== Body Introduction Wet electrospinning is a simple, versatile, and widely used technique for producing submicron and micron polymer fibers. This technique has considerable potential for low-cost and low-energy manufacturing with large fabrication area, providing high material utilization ratios and high positional accuracy.1,2 The electrospinning technique can instantaneously and continuously produce fibers with lengths of more than tens of centimeters that have an inherent cylindrical geometry similar to that of optical fibers.3 Furthermore, wet-electrospun (WES) fibers can be produced from a wide variety of spinnable polymers and composites, including highly transparent polymers and optically active composites.4−6 Thus, WES fibers are promising building blocks for small, flexible optical fiber devices, such as waveguides, sensors, and lasers.6−11 However, high propagation loss in WES fibers, normally on the order of tens or thousands of decibels per centimeter in the visible light region,6,7,9−15 has impeded their widespread use. Fasano et al. reduced the propagation loss caused by the surface morphology of WES conjugated polymer fibers by producing the fibers in a precisely controlled nitrogen atmosphere,14 but this approach could only be applied to specific materials. We previously reported that the high propagation loss in WES fibers could be attributed to (i) excess light scattering in the fibers resulting from their inhomogeneous density and (ii) loss at the interface between the fiber core and cladding, originating from nonuniformity of the fiber diameters.7 In this study, we reduced the propagation loss in WES polymer fibers by reducing excess light scattering in the fibers by a versatile heat treatment approach. The light transmission characteristics of single WES polymer fibers treated at different post-electrospinning temperatures revealed that the propagation loss was reduced significantly by 5.9 dB cm–1 at 532 nm wavelength when the fibers were heated above their glass transition temperature (Tg). Experimental Details Fiber Fabrication and Morphological Characterization Amorphous poly(lactic acid) composed of a racemic mixture of d- and l-lactic acid units (PDLLA) was used as the fiber material because it exhibits high transparency in the visible ranges16 and has a lower Tg than other well-known amorphous transparent polymers such as poly(methyl methacrylate) (PMMA) and polystyrene (PS).17 The lower Tg value enables easier investigation of the thermal properties of samples in this initial study. The as-received PDLLA pellets (Mw = 300 000–600 000, Polysciences, Inc.) were dissolved in chlorobenzene (99.0%, Wako Pure Chemicals Industries, Ltd.) with a concentration of 16 wt %. The solution was then loaded into a glass syringe equipped with a 0.18 mm-diameter stainless-steel needle and was supplied continuously at a rate of 0.02 mL h–1 using a syringe pump (KDS-100, KD Scientific, Inc.). A high-voltage power supply (HVU-30P100, MECC Co., Ltd.) was connected to the needle at 4.8 kV. The alternating biased-collector electrospinning method18 was used to fabricate single-aligned PDLLA fibers. Two stainless-steel collectors with 3 cm separation were placed 8 cm below the tip of the needle, one of which was biased with a negative voltage (−800 V) using a high-voltage power source (HJPM-1N3, Matsusada Precision Inc.). The electrospinning experiments were performed in air at temperatures of 24–25 °C with a relative humidity of 53–63%. The single-aligned electrospun PDLLA fibers were collected in two lengths. One length was placed on a silicon substrate to characterize the shape of each fiber before heating. The other length was attached to two tapered optical silica fibers (Fine Glass Technologies Co., Ltd.) to conduct waveguiding measurements (see Figure 1, STEP 1). The morphology of the single-aligned fibers was investigated using field-emission scanning electron microscopy (FESEM, SU8000, Hitachi) operating at 5.0 kV after a 4 nm-thick Os coating was applied. The mean diameter of each fiber was determined by averaging diameters taken from FESEM images at more than 12 800 positions along the 250 μm-long fibers using image analysis. A number of aligned WES PDLLA fibers were prepared by collecting the fibers for 30 min using a 15 cm-diameter gear-shaped rotating collector to conduct thermal, X-ray, and spectroscopic characterizations. The rotating speed of the collector was maintained at 100 rpm to prevent the as-spun fibers from being stretched by the collector. Figure 1 Schematic of the experimental setup for measuring the propagation loss and transmitted light intensity in a single PDLLA fiber. Waveguiding Measurements First, a collimated 532 nm laser (LCM-T-111, Laser-Export Co. Ltd.) beam with a 440 μm radius was irradiated to each fiber at different positions [Figure 1, STEP 1] to investigate the propagation loss in single WES PDLLA fibers. The laser beam was applied at a 43° angle to the fiber axis, and the polarization direction of the beam was perpendicular to the fiber axis. A portion of the laser beam was coupled to a guiding mode in the PDLLA fiber and collected via a right-tapered optical fiber after being guided through the PDLLA fiber. The intensity of the collected light was measured with a spectrometer (USB4000, Ocean Optics, Inc.). Then, a 632.8 nm He–Ne laser (GLG5370, NEC Corp.) beam was introduced to the PDLLA fiber from the left tapered optical silica fiber, and the intensity of the guided light collected via the right tapered optical fiber was monitored while using different heating temperatures (see Figure 1, STEP 2). After cooling of the heated PDLLA fibers by stopping the heating, the measurement performed in STEP 1 was repeated in STEP 3 (see Figure 1, STEP 3). After the measurement, each fiber was placed on a silicon substrate to characterize the fiber shape. The propagation loss was also investigated at the following wavelengths using collimated laser diode modules in the same manner as that in STEP 1 and STEP 3: 450 nm (CPS450, Thorlabs Inc.), 521 nm (CPS520, Thorlabs Inc.), 639 nm (CPS635, Thorlabs Inc.), and 849 nm (CPS850, Thorlabs Inc.). Each collimated laser beam was irradiated to the single PDLLA fiber though a 1.0 mm-diameter pinhole. Thermal, X-ray, and Spectroscopic Characterization The thermal properties of the WES PDLLA fibers and PDLLA pellets were investigated with a differential scanning calorimeter (DSC-60, Shimadzu Corp.) under a nitrogen gas flow at a rate of 50 mL min–1. Each sample of the WES PDLLA fibers (1.6 mg) or PDLLA pellets (3.0 mg) was heated at a rate of 10 °C min–1 from ambient temperature to 200 °C. The molecular structure of the PDLLA in the WES fibers was investigated using an X-ray diffractometer (RINT-2500, Rigaku Corp.) with a Cu Kα source (wavelength of 1.5418 Å), which was operated at 40 kV and 200 mA. The specific optical rotation of the PDLLA was measured in chloroform at a concentration of 10 g L–1 at 25 °C using a polarimeter (P-2100, JASCO) with a wavelength of 0.589 μm. The ultraviolet (UV)–visible (Vis)–near infrared (NIR) attenuation spectra were measured using a UV–Vis–NIR spectrophotometer (U-4100, Hitachi). A fused glass cell with a 1.0 cm path length was used to measure the transmittance spectra of pure PDLLA, chlorobenzene, and 16 wt % PDLLA solution in chlorobenzene. The pure PDLLA sample was prepared by filling the PDLLA pellets into the cell and melting them at 120 °C for 12 h. The polymer chain orientation in the WES PDLLA fibers was evaluated using a Fourier transform infrared (FT-IR) spectrometer (Thermo Nicolet 6700, Thermo Fisher Scientific Inc.) equipped with a wire grid polarizer (KRS-5 Wire Grid Polarizer, S. T. Japan). The FT-IR spectrometer resolution was 4 cm–1, and 128 scans were collected and averaged. In parallel polarization, the direction of the oscillating electric field of the incident IR beam is parallel to the aligned fiber axis; in perpendicular polarization, it is perpendicular to the fiber axis. Results and Discussion The specific optical rotation of the used PDLLA was measured to be approximately zero, while the optical rotations of poly(lactic acid) composed of pure optical isomers, namely poly(l-lactic acid) (PLLA) or poly(d-lactic acid), are reported to be −156 and 156° dm–1 g–1 cm3, respectively.19 This result confirmed that the PDLLA used in this study was comprised of equivalent amounts of l- and d-lactic acid20 to form an amorphous polymer. The X-ray diffraction patterns of the WES PDLLA fibers were also measured, but no obvious diffraction peaks were found at 2θ between 15° and 20°, even though crystallized PLLA exhibits diffraction peaks within this angle range.21 This result confirmed that the WES PDLLA fibers were amorphous. Five single-aligned WES PDLLA fibers, which were approximately 3 cm long, (Fibers A–E) were fabricated. One part of each fiber was placed on a silicon substrate to investigate the fiber morphology. Figure 2a shows an FESEM image of a single-aligned PDLLA fiber (Fiber A). The fiber was obtained with a smooth surface. The mean and standard deviation of the fiber diameters of each fiber are summarized in Table 1. The mean diameter ranges from 1.60 to 1.76 μm. Figure 2 (a) FESEM image of a single WES PDLLA fiber. (b) Bright- and (c) dark-field microscopy images of a single WES PDLLA fiber attached to two tapered optical fibers, where a 632.8 nm laser beam is introduced from the left tapered fiber to the PDLLA fiber. Table 1 Mean and Standard Deviation of the Fiber Diameters before and after Heating fiber A B C D E before heating mean diameter (μm) 1.68 1.69 1.67 1.60 1.76 standard deviation (μm) 0.29 0.14 0.32 0.15 0.18 after heating mean diameter (μm) 1.71 1.49 1.51 1.57 1.60 standard deviation (μm) 0.14 0.13 0.22 0.23 0.20 Then, a part of each fiber was attached to two tapered optical silica fibers, as shown in the bright-field microscopy image in Figure 2b. The PDLLA fibers were manually attached to the facing tapered fibers without much difficulty because electrostatic attraction appeared to act between the charged electrospun PDLLA fibers and tapered fibers. As shown in the bright- [Figure 2b] and dark-field [Figure 2c] microscopy images, a red laser beam with 632.8 nm wavelength was coupled to the PDLLA fiber from the left tapered fiber, although part of the laser beam was radiated at the tip of the left tapered fiber. The thin red line observed in Figure 2b,c along the PDLLA fiber position demonstrates that the light was guided in the fiber. Relatively bright dots observed along the PDLLA fiber in Figure 2c could be attributed to scattering of the guided light at the points with locally inhomogeneous morphology. Figure 3 shows dependence of the transmitted light intensity, normalized by the peak intensity, on the fiber temperature, using the same setup as that illustrated in Figure 1 STEP 2. The fiber temperature was increased from room temperature by 1.1–1.4 °C min–1 until the PDLLA fiber was broken. Even at room temperature (25–28 °C), the transmitted light intensity was more than 160 times higher than that after the PDLLA fiber was broken, which confirmed that direct light coupling from the left-tapered silica fiber to the right-tapered silica fiber, which was primarily caused by the radiated light at the tip of the left fiber, was negligible. Consequently, the introduced laser beam was mainly guided in the PDLLA fiber and was collected via the right-tapered optical fiber. The transmitted light intensity increased with increasing temperature, showing significant increases at around 45–55 °C. Moreover, the intensity showed peaks at around 55–60 °C. Over this temperature range, the intensity showed a saturated and/or unstable value until the intensity drop occurred because of the break of the PDLLA fiber. The transmitted light intensity was 3.5 times higher after heating than that before heating. These results indicated that the propagation loss in the PDLLA fibers was reduced by heating, but reduction of the connection loss between the PDLLA fiber and the tapered silica fibers remained a possible reason for the increased transmitted light intensity because the connection area was also heated in the present setup, as shown in Figure 1 STEP 2. Figure 3 Normalized intensity of the 632.8 nm light after transmission through each PDLLA fiber attached to the tapered optical fibers with increasing temperature. The different colored plots correspond to the different single fibers. The thermal properties of the WES PDLLA fibers and the as-received PDLLA pellets were investigated using DSC, as shown in Figure 4. The Tg value was determined to be 49.8 °C for the fibers (first heating), 53.1 °C for the fibers (second heating), and 53.5 °C for the pellets (second heating). Both samples from the second heating showed approximately equivalent Tg values because their erased thermal history allowed the thermal properties of the PDLLA material to dominate; however, the Tg value for the first heating fibers showed a slight decrease. Similar Tg decreases were previously reported for WES PDLLA fibers22 and thin polymer films.23,24 The evaluated Tg of the WES fibers corresponded to the temperature range of 45–55 °C, where the transmitted light intensity significantly increased. Excess light scattering loss in PMMA bulks has been reduced by polymerization or by heating the material above Tg;25,26 thus, the increase in the transmitted light intensity near and above the Tg value could be partially attributed to the reduction of excess light scattering loss in the PDLLA fibers. Figure 4 DSC thermograms of WES PDLLA fibers and as-received PDLLA pellets. To understand the change in propagation loss in each WES fiber due to heating and avoiding the effects of the connection loss, we measured the propagation loss before and after heating using the procedure shown in Figure 1. Here, the maximum heating temperature was determined to be around 55 °C because this temperature was above Tg of the WES PDLLA fibers and where the transmitted light intensity showed the maximum intensity (Figure 3). The propagation loss was determined by measuring the guided light intensity with different propagation lengths (l) using the procedure explained above. Here, l was determined as the length from the center of the spot at which the collimated laser was irradiated to the tip of the right tapered silica fiber. Figure 5a shows the normalized guided light intensity with different l, measured for a single WES PDLLA fiber (Fiber A) before and after heating. The wavelength of the irradiated light was 532 nm. The intensity decreased with increasing l, and the plots adequately fit the function e–al [solid lines in Figure 5a], where a is the loss coefficient. The propagation loss was evaluated using the following formula: −10 log(e–al\|l=1cm/e–al\|l=0cm) = 10a log(e) [dB cm–1]. Figure 5b summarizes the evaluated propagation loss of each PDLLA fiber before and after heating. The propagation loss was significantly reduced after heating for all five PDLLA fibers. For example, the propagation loss of Fiber A was reduced from 17 to 8.1 dB cm–1, which corresponded to an 8.2-fold increase in the transmitted light intensity when the light is guided through 1.0 cm of the fiber. These results demonstrated that heating of the WES PDLLA fibers above Tg significantly reduced the propagation loss in the fibers. The propagation loss of Fiber E was reduced to 5.9 dB cm–1, which was the lowest propagation loss reported for WES polymer fibers.6,7,9−15 Increase in fiber diameters normally decreases the propagation loss in the fibers,27−29 but significant increase in the mean diameter was not observed after heating, as shown in Table 1, although the propagation loss was significantly reduced for all five fibers. This result shows that some other loss factors were reduced after heating. Figure 5 (a) Normalized guided light intensity for Fiber A with different propagation lengths before and after heating, where the wavelength was 532 nm. Solid lines show single exponential fittings. (b) Propagation losses of the five single PDLLA fibers, evaluated before and after heating. The possible loss factors for WES fibers (from refs30−32) are summarized in Figure 6, separated into intrinsic and extrinsic factors. Figure 6 Possible loss factors for waveguiding in WES fibers summarized in refs.30−32 To investigate the absorption loss in the WES PDLLA fibers, UV–Vis–NIR measurement was conducted. Figure 7 shows the UV–Vis–NIR attenuation spectra of pure PDLLA, chlorobenzene, 16 wt % PDLLA solution in chlorobenzene, and water in a 1.0 cm path-length fused glass cell. Each attenuation spectrum was converted from the transmittance spectrum of the respective sample; thus, the measured attenuation included attenuation due to light reflection and scattering at each interface including that between air and the fused glass cell. Unique attenuation peaks due to the electronic transition absorption or molecular vibration absorption of materials were observed at wavelengths shorter than 450 nm and longer than 700 nm for pure PDLLA, chlorobenzene, and PDLLA solution. On the other hand, in the wavelength range from 450 to 680 nm, the attenuation spectra decreased with increasing wavelength as a result of the light reflection and scattering at each interface and the light scattering inside each filled material. Consequently, the intrinsic absorption loss in the wavelength range of 450–680 nm was estimated to be less than 0.02 dB cm–1 from the attenuation spectrum of pure PDLLA, by subtracting the gradually decreasing attenuation and considering the 1 cm pass length. The extrinsic absorption loss caused by transition metals and organic contaminants was estimated to be less than 0.02 dB cm–1 from the attenuation spectrum of the pure PDLLA, chlorobenzene, and PDLLA solution because no peaks exceeding 0.02 dB were observed at this wavelength range. Although the attenuation spectrum of water indicated that its absorption loss was less than 0.5 dB cm–1 in the wavelength range of 450–680 nm, as shown in Figure 7, the absorption loss of WES PDLLA fibers could decrease to less than 0.05 dB cm–1 if the fibers contained 10% water relative to the fiber volume. These results confirmed that the intrinsic and extrinsic absorption losses were much lower than the propagation loss in the WES PDLLA fibers. Figure 7 UV–Vis–NIR attenuation spectra of pure PDLLA, chlorobenzene, 16 wt % PDLLA solution in chlorobenzene, and water, which were placed in a 1.0 cm path-length fused glass cell. The intrinsic scattering loss originating from the intrinsic density inhomogeneity of the materials was the lower limit of scattering loss inherent to each material that remained even after the extrinsic scattering loss, namely the excess light scattering loss.33 Thus, the intrinsic scattering loss was not significantly reduced by the heat treatment. By roughly estimating the intrinsic loss from the measured attenuation spectrum of pure PDLLA [Figure 7], the intrinsic scattering loss was estimated to be less than 0.8 dB cm–1. However, this value of the intrinsic scattering loss should be an overestimation because both the light reflection loss and extrinsic light scattering loss were included in the measured attenuation spectrum. Moreover, the intrinsic scattering loss for other amorphous polymers, namely PMMA and PS, has been estimated to be less than 1 × 10–3 dB cm–1 at wavelengths of around 533 nm.32,34 Extrinsic scattering due to dust in the environment may occur during the measurements for transmitting light intensity and propagation loss; however, no large dust particles were observed in the bright-field microscope image of the PDLLA fiber [Figure 2b]. The dark-field microscope image of the fiber [Figure 2c] indicated relatively bright scattering points because of small dust particles or other factors not visible in the bright-field image, and the plots of the guided light intensity showed some variation [Figure 5a]. However, the variation was smoothed by fitting the plots with a single exponential function before the propagation loss was determined. Therefore, the local scattering loss due to dust should be negligible for the measured propagation loss. Moreover, the heat treatment could not reduce the scattering loss due to dust; thus, other factors caused the propagation loss reduction after heating. Additionally, the extrinsic radiation loss due to bends in the PDLLA fibers was negligible because the PDLLA fibers were kept straight during the optical measurements [Figure 2b]. The extrinsic scattering loss and radiation loss due to fluctuations in the fiber diameter are possible factors influencing the propagation loss in the WES PDLLA fibers, as previously indicated for WES PMMA fibers.7 Higher fluctuations in the fiber diameter cause higher propagation loss.29 To discuss the fluctuations before and after heating, the root-mean-square roughness (RRMS) for each fiber was calculated using the FESEM images of each 250 μm long PDLLA fiber; more than 12 800 readings for each fiber were recorded using image analysis. The RRMS was calculated using the following formula 1 where D, Dave, and l are the measured diameter of the fibers, the mean diameter of each fiber, and the position where the diameter was measured, respectively. Figure 8 summarizes RRMS of each fiber before and after heating. No clear reduction of the RRMS was observed after heating even though all five WES PDLLA fibers demonstrated reduced propagation loss [Figure 5b]. This result confirmed that extrinsic scattering loss and radiation loss due to the fluctuations in the fiber diameter must be excluded from the factors for propagation loss reduction due to heat treatment. Figure 8 RRMS of each PDLLA fiber before and after heating. Extrinsic scattering loss due to the extrinsic density inhomogeneity resulting from microvoids, microscopic density variation, and birefringence is the only possible remaining loss factor for the propagation loss reduction because of post-fabrication heat treatment. Consequently, extrinsic scattering loss must be the primarily reduced loss factor to account for the observed propagation loss reduction. Extrinsic density inhomogeneity causes excess light scattering, including Rayleigh, Mie, or geometric scattering, so that excess light scattering results in extrinsic scattering loss. Moreover, excess light scattering was recently reported to be one of the main factors for propagation loss in WES PMMA fibers,7 and the excess light scattering in PMMA bulks was reported to be reduced by heating them above Tg.25,26 These reports support the present reduction of propagation loss in WES PDLLA fibers because of heat treatment. Figure 9 shows the measured propagation loss in an WES PDLLA fiber with different wavelengths before and after heating. The propagation loss significantly decreased with the increasing wavelength, which was also observed in the as-WES PMMA fibers.7 This result confirmed that the propagation loss in the PDLLA fiber included extrinsic scattering loss. The propagation loss was significantly reduced after heating, especially at shorter wavelengths, which indicated that the enhanced excess light scattering at shorter wavelengths, for example, enhanced Rayleigh scattering35 proportional to (wavelength)−4, was significantly reduced after heating. Figure 9 Propagation loss of the PDLLA fiber with different wavelengths before and after heating. To investigate birefringence in the WES PDLLA fibers, polarized FT-IR spectra of aligned PDLLA fibers were measured, as shown in Figure 10a. The peak at 1755 cm–1 was assigned to C=O stretching, and the peaks at 1186 and 1090 cm–1 were assigned to C–O–C stretching.36,37 These stretchings are parallel to the polymer backbone. The main chains of PDLLA in the as-electrospun PDLLA fibers were modestly aligned along the fiber axis because the absorbance was higher with parallel polarization at around 1090, 1186, and 1755 cm–1 than that with perpendicular polarization. The dichroic ratio was calculated to be 2.14 by integrating the polarized absorbance in the range from 1650 to 1850 cm–1. The alignment of the PDLLA chains was relaxed after heating because the dichroic ratio after heating decreased to 1.35, as shown in Figure 10b. This alignment relaxation should reduce the birefringence in the PDLLA fibers and partially decrease the extrinsic scattering loss. The effects of microvoids and microscopic density variation are still unclear, but wet-electrospinning produces fibers with rapidly evaporating solvents so that microvoids and microscopic density variation are generated. Several papers reported inhomogeneous inner structures of polymers in WES fibers,5,38 and the WES PDLLA fibers in the present study showed higher propagation loss at shorter wavelength, reflecting the extrinsic density inhomogeneity. A previous work reported that PMMA bulks without heat treatment above Tg included inhomogeneous structures with a dimension of about 100 nm, which caused excess light scattering and increased scattering loss.25,26 Additionally, the inhomogeneous structures disappeared after heat treatment above Tg, and the scattering loss was reduced. On the basis of these results, inhomogeneous structures in the WES PDLLA fibers, microvoids, and microscopic density variation, should be reduced after heat treatment above Tg. Figure 10 Polarized FT-IR spectra of the aligned PDLLA fibers (a) before and (b) after heating. Here, parallel and perpendicular represent the polarized directions of the oscillating electric field of the incident IR beam to the fiber direction. We demonstrated that heating amorphous WES fibers above Tg is a versatile approach for reducing their propagation loss. This technique should be applicable to a variety of WES fibers composed of amorphous polymers. Moreover, optimizing the heating conditions should reduce the propagation loss below the loss observed in this study (lower than 5 dB cm–1 at 533 nm wavelength). Conclusions We demonstrated a comprehensive and versatile approach for reducing propagation loss in WES polymer fibers by heating the fibers above the glass transition temperature of PDLLA. This heat treatment significantly reduced the propagation loss in amorphous WES PDLLA fibers from 17.0 to 8.1 dB cm–1 at a wavelength of 533 nm. The propagation loss reduction could be attributed to the reduction of extrinsic excess scattering losses due to the extrinsic density inhomogeneity in the fibers. These findings pave the way for low-loss WES fiber waveguides and their application in small, flexible optical fiber devices such as waveguides, sensors, and lasers. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. 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PMC006xxxxxx/PMC6644406.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145904810.1021/acsomega.8b00877ArticleLarge-Scale Fabrication of High-Performance Ionic Polymer–Metal Composite Flexible Sensors by in Situ Plasma Etching and Magnetron Sputtering Fu Ruoping †‡Yang Ying ‡Lu Chao ‡Ming Yue ‡Zhao Xinxin ‡Hu Yimin ‡Zhao Lei ‡Hao Jian †Chen Wei ‡§† Department of Chemistry, College of Sciences, Shanghai University, Shanghai 200444, P. R. China‡ i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P. R. China§ Nanotechnology Centre for Intelligent Textiles and Apparel, Institute of Textiles and Clothing, The Hong Kong Polytechnic University, Kowloon, Hong Kong 999077, P. R. China E-mail: wchen2006@sinano.ac.cn (W.C.).15 08 2018 31 08 2018 3 8 9146 9154 02 05 2018 01 08 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Flexible electronics has received widespread concern and research. As a most-fundamental step and component, polymer metallization to introduce conductive electrode is crucial in successful establishment and application of flexible and stretchable electronic system. Ionic polymer–metal composite (IPMC) is such an attractive flexible mechanical sensor with significant advantages of passive and space-discriminative capability. Generally, the IPMC sensor is fabricated by the electroless plating method to form structure of ionic polymer membrane sandwiched with two metallic electrodes. In order to obtain high-quality interface adhesion and conductivity between polymer and metal, the plating process for IPMC sensor is usually time-consuming and uncontrollable and has low reproducibility, which make it difficult to use in practice and in large-scale. Here, a manufacturable method and equipment with short processing time and high reproducibility for fabricating IPMC sensors by in situ plasma etching and magnetron sputtering depositing on flexible substrates is developed. First, the new method shortens the fabrication period greatly from 2 weeks to 2 h to obtain IPMC sensors with sizes up to 9 cm × 9 cm or arrays in various patterns. Second, the integrated operation ensures all sample batch stability and performance repeatability. In a typical IPMC sensor, nearly 200 mV potential signal due to ion redistribution induced by bending strain under 1.6% can be produced without any external power supply, which is much higher than the traditional electroless plating sensor. This work verified that the in situ plasma etching and magnetron sputtering deposition could significantly increase the interface and surface conductivity of the flexible devices, resulting in the present high sensitivity as well as linear correlation with strain of the IPMC sensor. Therefore, this introduced method is scalable and believed to be used to metalize flexible substrates with different metals, providing a new route to large-scale fabrication of flexible devices for potential wearable applications in real-time monitoring human motion and human–machine interaction. document-id-old-9ao8b00877document-id-new-14ao-2018-00877uccc-price ==== Body Introduction In order to overcome application problems of the electronic system in the large deformation and arbitrarily curved surfaces, flexible electronics that is compatible with movable parts has been driven to the advent and growth over the last few decades. Flexible electronics create a wide range of revolutionary functional devices, including sensors, actuators, robots, and other electronic devices that are bendable, curved, and even stretchable.1−9 As the most basic component of electronics, the thin-film conductive electrode of polymer metallization fabrication has become a key part of the successful application of flexible and stretchable electronic systems. Recently, ionic polymer–metal composites (IPMCs) have extensively been studied for their excellent properties and potential applications in the areas of science and technology as flexible sensors.10−16 An IPMC sample typically consists of a thin ion-exchange membrane (e.g., Nafion) and metallic electrodes on both surfaces with a noble metal.17 Also, IPMC can induce voltages by bending or stretching the membrane, which makes IPMC an ideal flexible sensor. Compared with electronic sensors,18−20 these IPMC sensors show significant advantages of no power supply and the ability to distinguish bending directions. IPMC sensors are generally fabricated by electroless plating21 to achieve polymer metallization on the flexible Nafion membrane. Polymer metallization use conventional conductive materials, such as rigid metal nanoparticles (mainly including Cu, Ag, or Au) on polymer film to realize flexibility and stretchability. However, these conductive materials may not be suitable for flexible electronics. The flexible polymeric material (from fractions to hundreds of MPa) and the hard electrode material (from tenths to hundreds of GPa)22 have the large Young’s modulus mismatch, which causes electrode cracking, stripping, and delamination during multicycle bending and further limits the lifetime and the bending stability of the IPMC sensors.23 Thus, in view of applications, fabrication of compliant and well-adherent electrodes that are closely integrated with the polymer layer is crucial.24 Besides, fabrication of IPMC sensors still remains a big challenge mainly because the traditional electroless plating method is manual operation. This process not only consumes a long period, but it also has many uncontrollable factors. Most importantly, reliability of manual operation is not strong, resulting in low reproducibility. What’s more, they have difficulty for batch production. Therefore, in order to develop production techniques of industrial interest, a non-manual preparation method with high efficiency should be developed. Here, in order to solve the aforementioned problems, we develop a manufacturable and integrative method and equipment with short processing time and high reproducibility for fabricating IPMC sensors by in situ plasma etching and magnetron sputtering depositing on flexible substrates. Some literature studies have only used plasma to etch Nafion membrane. Omosebi and Besser25 used plasma to etch the Nafion membrane for multiple electrochemical applications. Van Nguyen26 etched with argon on the surface Nafion membrane and investigated its performance in a proton-exchange membrane fuel cell. Also, some literature studies have only reported to prepare IPMC using magnetron sputtering. Zhou27 sputter-coated a gold seed layer of 0.4 μm thickness. Siripong28 employed sputter coating for deposition of gold on Nafion for fast and economical fabrication of IPMC. Thus, we place two devices into a single chamber to rapidly and large-scale prepare IPMC. Plasma etching is a potentially attractive method to control polymer surface modification without affecting the bulk properties of the material.29 Plasma treatment depends heavily on the adjustment of parameters such as power and treatment duration.30 We can quantify and repeat the degree of etching with defined parameters. Magnetron sputtering has developed rapidly over the last decades to the point where it has become established as the process of choice for the deposition of a wide range of industrially important coatings on plastic substrates at room temperature.31 For equipment, plasma etching filament and magnetron sputtering target are installed in one chamber and the control panel was operated to achieve the integrative fabrication. Plasma etching increases the roughness of the intermediate layer surface to improve the adhesion of the electrode layer. Magnetron sputtering deposits silver particles to metallize Nafion membrane. Silver is considered as a high conductive material and is much cheaper than gold, platinum, or other precious metals. The integrative preparation method provides the possibility of batch and reproducible production of flexible IPMC sensors. First, the new method shortens the fabrication period greatly from 2 weeks to 2 h to obtain IPMC sensors with sizes up to 9 cm × 9 cm or arrays in various patterns. Second, the integrated operation ensures all sample batch stability and performance repeatability. In a typical IPMC sensor, nearly 200 mV potential signal under a bending-induced strain as small as 1.6% can be produced without any external power supply, which is 63 times higher than that of the sensor fabricated by traditional electroless plating. We verified that the in situ plasma etching and magnetron sputtering depositing could significantly increase the interface and surface conductivity of the flexible devices. Plasma etching processing improves the degree and uniformity of etching on the Nafion film surface, resulting in a much lower equivalent series resistance. The magnetron sputtering-based sensor shows a good interface contact between the electrolyte and electrode as well as excellent electronic conductivity of the electrode material. Moreover, this flexible IPMC sensor has better bending cycle stability and high sensitivity as well as linear correlation with strain. Then, we mainly optimize the parameters of etching power, etching time and magnetron sputtering, sputtering time, sputtering power, working pressure, and working argon flow rate to get an IPMC sensor with excellent performance. In a word, this introduced method is scalable and believed to be used to metalize flexible substrates with different metals, providing a new route to large-scale fabrication of flexible devices for potential wearable applications in real-time monitoring human motion and human–machine interaction. Results and Discussion Preparation and Characterization The equipment used to fabricate the flexible IPMC sensor is shown in Figure 1a. The sample holder (①), the filament (③) for plasma etching, and the magnetron sputtering target (④) are installed in the chamber of the equipment. The equipment is also equipped with a mechanical handle (②) for inverting the sample on the sample holder, which ensures that both sides of the sample are plasma-etched and magnetron sputtering-coated with silver particles to achieve integrative fabrication. In the fabrication process, we only need to operate and set the plasma etching and magnetron sputtering parameters on the control panel instead of manual operation, which is a simple and controllable process. A schematic representation of the fabrication process of the flexible IPMC sensor is presented in Figure 1b. Before plasma etching, the Nafion substrate (suitable for different sizes and array pattern) is fixed on the hollow part of rotatable sample holder allowing the rotation of the substrate along a direction, which assures sample uniformity of etching and metalizing over the entire surface. The picture of simple holder is shown in Figure S1. Then, plasma etching with defined energy is used to control Nafion membrane surface modification. After completing, the holder is turned over 180° by the mechanical handle, and the opposite side of the Nafion polymer is processed. Finally, magnetron sputtering deposited silver particles on the metallic Nafion polymer. Once the electrode with appropriate conductivity has been deposited, the holder is turned over 180° by the mechanical handle, and the opposite side of the Nafion polymer is deposited with silver particles. The details can be found in the Experimental Methods section. Figure 1 Schematic of the fabrication process. (a) Equipment used to manufacture the IPMC sensor. (b) Schematic of the fabrication process of the IPMC sensor. The pictures of fabricated IPMC sensors are shown in Figure 2. The IPMC sensor is flexible and can be easily bent. What’s more, the specular IPMC sensor surface is shiny and bright; in other words, the fabricated silver electrode layer has very high quality. We can obtain IPMC sensors with size up to 9 cm × 9 cm (Figure 2a) or arrays in various patterns (Figure 2b–d). Moreover, mechanical operation ensures high performance of each batch samples. Figure 2 Pictures of the fabricated IPMC sensors. (a) Pictures of large-scale IPMC sensors. (b–d) Pictures of various array patterns of IPMC sensors. Then, the observation of microscopic morphology of the IPMC sensor is done by using an electron microscope. Figure 3a displays the scanning electron microscopy (SEM) image of the IPMC sensor surface, which clearly shows the homogeneous silver layer coated on the surface of the polymer membrane layer and the silver nanoparticles are closely integrative, having a diameter between 50 and 160 nm. Figure 3b displays a detail of the cross-sectional SEM image of the IPMC sensor, which clearly indicates that the silver nanoparticle layer and the Nafion membrane interlayer are well-interconnected. The structure benefits the cyclic stability of the sensor. In the inset of Figure 3c, the Nafion membrane interlayer with an average thickness of 183 μm is sandwiched between two silver electrodes, forming the IPMC sensor. In order to verify the distribution of the Ag electrode layer in the IPMC sensor, Figure 3c shows the energy-dispersive X-ray spectroscopy (EDX) signal, which is collected from the vertical cross section of the IPMC in the inset SEM image. Ag is mainly concentrated on both sides of IPMC. Moreover, typical X-ray diffraction (XRD) powder diffraction pattern of the surface layer of the IPMC sensor is shown in Figure 3d. The peaks at 38.1°, 44.21°, 64.44°, and 77.33° correspond to the (111), (200), (220), and (311) crystalline planes of the face-centered cubic crystal structure of Ag (JCPDS, file no. 04-0783), which demonstrates the existence of Ag electrode layer. Figure 3 Characterization of the morphology of the IPMC sensor. (a) SEM image of the IPMC sensor surface. (b) Cross-sectional SEM image of the IPMC sensor. (c) EDX line scan of the cross section of the IPMC sensor. The inset shows the corresponding SEM image. (d) Typical XRD pattern of the surface layer of the IPMC sensor. Sensing Performance of the IPMC Sensor An IPMC sample typically consists of a thin ion-exchange membrane and a noble metal as electrodes on both surfaces. Figure 4a displays the sensing mechanism of the IPMC sensor. As we know, the Nafion membrane contains the sulfonic acid group and can be ionized and release the mobile hydrions with adsorbed water. When the sensor is in a flat state, mobile hydrions uniformly disperse in the Nafion membrane. When a bend deformation is applied on the sensor, an elastic stress gradient is generated along the thickness. The mobile hydrions on the compressed side of the membrane migrate and accumulate on the lower hydraulic pressure side of the membrane. More hydrions accumulate on the stretched side, resulting in a positive potential. The voltage signal is generated from ion movement and accumulation in the deformation process, so the sensor could generate electrical signal without external energy supply. Figure 4 Sensing mechanism and sensing performance of IPMC sensors. (a) Schematic of the sensing mechanism. (b) Potential of the in situ plasma etching and magnetron sputtering depositing silver particle-based IPMC sensor and the electroless plating-based IPMC sensor. (c) Voltage responses of the IPMC sensor to the recognization of the bending direction. (d) Potential of the sensor with variations of the bending deformation of 1, 2, 3, 4, 5, 6, and 7 mm. (e) Potential signal of the sensor vs strain (ε). (f) Bending and recovery of the IPMC sensor for 3000 cycles of 3 mm displacement. For the sensing mechanism of the sensor, we buit a corresponding test platform and obtained signals of sensors under different strains. The test method can be found in the Experimental Methods section. The output electrical signals of magnetron sputtering depositing silver particle-based sensor and conventional electroless plating-based sensor are shown in Figure 4b. Output voltage shows a periodic alternation of positive voltage, and output voltage of the magnetron sputtering sensor is 112.55 mV. However, when the IPMC sensor returns back after bending, hysteresis is produced because the movement of the ions is not as fast as electron transport. Thus, the signal peak is asymmetric. Output potential of the electroless plating sensor is 1.80 mV. The signal of magnetron sputtering is much larger than that of the electroless plating sensor. The ability of the IPMC sensor to recognize the orientation is shown in Figure 4c. The insets in Figure 4c display the real operation scenes of upward-bending and downward-bending in the test process. For the first six times, the sensor bends downward to get the negative voltage, and for the latter six times, the sensor bends up and gets a positive voltage. As the sensor bends to opposite directions, positive and negative relative potentials appear in turn, respectively. A series of bending deformations of 1, 2, 3, 4, 5, 6, and 7 mm are repeatedly measured (Figure 4d). Different displacements produce different signals, indicating high repeatability and sensibility of IPMC sensors. We use the Euler–Bernoulli theory to build a model to calculate the strain at the corresponding displacement. The film sensor has a thickness of h and a length of L. When the displacement platform moves x, the film is curved. A schematic diagram is shown in Figure S2. Assuming that the central angle of the curved arc is θ, the radius is R. According to the geometric relations, we can list the equations 1 2 We know L and x and can get θ and R from eqs 1 and 2. Finally, the Euler–Bernoulli approximation theory is used to calculate the bending strain, and the strain corresponding to the displacement is obtained. 3 Using this method, the strain of our IPMC is 1.03% at a displacement of 3 mm. Potential signal as a function of a series of strain is given in Figure 4e, which exhibits an approximately linear correlation. Small error bars (standard deviations) are taken from 10 times measurement. Therefore, the IPMC sensor has perfect repeatability. The sensor potential under the maintained extended state is also detected (Figure S3). When the sensor keeps bending, the signal continues to increase and then begins to fall slowly. The output signal declines around 5% slowly when the sensor maintained 10 s bending state (Figure S3a). The output signal declines around 27.5% while maintaining 60 s bending state (Figure S3b). In order to analyze the durable performance of the IPMC sensor, cyclical bending deformation and recovery of the sensor was measured for 3000 cycles of 3 mm displacement and is shown in Figure 4f. The insets in Figure 4f show the details of the cyclic test. The output potential signals of the sensor are stably maintained with a relative small variation. Although the signal has slight attenuation, the signal is still much larger than the signal of the electroless plating-based sensor, and the peak type is consistent. It is proved that the sensors have a relatively good cycle stability. Therefore, the sensor fulfills the requirement for practical application. This is the result of our preliminary experimental stage. In the future, we will continue to explore and improve its durable performance. The electromechanical behavior of IPMC under electrical voltage stimulation of ±1 V is also investigated (Figure S4). The bending displacement is monitored by a laser displacement meter. Bending displacement reaches 11.0 μm. The size of the mobile ions in the IPMC is small. If we increase the number of mobile ions and soak large-size ions in the Nafion membrane, the electromechanical behavior of IPMC will be improved. The traditional method for preparing an IPMC sensor is manual processing for roughing Nafion film and electroless plating for electrode layers. The signal of our sensor (112.55 mV, Figure 4b) is about 63 times compared with that of electroless plating-based sensor (1.80 mV, Figure 4b). We read the relevant literature studies and found that the signal of the sensor made by electroless plating is relatively low. The IPMC sensor prepared by Song32 can generate a voltage signal of 20 mV. This is the largest signal of IPMC we have seen from the literature so far. However, the signal of our IPMC sensor is much larger than this value. The electrode layer thickness of the electroless plating-based IPMC sensor is about 3–5 μm. The electrode layer thickness of the magnetron sputtering-based IPMC sensor is 280–300 nm. For the electroless plating-based IPMC sensor, the impregnation–reduction steps were repeated five times. The reduction step is a process of electrode layer growth, and the reduction takes a total of 25 h. The growth rate is 0.12–0.2 μm/h. For the magnetron sputtering-based IPMC sensor, magnetron sputtering lasts for 30 min and the electrode layer thickness increases to 280–300 nm. The growth rate is 0.56–0.6 μm/h. In order to evaluate the effect of the in situ etching and magnetron sputtering depositing method to increase potential signal, first, the average root mean square (rms) surface roughness (Rq) value of the treated Nafion membrane is measured and analyzed. Figure 5a,b shows 3D atomic force microscopy (AFM) images of Nafion membrane of plasma etching processing and manual processing, respectively. The average Rq value of Nafion membrane of plasma etching processing is about 72.9 nm. The measured Rq value for manual processing Nafion membrane is about 65.6 nm and is relatively lower. It is clear from the AFM image that surface grooves of the plasma etching-treated membrane are evenly distributed and have comparatively consistent depth compared with those of the manual processing membrane, which greatly benefits for the proper microstructural development. The observed more uniform rough surface is a desirable two-dimensional layer by layer growth mode (Frank–van der Merwe mode).33Figure 5c shows Nyquist plots of the IPMC sensors. The equivalent series resistance of the magnetron sputtering-based IPMC sensor is 2 Ω, which is much lower than that of electroless plating-based sensor (equivalent series resistance of is 145 Ω). The magnetron sputtering-based IPMC sensor shows a good interface contact between the electrolyte and electrode as well as excellent electronic conductivity of the electrode material. The surface sheet resistance of the electrode film is as high as 0.08–0.15 Ω/□, further confirming excellent electronic conductivity. In summary, it is proved that the in situ plasma etching and magnetron sputtering depositing method is effective for fabricating high-performance IPMC sensors. Figure 5 (a) 3D AFM image of the plasma etching-based Nafion membrane. (b) 3D AFM image of the manual processing-based Nafion membrane. (c) Nyquist plots of three electrodes of the IPMC sensor with electroless plating and the IPMC sensor with magnetron sputtering. The inset picture clearly displays Nyquist plots of three electrodes of the IPMC sensor with magnetron sputtering. However, the plasma etching power and duration have a great influence on the degree and uniformity of the film surface etching. In addition, magnetron sputtering time, sputtering power, working pressure, and working argon flow rate also have great impact on the performance of the sensor. Consequently, the conditions of the preparation process are optimized to obtain excellent IPMC sensors. Cho et al.34 studied that the etching procedure did not alter the chemical character of the membrane. Only the physical roughness does not affect performance. The surface morphologies of the modified Nafion membranes are investigated by using SEM, as shown in Figure 6a–i. The surface roughness enhances with increase of power (Figure 6a–f) or time (Figure 6g–i) because of the effect of the plasma etching treatment. Output potential signals of IPMC sensors of different plasma etching powers or time can be used as a parameter to determine the best conditions, which is shown in Figure 7a,b. When the etching power is 8 or 10 W, the prepared sensor cannot obtain a stable output signal. The output signal increases to the maximum and then significantly goes down as the etching power increases. When the etching time is 360 s, the sensor has maximum output signal. The Nafion membrane surface can be etched efficiently, which affects the output signal of IMPC. Figure 6 SEM images of different plasma etching powers and constant 180 s etching duration on the Nafion membrane surface: (a) 8 W; (b) 10 W; (c) 12 W; (d) 14 W; (e) 16 W; and (f) 18 W. SEM images of different plasma etching durations and constant 14 W etching power on the Nafion membrane surface: (g) 180 s; (h) 360 s; and (i) 540 s. Figure 7 (a) Output potential signals of IPMC sensors of different plasma etching powers and constant 180 s etching duration on the Nafion membrane surface. (b) Output potential signals of IPMC sensors of different plasma etching durations and constant 14 W etching power on the Nafion membrane surface. In the same method, the effects of sputtering time, sputtering power, working pressure, and working argon flow rate on the film performance are tested with indicators such as electrode layer thickness and output potential signal while keeping other parameters constant. Figure 8a shows output potential signals and electrode layer thicknesses of sensors under different sputtering power. In a certain range, the greater the sputtering power, the deposited particles have higher energy and the metal element deposition on the substrates is accelerated. Therefore, the particles have higher adhesion with the substrate. The film thickness increases with the increase of sputtering power. Output potential signal reaches the maximum value at 100 W power; the value of the output potential is an important parameter of device performance. Therefore, the sputtering power at 100 W is considered as a better value to ensure outstanding sensor performance. Figure 8 Curve of output potential signals and electrode layer thicknesses of sensors under different magnetron sputtering conditions: (a) 90–130 W different sputtering power and constant sputtering 30 min duration, 1.0 Pa working pressure, 30 sccm argon flow rate; (b) 5–60 min different sputtering time and constant sputtering 120 W power, 1.0 Pa working pressure, 30 sccm argon flow rate; (c) 0.25–3.5 Pa different sputtering working pressure and constant sputtering 120 W power, 30 min duration, 30 sccm argon flow rate; and (d) 20–40 sccm different sputtering argon flow rate and constant sputtering 120 W power, 30 min duration, 30 sccm argon flow rate. Figure 8b depicts output potential signals and electrode layer thicknesses of sensors under different sputtering times. It is observed that the electrode layer thickness increases with increase of sputtering time. To be more specific, with the increase of sputtering time, the content of silver increases. The number of particles deposits onto the Nafion substrate increases. ZAO films were prepared by magnetron sputtering conducted by Song,35 who has already shown that for a longer sputtering time, the thickness of the film increases as well, and interestingly shows a linear correspondence to sputtering time. Output potential signal reaches the maximum value at 30 min. With a shorter deposition time, there are less particles on the substrate. As the deposition time increases, the number of particles increases, and the layer thickens. The electrode layer has good compactness. However, with a longer deposition time, the electrode layer thickness increases, which is not conducive to sensors bending deformation. The electrode is easy to peel and split as the thickness increases. Thus, the sputtering time at 30 min is considered as a better value to ensure sensor performance excellent. It can be seen from Figure 8c that with the increase of working pressure, the electrode layer thickness decreases. With a lower deposition pressure, there are less collisions in the path of particles to the substrate. Particles can easily reach the substrate to form a film and the film has good performance. However, when the deposition pressure increases, there are more collisions in the path of particles to the substrate, thus the particles energy decreases. The amount of particles reaching the substrate decreases, which causes more defects in the films and leads to the decrease of electrode layer thickness. Output potential signal of sensor increases from 2.2 mV (0.25 Pa) to a maximum value at 29.4 mV (1.0 Pa) and then goes down to 9.5 mV at 3.4 Pa. Considering appropriate electrode layer thickness and electrical signals, the sensor performance is more excellent when the deposition pressure at 1.3 Pa. Figure 8d shows the variety of electrode layer thicknesses and output potential signals of sensors as a function of different the argon flow rate. Apparently, the electrode layer thickness decreases with the increase of argon flow rate. When the magnetron sputtering starts, the electrons collide with the argon atoms in the process of flying to the substrate under the action of the electric field and produce argon ions and new electrons. Argon ions in the electric field accelerate toward the cathode target and high-energy bombard the target surface, and the target sputtering occurred. Neutral target atoms or molecules are deposited on the substrate to form a thin film. The higher argon flow rate increases the working pressure, and there is lower mobility and a great number of native defects. The output potential signal of the sensor rises to a peak value at an argon flow rate of 30 sccm and then decreases as the argon flow rate further increases. From inspection, the maximum voltage signal is found to be 29.55 mV for the sample grown using an argon flow rate of 30 sccm. Considering the stability of the sensors and the output of the electrical signal, the optimal argon flow rate is 28 sccm. Conclusions In this work, an Ag electrode-based IPMC sensor is fabricated on a flexible Nafion substrate by the in situ plasma etching and dc balanced magnetron sputtering deposition technique. The new method shortens the fabrication period greatly from 2 weeks to 2 h to obtain IPMC sensors with sizes up to 9 cm × 9 cm or arrays in various patterns. The integrated operation ensures all sample batch stability and performance repeatability. These IPMC sensors can generate electrical signals under the external bending deformation for mobile hydrions migrate and accumulate on the lower hydraulic pressure side. IPMC sensors we fabricated can generate nearly 200 mV potential signal without external power supply under a 1.6% bending-induced strain and recognize different directions of the bending strain. The signal of our sensor is much larger than that of the traditional electroless plating-based sensor. What’s more, this IPMC sensor is suitable for in long-term and large-scale bending and have high sensitivity as well as linear correlation with strain. The plasma etching-based Nafion membrane has higher rms surface roughness (Rq) values. Surface grooves of membrane are evenly distributed and have comparatively consistent depth. The equivalent series resistance of the magnetron sputtering-based IPMC sensor is 2 Ω, which is much lower than that of the electroless plating-based sensor (equivalent series resistance of is 145 Ω). The magnetron sputtering-based sensor shows a good interface contact between the electrolyte and electrode as well as excellent electronic conductivity of the electrode material. Optimal properties of the IPMC sensor are obtained with plasma etching power at 14 W, plasma etching duration at 360 s, magnetron sputtering power at 100 W, magnetron sputtering time at 30 min, magnetron sputtering working pressure at 1.3 Pa, and a magnetron sputtering working argon flow rate at 28 sccm. From these data, the IPMC sensor fabricated by in situ plasma etching and magnetron sputtering has high performance of large and stable output signal. The study provides a manufacturable and integrative method and equipment with short processing time and high reproducibility for large-scale preparing flexible sensors of mechanical bending durability on flexible substrates. This preparation method is scalable and silver electrodes can be replaced with other metallic materials such as gold, copper, chromium, indium tin oxide (ITO), aluminum oxide, and so forth. We believe that this method provides a new route to short-time and quantifiably fabricate IPMC sensors for potential wearable applications in real-time monitoring human motion and human–machine interaction. Experimental Methods Materials and Instruments Hydrogen peroxide, sulfuric acid, HAuCl4, 1,10-phenanthroline monohydrate (Phen), and sodium sulfite were purchased from Sinopharm Chemical Regent Co. Ltd. The Nafion 117 membrane was purchased from the Dupont. Equipment with the commercial dc balanced magnetron sputtering system and the inductive coupled plasma-reactive ion etcher was customized. Preparation of the IPMC Sensor by in Situ Plasma Etching and Magnetron Sputtering Depositing Before etching, the Nafion membrane was cleaned by hydrogen peroxide (5% mass fraction) and diluted sulfuric acid (1 mol L–1). Then, the Nafion membrane (suitable for different size) was fixed on the hollow part of the rotatable sample holder in the chamber allowing the rotation at a speed of 5 rpm of the substrate along a direction, which assures sample uniformity of etching and metallizing over the entire surface. The chamber pressure was pumped down to below 3 × 10–4 pa in order to prevent residual atmosphere effects on the composition of the Nafion films. Then, the argon valve was opened and the argon flow rate was set as 8 sccm. All of the specimens were grown by using pure argon (99.99%) as etching and sputtering gas. We controlled the screen voltage, the screen current, and etching duration during plasma etching to get a sample with uniform etching and excellent performance. After completing treatment of one side of the Nafion membrane, the holder was turned over 180° by the mechanical handle, and the opposite side of the membrane was etched. Then, magnetron sputtering deposited silver particles on the metallic polymer. The sputtering process was performed at room temperature. The chamber pressure was adjusted to appropriate value. The argon flow rate was set to what we want. Then, the deposition power and duration parameters were set. Once the deposition was completed, the holder was turned over 180° by the mechanical handle, and the opposite side of the polymer was processed. No post annealing was performed after deposition. The substrate temperature was maintained at 20 °C approximately during the deposition process. The Nafion membrane is cleaned with ionized water, vacuumed in the chamber, etched, and sputtered to plate the electrodes. The Nafion membrane retains its intrinsic water, which assures the migration of ions in the membrane, thereby generating the sensing signal. The IPMC sensor was cut into a size of 25 mm × 5 mm for the experimental test. Preparation of the Electroless Plating-Based Sensor The Nafion membrane was roughed by 1200 sandpaper polishing 800 times both sides. Then, the Nafion membrane was cleaned. Next step, the Nafion membrane was placed into a solution of [Au(Phen)Cl2]Cl for 24 h to allow the sufficient impregnation of cations. After that, the Nafion membrane was immersed into deionized water in water-bath heating. A Na2SO3 solution of 5 mmol L–1 was slowly dropped into the water till the Au cations at the membrane’s surface are reduced to Au particles totally. The impregnation–reduction steps were repeated five times. Sensing Measurement The IPMC sensors need to be deformed to produce a voltage signal. We built a corresponding test platform, including signal generation section and signal collection section. The signal generation section includes fixtures, displacement platform (MTS121), and displacement stepper motor. The signal collection section includes an electrochemical workstation and a controller computer. The displacement platform can realize precise displacement control in the X-axis direction, with an accuracy of 0.005 mm. Moreover, the displacement platform can realize and control different movement rates, the number of cycle displacements, and the distance of displacement, including any distance of 0–7 mm. The electrochemical workstation can collect and display on the computer the electrical signals generated by the deformation of the sensor. Therefore, we can use this measurement platform to realize the basic bending performance test of our sensor. The IPMC sensor in response to the cyclic bending and resuming flat state is tested. The sensor is placed on the displacement platform and is clamped by fixtures. The sensors we test have an effective length of 1.8 cm. Also, the maximum displacement of the platform is set as 3 mm. The displacement platform gradually moves from 0 to 3 mm, the signal generated by the sensor gradually increases from 0, and then the displacement platform gradually returns from 3 to 0 mm. The signal generated by the sensor gradually decreases from the maximum value. Characterization Surface morphology and cross-sectional SEM images were obtained with a Hitachi S-4800 filed emission scanning electron microscope. The 3D AFM images and the average rms surface roughness (Rq) values were obtained with a Dimension 3100 atomic force microscope. EDX was performed on Quanta 400FEG (FEI). XRD patterns were obtained on a X’Pert-Pro MPD (Cu Kα). The surface sheet resistance of the electrode film was tested by using a multifunctional digital four-probe tester (ST-2258C). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00877.Picture of a simple holder; schematic diagram of analytical model of the sensor; potential change of IPMC under overtime state maintaining; and electromechanical behavior of IPMC under electrical voltage stimulation of ±3 V (PDF) Supplementary Material ao8b00877_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was supported by the External Cooperation Program of BIC from Chinese Academy of Sciences (grant no. 121E32KYSB20130009) and the Science and Technology of Jiangsu Province (grant no. BE2016086). ==== Refs References Ge J. ; Sun L. ; Zhang F.-R. ; Zhang Y. ; Shi L.-A. ; Zhao H.-Y. ; Zhu H.-W. ; Jiang H.-L. ; Yu S.-H. A Stretchable Electronic Fabric Artificial Skin with Pressure-, Lateral Strain-, and Flexion-Sensitive Properties . Adv. 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PMC006xxxxxx/PMC6644407.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145799710.1021/acsomega.8b00813ArticleSolution-Processed BiI3 Films with 1.1 eV Quasi-Fermi Level Splitting: The Role of Water, Temperature, and Solvent during Processing Williamson B. Wesley Eickemeyer Felix T. Hillhouse Hugh W. Department of Chemical Engineering, Clean Energy Institute, and Molecular Engineering & Sciences Institute, University of Washington, Seattle, Washington 98105, United States E-mail: h2@uw.edu (H.W.H.).05 10 2018 31 10 2018 3 10 12713 12721 25 04 2018 08 05 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. We present a mechanistic explanation of the BiI3 film formation process and an analysis of the critical factors in preparing high-quality solution-processed BiI3 films. We find that complexation with Lewis bases, relative humidity, and temperature are important factors during solvent vapor annealing (SVA) of films. During SVA, water vapor and higher temperatures limit the formation of the BiI3–dimethylformamide coordination complex. SVA with an optimized water content and temperature produces films with 300–500 nm grains. Films that formed solvent coordination compounds at lower temperatures showed preferential crystal orientation after solvent removal, and we elucidate its implications for carrier transport. Addition of dimethyl sulfoxide to highly concentrated tetrahydrofuran–BiI3 inks prevents film cracking after spin-coating. We have measured a quasi-Fermi level splitting of 1.1 eV and a diffusion length of 70 nm from films processed with optimal temperature and humidity. The best device produced by optimized SVA has a power conversion efficiency of 0.5%, Isc of ∼4 mA/cm2, and VOC of ∼400 mV. The low photocurrent and voltage we attribute to the low diffusion length and the unfavorable band alignment between the absorber and the adjacent transport layers. The deep understanding of the relationship between morphology/crystal structure and optoelectronic properties gained from this work paves the way for future optimization of BiI3-based solar cells. document-id-old-9ao8b00813document-id-new-14ao-2018-00813rccc-price ==== Body Introduction Photovoltaic (PV) materials such as copper zinc tin sulfoselenide (CZTS),1−3 copper indium gallium sulfoselenide (CIGS),4−6 and, more recently, hybrid perovskites (HPs)7−9 are all solution-processable. Solution-processed materials are exciting because they can be spray-coated, blade-coated, or slot die-coated onto large substrates, often at low temperatures,10 and thus may reduce the upfront capital expenditure (CAPEX) to begin manufacturing.11 The CAPEX of roll-to-roll processing may be as low as 0.06$/WaCap versus 0.68 to 1.01$/WaCap for CdTe and polysilicon processes ($/WaCap is $ per annual production capacity in watts). However, materials such as CZTS are not yet commercializable as device efficiencies are still less than 15%, mainly because of low minority carrier diffusion lengths12 and low open-circuit voltages. However, CIGS cells are commercial and have demonstrated module efficiencies of 16.5% for vacuum-deposited absorbers.13 Solution-processed CIGS is also being developed with laboratory-scale efficiencies of 14.7%.6 The process still requires a high-temperature step, and indium (used in CIGS) is considered by some to add financial risk because of indium price volatility or constraints on the manufacturing growth rate.14 Solution-processed HP efficiencies have increased rapidly and are over 20% with the added benefit of requiring no high-temperature steps.15 As promising as these material are, they possess several shortcomings. All high-efficiency HP materials contain lead, which has environmental health risk.16 HP materials also have limited lifespans owing to low thermodynamic stability of the compound,17 degradation in the presence of light and moisture,18 or light-induced phase segregation.17 However, these materials have remarkable properties and defect tolerance. The origin of this defect tolerance is believed to come from a partial oxidation of the Pb2+ cation, withholding its 6s2 electrons.19 These 6s2 electrons give rise to a valence band maximum consisting of antibonding orbitals. In addition, relativistic spin–orbit effects increase the width of the conduction band considerably. As a result, dangling bonds from vacancy formation appear as resonances within the bands, rather than as trap states in the band gap.20 Bi3+ likewise possesses this beneficial electronic structure, and it is expected to possess a similar defect tolerance.20 BiI3 is composed of layers of edge-sharing BiI6 octahedra held together in part by van der Waals interactions.21 Lehner et al. have found that BiI3 has a large static dielectric constant with principal components of the static dielectric tensor of εxx∞ = 18.9 and εzz∞ = 15.0, which may indicate effective screening of charged point defects, thus improving transport.22 BiI3 has also shown lifetimes of over 7 ns.19 Materials with lifetimes of at least 1 ns (such as CZTS, InP, and CIGS) have achieved device efficiencies of greater than 10%.19 Furthermore, materials such as BiI3 are well-investigated materials for room-temperature gamma-ray detectors23 owing to the high interaction cross section. One of the biggest advantages of Bi3+ over Pb2+ is that it is less toxic. For instance, bismuth is already in use as a component of lead-free solders. Compared to lead, mercury, and arsenic, bismuth is the least toxic because of its low water solubility.24 While the World Health Organization (WHO) has set a standard of 10 ppb for lead in drinking water,25 in contrast, no limits have been imposed for bismuth. Finally, BiI3 has a 1.8 eV band gap and high absorption coefficient.21 Using the absorption coefficient data presented by Brandt et al.,21 a 500 nm thick film would be sufficient to absorb 95% of incident above-band gap photons. A 1.8 eV-band gap BiI3 top cell would be well-matched with a 1.12 eV Si bottom cell, which would give a maximum theoretical efficiency of 45.3% in a four-terminal tandem configuration.26 Processing variables such as substrate temperature during physical vapor deposition or choice of solvent for solution processing21 greatly impact the morphology of the BiI3 film. Bridgman growth23 and vacuum-based methods21 are well-developed techniques for making BiI3 films. Solution-processing routes are less well-developed. BiI3 has been solution-deposited from dimethylformamide (DMF),21,27 tetrahydrofuran (THF),27 and dimethyl sulfoxide(DMSO).28 In fact, bismuth trihalides are known to form coordination compounds with all of these solvents.28,29 These solvents are similar in that they possess Lewis base sites, which result in the solvent and BiI3 forming Lewis acid-base adducts. Hamdeh et al. have applied DMF solvent vapor annealing (SVA) on a spin-coated BiI3 film from THF solution to grow large grains, and by this, they have achieved the highest power conversion efficiency (PCE) to date for a BiI3-based solar cell of 1.0%.27 In SVA, films are heated in the presence of solvent vapor using solvents that have affinity for BiI3. The presence of solvent vapor increases the mobility of molecular species in the film and allows the film to reorganize and achieve larger grains. DMF SVA has been also used previously in the hybrid perovskites to grow grains of 1 μm in diameter.30,31 However, SVA is sensitive to the environment, which causes severe reproducibility issues, and the key parameters, which have an impact on the morphology, are neither known nor understood. Further, state-of-the-art BiI3 solar cells with a PCE of around 1% suffer from significant current and voltage losses. The materials chemistry reasons for these deficiencies are not known to date. Here, we address both issues. We present an investigation of important variables affecting SVA of BiI3 films, such as temperature and water concentration. We used DMF as the SVA solvent as it forms a mildly stable BiI3–DMF complex,28 which plays a role in growing good morphologies. We show that tuning of water vapor concentration and temperature is crucial to grow large-grained, void-free solution-processed BiI3 films and that the presence of ambient oxygen is less crucial. We present a mechanism for the action of both water and temperature. Through this understanding, we present an optimized SVA process for growing continuous BiI3 films on TiO2 substrates. For the first time, we apply absolute intensity photoluminescence (AIPL) measurements on BiI3 films to assess the maximum possible open-circuit voltage and lateral dc photoconductivity measurements to assess the carrier transport. These measurements give us access to the two key parameters for solar cell operation, the quasi-Fermi level splitting (QFLS) and the average electron–hole diffusion length. By correlating different SVA processes with these material property measurements and PV device performance, we reveal the limiting factors for the low device performance. Results and Discussion Effect of Water, Temperature, and Solvent on Morphology Pre-SVA films are dense but small-grained (Figure 1a). SVA grows the grains (Figure 1b,c), but the morphologies are not always the same. We noticed that films prepared in the fume hood with SVA at 100 °C on different days have very different morphologies. These films were prepared under the exact same conditions, with the only difference being the humidities on these days. Indeed, it is known that relative humidity (RH) can have a significant influence on SVA.36 We see disconnected disk-shaped grains (Figure 1b), whereas films prepared at a different day showed large, uniform, and dense grains (Figure 1c). To better understand this, we treated BiI3 films with SVA at different temperatures and RHs. Figure 1 Scanning electron microscopy (SEM) images of BiI3 films grown on FTO/TiO2 substrates (a) after spin-coating and before SVA; post DMF SVA on days with (b) lower RH and (c) higher RH. We prepared films treated with SVA at 0, 35, and 70% RH at 80, 100, and 120 °C both in a fume hood but also inside a glovebox to investigate the effect of ambient atmosphere. The relevant parameter behind RH is the activity of water, which is proportional to the vapor phase mole fraction. The vapor phase mole fraction is related to the water concentration, assuming that all water introduced during the start of SVA goes into the vapor phase. Water concentrations for a RH/T combination are given in Table S1. Multiplying this number by the SVA inner Petri dish volume gives the total mass of water needed for SVA. It is this mass of water that was mixed with DMF to create the water–DMF SVA solvent mixtures. We observe that the SVA solvent is completely evaporated throughout the SVA process. Some minor solvent and water loss is expected as the SVA Petri dishes are not sealed. SVA is performed inside a smaller Petri dish enclosed in a larger Petri dish to maintain the temperature, reduce airflow effects, and limit material loss. It is important to note that films prepared in ambient air have additional water exposure from ambient RH. On the days we prepared our films in air, RH was 42% at 20 °C, which corresponds to an additional water content of 7.2 × 10–6 g/cm3, which is between 1 and 7% of the total water concentration for the 35 and 70% RH conditions at 80–120 °C and will hence be neglected in the following. Films prepared from concentrated inks exhibited cracking after spin-coating (Figure S2a). Note that the BiI3 concentration in this study is twice that in previous work.27 This cracking comes from tensile stresses in the film as the THF rapidly evaporates.37,38 We developed a modified ink to prohibit cracking. Addition of 1–2 vol % DMSO to the THF inks prevented cracking and resulted in smooth films (Figure S2b). The colors of the pure THF and THF + DMSO films are different. The pure THF films are black after spin-coating owing to the rapid THF evaporation leaving a pure BiI3 phase behind. The THF + DMSO films are dark red after spin-coating. This red color indicates the presence of a BiI3–DMSO complex in the film.28 Within 20 min of exposure to ambient air, these films gradually turn black, indicating that the BiI3–DMSO complex is not stable at room temperature and thus decomposes to leave behind the pure BiI3 phase. This slow decomposition process accompanied by the evaporation of DMSO helps in obtaining uniform BiI3 film nucleation and growth, which is similar to the behavior observed in hybrid perovskites.7 We also tested DMF as an additive to the BiI3–THF inks, but this resulted in films that were not dense (Figure S3). SEM images of films after SVA treatment in a fume hood with different RHs and temperatures are presented in Figure 2. Each of the panels in Figure 2 represents one film, prepared at a specific SVA condition. For some SVA conditions, the films turned orange after some time during solvent vapor exposure in the second step of SVA. This is indicated by the colored swatch in the lower right-hand corner of each panel. A brighter orange swatch means the film turned orange early on during the second step of SVA. The darker the swatch, the longer the film took to turn orange. All black swatches mean the films never changed color. The times listed in the orange swatches are the durations the films were orange. The films prepared at 80 °C and 0% RH (Figure 2a) remained orange until it was uncovered. This indicates that even though the Petri dishes are not sealed, they retain SVA solvent for the duration of SVA. Figure 2 SEM images of BiI3 films on FTO/TiO2 prepared in air with the given RH (rows) and temperature (columns) during SVA [(a) 0% RH/80 °C, (b) 0% RH/100 °C, (c) 0% RH/120 °C, (d) 70% RH/80 °C, (e) 70% RH/100 °C, and (f) 70% RH/120 °C]. The upper and lower halves of each panel are low- and high-magnification images of the same sample made at the given RH and T, respectively. Scale bars are labeled in each image. The colored swatches in the lower right corners of each panel represent the approximate amount of time the sample remained orange during the second step of SVA. Swatches that are primarily orange mean the sample turned orange early on, whereas all black swatches mean the sample never changed color. Times inside the swatches indicate the duration for which the films remained orange. All films prepared with SVA at 80 °C turned orange during step 2 (solvent exposure) of the SVA process, independent of RH. However, the time it took the films to turn orange increases with RH. The film prepared at 80 °C and 0% RH (Figure 2a) turned completely orange within 30 s of solvent addition. For the same temperature, films prepared at 35% (Figure S5b) and 70% RH (Figure 2d) turned completely orange 7 and 9 min into SVA, respectively. The film prepared at 100 °C and 0% RH showed a nonuniform behavior: part of the film turned orange 60 s into SVA and part remained black (Figure S6a). The SEM images of the orange part of this film are shown in Figures 2b and S6b. This is in good agreement to our initial findings where we observed morphological differences with films treated with 100 °C SVA under different ambient humidities. The partial color change indicates that the portion of the substrate that turned orange experienced a higher DMF partial pressure than the portion that stayed black. The region that turned orange was the side of the substrate closest to the DMF source. During the final step of SVA, where the lid is opened so that the solvent could escape, all of the films that had turned orange during SVA immediately turned black again. No color change was observed for 100 °C + 35% RH (Figure S5e), 100 °C + 70% RH (Figure 2e), or any of the films made at 120 °C (Figure 2c,f). Visually, there is a difference between films that turned orange during SVA and those that stayed black: films that turned orange are hazy, whereas those that did not change color remain relatively shiny. The haziness of the films that turned orange during SVA comes from light scattering at larger domains (1–20 μm) that developed in the film (Figures 2a,b,d and S6b). The films that changed color during SVA at 80 °C (Figures 2a,d and S5) have larger domains sitting on top of the films. Looking at the higher magnification images, we see that the film is primarily made of clusters of small (<100 nm diameter) grains. The film that experienced the plume of DMF (Figure 2b) consists of large platelets (∼15 μm diameter). The differences between this case and the 80 °C cases are attributable to the high DMF concentration in the plume. All of the films that did not change color are similar (Figure 2c–f), consisting of apparent grain sizes of 300–500 nm diameter, as determined by SEM. This means that as long as the films do not change color during SVA, dense, large-grained films can be grown. Films made inside and outside of a glovebox with the same SVA condition have similar morphologies. For instance, films made with SVA at 120 °C and 70% RH inside (Figure S7a) and outside (Figure S7b) a glovebox show similar SEM grain size (300–500 nm) and polydispersity. This shows that the ambient environment does not have a big impact on morphology. We will focus on films prepared outside a glovebox for the rest of our discussion. Full comparison of glovebox and air films is given in the Supporting Information (Figures S4 and S5). Mechanism behind the Effects of Water, Temperature, and Solvent We explain the role of SVA temperature and water concentration by considering the BiI3–DMF complexation chemistry. Nørby et al.28 have determined via single-crystal X-ray diffraction (XRD) that the carbonyl oxygen in DMF complexes with the Bi3+ center to give a solvent complex crystal with a unit cell of Bi(DMF)8–Bi3I12. Octahedral complexes such as BiI3 typically undergo ligand exchange via a dissociative interchange mechanism (Id).39 In an Id mechanism, the bond with the incoming group is formed at the same time the bond with the leaving group is broken. In our case, the incoming group is the carbonyl group on the DMF and the leaving group is an iodide anion. Bi3+ is a Lewis acid of intermediate hardness. The oxygen in the DMF carbonyl group is a hard base, and the iodide anion is a soft base.40 As a result, Bi3+ is expected to have a similar ability to complex with the carbonyl oxygen in DMF and the iodide. Furthermore, as has been shown by Sanderson et al., the bond dissociation enthalpy of the Bi–I bond is the smallest for the Bi–halides (i.e., the Bi–I bond is easier to break than other Bi–halide bonds).41 The character of the bond is largely ionic, and the large ionic radius of the iodide anion means it is held less strongly by the Bi3+ center because of electron–electron repulsion between Bi and I. The ionic character of the Bi–I bond is critical for facile DMF complexation. The Id mechanism requires a good leaving group. Because the Bi–I bond is more ionic than covalent and thus more easily broken, the iodide makes a good leaving group, allowing the DMF to readily complex with the Bi3+ center. We first consider the water-free (DMF vapor only) 80 °C case. The spin-coated crystalline BiI3 film is made up of BiI6 octahedra arranged in two-dimensional (2D) sheets.21 Consider a single BiI6 octahedron exposed to DMF vapor. The DMF approaches the Bi3+ center and begins to form a Bi–OR complex with the DMF carbonyl oxygen. At the same time, the Bi–I bond is being broken and the I– leaves. This reaction continues to take place until the Bi center is coordinated to eight DMF molecules [Bi(DMF)8]3+, which is charge-balanced by a [Bi3I12]3– group. If enough DMF is present, the entire film is transformed to a DMF–BiI3 coordination compound. This compound may crystallize with a unit cell composed of four Bi(DMF)8–Bi3I12 pairs, as shown by Nørby et al.28 We note that BiI3 also coordinates with DMSO and THF, the solvent system for our inks (a more detailed discussion about this can be found in the Supporting Information). The BiI3–DMF coordination compound is the orange phase witnessed under low RH and low T. The solvent complex film is thicker than the BiI3 film because of DMF uptake. The unit cell volume of the BiI3–DMF solvent crystal is 6507.2 Å3, and it contains four Bi(DMF)8–Bi3I12 moieties, that is, a volume of 406.7 Å3 per BiI3.28 Pure BiI3, in contrast, has a unit cell volume of 1016.7 Å3 with six BiI3 moieties per unit cell,42 that is, a volume of 169 Å3 per BiI3. This means that there is a volume increase and, concomitantly, a thickness increase of the orange phase of a factor of 2.4 compared with the BiI3 film. Upon drying, this film collapses back to the BiI3 film, which results in a different morphology relative to the initial (pre-SVA) BiI3 film. This can lead to very rough films with large BiI3 clusters, as shown in Figure 2a,d or to films with large disconnected platelets, as shown in Figure S6b. If a BiI3 film is exposed to DMF vapor for a much longer time than 9 min in our SVA process, then macroscopic morphologies can form, as shown in Figure S9. Now, consider the case of SVA at higher temperatures (T ≈ 120 °C). We did not observe the orange phase formation during SVA at this temperature with the DMF concentrations considered. To better understand this, we collected data to perform a van’t Hoff analysis (details in the Supporting Information). The reaction considered is 4BiI3 + 8DMF ↔ [Bi(DMF)8]3+[Bi3I12]3–. BiI3 films were placed in sealed vials with different DMF dosings. The BiI3 films were allowed to complex with the DMF at room temperature until the orange solvent complex formed. The vials were then heated in a box oven, slowly increasing the temperature until the films turned black again. From these temperatures, we get the equilibrium constant for the BiI3–DMF complexation. By plotting ln(Keq) versus 1/T, we can extract ΔHrxn and ΔSrxn for the system. On the basis of the stoichiometry of the above reaction, this yielded values of ΔHrxn of–3 ± 1 eV and ΔSrxn of −0.006 ± 0.003 eV/K per molecular of the coordination compound. If one assumes that each successive DMF addition has similar thermodynamics, the enthalpy and entropy are ΔHrxn of −0.4 eV and ΔSrxn of −0.0007 eV/K per DMF, respectively. This means that this reaction is enthalpically favorable up to 140 °C, at which point the entropic term begins to dominate. At temperatures exceeding 140 °C, no concentration of DMF would result in the films turning orange. The important point here is that at elevated temperatures, it is more favorable for the DMF to be in the vapor phase rather than to be complexed with BiI3. It is this effect that prevents the orange solvent complex from forming at higher SVA temperatures, independent of RH. For an efficient crystal growth during SVA, the maximum DMF concentration below the level which leads to the orange phase is desirable. Thus, our DMF concentration (10 ppm) during SVA is chosen so that the orange solvent complex forms up to a temperature of 100 °C, which is the ideal temperature for the SVA process shown previously.27 Larger DMF concentrations would lead to the formation of the orange phase at higher temperatures; for smaller DMF concentration, the films would stay black at lower temperatures. Now, we discuss the effect of water on the SVA process. At 80 °C, RH slows down the transformation of the BiI3 into the orange solvent complex. At 100 °C, the presence of water (≥35% RH) prevents the film from turning orange at all and results in more uniform grain growth. To understand this, we note that hydrogens in water will hydrogen bond with the oxygen in DMF. As a result, the electron-donating strength (i.e., the Lewis basicity) of the carbonyl oxygen is reduced. Thus, the DMF is rendered less reactive and will complex less readily with the BiI3. This would be expected for cases where the water concentration is equal to or greater than the DMF concentration, which is true at the DMF–water dosings used during SVA. This explains the difference we see between films prepared at 100 °C with 0 and 70% RH. Effect of Substrates on Film Morphology We spin-coated 200 mg/mL BiI3 in THF + 1–2 vol % DMSO onto FTO/TiO2, FTO/ZnO, and ITO/Cu/NiOx substrates. We observe significantly different film morphologies with the different substrates. Films grown on TiO2 (Figure 3a) are made of 300–500 nm densely packed grains. Films grown on ZnO (Figure 3b) are more polydisperse and contain small and large platelets with sharp edges. Films prepared on copper-doped nickel oxide (Cu/NiOx) (Figure 3c) are also more polydisperse than the films grown on TiO2 but also contain large (1 μm dia.) platelets. We rationalize these observations by noting that film–substrate surface energy differences can influence film growth. In the cases where substrate surface energy is smaller than film + interface surface energies, island growth is expected.43 Other factors such as deposition temperature also can impact crystal growth.21 This means that by optimizing parameters such as SVA solvent, water concentration, and temperature, we might be able to obtain dense films with the desired grain size for each choice of substrate. Figure 3 SEM images of BiI3 films grown with SVA at 120 °C and 70% RH on (a) FTO/TiO2, (b) FTO/ZnO, and (c) ITO/Cu/NiOx. Crystallographic Orientation XRD spectra for BiI3 films grown at different SVA temperatures and water vapor concentrations are shown in Figure 4a). The films that turned orange during SVA, that is, those grown at 80 °C (0% RH and 70% RH) and 100 °C (0% RH) show a pattern significantly different from the black ones. The most pronounced XRD peaks for those films are due to reflections at the (003), (006), and (009) planes, which belong to the same family of (00l) planes. This indicates a texturing of those films. In contrast, the films that did not change color during SVA show much smaller peaks for the (00l) planes. We observe pronounced reflections at (113) and (300) in these films, indicating less texturing. Figure 4 (a) XRD spectra as a function of SVA temperature and RH. The green and blue curves show the spectra for those areas that turned orange and that stayed black, respectively (see Figure 2). The dotted vertical lines indicate BiI3 powder peaks. The solid black vertical lines indicate the peaks of the FTO substrate. (b) TCs related to the (300) plane (green), (113) plane (blue), and (003) plane (red) as a function of SVA temperature for 0% RH (open squares) and 70% RH (open pyramids). To further study this texturing effect, we evaluate the texture coefficient (TC). The TC of a plane (hkl) is defined as , where I(hkl) is the measured intensity, I0(hkl) is the intensity of the powder diffraction, and N is the number of planes considered.44 A TC equal to 1 indicates no preferred orientation. The TCs for the (300), (113), and (003) planes are shown in Figure 4b for different SVA temperatures and water concentrations. All films that turned orange during SVA, that is, at 80 °C show a TC of around 3 for the (003) plane and of close to 0 for the (113) and (300) planes. We note that the same TCs are also observed for the films that turned orange during SVA at 100 °C, 0% RH (not shown in Figure 4b). This indicates a strong texturing: these films are oriented with their (003) plane predominantly parallel to the substrate; that is, the BiI3 film is layered in such a way that the planes which contain edge-shared BiI6 octahedra are parallel to the substrate. The films that did not change color do not show this texturing: TCs for the (003) and (113) planes are between 0.5 and 0.9, whereas the TC for the (300) plane is around 1.5. This indicates a slightly preferred orientation in such a way that the edge-shared BiI6 planes are perpendicular to the substrate. Similar texturing has been found in BiI3 films that have formed from BiI3–DMSO complexes.28 Because the 2D sheets of edge-shared BiI6 octahedra are only weakly connected by van der Waals forces, it is expected that in-plane transport should exhibit larger mobility than perpendicular to the 2D sheets. Hence, the films that turned orange during SVA should show a better carrier transport parallel to the substrate and worse transport perpendicular to the substrate plane than the films that did not change color. We will come back to this point later. Optoelectronic Quality We performed AIPL measurements at a PV device relevant excitation laser intensity comparable to one sun. PL measurements on BiI3 films have been carried out previously;21,27 however, because the PL quantum yield (PLQY) is very low, those measurements used laser excitation intensities comparable to >100 sun. Here, we were able to measure BiI3 AIPL at 1 sun effective intensity. This was enabled by both the high quality of the films and a modification to our measurement technique (see the Supporting Information for details). The AIPL is peaked at 1.78 eV with a full width at half-max of 180 meV (Figure 5a, blue trace). This is very close to the measured band gap of 1.79 eV determined from UV–vis absorption measurements (Figure 5a, red trace). The fact that the PL peak is right at the band edge indicates that sub-band gap states should not cause a significant loss of voltage. From AIPL, we get the PLQY, which we used to calculate the QFLS (Figure 5b) with the method described in Braly and Hillhouse.33 The QFLS is an indication of the maximum voltage Voc achievable by a solar cell device. QFLS is proportional to kT ln(1/PLQY). Therefore, what may appear as larger differences in PLQY does not correspond to as large differences in QFLS. In Figure 5b, we also show the ratio of the measured QFLS to the Shockley–Queisser QFLS (QFLSSQ) for a material with the same band gap, χ = QFLS/QFLSSQ.33 χ may be considered to be the optoelectronic quality of the material. All films prepared at 80 °C as well as the film prepared at 100 °C with 0% RH have PLQY < 1 × 10–7. Those were the films that turned orange during SVA. The films prepared at 100 °C with 35 and 70% RH and all 120 °C films have PLQY values of 2 × 10–7%. These films remained black during SVA. Figure 5 (a) Absorption coefficient (red curve) and PL spectral flux spectrum of BiI3 with a peak at 1.78 eV (blue curve). (b) PLQY, QFLS, and χ from 1 sun AIPL of BiI3 films prepared with different SVA temperatures and RHs. (c) Two-point lateral dc photoconductive diffusion length measurements of BiI3 films grown with different SVA temperatures and RHs. The lower PLQY films were the films that had turned orange during SVA, whereas the higher PLQY values were obtained from the films that did not change color during SVA. PLQY was greater by 2–6 times in the films that did not change color during SVA. This may be understood as an effect of the grain size. The films that turned orange during SVA have grains with diameters ≤100 nm. The films that remained black have grains 300–500 nm in size. The differences in PLQY may be attributed to nonradiative recombination at the grain boundaries. The smaller grained films have a greater number of grain boundaries, meaning that nonradiative recombination would be greater. However, we cannot exclude differences in the bulk. Films that turned orange during SVA may have entrained DMF within the crystalline BiI3. This may give rise to different point defects and contribute to nonradiative recombination. Coming back to our discussion on transport, a lateral dc photoconductivity method34,35 was used to estimate the lateral diffusion length to assess the impact of SVA on carrier transport. The diffusion length (LD) determined from this technique is an average (root mean square) of the electron and hole diffusion lengths. A slight trend is observed toward higher diffusion lengths with increasing temperature and RH from 40 nm at 80 °C/0% RH to 55 nm at 120 °C/0% RH and 70 nm at 120 °C/70% RH, as shown in Figure 5c. Because these are lateral diffusion lengths, that is, the carriers flow parallel to the substrate, this behavior is contradictory to what we would expect from the above discussion on texturing of the films: carrier transport is expected to be better for films that turned orange during SVA (i.e., at 80 °C/0% RH and 80 °C/70% RH), which is obviously not the case. One likely reason for this is the much lower grain size of the films that changed color during SVA compared to the films that did not change color (see Figure 2), giving rise to significantly more grain boundaries, which reduces the carrier mobility. In addition, the lifetime of photoexcited carriers in the films that turned orange during SVA is significantly lower than that in the films that did not change color because of the higher recombination rate as indicated by the lower PLQY. Because the diffusion length is proportional to the mobility-lifetime product, the latter two effects counterbalance the beneficial texturing effects of the films that turned orange during SVA as compared to the films that did not change color. PV Devices We made PV devices to assess the impact of SVA on PV performance. An ideal band diagram (assuming no ion migration, intrinsic BiI3, no mass transport across the interfaces, and no surface chemistry/interface dipoles) and a device architecture schematic are shown in Figure 6a and the inset of 6b, respectively. We report current density versus voltage (JV) curves and time-dependent PCE (using maximum power-point tracking45) under continuous simulated AM1.5G illumination. The best solar cell with the optimized SVA process (120 °C + 70% RH) has a stabilized PCE (Figure 6b, blue curve) of 0.51%. A device made from a film that turned orange during SVA (e.g., 80 °C + 70% RH) in the same architecture shows a much lower stabilized PCE of 0.04% (Figure 6b, orange curve). Figure 6 (a) Equilibrium band diagram of FTO/TiO2/BiI3/spiro-OMeTAD (SOMT) at short circuit. The dashed red line indicates the Fermi level. (b) Maximum power-point tracking curves for devices made with 120 °C/70% RH (blue curve) and 80 °C/70% RH (orange curve). The inset shows device stack. (c) Light (blue) and dark (orange) JV curves for the 120 °C and 70% RH device. (d) Light (blue) and dark (orange) JV curves for the 80 °C and 0% RH device. This more than 1 order of magnitude lower PCE comes mainly from two factors, a significantly lower photocurrent and a much lower fill factor due to the s-shaped JV. We note that the lower photocurrent very likely stems from a much lower carrier diffusion length perpendicular to the substrate for the films that turned orange during SVA. This is expected from the texturing effects discussed above, the lower carrier lifetime due to the lower PLQY, and the lower mobility due to the increased number of grain boundaries. While the band alignment shown in Figure 6a suggests that an electric field should be present across the device to assist with carrier collection, if charged iodide vacancies migrate (as in hybrid perovskite solar cells) to screen the internal field, then carrier diffusion would have to be relied on for carrier collection.23,46 The relatively low diffusion lengths measured (70 nm) could then partly explain the low short-circuit current (Jsc) of 4.14 mA/cm2. Another contributing factor to the low photocurrent could be the misalignment of the conduction band edges (CBES) of TiO2 and BiI3. An ab initio calculated value for the CBE for BiI3 is 4.1 eV, and the accepted value for TiO2 is 4.2 eV below vacuum.22 These are very close, and if correct would not yield a barrier for electron extraction. However, if the actual band edge position of BiI3 is different, it may cause a barrier for electron injection from BiI3 into TiO2. The large differences between the light and dark curves suggest further device issues, such as a light-dependent saturation current, photoexcited carrier screening of electrostatic barriers at the interfaces (not shown in the band diagram), voltage-dependent photocurrent, and light-dependent shunt and series resistances. The table in Figure S10b shows the results of fitting the nonideal diode equation to the light and dark curves for the device made at 120 °C and 70% RH (Figure S10a). The reverse saturation currents (J0), series resistances, and shunt resistances all differ by more than an order of magnitude. With regard to the low voltage, the low diffusion length may play a role as well, as it signifies significant nonradiative recombination. Furthermore, the band alignment also likely plays an important role. With regard to the hole-extracting contact, the band edge of the spiro-OMeTAD lies 5.1 eV below vacuum,47 and the position of the BiI3 valence band is 6.0 eV below vacuum.22 While one would not expect a barrier for hole extraction, voltage could be lost because of the large offset between the BiI3 valence band and the spiro-OMeTAD Fermi level. Conclusions We have explored the mechanisms behind the BiI3 film formation process and demonstrated the roles that temperature and water play in the solvent annealing process for BiI3 layers. We developed a reliable process for growing large-grained, continuous films. Films with small grains and poor morphologies result in reduced device performance and optoelectronic quality. Performing SVA with precise dosing of water and DMF grows uniform films with a SEM-based morphological grain size of 300–500 nm. Water acts to moderate the influence of DMF during the SVA process, allowing more controlled growth to occur. Higher temperatures also moderate the action of DMF. We used highly concentrated BiI3–THF inks (200 mg/mL) and observed severe cracking of the films upon spin-coating and drying. Modifying the ink chemistry by addition of 1–2 vol % DMSO, we successfully prevented film cracking and obtained homogeneous and smooth films. Formation of the orange BiI3–DMF complex during SVA at low temperatures results in films with a preferred orientation: the planes corresponding to the 2D sheets of BiI6 octahedra are oriented parallel to the substrate. From diffusion length measurements and device characterization, we obtained clear indication for very poor transport in the direction perpendicular to these planes. The reason is the weak van der Waals interaction of these 2D sheets. This shows that for the structurally two-dimensional material BiI3, the film texture is an essential parameter for its application in optoelectronic devices. From AIPL measurements, we determined a QFLS of 1.1 eV at 1 sun, which is a measure for the maximum photovoltage that can be obtained in a solar cell. This is a promising result because an optimized solar cell with BiI3 as the absorber material only limited by this QFLS could achieve >15% PCE. A QFLS of 1.1 eV is 73% of the Shockley–Queisser limit for BiI3, which indicates that there is still room for improvements via defect passivation. However, it also indicates that bulk nonradiative recombination is not the main reason for the low voltage of state-of-the-art BiI3 solar cells. Both the large voltage deficit and current deficit seen in BiI3 devices are a consequence of low diffusion length and nonoptimal band alignment. This work reveals the mechanisms behind the film formation process of BiI3 films, which are potentially also applicable to other metal halide materials. It further expands the understanding of the relationship between the chemistry and physics of BiI3 films and devices, opening the door for improving device efficiencies. Further development of this material could enable it to become a competitor to PbI2-based hybrid perovskites while avoiding the toxicity and potentially also the stability issues of the perovskite material. Experimental Section Materials BiI3 inks (200 mg/mL) were prepared by dissolving BiI3 (99.999%, Alfa Aesar) in anhydrous THF (99.9%, inhibitor-free, Sigma-Aldrich) inside a N2-filled glovebox. The inks were sealed and then mixed with an ultrasonicator immediately before use. Fluorine-doped tin oxide (FTO) substrates (14 × 14 mm2) (TEC 7, Sigma-Aldrich) were sequentially cleaned by sonication in a detergent solution, DI water, acetone, and 2-propanol for 10 min. Substrates were cleaned with an Ar plasma immediately before use. TiO2 solutions were prepared by mixing 10 mL of ethanol (200 proof, Sigma-Aldrich), 69 μL of HCl (37 wt %, aq, Macron Fine Chemicals), and 0.727 mL of Ti(IV) ethoxide (Aldrich) and sonicating it for 30 min. This solution was spin-coated onto the FTO substrates in air at 2000 rpm for 15 s. Substrates were coated twice, with a 500 °C anneal in a box oven between layers. BiI3 inks were deposited on the TiO2-coated substrates by spin-coating at 2000 rpm for 35 s inside a N2-filled glovebox and in a fume hood. After spin-coating, the films were solvent vapor-annealed for 10 min with DMF–water mixtures. SVA consists of three steps: (1) the as spin-coated BiI3 film is set inside a Petri dish on a hot plate; (2) after 60 s, the solvent is added to the Petri dish with the film and the lid is closed; and (3) after 9 min, the lid is removed so that the film is exposed to the ambient atmosphere for 60 s to remove residual DMF and water. DMF volume was held constant at 6.4 μL, and water content was varied to achieve 0, 35, or 70% RH at 80, 100, or 120 °C. See Figure S1 and Table S1 in the Supporting Information for details on the SVA setup and water concentrations. hole transport layers were prepared by spin-coating spiro-OMeTAD, as described elsewhere.9 The spiro-OMeTAD was doped with Li and Co and oxidized overnight. Devices were finished by thermal evaporation of 100 nm Au at a base pressure of 1 × 10–6 Torr. Characterization SEM images were collected at a 5 kV accelerating voltage with an FEI XL830 Dual Beam SEM/FIB. Crystallographic measurements were collected with a Bruker D8 Discover XRD. AIPL measurements were collected at 1 sun photon flux with an apparatus described previously.32,33 The 1 sun condition used corresponds to a photon flux of 1.28 × 1021 photons/s m2 at a laser wavelength of 532 nm. A 150 lines/mm Czerny–Turner monochromator blazed at 500 nm was used to collect the signal. PLQY and the peak position were used to calculate the QFLS and the optoelectronic quality (χ).33 PV devices were tested in air using an AAA solar simulator (Oriel Sol3A) and a Keithley 2400 SMU. A photoconductivity method was used to determine the mean carrier diffusion length34,35 using a bias of +20 V with a 432 nm blue light-emitting diode at 3.66 × 1021 photons/s m2 flux, which is 2.8 sun equivalent for a 1.8 eV band gap material. Photoconductivity measurements were made inside a temperature-controlled stage held at 20 °C. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00813.Additional experimental details, BiI3 complexation with DMSO and THF, SEM images, and LambertW fitting of IV curves (PDF) Supplementary Material ao8b00813_si_001.pdf Author Contributions B.W.W. and F.T.E. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. Acknowledgments This research was supported by the U.S. Department of Energy SunShot Initiative, Next Generation Photovoltaics 3 program, Award DE-EE0006710. We also acknowledge partial support from the University of Washington Molecular Engineering Materials Center (UW MEM-C): an NSF MRSEC under award number DMR-1719797. ==== Refs References Xin H. ; Katahara J. K. ; Braly I. L. ; Hillhouse H. W. 8% Efficient Cu2ZnSn(S,Se)4 Solar Cells from Redox Equilibrated Simple Precursors in DMSO . Adv. Energy Mater. 2014 , 4 , 1301823 10.1002/aenm.201301823 . Ki W. ; Hillhouse H. W. Earth-Abundant Element Photovoltaics Directly from Soluble Precursors with High Yield Using a Non-Toxic Solvent . Adv. Energy Mater. 2011 , 1 , 732 –735 . 10.1002/aenm.201100140 . Guo Q. ; Ford G. M. ; Yang W.-C. ; Walker B. C. ; Stach E. A. ; Hillhouse H. W. ; Agrawal R. 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PMC006xxxxxx/PMC6644408.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145805610.1021/acsomega.8b01949ArticleCovalently Crosslinked 1,2,3-Triazolium-Containing Polyester Networks: Thermal, Mechanical, and Conductive Properties Tracy Clayton A. Adler Abagail M. Nguyen Anh Johnson R. Daniel Miller Kevin M. Department of Chemistry, Murray State University, 1201 Jesse D. Jones Hall, Murray, Kentucky 42071, United States E-mail: kmiller38@murraystate.edu. Tel: +1 270 809 3543. Fax: +1 270 809 6474.18 10 2018 31 10 2018 3 10 13442 13453 08 08 2018 05 10 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Azide–alkyne “click” cyclization was used to prepare a series of polymerizable acetoacetate monomers containing a 1,2,3-trizolium ionic liquid group. The monomers were subsequently polymerized using base-catalyzed Michael addition chemistry, producing a series of covalently crosslinked 1,2,3-triazolium poly(ionic liquid) (TPIL) networks. Structure–activity relationships were conducted to gauge how synthetic variables, such as counteranion ([Br], [NO3], [BF4], [OTf], and [NTf2]), and crosslink density (acrylate/acetoacetate ratio) effected thermal, mechanical, and conductive properties. TPIL networks were found to exhibit ionic conductivities in the range of 10–6–10–9 S/cm (30 °C, 30% relative humidity), as determined from dielectric relaxation spectroscopy, despite their highly crosslinked nature. Temperature-dependent conductivities demonstrate a dependence on polymer glass transition, with free-ion concentrations impacted by various ions’ Lewis acidity/basicity and ion mobilities impacted by freely mobile anion size. document-id-old-9ao8b01949document-id-new-14ao-2018-01949mccc-price ==== Body Introduction Poly(ionic liquid)s (PILs) remain a fascinating and important class of polyelectrolytes, combining the unique properties of ionic liquids (ILs) (high thermal and electrochemical stability, variable solubility, viscosity, and ionic conductivity) with the tunable functionality and mechanical stability of various macromolecular architectures.1−4 The thermal, mechanical, and conductive properties of PILs are primarily varied by changing the chemical structure of the cation (ammonium, phosphonium, imidazolium), anion (halides, inorganic fluorides, perfluoronated sulfonimides), or both. As a result of their synthetic versatility, PILs have been targeted for a number of important modern-day applications, examples of which include actuators,5 batteries,6,7 fuel cells,8,9 and separation membranes.10−13 PILs have been generally prepared through the polymerization monomers containing ILs, the postpolymerization quaternization of neutral polymers or quaternization resulting from step-growth polymerization of neutral monomers. Contributions from our laboratories to the field of PILs have centered on the preparation of covalently crosslinked polyester networks following either a thiol–ene photopolymerization14 or a base-catalyzed Michael addition polymerization strategy.15−19 Michael addition reactions are extremely useful for forming new carbon–carbon bonds and readily occur under base-catalyzed conditions between a Michael donor (an acetoacetate) and an α,β-unsaturated carbonyl compound, such as an diacrylate ester (the Michael acceptor).15,16 Upon deprotonation of an acetoacetate, the resulting enolate anion undergoes addition to an acrylate, resulting in a new carbon–carbon bond after neutralization. Since each acetoacetate contains two acidic protons, an additional Michael addition will occur in the presence of excess diacrylate, resulting in covalent crosslinking. The degree of crosslinking (and thus the thermal and mechanical properties of the network) can be easily controlled through the manipulation of the acrylate/acetoacetate ratio.16 Previous work from our research group has demonstrated the versatility of the Michael addition polymerization as a method for the preparation of imidazolium17,18 and 1,2,4-triazolium PIL networks.19 Variations in network architecture (crosslink density, counteranion, cation) have led to a library of PILs that display a wide range of thermal and mechanical properties. Although the majority of the PIL literature has focused on ammonium, phosphonium, and imidazolium polycations (with mobile counteranions), 1,2,3-triazolium-based poly(ionic liquid)s (TPILs) have become more prominent due to the development of the copper(I)-catalyzed azide–alkyne “click” cycloaddition reaction.20,21 Drockenmuller et al. have pioneered efforts in the synthesis of a variety of TPILs, several approaches of which have been recently reviewed.22 Such TPIL synthetic efforts have ranged from the direct step-growth polyaddition of azides and alkynes to the chain-growth polymerization of 1,2,3-triazolium-containing (meth)acrylic monomers. Within their library of TPILs, several covalently crosslinked networks were prepared and evaluated for various applications. For example, an epoxy-functionalized IL monomer was reacted with a poly(propylene glycol)-based diamine, providing TPIL epoxy–amine networks with relatively high anhydrous ionic conductivities (∼10–7 S/cm at 30 °C).23 In another example, covalently crosslinked polyether-based TPIL membranes with conductivities of up to 10–6 S/cm at 30 °C under anhydrous conditions and CO2 permeability values of 59–110 barrer with CO2/N2 selectivities of 25–48 were also synthesized.24 TPIL vitrimer networks utilizing thermally reversible C–N bond transalkylation exchanges have also been reported and were found to exhibit ionic conductivities up to 10–8 S/cm (30 °C, anhydrous) as well as reshaping and recycling behavior.25,26 TPIL polyester networks, which contained an asymmetrically substituted 1,2,3-triazolium group with a limited set of counteranions, were recently communicated by our laboratory.27 Electrochemical impedance spectroscopy, utilizing a four-electrode cell, showed that the resulting TPIL networks exhibited good conductivities between 10–6 and 10–8 S/cm (25 °C, 30% relative humidity (RH)), indicating the promising nature of this class of PILs. Most notable was the appearance of a common “crossover” temperature (∼85 °C) in the ionic conductivity curves, given the same monomer ratio. At temperatures below the crossover point, Tg/chain dynamics were found to correlate well to ionic conductivity. Above the crossover temperature, however, ionic conductivities correlated better to anion size, alluding to the importance of “free” ion mobility. Though these correlations are noteworthy, a much more in-depth investigation utilizing a wider range of counteranions (in terms of size and lipophilicity) in combination with dielectric relaxation spectroscopy (DRS) is necessary to further decipher the complex relationships that exist between chain dynamics, free-ion concentration, and conducting ion mobility. Toward this end, the synthesis of a more focused series of symmetrically substituted TPILs is reported herein where DRS has been employed to determine ionic conductivities (at 30% RH) and to explore chain dynamics and ion-transport properties. A comparative analysis of these properties across various anions, crosslink density (acrylate/acetoacetate ratio), and cations (imidazolium 1,2,4-triazolium, 1,2,3-triazolium) is discussed. Due to the presumed hydrophilic nature of the TPIL [Br] network, coupled with a minimum achievable RH of 30%, the effect of relative humidity on conductivity and water uptake was also explored. Results and Discussion Synthesis of the 1,2,3-triazolium acetoacetate monomers began by first converting commercially available ethyl-6-bromohexanoate to the azide with sodium azide (Scheme 1). Reaction with trimethylsilylacetylene under Cu-catalyzed click cyclization conditions resulted in the tetramethylsilane-substituted triazole ring (not isolated), which was subsequently treated with a fluoride source (tetrabutylammonium fluoride, TBAF) to provide the desired 1,2,3-triazole ester 1.21 Reduction of the ester was then accomplished with diisobutylaluminum hydride (DIBAL-H), resulting in 1-(6′-hydroxyhexyl)-1,2,3-triazole 2. Transesterification with tert-butylacetoacetate, followed by coupling with 6-bromohexylacetoacetate 4, resulted in the targeted acetoacetate [Br] monomer 5. Various anion metathesis reactions (AgNO3, AgBF4, AgOTf, LiNTf2) were then employed to gain access to other counteranions of interest (monomers 6–9). All of the 1,2,3-triazolium acetoacetate monomers were clear, room-temperature ionic liquids with varying degrees of yellow to orange color. 1H and 13C NMR spectroscopies, as well as elemental analysis, were used to identify the products and determine purity, respectively, and residual [Br] for PILs 6–9 was found to be <0.1% w/w by ion chromatography.28 Scheme 1 Preparation of 1,2,3-Triazolium Acetoacetate Monomers 5–9 Analysis of the thermal properties of acetoacetate monomers 5–9 was conducted prior to polymerization. Differential scanning calorimetry (DSC) Tg values (Table 1, Figure S18) correlated inversely to the size of the counteranion on the order of [Br] > [NO3] ≈ [BF4] > [OTf] > [NTf2]. Here, assessment of the relative anion sizes is based upon molar masses and thermochemical radii.29 The ability of ionic liquids to exhibit glass-forming behavior has been previously described using techniques such as DSC, X-ray diffraction, and Raman spectroscopy.30,31 It is also worth noting that the observed relationship between anion size and Tg has been observed previously with imidazolium- and 1,2,4-triazolium-containing acetoacetate monomers.17−19 Table 1 Thermal Properties of 1,2,3-Triazolium-Containing Acetoacetate Monomers compound anion DSC Tg (°C) TGA Td5% (°C) 5 Br –46.3 188 6 NO3 –54.9 194 7 BF4 –55.4 223 8 OTf –60.5 239 9 NTf2 –67.9 260 Thermal stability, as defined by Td5% (the temperature at which 5% of the material has decomposed), correlates inversely to the Lewis basicity of the counteranion on the order of [NTf2] > [OTf] > [BF4] > [NO3] > [Br] (Table 1 and Figure S19).32 All of the acetoacetate monomers exhibited a one-step degradation except for [BF4] 7 where a two-step process was observed. Recent work by Clarke et al. using direct insertion mass spectrometry investigated the decomposition products of 1-alkyl-3-methylimidazolium [BF4] ILs and found fluoride to be a thermally induced product of the reaction between tetrafluoroborate and the imidazolium cation.33 We hypothesize here that as IL monomer 7 is being heated, fluoride is generated. As fluoride is a stronger Lewis base (compared to tetrafluoroborate), the first step results from decomposition of the monomer from [F] (the exact nature of the decomposition was not determined as part of this study). The second step would represent decomposition as a result of [BF4]. To support this hypothesis, the production of fluoride from [BF4] acetoacetate monomer 7 was observed as a function of time using isothermal thermogravimetric analysis (TGA) (at 200 °C) followed by analysis of the residue by ion chromatography. Fluoride content of monomer 7 (determined from the [F]/[BF4] ratio) was found to be negligible, however, a 20-fold increase in fluoride concentration was observed during the first hour at 200 °C. Additional time found the amount of [F] and [BF4] to decrease, presumably because both anions are being consumed as part of monomer decomposition. The results from this study clearly show that fluoride is thermally generated during the decomposition of [BF4] monomer 7 and thus could influence the observed decomposition temperature. 1,2,3-Triazolium-containing acetoacetates 5–9 were polymerized following a standard Michael addition polymerization protocol developed by our laboratory. The appropriate amount of acetoacetate was allowed to react with commercially available 1,4-butanediol diacrylate in the presence of catalytic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2 mol %) in a solution of dichloromethane (50 wt %) (Scheme 2).17−19 Each solution was placed into a poly(tetrafluoroethylene) (PTFE) mold and cured under ambient conditions for 48 h, followed by 48 h in a 60 °C. The films were then dried in a vacuum oven at 60 °C for an additional 48 h to ensure removal of trace solvent residue and moisture. Since conductive properties of the analogous imidazolium and 1,2,4-triazolium PIL Michael networks had not been previously measured, comparison networks were prepared fresh, which utilized previously reported imidazolium and 1,2,4-triazolium [NTf2] acetoacetate monomers (10 and 11, respectively).17,19 For PILs where a variation in cation or anion was of interest, the acrylate/acetoacetate ratio was held constant at 1.5:1.0. For PILs in which the cation or monomer ratio was varied, the anion was held constant at [NTf2]. Scheme 2 Michael Addition Polymerization of 1,2,3-Triazolium-Containing Acetoacetate Monomers Gel fraction and % swelling of the PIL networks were determined from Soxhlet extraction experiments using dichloromethane (Table 2). High gel fraction values (>92%) were observed for all of the networks studied here. Higher swelling values were observed with increasing anion size, presumably due to an increase in free volume within the network as a result of anion plasticization, although it is possible that the use of a more hydrophobic anion ([OTf] and [NTf2]) could result in stronger polymer–solvent interactions. As expected, an increase in the acrylate concentration (polymers 16–18) decreased the % swelling due to the increase in covalent crosslinking and a decrease in free volume within the network. Gel fraction and % swelling values were similar across the series in which the cation was varied (networks 16, 19, and 20). Table 2 Gel Fraction and Thermal Properties for TPIL Networks polymer anion (cation) Acr/AcAc ratio gel fraction (%) % swelling DSC Tg (°C) TGA Td5% (°C) 12 Br 1.5:1.0 97.6 ± 0.4 129.0 ± 0.9 7.7 195 13 NO3 1.5:1.0 95.6 ± 0.1 131.6 ± 1.0 3.9 203 14 BF4 1.5:1.0 97.4 ± 0.2 130.4 ± 3.3 2.9 256 15 OTf 1.5:1.0 95.2 ± 0.3 139.8 ± 0.8 –5.2 302 16 NTf2 1.5:1.0 92.2 ± 0.3 155.1 ± 2.2 –15.6 318 17 NTf2 1.2:1.0 93.1 ± 0.3 151.4 ± 2.5 –22.0 312 18 NTf2 1.8:1.0 93.8 ± 0.8 140.2 ± 3.2 –2.9 318 19 (IM) 1.5:1.0 93.8 ± 0.1 151.5 ± 0.3 –14.4 321 20 (124TRI) 1.5:1.0 95.1 ± 0.4 155.7 ± 0.5 –9.2 315 PIL networks were initially analyzed by DSC to evaluate Tg and to correlate to changes in network structure (Table 2 and Figures S20–S22). An increase in the size of the counteranion led to a decrease in Tg on the order of [Br] > [NO3] ≈ [BF4] > [OTf] > [NTf2], likely due to a plasticizing effect that is often observed in PILs with larger, more hydrophobic anions. As expected, upon increasing the acrylate concentration and thus the crosslink density, the Tg increased, given the same counteranion [NTf2], with the 1.8:1.0 acrylate/acetoacetate ratio PIL exhibiting the highest value of −2.9 °C. Changing the cation led to little difference in Tg between the imidazolium and 1,2,3-triazolium PIL networks, however, the 1,2,4-triazolium [NTf2] PIL was observed to have a higher Tg value of −9.2 °C, presumably due to the higher Lewis acidity of the cationic heterocycle, thereby creating stronger ionic interactions within the network.34,35 TGA was utilized to investigate changes in thermal stabilities (Td5%) as a function of structural changes (Table 2 and Figures S23–S25). Analysis of the TPILs with variable counteranion indicated once again reflected the correlation between Lewis basicity and relative thermal stability in ILs and PILs. Overall, [Br] PIL 12 was observed to have the lowest Td5% value (195 °C) than any of the other counteranion systems due to its relatively high Lewis basicity. In fact, Td5% values were found to be inversely related to Lewis basicity on the order of [NTf2] > [OTf] > [BF4] > [NO3] > [Br]. This trend is analogous to the previously discussed acetoacetate monomers, however, it is worth noting that the PIL networks exhibit higher thermal stabilities overall. Neither a change in crosslink density (acrylate/acetoacetate ratio) nor in cation structure appeared to adversely affect thermal stability, with all of the [NTf2] PIL networks exhibiting Td5% values in excess of 310 °C. The mechanical properties of the 1,2,3-triazolium-containing polyester networks were analyzed using dynamic mechanical analysis (DMA). Networks which employed different anions were compared initially (Figure 1a) and it was observed that in general all of the TPILs exhibited similar rubbery plateau moduli E′ above the Tg (Table 3). Such a finding was further supported by apparent crosslink density (ρx) values, which were calculated from rubbery elasticity theory according to the following equation 1 where E′ is the storage modulus at a temperature above the Tg (Pa), R is the gas constant (8.314 J mol/K), and T is the temperature (K).36 All ρx values were found to be in the range of (5.30–6.76) × 10–4 mol/cm3 with no discernible correlation with anion size or Lewis basicity (Table 3). In line with the DSC Tg data, the DMA Tg values, as determined from the maximum of the corresponding tan δ curves (Figure S26), showed a correlation with anion size, with PILs employing the largest anions [NTf2] 16 and [OTf] 15 resulting in the lowest DMA Tg values (−2.40 and 13.8 °C, respectively) and the network with the smallest anion, [Br] 12, exhibited the highest DMA Tg (37.5 °C). Figure 1 DMA storage modulus (E′) comparison of TPIL networks: with (a) variation in counteranion; (b) variation in acrylate/acetoacetate ratio; (c) variation in cation. Table 3 Mechanical Properties of the TPIL Networks polymer anion Acr/AcAc ratio tan δ max Tg (°C) E′@100 °C (MPa) ρx × 10–4 (mol/cm3) 12 Br 1.5:1.0 37.5 6.29 6.76 13 NO3 1.5:1.0 23.7 5.08 5.46 14 BF4 1.5:1.0 24.3 6.18 6.64 15 OTf 1.5:1.0 13.8 5.79 6.22 16 NTf2 1.5:1.0 –2.4 4.93 5.30 17 NTf2 1.2:1.0 –7.6 2.57 2.76 18 NTf2 1.8:1.0 15.7 6.08 6.53 19 (IM) 1.5:1.0 –6.4 4.67 5.02 20 (124TRI) 1.5:1.0 5.6 5.67 6.09 Variation in acrylate/acetoacetate ratio gave a predictable response in the corresponding mechanical properties, where an increase in acrylate concentration led to an increase in crosslink density, an increase in E′ rubbery plateau modulus (Figure 1b), and an increase in DMA Tg (Figure S27). Interestingly, a reduction in crosslink density led to an increase in the glassy plateau modulus. Although additional structural characterization is necessary, it is speculated that lower crosslinking may increase anion aggregation resulting from improved ion mobility, leading to a higher glassy plateau modulus. Variation in cation led to minimal change in crosslink density or E′ rubbery plateau modulus (Figure 1c); however, the 1,2,4-triazolium PIL network 20 was found to have a higher DMA Tg (Figure S28). As with the previously described DSC Tg data, this trend is presumably due to the higher Lewis acidity/coordinating ability of the 1,2,4-triazolium cation. To evaluate the ionic conductivity of the networks, frequency-dependent dielectric spectra were collected on each TPIL over a range of temperatures. Direct current conductivities (σ) were determined by first converting dielectric loss spectra (ε″) to the real conductivity (σ′) domain via the equation σ′ = ε″εoω, where εo is vacuum permittivity and ω is angular frequency in rad/s. The real conductivity spectra are characterized by a frequency-independent plateau region, and values of σ were taken to be the plateau value. Exemplar real dielectric (ε′), ε″, and σ′ spectra for TPIL 14 at 80 °C are provided (Figure S29). Once values were extracted from all spectra, conductivities were plotted for each TPIL as a function of temperature. These temperature-dependent conductivities are shown in Figure 2. Much to our surprise, the TPIL networks exhibited promising conductivities (10–6–10–9 S/cm at 30 °C, 30% RH) overall despite their crosslinked nature and were comparable to several previously reported TPILs, including those from our own laboratories (10–5–10–11 S/cm at 30 °C, 0–30% RH).22−27 Our initial hypothesis is that by anchoring the ionic groups into a crosslinked network, a more uniform ionic material where ionic aggregation is severely limited has been created, leading to better than expected conductivities. The concept of uniform ion distribution (termed “percolated aggregates”) has been proposed from experimental data37 and theorized in simulations38−40 for PILs where ionic groups are polymerized into the backbone rather than left pendant where aggregation is more probable. When comparing the behavior of PILs having different anions (Figure 2a), it is clear that conductivity depends strongly on polymer dynamics at lower temperatures. More precisely, lower temperature conductivities vary inversely with PIL Tg. However, with elevating temperature, the conductivity curves for various anions converge or crossover, highlighting an increasing dependence on additional factors. A deeper exploration of the relative contribution of TPIL properties to conductivity is presented later. Figure 2 Ionic conductivity curves (log σ vs 1000/T) for TPIL networks with (a) variable counteranion; (b) variable acrylate/acetoacetate ratio; (c) variable cation. The dashed curves represent Vogel–Fulcher–Tamman (VFT) fitting curves. When the anion is held constant, but the acrylate/acetoacetate ratio is varied (Figure 2b), the highest-Tg TPIL again displays the lowest conductivity, whereas the lowest Tg PIL’s conductivity is greatest. Unlike the trend observed with various anions, however, this Tg-dependent pattern is preserved across the entire measured temperature range. In addition, the relative differences in conductivities for the three TPILs at any given temperature are reasonably consistent across all temperatures. It should be noted that the ordering of the PILs having different acrylate/acetoacetate ratio also matches what would be expected based on ion content, i.e., the 1.2:1.0 ratio contains the greatest ionic monomer content and displays the largest conductivity, whereas the 1.8:1.0 ratio contains the least ionic monomer and concomitantly the smallest conductivity. Therefore, the overall ion content of these three PILs may also be contributing to observed behavior. Similarly, Figure 2c depicts the temperature-dependent conductivities of PILs where the anion and ratio are held constant and the nature of the cation is changed. As with the previous data, the three PIL’s conductivities follow the inverse relationship to Tg across the entire temperature range. The conductivity curve for the imidazolium PIL does rise more steeply with temperature than either of the triazolium PILs indicating there may be additional factors that govern ion transport. For instance, the conductivity trend for cations also follows a pattern associated to Lewis acidity of the cations (increasing conductivity with decreasing acidity), which can influence strengths of ionic interactions and thereby numbers of freely mobile anions in the PIL. All conductivity curves were fitted to the Vogel–Fulcher–Tamman (VFT) equation 2 where σ∞ is the infinite conductivity limit, To is Vogel temperature where ion motion stops, T is experimental temperature, and D is the strength parameter, which is inversely related to the fragility of polymer dynamics.14 The results from this fitting are shown in Table 4, and each dashed curve in Figure 2 depicts the VFT fit. Table 4 Ionic Conductivity Data and VFT Fitting Parameters for TPIL Networks PIL network anion (cation) Acr/AcAc ratio σ at 30 °C (S/cm) σ∞ (S/cm) D To (K) 12 Br 1.5:1.0 6.5 × 10–9 9.0 8.4 217 13 NO3 1.5:1.0 1.3 × 10–7 1.2 7.5 207 14 BF4 1.5:1.0 6.2 × 10–8 10.7 8.1 213 15 OTf 1.5:1.0 2.5 × 10–7 2.8 8.2 202 16 NTf2 1.5:1.0 9.3 × 10–7 0.8 6.8 203 17 NTf2 1.2:1.0 1.7 × 10–6 10.7 11.5 175 18 NTf2 1.8:1.0 2.2 × 10–7 1.5 8.8 195 19 (IM) 1.5:1.0 4.1 × 10–7 5.6 10.8 183 20 (124TRI) 1.5:1.0 2.5 × 10–7 0.6 7.0 206 To elucidate the relative contributions of Tg versus other factors to PIL conductivity, each graph from Figure 2 was replotted using an x-axis of Tg/T; these plots are shown in Figure 3. Such scaling of conductivity and other transport data has been used to de-emphasize differences in polymer segmental motion, to more clearly identify the extent to which other factors impact results. The more strongly that chain dynamics impact conductivity, the more that the individual conductivity plots will converge into something akin to a master curve. This convergence is most apparent with the data presented herein with conductivities for TPILs in which acrylate/acetoacetate ratio is varied, Figure 3b. So, although those TPILs vary in percentage of ionic subunits within the polymer, their conductivities appear to depend primarily on polymer dynamics within the range of temperatures studied. On the other hand, the conductivity profiles for anion variation and cation variation, Figure 3a,c, do not converge well indicating that other factors beyond polymer motion are contributing to observed behaviors. Figure 3 Tg-normalized ionic conductivity curves (log σ vs Tg/T) given (a) variable counteranion; (b) variable acrylate/acetoacetate ratio; (c) variable cation. The dashed curves represent VFT fitting parameters. Models of electrode polarization have been developed that can be applied to dielectric spectra (both ε′ and ε″) that allow the relative contributions of free-ion concentration (p) and free-ion mobility (μ) toward conductivity to be determined. Electrode polarization results from an accumulation of mobile ions at the electrodes and is most clearly manifested via the rapid rise then plateau of ε′ when moving toward the lower frequency end of that spectrum. The region of the spectra where polarization dominates, then, can be described by modified MacDonald’s theory using a Debye-type relaxation function 3 where εEP* is the complex dielectric function, ΔεEP is the relaxation strength, τEP is the relaxation time (or time scale for full polarization), and n is an exponent dependent upon electrode roughness.41−43 The relaxation time, then, can be correlated to μ and p through the following relationship 4 and relaxation strength is equivalent to the following 5 where LD, the Debye length, is defined as 6 L is sample thickness/electrode spacing, εs is the static dielectric constant, e is elementary charge, and k is Boltzmann’s constant. Therefore, simultaneous fitting of temperature-dependent ε′ and ε″ spectra with eq 3 allows for μ and p to be calculated as a function of temperature from fitting parameters.43 All of the obtained dielectric spectra were well fit by the modified Macdonald model except in the case of bromide-containing PIL 12. Example fits are shown in Figure S29 and a plot illustrating τEP as a function of temperature for the variable cation and variable anion PILs is shown in Figure S30; values of n for fitting ranged from 0.67 to 0.82. Applying multiplicative shift factors to ε′ spectra for each PIL resulted in well-converged master curves except in the case of [Br] 12 (Figure S31), and it appears that its polarization region should be modeled by a modified or additional relaxation functions. Attempts to include an interfacial polarization function in the fitting for [Br] 12 proved unsuccessful in fully describing its behavior.44,45 It should also be noted that fitting dielectric spectra with Havriliak–Negami functions at frequencies higher than the onset of electrode polarization were also attempted to explore polymer segmental relaxations. However, high-frequency features were subtle/weak (substantially dominated by electrode polarization) to nonexistent in the studied frequency range. The inability to observe such relaxations was confirmed via the use of derivative spectra (εder), which is supposed to aid in such evaluation by eliminating contributions to the spectra from ion conduction.44 Plots of temperature-dependent free-ion concentration and ion mobilities obtained from dielectric fitting are shown in Figure 4. The temperature dependence of free-ion concentration, or number density of conducting ions, is described by an Arrhenius function 7 where p∞ is the high-temperature limiting concentration and Ea is the activation energy for conducting ions, which can be thought of as a binding energy for an ion pair. Therefore, the fitted line in Figure 4a was used to determine log p∞ from the y-intercept and Ea and from the slope. These values are shown in Table 5. The determined values of log p∞ are generally within the 21–22 cm–3 range, which is sensible based on a calculated value for total possible free ions of approximately 21.1 cm–3 obtained from monomer stoichiometries and molar masses. Furthermore, the activation energies of PILs 13–16 follow a pattern defined by the Lewis basicity of the anion, with the most weakly basic [NTf2] forming the weakest ion pairs and thereby having the lowest Ea, whereas the most strongly basic [NO3] forms the strongest ion pairs and requires the largest Ea.32 Similarly, variations in monomer cation (PILs 16, 19, and 20) result in Ea values that trend with the cations’ Lewis acidities, again corresponding to strength of ion pairing interactions. Figure 4 Temperature-dependent (a) free-ion concentrations and (b) ion mobilities calculated from dielectric spectral fitting using the modified Macdonald model. The dashed lines/curves illustrate the (a) Arrhenius or (b) VFT fit to each data set. Table 5 Free-Ion Concentrations and Ion Mobilities for TPILs PIL network anion (cation) log p∞ (cm–3) Ea (kJ/mol) log μ∞ (cm2/(V s)) D To (K) 13 NO3 21.59 25.28 –0.13 7.62 190.4 14 BF4 21.62 19.83 1.23 10.88 189.3 15 OTf 21.51 18.92 –1.54 2.98 237.1 16 NTf2 21.66 16.07 –2.57 2.09 242.8 19 (IM) 20.95 17.06 –1.74 2.14 247.4 20 (124TRI) 22.65 29.53 –2.54 1.26 254.0 Temperature-dependent ion mobilities, like conductivities, are described by a VFT fitting 8 where the parameters are defined similarly to eq 2. The data and VFT fit are shown graphically in Figure 4b, whereas obtained fitting parameters are listed in Table 5. With PILs that vary by anion, limiting mobilities increase with decreasing anion size, except with [BF4] behaving as an outlier. Mobilities of [BF4]-containing PILs that are 2 orders of magnitude greater than [NTf2] PILs have been reported in the past, though no other counteranions were included in that study.46 Furthermore, the [BF4] anion has demonstrated limited stability in ILs, particularly at elevated temperature and in the presence of water.47 Since high temperatures were employed in this study, and measurements were not made under anhydrous conditions, it is possible that [BF4] decomposition/hydrolysis may contribute to the behavior of TPIL 14, however, such analyses were beyond the scope of this work. With the PILs where anion is constant, but cation is varied, the limiting mobilities with [123TRI] and [124TRI] cations are very similar, but [IM] is nearly an order of magnitude greater. It is likely that this observed difference is due to crosslink density for [IM] being lower than with the two triazolium PILs (see Table 3). With higher crosslink density, it is expected that free ions would be required to take more tortuous paths during conduction, thereby lowering their apparent mobilities. Figure 5 shows the ionic conductivity of TPIL [Br] 12 as a function of temperature at three different relative humidity values (RH 30, 60, and 90%), whereas Table 6 summarizes the water uptake (wt %) of TPIL [Br] 12 at each of these RH conditions after an overnight (16 h) soak (samples tested in triplicate). The bromide conductivity increases approximately 4 orders of magnitude when the RH was increased from 30 to 60%. A further increase of another order of magnitude was observed when the RH was held at 90%. These increases can be attributed to a water-assisted ion-transport mechanism, commonly seen in PILs, which contain hydrophilic ions, such as halides. Such a transport phenomenon has been likened to water–Nafion systems and has been observed with other bromide-containing PIL co-polymers, prepared from acrylic-functionalized imidazolium monomers.48 The strong likelihood of water-assisted bromide transport is further supported by their noticeably large water uptake (0.64–27.2 wt %) over the humidity range of interest (Table 6). Such a large increase in ionic conductivity, as expected, was not observed for the highly hydrophobic TPIL [NTf2] 16. For the more hydrophobic PIL [NTf2] 16, ion transport would be more strongly dictated by segmental motion and polymer dynamics. Although their conductivities at higher % RH were not examined, it must be noted that both the [NO3] and [BF4] TPIL networks did exhibit some water absorption at 30% RH (Table S1), though not nearly to the extent that TPIL [Br] 12 did. Regardless, this small amount of water absorbed could result in a slight enhancement of ionic conductivity that otherwise might not be observed under anhydrous conditions. Figure 5 Ionic conductivity curves for TPIL [Br] 12, reflecting the effect of variable relative humidity. Table 6 Effect of Relative Humidity on Ionic Conductivity and Water Uptake of the [Br] and [NTf2] TPIL Networksa 30% RH 60% RH 90% RH anion σ (S/cm) water uptake (%) σ (S/cm) water uptake (%) σ (S/cm) water uptake (%) Br 6.46 × 10–9 0.64 ± 0.05 3.16 × 10–5 7.90 ± 0.10 2.45 × 10–4 27.2 ± 0.4 NTf2 9.33 × 10–7 <0.01 9.98 × 10–7 0.05 ± 0.02 1.23 × 10–6 0.67 ± 0.13 a Data were obtained after a 16 h soak at 30 °C at the specified RH. Ion mobility (μ) and free-ion concentration (p) were investigated at 30 and 60% RH for the [Br] and [NTf2] TPIL networks (Figure S32). Similarly to what was noted above, the modified Macdonald model fit all data well, except for [Br] at 30% RH. For [Br] at 30% RH, an interfacial polarization function was added and while the addition still did not fully describe the PIL’s behavior, it did provide enough information such that reasonable humidity comparisons could be made. When humidity was increased, ion mobility was found to increase for both TPIL networks, presumably due to the plasticizing ability of water. Free-ion concentration for the [Br] TPIL network was found to increase, but decrease for the [NTf2] networks given an increase in humidity. This observation is attributed to the ability of water to solvate bromide, while causing the much more hydrophobic NTf2 anion to aggregate. Unfortunately, the data acquired at 90% RH were such that these did not allow for a proper fitting to determine μ and p parameters. Conclusions A series of 1,2,3-triazolium-based bisacetoacetate monomers were synthesized using an azide–alkyne click cyclization strategy and subsequently polymerized using base-catalyzed Michael addition. The resulting TPIL networks were analyzed for their thermal (DSC, TGA), mechanical (DMA), and electrochemical (DRS) properties. The counteranion, crosslink density (acetoacetate/acrylate ratio), and cation were varied to observe changes in these properties. DSC thermal analysis of the TPILs indicated an inverse correlation between Tg and counteranion size, with [NTf2] TPIL exhibiting the lowest Tg. An increase in the Tg was observed when the acrylate concentration was increased due to an increase in covalent crosslinking. An increase in thermal stability (Td5%) was observed with decreasing Lewis basicity of the anion on the order [NTf2] > [OTf] > [BF4] > [NO3] > [Br]. Changes in crosslink density brought about by varying the acrylate/acetoacetate ratio did not appear to affect thermal stability. Dynamic mechanical analysis indicated that changes in counteranion did not influence the rubbery plateau storage modulus (E′) or the apparent crosslink density (ρx) in any discernable way, whereas DMA Tg (tan δ max) was observed to be inversely related to counteranion size (supporting DSC Tg results). An increase in acrylate concentration did result in an increase in E′ and crosslink density. Despite the network nature of the TPILs, obtained conductivities of 10–6–10–9 S/cm at 30 °C were comparable to a number of previously reported PILs. Ion conduction demonstrated a reliance on polymer fluidity, especially at lower measured temperatures. Analysis of dielectric data highlighted a further dependence of conductivity on both Lewis acidity/basicity of the employed cations/anions and on free anion size via those properties influence on free-ion concentration and ion mobility, respectively. Experimental Section Materials All commercial reagents and solvents were purchased from either Acros Organics or Sigma-Aldrich and used as received. Tetrahydrofuran (THF) (99.9%) and N,N-dimethylformamide (DMF) (99.9%) were purchased as anhydrous from Acros Organics and used as received. Ultrapure water having a resistivity of 18 MΩ cm was produced using an ELGA Purelab Ultra filtration device. A JEOL-ECS 400 MHz spectrometer was utilized to obtain 1H and 13C NMR spectra, and chemical shift values reported are referenced to residual solvent signals (CDCl3: 1H, 7.24 ppm; 13C, 77.16 ppm; DMSO-d6: 1H, 2.50 ppm; 13C, 39.52 ppm). Elemental analyses were completed by Atlantic Microlab, Inc. The syntheses of 1-acetoacetoxy-6-bromohexane 4,19 1,4-bis(6′-acetoacetoxyhexyl)-1,2,4-triazolium bis(trifluoromethylsulfonyl)imide 10,19 and 1,4-bis(6′-acetoacetoxyhexyl)-imidazolium bis(trifluoromethylsulfonyl)imide 11(17) have been previously reported. Synthesis of Ethyl Ester 1 Ethyl-6-bromohexanoate (10.0 g, 44.8 mmol), sodium azide (5.83 g, 89.6 mmol), and anhydrous DMF (100 mL) were charged to a 250 mL round-bottom flask equipped with a magnetic stir bar. The resulting mixture was stirred for 24 h at 60 °C. The reaction was then cooled to room temperature, and trimethylsilylacetylene (5.72 g, 58.3 mmol) and copper(I) iodide (1.11 g) were sequentially added. The reaction was stirred at room temperature for 2 h, then warmed to 50 °C, and held for 24 h. The mixture was then cooled to room temperature and poured onto ethyl ether (300 mL). The organic phase was separated and washed with 150 mL portions of a 3:1 mixture of saturated NH4Cl/NH4OH until the aqueous wash layer was colorless (i.e., no blue color). The organic phase was then further washed with brine, dried over a mixture of Na2SO4/MgSO4, filtered, and the solvent removed under reduced pressure to afford an orange oil, which was immediately dissolved in anhydrous THF (50 mL). A 1.0 M solution of TBAF in THF (53.8 mL, 53.8 mmol of TBAF) was added, and the resulting solution was stirred at room temperature overnight. The residuals were removed under reduced pressure, and the crude oil was dissolved in ethyl acetate and washed sequentially with 5% NaCl solution twice and brine. The organic phase was separated, dried over Na2SO4/MgSO4, filtered, and the solvent removed to give a brown oil, which was purified by column chromatography on silica gel with a gradient elution of 0–50% ethyl acetate in hexanes. Purification resulted in 5.77 g of a light yellow oil (61%). 1H NMR (400 MHz, CDCl3): δ 1.17 (t, 3H), 1.29 (m, 2H), 1.58 (m, 2H), 1.86 (m, 2H), 2.21 (t, 2H), 4.03 (t, 2H), 4.32 (q, 2H). 13C NMR (100 MHz, CDCl3): δ 14.29, 24.30, 25.96, 30.10, 33.98, 49.99, 60.39, 123.36, 133.81, 173.48. Anal. calcd for C10H17N3O2: C 56.85, H 8.11, N 19.89. Found: C 56.78, H 8.29, N 19.86. Synthesis of 1-(6′-Hydroxyhexyl)-1,2,3-triazole 2 In a 500 mL, three-necked round-bottom flask was dissolved the 1,2,3-triazole ester 1 (6.60 g, 31.2 mmol) in dichloromethane (60 mL). The magnetically stirred solution was cooled to 0 °C under N2 whereupon DIBAL-H (93.7 mL of a 1.0 M solution in hexanes, 93.7 mmol) was added dropwise over a 45 min period, followed by warming to room temperature where stirring continued for 24 h. The reaction was then slowly quenched with 3.0 M HCl (30 mL), followed by the transfer of the reaction mixture into a separatory funnel where the organic layer was separated. The aqueous layer was extracted once with dichloromethane, and the organic layers were combined, dried over Na2SO4/MgSO4, filtered, and the solvent removed under reduced pressure to afford 4.45 g of a light yellow oil (84%). 1H NMR (400 MHz, DMSO-d6): δ 1.21 (m, 2H), 1.27 (m, 2H), 1.36 (m, 2H), 1.80 (m, 2H), 3.35 (t, 2H), 4.33 (t, 2H), 7.70 (s, 1H), 8.11 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 24.86, 24.99, 29.79, 32.28, 49.06, 60.51, 124.53, 133.14. Anal. calcd for C8H15N3O: C 56.78, H 8.93, N 24.83. Found: C 56.49, H 9.17, N 24.56. Synthesis of 1-(6′-Acetoacetoxyhexyl)-1,2,3-triazole 3 1-(6′-Hydroxylhexyl)-1,2,3-triazole 2 (4.29 g, 25.4 mmol) was dissolved in acetonitrile (100 mL) in a 250 mL round-bottom flask. tert-Butylacetoacetate (12.03 g, 76.1 mmol) was then added, and the resulting magnetically stirred solution was warmed to reflux and held for 2 h. Volatiles were then removed under reduced pressure to afford 5.97 g (93%) of a light orange oil. 1H NMR (400 MHz, DMSO-d6): δ 1.21 (m, 2H), 1.30 (m, 2H), 1.54 (m, 2H), 1.79 (m, 2H), 2.16 (s, 3H, CH3 on AcAc group), 3.57 (s, 2H, CH2 on AcAc group), 4.01 (t, 2H, J = 6.7 Hz), 4.33 (t, 2H, J = 7.2 Hz), 7.70 (s, 1H), 8.11 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δ 24.63, 25.37, 27.78, 29.58, 30.10, 49.25, 49.55, 64.25, 124.53, 133.14, 167.14, 201.63. Anal. calcd for C12H19N3O3: C 56.90, H 7.56, N 16.59. Found: C 56.58, H 7.56, N 16.32. Synthesis of 1,3-Bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium Bromide 5 1-(6′-Acetoacetoxyhexyl)-1,2,3-triazole 3 (2.96 g, 10.9 mmol) and 1-acetoacetoxy-6-bromohexane 4 (2.97 g, 11.2 mmol) were added to a 25 mL round-bottom flask and stirred at 60 °C for 48 h under an atmosphere of nitrogen. The mixture was then cooled to room temperature and washed with ethyl ether (4 × 20 mL). The product was dried under reduced pressure, resulting in 4.30 g of an orange oil (91%). 1H NMR (400 MHz, DMSO-d6): δ 1.31 (m, 8H), 1.56 (m, 4H), 1.92 (m, 4H), 2.17 (s, 6H, CH3 on AcAc group), 3.59 (s, 4H, CH2 on AcAc group), 4.04 (t, 2H, J = 6.6 Hz), 4.64 (t, 2H, J = 7.1 Hz), 8.99 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 24.50, 24.92, 27.70, 28.47, 30.00, 49.59, 52.95, 64.19, 130.87, 167.15, 201.49. Anal. calcd for C22H36N3O6Br: C 50.97, H 7.00, N 8.11. Found: C 50.85, H 7.03, N 8.02. Synthesis of 1,3-Bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium Nitrate 6 To a 50 mL round-bottom flask equipped with a magnetic stir bar was dissolved 1-(6′-acetoacetoxyhexyl)-1,2,3-triazolium bromide 5 (1.80 g, 3.47 mmol) in deionized (DI) water (10 mL). A solution of sliver nitrate (0.62 g, 3.65 mmol) in DI water (5 mL) was added, and the reaction was stirred at room temperature overnight in the dark. The mixture was then filtered through Celite and dried under vacuum, resulting in 1.70 g of a light amber oil (98%). 1H NMR (400 MHz, DMSO-d6): δ 1.32 (m, 8H), 1.54 (m, 4H), 1.91 (m, 4H), 2.17 (s, 6H, CH3 on AcAc group), 3.59 (s, 4H, CH2 on AcAc group), 4.04 (t, 2H, J = 6.6 Hz), 4.62 (t, 2H, J = 7.1 Hz), 8.94 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 24.48, 24.91, 27.69, 28.45, 30.06, 49.55, 51.50, 64.17, 130.79, 167.26, 201.62. Anal. calcd for C22H36N4O9: C 52.79, H 7.25, N 11.19. Found: C 52.55, H 7.13, N 11.04. Synthesis of 1,3-Bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium Tetrafluoroborate 7 To a 50 mL round-bottom flask equipped with a magnetic stir bar was dissolved 1-(6′-acetoacetoxyhexyl)-1,2,3-triazolium bromide 5 (2.00 g, 3.85 mmol) in DI water (20 mL). A solution of sliver tetrafluoroborate (0.76 g, 3.89 mmol) in DI water (5 mL) was added, and the reaction stirred overnight, in the dark, at room temperature. The mixture was then filtered through Celite and dried under vacuum, resulting in 1.91 g of a light yellow oil (94%). 1H NMR (400 MHz, DMSO-d6): δ 1.32 (m, 8H), 1.56 (m, 4H), 1.91 (m, 4H), 2.17 (s, 6H, CH3 on AcAc group), 3.59 (s, 4H, CH2 on AcAc group), 4.04 (t, 2H, J = 6.4 Hz), 4.62 (t, 2H, J = 7.1 Hz), 8.92 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 24.47, 24.91, 27.69, 28.45, 30.07, 49.55, 53.02, 61.17, 130.77, 167.27, 201.63. Anal. calcd for C22H36BF4N3O6: C 50.30, H 6.91, N 8.00. Found: C 50.12, H 6.88, N 7.88. Synthesis of 1,3-Bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium Triflate 8 To a 50 mL round-bottom flask equipped with a magnetic stir bar was dissolved 1,3-bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium bromide 5 (1.20 g, 2.31 mmol) in DI water (15 mL). A solution of silver triflate (0.61 g, 2.36 mmol) in DI water (5 mL) was added. The mixture was stirred overnight at room temperature in the dark and then filtered through a short pad of Celite, followed by solvent removal under reduced pressure to afford 1.21 g (89%) of a light yellow oil. 1H NMR (400 MHz, DMSO-d6): δ 1.31 (m, 8H), 1.56 (m, 4H), 1.93 (m, 4H), 2.17 (s, 6H, CH3 on AcAc group), 3.59 (s, 4H, CH2 on AcAc group), 4.05 (t, 2H, J = 6.6 Hz), 4.62 (t, 2H, J = 7.1 Hz), 8.92 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 24.47, 24.91, 27.69, 28.45, 30.07, 49.56, 52.98, 64.19, 120.68 (q, J = 322 Hz, −CF3), 130.82, 167.27, 201.61. Anal. calcd for C23H36F3N3O9S: C 47.01, H 6.18, N 7.15. Found: C 46.98, H 6.14, N 7.25. Synthesis of 1,3-Bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium Bis(trifluoromethylsulfonyl)imide 9 1,3-Bis(6′-acetoacetoxyhexyl)-1,2,3-triazolium bromide 5 (1.50 g, 2.89 mmol) was added to a 50 mL round-bottom flask and dissolved in DI water (15 mL). Lithium bis(trifluoromethylsulfonyl)imide (0.87 g, 3.04 mmol), dissolved in DI water (5 mL), was added to the reaction, and the resulting mixture was stirred at room temperature overnight. Dichloromethane (25 mL) was then added and the organic phase was separated, washed with DI water (3 × 10 mL), and then the solvent was removed under reduced pressure to afford 2.00 g (96%) of a light yellow oil. 1H NMR (400 MHz, DMSO-d6): δ 1.32 (m, 8H), 1.56 (m, 4H), 1.91 (m, 4H), 2.17 (s, 6H, CH3 on AcAc group), 3.59 (s, 4H, CH2 on AcAc group), 4.04 (t, 2H, J = 6.4 Hz), 4.62 (t, 2H, J = 7.1 Hz), 8.92 (s, 2H). 13C NMR (100 MHz, DMSO-d6): δ 24.48, 24.94, 27.71, 28.45, 30.08, 49.57, 53.00, 64.17, 119.43 (q, J = 319 Hz, −CF3), 130.84, 167.25, 201.55. Anal. calcd for C24H36F6N4O10S2: C 40.11, H 5.05, N 7.80. Found: C 40.09, H 5.01, N 7.93. Procedure for Michael Addition Polymerizations All of the polymerizations were completed in dichloromethane (50 wt %). In a typical procedure, 1,3-bis(2′-acetoacetoxyethyl)imidazolium bis(trifluoromethylsulfonyl)imide 9 (0.90 g, 1.25 mmol) and 1,4-butanediol diacrylate (0.37 g, 1.88 mmol, 1.5 M equiv) were dissolved in dichloromethane (1.27 g). DBU catalyst (9.5 mg, 2 mol %) was then added, and the resulting solution was mixed for 2 min. The solution was poured into a PTFE mold and cured for 48 h at ambient temperature, followed by curing in a 60 °C oven for 48 h. Complete solvent removal and sufficient dryness prior to analysis were ensured by placing the sample in a vacuum oven (<0.1 mmHg) for 24 h at 50 °C. Polymer Analysis All acetoacetate monomers and PIL network films were stored in a vacuum oven at 40 °C for 48 h prior to any analytical testing. Differential scanning calorimetry (DSC) was performed using a TA Instrument Q200 Differential Scanning Calorimeter with a heating rate of 5 °C/min on 4–8 mg samples. Glass transition temperatures (Tg) were determined from the second heating by the inflection point of the curve observed. A TA Instrument Q500 Thermogravimetric Analyzer was used to determine Td5%, the temperature at which 5% weight loss was observed, at a heating rate of 10 °C/min under an inert nitrogen atmosphere. The mechanical properties of the TPIL networks were analyzed using a TA Instruments Q800 Dynamic Mechanical Analyzer (DMA) in film tension mode (single frequency of 1 Hz) at a heating rate of 5 °C/min. Soxhlet extraction in refluxing CH2Cl2 (24 h) was completed on all TPIL networks in duplicate. The swollen sample was weighed (mwet) and then placed in a vacuum oven (60 °C, 24 h) to obtain the dry weight (mdry). Gel fraction was determined as follows: gel fraction = (mdry/mo) × 100 where mo is the original mass of the sample. Percent swelling was calculated as follows: % swelling = (mwet/mo) × 100.16 The influence of water absorption was determined for each TPIL network (in triplicate) by conditioning the sample in the humidity chamber (Espec BTL-433 benchtop temperature/humidity oven at 30, 60, 90% RH) for 16 h, followed by conductivity testing. Water absorption was determined using the following equation: water absorbed = ((mwet – mdry)/mdry) × 100. Dielectric Relaxation Spectroscopy DRS was performed with a Metrohm FRA32M frequency response analyzer coupled to an ECI10M impedance interface and a custom-built (in-house) two-electrode cell (Figure S17). The cell was placed inside the aforementioned Espec BTU-433 controlled-temperature/humidity chamber to maintain constant conditions (between 30 and 150 °C and 30% RH) during each measurement. Sample membranes of ∼12 mm diameter and 1 mm thickness were sandwiched between 304L stainless steel electrodes separated by a 1 mm thick PTFE spacer. Electrodes were polished to a mirror finish with 8000-, 14 000-, and 60 000-mesh diamond paste (Sandvik Hyperion; Worthington, OH) prior to use. For each sample, dielectric/impedance spectra were collected at various temperatures using a frequency range of 1–107 Hz and alternating current amplitude of ±0.01 V. The stray admittance and residual impedance of the test cell were evaluated by open cell (no sample) and shorted cell measurements, respectively, using the same experimental conditions outlined above; shorted measurements were performed using a 12 mm dia by 1 mm thick disk of 99.99% pure Cu as the sample. Compensations for stray admittance and residual impedance were applied to all data via Excel, as outlined elsewhere.49 Additionally, all measured values of real dielectric (ε′) were corrected for contribution from the spacer according to the method described by Johari.50 Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01949.1H and 13C NMR spectra for compounds 1–3 and 5–9 are provided along with representative DSC and TGA data for each of the diacetoacetate monomers 5–9; DSC thermograms; TGA traces and DMA tan δ curves for TPIL networks 12–20; two-electrode cell used in DRS measurements (PDF) Supplementary Material ao8b01949_si_001.pdf The authors declare no competing financial interest. Acknowledgments Acknowledgment is made to the donors of the American Chemical Society Petroleum Research Fund for support of this research (ACS PRF# 53097-UNI7). 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PMC006xxxxxx/PMC6644409.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145782610.1021/acsomega.7b00891ArticleSelective Adsorption of Coronene atop the Polycyclic Aromatic Diimide Monolayer Investigated by STM and DFT Geng Yanfang †§Wang Shuai †§Shen Mengqi †Wang Ranran ‡Yang Xiao ‡Tu Bin †Zhao Dahui ‡Zeng Qingdao †† CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology (NCNST), 11 Zhongguancunbeiyitiao, Beijing 100190, P. R. China‡ Beijing National Laboratory for Molecular Sciences, The Key Laboratory of Polymer Chemistry and Physics of the Ministry of Education, College of Chemistry, Peking University, Beijing 100871, P. R. China E-mail: tub@nanoctr.cn (B.T.).* E-mail: dhzhao@pku.edu.cn (D.Z.).* E-mail: zengqd@nanoctr.cn (Q.Z.).08 09 2017 30 09 2017 2 9 5611 5617 29 06 2017 25 08 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. The self-assemblies of polycyclic aromatic diimide (PAI) compounds on solid surfaces have attracted great interest because of the versatile and attractive properties for application in organic electronics. Here, a planar guest species (coronene) selectively adsorbs on the helicene-typed PAI1 monolayer strongly, depending on the conjugated cores of these PAIs. PAI1 molecule displays evidently a bowl structure lying on the highly oriented pyrolytic graphite surface due to the torsion of the “C”-shaped fused benzene rings. In combination with density functional theory calculation, the selective inclusion of coronene atop the backbone of the PAI1 array might be attributed to the bowl structure, which provides a groove for immobilizing coronene molecules. On the other planar densely packed arrays, it is difficult to observe the unstable adsorption of coronene. The selective addition of coronene molecules would be a strategic step toward the controllable multicomponent supramolecular architectures. document-id-old-9ao7b00891document-id-new-14ao-2017-00891yccc-price ==== Body Introduction Two-dimensional (2D) self-assemblies on a solid surface are an interesting subject due to the promising perspectives in the nanotechnology field.1−3 Although the exploitation of controlled supramolecular chemistry has reached a maturity level where the specific self-assembly can be predicted to high accuracy, controllable ordering in the multicomponent architectures at the molecular scale remains a challenge. To obtain well-defined complex structures, 2D multicomponent nanostructures through the selective accommodation of many types of guest molecules in various host template networks have attracted a great deal of attention over the past decades.4,5 In most cases, the host molecules formed 2D solid networks with suitable cavities for guest molecules, and the inclusion of guest molecules cannot disturb the already existing host network. The shape and size complementarities between the cavity and guest molecule play an important role in the nanostructure. On the other hand, there are a few cases of coassembly in which host nanostructures on the solid surface were tuned by the inclusion of guest molecules.6,7 The applications of host–guest systems for the fixation of functional compounds have been hampered by the inefficient understanding of the behavior of incorporation of guest species on the host molecules. Among the most studied guest molecules, coronene (Cor), with sixfold symmetry, displayed a stable adsorption structure on unmodified solid surfaces, including various metal surfaces8,9 and the highly oriented pyrolytic graphite (HOPG) substrate.10−12 In most reports, a single coronene molecule or clusters were confined within various networks.13−27 Note that the uses of coronene to transform the self-assembled surface structures have also been observed.16,17,19 In addition to the location of coronene in the host cavity, there are some cases in which coronene molecules are partly or completely located on the host molecules. Horn et al. found an ordered second coronene layer on top of the first coronene layer due to the coronene layer and substrate underneath.28,29 Zhang et al. observed a coronene layer on top of HPB-6pa network, which might be attributed to the electronic interaction and van der Waals interactions.30 Note that the cavity of HPB-6a is slightly smaller than the size of the coronene molecule by around 0.12 nm. Therefore, the adsorption site of coronene might be attributed to many factors, like the substrate, the cavity of host network, the host structure, and so on. In this study, we designed four polycyclic aromatic diimide (PAI) derivatives, namely, PAI1 (N,N′-di(2-octyldodecyl)dibenzo[c,g]phenanthrene-1,2,5,6-tetracarboxyldiimide), PAI2 (N,N′-di(2-octyldodecyl)benzo[k]tetraphene-5,6,12,13-tetracarboxyldiimide), PAI6 (N,N′-di(2-octyldodecyl)benzo[c]picene-7,8,15,16-tetracarboxyldiimide), and PAI8 (N,N′-di(2-octyldodecyl)benzo[c]naphtho[2,1-k]tetraphene-5,6,14,15-tetracarboxyldiimide), as the target host molecules (Figure 1), which have the same alkyl chains but different backbone blocks.31 The branched alkyl side chains attached on the imide nitrogen atoms help in ensuring the stable adsorption on the solid surface and the recognizable 2D structure. As a powerful tool to study the molecular systems adsorbed on flat and conductive solid substrates, scanning tunneling microscopy (STM) showed 2D self-assembled monolayers formed by PAI derivatives at the 1-phenyloctane/HOPG interface. Coronene molecules then periodically located on the specific highly ordered PAI1 host array. In combination with density functional theory (DFT) calculations, the results showed that coronene can only be immobilized in the bowl structure, whereas it is unstable on the planar PAI2, PAI6, and PAI8 monolayers. The formed Cor/PAI1 bilayer structure is more thermodynamically favorable than that of the PAI1 system. It is expected that systematic investigations may lead to control the distribution and dispersion of guest molecules on or in the preformed arrays on surface. Figure 1 Chemical structures of four PAI-based compounds. Results and Discussion Self-Assembled 2D Structure of PAIs First, self-assemblies of four PAI derivatives were investigated. Figure 2 displays the high-resolution STM images of 2D molecular organizations. In each STM image, the brighter features correspond to the conjugated backbone cores of the PAI derivatives, whereas the darker areas are occupied by the alkyl chains. The orientation of the backbones is parallel to each other, showing the formation of striped patterns. It is noteworthy that PAI1 exhibits bright “V”-shaped protrusion, indicating that the backbone of PAI1 is not parallel with respect to the substrate surface.31−33 Such nonplanar geometry might be attributed to the distortion of the C-shaped polycyclic skeletons. The V-shaped configuration on the HOPG surface might come from the interaction between the interdigitated alkyl chains as well as the interaction between alkyl chains and the substrate. For PAI2, PAI6, and PAI8 molecules, they all exhibit rodlike protrusions in the STM images, indicating that their backbones are parallel to the HOPG surface. Therefore, the interactions between adsorbates and the substrate determine the stable adsorption of molecules on the surface. The unit cells containing one PAI molecule are superimposed on the STM images, and the unit-cell parameters are summarized in Table S1. Figure 2 High-resolution STM images (15 nm × 15 nm) of molecule PAI1 (a), PAI2 (b), PAI6 (c), and PAI8 (d) at the 1-phenyloctane/HOPG interfaces. The unit cells are inserted on the STM images. Tunneling parameters: (a) 298.8 pA, 511.2 mV; (b) 299.1 pA, 567.3 mV; (c) 299.1 pA, 601.5 mV; and (d) 299.1 pA, 623.8 mV. Although the distances between adjacent molecules are slightly different, there are great changes in the parameter α in the range of (82–99) ± 2°, indicating that the relative orientations of these PAI compounds are largely different. The orientation of the conjugated cores may be due to the hydrogen bond between −C=O and H–C of the phenyl ring from adjacent two molecules. One carbonyl group in PAI1 cannot form a hydrogen bond with the other adjacent PAI1 molecule because the neighboring backbone of PAI1 rotates out of the substrate surface. Additionally, the elastic alkyl chains tend to be out of the surface upon the close-packed surface.34 In the cases of PAI2, PAI6, and PAI8, the carbonyl groups are on the two sides of the conjugated center; therefore, there might be two hydrogen bonds marked with red circles between two adjacent molecules. In these STM images, the widths of dark stripes are measured to be 1.2 ± 0.2 nm, which are consistent with the length of the alkyl side chains. The molecular models (Figure 3) corresponding to STM images were obtained through DFT calculation, which further confirm the molecular geometry on the surface. The calculated interaction energies between adsorbates and interaction energies between adsorbates and the substrate in these systems are shown in Table 1. In these four systems, the interaction energies between adsorbates are around 20 kcal mol–1, which result from the interactions between interdigitated alkyl chains. Compared with the other three systems, the slightly smaller intermolecular interaction in PAI1 might be attributed to the curled alkyl chain induced by the rotation of the backbone. The much larger interaction energies between adsorbates and substrates of PAI2, PAI6, and PAI8 than those of PAI1 are consistent with the supposed configuration of PAI1 above. The differences of adsorbate–substrate interaction energies between PAI2, PAI6, and PAI8 might be due to the backbones. These results provide evidence that the molecule–substrate interaction is the predominant factor relative to the molecule–molecule interaction. The low total energy per unit area in PAI1/HOPG systems compared to that of other systems supports the only inclusion of coronene molecules. Figure 3 Molecular models (a)–(d) of self-assembled monolayers corresponding to the STM images (a)–(d) in Figure 2, respectively. The unit cells are inserted on the models. Table 1 Calculated Interaction Energies in the Systems of PAI1/HOPG, PAI1 + Cor/HOPG, PAI2/HOPG, PAI6/HOPG, and PAI8/HOPGa interaction energies between adsorbates (kcal mol–1) interaction energies between adsorbates and substrate (kcal mol–1) total energy (kcal mol–1) total energy per unit area (kcal mol–1 Å–2) PAI1 –20.121 –56.286 –76.407 –0.237 PAI1 + Cor –34.262 –133.794 –168.056 –0.458 PAI2 –21.712 –291.473 –314.185 –1.006 PAI6 –24.629 –332.837 –357.466 –1.058 PAI8 –23.966 –394.992 –418.958 –1.108 a Note that the more negative energy means the system is more stable. Selective Adsorption of Coronene on PAI1 Surface Next, the coadsorption behavior of the guest coronene in these self-assembled patterns of PAI molecules was investigated. Except for the PAI1 monolayer, STM observation showed no evidence that the coronene molecule can be immobilized in other monolayers. In the large-scale STM image of coronene/PAI1, as shown in Figure S1, the stripes brighter than those of PAI1 were clearly visible, obviously indicating the localization of coronene molecules. The high-resolution STM image, as shown in Figure 4, shows that the V-shaped protrusion of PAI1 becomes circular spots. A unit cell is superimposed upon the STM image with a = 2.9 ± 0.1 nm, b = 1.3 ± 0.1 nm, and α = 99 ± 2°, which is significantly different from unit parameters of the parent PAI assembly (a = 2.7 ± 0.1 nm, b = 1.1 ± 0.1 nm, and α = 92 ± 2°). The width of the bright stripe is consistent with the diameter of the coronene plane parallel to the HOPG surface. The cross-sectional profiles (Figure 4b) show that the bright stripe is located higher than the darker stripe by around 0.1 nm in the PAI1 array, whereas the bright stripe is located higher than the darker stripe by around 0.2 nm after deposition of coronene. Additionally, competitive adsorption of coronene on the HOPG surface could not happen because there is no coronene phase, as has been observed. If coronene molecules preferentially adsorb on the HOPG surface, the structure of the PAI1 monolayer might be disturbed. From these factors, the adsorbed coronene molecule is expected to be located on the PAI1 conjugated cores. Figure 4 (a) High-resolution STM image of the self-assembled densely packed structure of Cor/PAI1/HOPG with the chemical structure of coronene (15 nm × 15 nm; Iset = 268.8 pA; Vbias = 580.4 mV), (b) line profile of the PAI1 and Cor/API1 monolayer on the HOPG surface corresponding to the STM images, (c) molecular model in image (a), (d) side view of the molecular model in (c). The coronene and conjugated core of PAI1 cannot be resolved separately due to the limited resolution. The appropriate position of the coronene molecules can be identified by DFT calculation. The molecular model, as shown in Figure 4c, reveals the formation of a two-layer densely packed structure by specific adsorption of the coronene molecule on the bowl-conjugated core of PAI1. Coronene displayed parallel configuration to maximize the interaction between coronene and the substrate.35 The interaction energies, including intermolecular and molecule–substrate, give more self-assembled information in the whole system, as summarized in Table 1. The contributions for the stable assembly of PAI compounds include the intermolecular interaction mainly coming from the interdigitated alkyl chains and the interaction between PAI1 and the substrate. After addition of coronene molecules, the total intermolecular interaction energy, including that between PAI1 molecules and that between coronene and PAI1, was increased to −34.262 kcal mol–1. This might be attributed to the interaction between the carboxyl oxygen atom and coronene. In other words, the interaction between coronene and PAI1 contributes to the location position of coronene. Note that the interaction energies between adsorbates and the substrate largely increase up to −133.794 kcal mol–1, which indicates that there is a strong interaction between the coronene/PAI1 system and HOPG substrate. The adsorption of coronene molecules on PAI1 induced the increased stability of PAI1 on the HOPG substrate, leading to the increased interaction between PAI1 and the substrate. Therefore, the ordered coronene layer might be owing to the PAI1 layer and the HOPG underneath. On the monolayers of PAI2, PAI6, and PAI8, coronene molecules may tend to move because there is no groove structure to immobilize the molecules, resulting in that stable adsorption cannot be observed in STM images. In addition, DFT calculations showed smaller interaction energies for coronene on these monolayers, indicating that these are not preferable structures. The total energy per unit area obtained through considering all possible interactions in the system offers an effective factor to evaluate the thermodynamic stability of different arrays. The selectivity of immobilization coronene in PAI1 can be explained by smaller total energy per unit area of PAI1 + Cor/HOPG (−0.458 kcal mol–1 Å–2) system than that of PAI1/HOPG (−0.237 kcal mol–1 Å–2), indicating the inclusion of coronene make the PAI1 monolayer more table. This function of stability can be explained by the energy gain. The resulting interaction between physisorbed host and coronene with substrate overcomes the instability of the lower density of host matrix. In presence of coronene guest molecules, the formation of honeycomb network is thermodynamically favored.6,7 As a result, the inclusion of coronene molecules in PAI1 monolayer should depend on the properties of molecular template monolayers. To study the electronic interactions between the coronene molecules and PAI1 monolayer as well as the substrate, we also calculated the density of states (DOS) to discuss the energy level alignment at the coronene/PAI1 interface.36,37 In Figure 5, the DOS for PAI1/HOPG and Cor + PAI1/HOPG are shown. The increased total DOS structures indicate that strong interaction between coronene molecules and PAI1/HOPG system. The interaction between PAI1 and coronene molecule is calculated to be −22.717 kcal mol–1. As shown in the enlarged DOS diagram, the adsorption of coronene molecules induced the change of energy-level alignment, which is expected to be used in organic electronics. Figure 5 Density of states (DOS) for the HOPG slab and PAI/HOPG system before and after adsorption of coronene molecules. Fermi energy is aligned to 0 eV. Conclusions A series of PAI derivatives assembled into densely packed monolayers, in which only PAI1 can serve as a template for the immobilization of coronene molecules. In contrast to the spatial selection in the cavity, a close-packed coronene molecule atop the PAI1 monolayer has been observed. The selective incorporation of coronene is explained by the interaction energy on the basis of the theoretical calculations. Many systems for further symmetrical studies are needed to study the structures of templates which can adsorb coronene molecules, and it is necessary to further explore efficient recipes to distinguish clearly the specific position of guest molecules in the close-packed monolayer. Experimental Section Materials The syntheses of PAI derivatives were carried out according to the previous report.31 Self-assembled PAI monolayers were prepared by directly dropping the dilute solution of PAI derivative in 1-phenyloctane onto the freshly stripped HOPG (grade ZYB) surface, which was purchased from Agilent. The concentration of the solution was prepared to be less than 1.0 × 10–4 M to ensure the formation of highly ordered structures. The solution of coronene in 1-phenyloctane with the concentration of 1.0 × 10–2 M was prepared and then was directly dropped onto the PAI monolayer. After a while, a drop of 1-phenyloctane solution was deposited onto the preprepared surface followed by STM record. Measurement STM measurements were performed at the 1-phenyloctane/HOPG interface at room temperature (22–25 °C) by using a Nanoscope IIIa (Agilent) with tips mechanically formed from Pt/Ir (80/20). To obtain the topography image, constant-current STM was performed by continuously adjusting the vertical position of the STM tip. All of the tunneling conditions were given in the corresponding figure captions. Calculations Theoretical calculations were performed using the DFT-D scheme provided by DMol3 code.38 We used the periodic boundary conditions (PBC) to describe the 2D periodic structure on the graphite in this work. The Perdew–Burke–Ernzerhof parameterization of the local exchange correlation energy was applied in local spin density approximation to describe exchange and correlation. All-electron spin-unrestricted Kohn–Sham wave functions were expanded on a local atomic orbital basis. For the large system, the numerical basis set was applied. All calculations were all-electron ones and performed with the medium mesh. The self-consistent field procedure was done with a convergence criterion of 10–5 au on the energy and electron density. Combined with the experimental data, we have optimized the unit-cell parameters and the geometry of the adsorbates in the unit cell. When the energy and density convergence criterion are reached, we could obtain the optimized parameters and the interaction energy between adsorbates. To evaluate the interaction between the adsorbates and HOPG, we design the model system. In our work, adsorbates consist of π-conjugated benzene rings. Because adsorption of benzene on graphite and graphene should be very similar, we have performed our calculations on infinite graphene monolayers using PBC. In the superlattice, graphene layers were separated by 35 Å in the normal direction. When modeling the adsorbates on graphene, we used graphene supercells and sampled the Brillouin zone by a 1 × 1 × 1 k-point mesh. The interaction energy Einter of adsorbates with graphite is given by Einter = Etot(adsorbates/graphene) – Etot(isolated adsorbates in vacuum) – Etot(graphene). For the DOS calculations, k-point samplings for the Brillouin zone were performed using the 5 × 5 × 1 Monkhorst–Pack k-point mesh. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00891.Experimental and calculated unit-cell parameters, large-scale STM images of PAI1 and PAI1/coronene structure (PDF) Supplementary Material ao7b00891_si_001.pdf Author Contributions § Y.G. and S.W. equally contributed to this work. The authors declare no competing financial interest. Acknowledgments The authors are sincerely thankful to the support from the National Basic Research Program of China (2016YFA0200700) and National Natural Science Foundation of China (Nos. 21472029, 21773041, and 51473003). ==== Refs References De Feyter S. ; De Schryver F. C. Two-dimensional supramolecular self-assembly probed by scanning tunneling microscopy . Chem. Soc. 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PMC006xxxxxx/PMC6644410.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145907810.1021/acsomega.8b00934ArticleSuperstructure-Dependent Loading of DNA Origami Nanostructures with a Groove-Binding Drug Kollmann Fabian †Ramakrishnan Saminathan †Shen Boxuan ‡Grundmeier Guido †Kostiainen Mauri A. ‡Linko Veikko †‡Keller Adrian †† Technical and Macromolecular Chemistry, Paderborn University, Warburger Str. 100, 33098 Paderborn, Germany‡ Biohybrid Materials, Department of Bioproducts and Biosystems, Aalto University, P.O. Box 16100, FI-00076 Aalto, Finland* E-mail: veikko.linko@aalto.fi (V.L.).* E-mail: adrian.keller@uni-paderborn.de (A.K.).20 08 2018 31 08 2018 3 8 9441 9448 08 05 2018 03 08 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. DNA origami nanostructures are regarded as powerful and versatile vehicles for targeted drug delivery. So far, DNA origami-based drug delivery strategies mostly use intercalation of the therapeutic molecules between the base pairs of the DNA origami’s double helices for drug loading. The binding of nonintercalating drugs to DNA origami nanostructures, however, is less studied. Therefore, in this work, we investigate the interaction of the drug methylene blue (MB) with different DNA origami nanostructures under conditions that result in minor groove binding. We observe a noticeable effect of DNA origami superstructure on the binding affinity of MB. In particular, non-B topologies as for instance found in designs using the square lattice with 10.67 bp/turn may result in reduced binding affinity because groove binding efficiency depends on groove dimensions. Also, mechanically flexible DNA origami shapes that are prone to structural fluctuations may exhibit reduced groove binding, even though they are based on the honeycomb lattice with 10.5 bp/turn. This can be attributed to the induction of transient over- and underwound DNA topologies by thermal fluctuations. These issues should thus be considered when designing DNA origami nanostructures for drug delivery applications that employ groove-binding drugs. document-id-old-9ao8b00934document-id-new-14ao-2018-00934fccc-price ==== Body Introduction During the last three decades, the field of structural DNA nanotechnology has developed a variety of techniques to assemble DNA into increasingly complex two-dimensional (2D) and three-dimensional (3D) nanostructures.1−3 Currently, DNA nanostructures and especially DNA origami4,5 are widely investigated with regard to their applicability in fields as diverse as nanoelectronics,6−10 molecular sensing,11−14 and drug delivery.15−26 For the latter application, drug loading of the DNA origami delivery systems has been achieved mostly via intercalation between the base pairs of the DNA origami’s double helices.16−19,23,25,26 Intercalation induces unwinding of the double helices and may therefore lead to structural distortions of the DNA origami nanostructures.16,27 By employing DNA origami designs with deliberately underwound double helices, however, intercalator loading of the DNA origami can be enhanced.16,28 The interaction of DNA origami with other chemical species that undergo nonintercalative binding to DNA is less studied. Opherden et al. investigated Mg2+ and Eu3+ coordination of two different DNA origami nanostructures, that is, 2D triangles and 3D six-helix bundles (6HBs).29 By employing a variety of spectroscopic techniques, a superstructure-specific geometry of Eu3+-binding sites was revealed in the two DNA origami designs that furthermore deviates from that of genomic double-stranded DNA (dsDNA). In this work, we investigate the binding of the drug methylene blue (MB) to different 2D and 3D DNA origami nanostructures under conditions favoring minor groove binding and observe a strong dependence on DNA origami superstructure. MB is a fluorescent azine dye that is widely used as an optical and electrochemical probe molecule in the study of DNA-based systems and reactions.30−33 In clinical practice, MB has been used extensively as a therapeutic agent to treat numerous diseases, including malaria and methemoglobinemia.34,35 More recently, MB has been rediscovered as a photosensitizer for the photodynamic therapy of various viral, bacterial, and fungal infections, as well as cancers.36−45 Consequently, also the delivery and controlled release of MB by various carrier systems has received significant attention in recent years.46−53 Results and Discussion MB can interact with dsDNA via different binding modes. At low salt concentrations, intercalation into the G–C base pairs is favored, whereas at the high salt concentrations typically employed in DNA origami experiments, for example, 10 mM MgCl2 as used in the following experiments, a transition to nonintercalative binding occurs.54−57 Irrespective of the binding mode, however, interaction of MB with DNA is in general accompanied by a decrease in its absorption at 668 nm.57−59 In order to elucidate the nature of this nonintercalative binding mode, UV–vis absorbance spectra of MB with and without genomic dsDNA from salmon testes were recorded at different ionic strengths. This dsDNA has a GC content of 41% and is thus comparable to the fully hybridized M13mp18-based DNA origami scaffold with a GC content of 42%. As can be seen in the spectra presented in Figure 1a, the absorbance at 668 nm clearly decreases upon addition of dsDNA, both in water and in 1× TAE (Tris base, acetic acid, and ethylenediaminetetraacetic acid) buffer supplemented with 10 mM MgCl2. In water, this hypochromicity is accompanied by a significant red shift of the absorbance peak that results in a crossing of both spectral signatures at a wavelength of about 680 nm. In TAE/MgCl2 buffer, however, this shift is absent, indicating the nonintercalative binding of MB, which is in agreement with previous reports.58,59 Figure 1 (a) UV–vis absorbance spectra of 20 μM MB with and without genomic dsDNA from salmon testes in water and MgCl2-containing TAE buffer. DNA concentrations were 13 μM (water) and 11 μM (TAE + 10 mM MgCl2) in phosphates. In order to enhance the signal-to-noise ratio and detect even small bathochromic shifts, the shown spectra have been averaged over 10 individual absorbance measurements. Individual spectra can be found in the Supporting Information. (b) UV–vis spectra of the competition between 20 μM MB and 500 μM spermidine (left) and netropsin (right), respectively, in 1× TAE buffer supplemented with 10 mM MgCl2. The spectra of the spermidine-containing samples exhibited a broad background which has been subtracted in the above plot. The concentration of the genomic dsDNA was 308 μM in phosphates. In order to identify the nonintercalative binding mode of MB, competition assays were performed, employing spermidine and netropsin. While spermidine can bind to both the minor and major grooves,60,61 netropsin is a potent minor groove binder.62,63 Depending on the binding modes of MB and the competing groove binder, addition of a large excess of these groove binders will result in its displacement from the minor groove, the major groove, or both, and thereby reverse the hypochromicity observed upon MB binding to the dsDNA. Indeed, as can be seen in Figure 1b, addition of both groove binders results in a drastic increase in MB absorbance, which almost reaches the value of free MB. This verifies the binding of MB to the minor groove of dsDNA under the current buffer conditions, which also agrees with previous theoretical predictions.64 Next, we set out to investigate the interaction of MB with a selection of representative DNA origami nanostructures (see Figure 2). In particular, we chose two 3D DNA origami nanostructures, that is, 6HBs65 and 60-helix bundles (60HBs).66 The 6HBs have previously been found to have an extraordinarily high structural stability under physiological conditions, whereas more complex 3D structures such as the 60HB are typically less stable.67,68 Furthermore, we also selected three differently shaped 2D DNA origami. The Z shape69 is also designed on the honeycomb lattice and features a chiral shape that may serve as an indicator for visualizing MB-binding-induced structural distortions by atomic force microscopy (AFM).27 The Rothemund triangle,4 on the other hand, has previously been identified as a potent drug delivery vehicle in vivo.18 It is designed on the square lattice and appears highly strained (see the corresponding CanDo70,71 simulation in Figure 2). In order to evaluate the effect of such strain on MB binding, we have also evaluated a more relaxed DNA origami structure in the form of a bow tie that is designed on the same lattice but twist-corrected.69 These DNA origami nanostructures are characterized by AFM in Figure 2, both directly after assembly and after subsequent exposure to 20 μM MB. Obviously, all DNA origami nanostructures remain intact upon MB exposure. Furthermore, no major structural distortions induced by MB binding can be observed by AFM. This is in agreement with the minor groove binding of MB, which does not require any major structural alterations of the DNA helices.54,55,64,72 Intercalators, on the other hand, may induce unwinding of the helices and thereby twisting of the DNA origami.16,27,28 Such a twisting should be visible at least for the Z-shaped DNA origami.27 Figure 2 CanDo70,71 simulations and AFM images (1 × 1 μm2) of 6HBs (height scales 4.5 nm), 60HBs (height scales 12 nm), Z-shaped DNA origami (height scales 3 nm), triangles (height scales 3 nm), and bow ties (height scales 2 nm) before and after exposure to 20 μM MB. The color coding in the CanDo simulations indicates the lattice type (red—honeycomb, blue—square). The binding of MB to these DNA origami nanostructures was investigated by UV–vis spectroscopy. In addition to the DNA origami, we also used synthetic dsDNA (15 bp) with a GC content close to that of the M13 scaffold (40%) as a reference. All the UV–vis spectra shown in Figure 3a were characterized by a decrease in the absorbance of MB at 668 nm with increasing DNA concentration. As in the case of genomic dsDNA, no bathochromic shift of the absorption peak is observed, indicating the binding of MB to the minor groove. Figure 3 (a) Representative UV–vis spectra of 20 μM MB in 1× TAE buffer containing 10 mM MgCl2 and different phosphate concentrations of dsDNA and different DNA origami nanostructures, respectively. The spectra are normalized to the absorbance at 668 nm in the absence of any DNA. (b) Representative normalized MB absorbance at 668 nm as a function of phosphate concentration. (c) Corresponding binding isotherms. The solid red lines in (c) are linear fits to the data that have been used for the determination of Kd values. R2 values of the fits are given in the plots. Error bars in (b,c) represent standard deviations obtained by averaging over five individual measurements. For Kd determination, these concentration-dependent measurements have been repeated at least once (see Methods). The normalized absorbance values shown in Figure 3b are found to saturate at high DNA concentrations, indicating that at these concentrations, all MB in solution is DNA-bound. For all DNA origami nanostructures investigated here, saturation occurs at phosphate concentrations between 35 and 55 μM and with different saturation values of the absorbance. Also, the obtained loading efficiencies differ significantly for the different DNA origami nanostructures (see Table S1 for base pair contents) and range from about 5700 MB molecules per DNA origami for the 60HB to about 7400 for the bow tie (see Table S1). The differences in the MB–DNA interaction observed in Figure 3a,b were assessed more quantitatively by converting the concentration-dependent absorbance values at 668 nm into binding isotherms (see Figure 3c) as described by Zhang and Tang.58 From the linear fits to the data shown in Figure 3c, dissociation constants Kd can be calculated as the ratio of intercept and slope.58 This approach is frequently employed for the quantitative investigation of the interaction of DNA with both intercalators58,73 and groove binders.74,75 All the determined Kd values are compared in Figure 4. Obviously, DNA origami 6HBs present a Kd value virtually identical to that of synthetic dsDNA with the same GC content. This is to be expected because the 6HBs have been designed on a honeycomb lattice with 10.5 bp per helical turn and should thus exhibit a DNA topology close to the canonical B-form. The same argument of DNA topology also holds true for the 60HB, which indeed has a similar Kd as the 6HB and the dsDNA. Although in such compact 3D DNA origami nanostructures, access of MB to the inner helices may be restricted because of the shielding by the outer helices, this effect appears to be of minor importance in the 60HB. Figure 4 Determined Kd values for the investigated DNA structures. Values are presented as averages over n independent concentration series with the standard error of the mean as error bars (see Methods). Such geometric effects, however, should be entirely absent in structures that consist just of a single honeycomb “unit cell” such as the 6HB or in objects that are purely two-dimensional. The latter is true for the Z-shaped 2D DNA origami that was constructed using the honeycomb lattice and is thus designed to exhibit 10.5 bp/turn just as the 6HB and the 60HB. Surprisingly, however, the Kd determined for the Z-shaped DNA origami is more than twice as large as those of the 6HB and the dsDNA (see Figure 4). On the basis of the overlap rule of standard error bars,76 the difference between the Z shape and the dsDNA is estimated to be statistically significant with p < 0.05. In addition to the honeycomb lattice-based DNA origami, we have also evaluated MB binding to two 2D DNA origami nanostructures based on the square lattice.4,69 These structures are designed with 10.67 bp/turn and thus exhibit DNA topologies different from the canonical B-form of dsDNA.4 Efficient minor groove binding of MB64,72 and other molecules77,78 typically requires a tight fit. Therefore, underwinding of the helices will result not only in an increased spacing of base pairs but also in a widened minor groove79 and should thereby reduce the binding affinity of MB. This is in contrast to the case of intercalation, where underwinding results in lower Kd values because of easier access to the widened gaps between base pairs.16,28 In agreement with this hypothesis, both DNA origami triangles and twist-corrected bow-tie structures show Kd values slightly higher than that of dsDNA, although the effect is surprisingly small (see Figure 4). Of all the DNA origami nanostructures studied in this work, the Z shape shows the strongest deviation from B-DNA, even though it is designed on the same honeycomb lattice as the 6HB and the 60HB, both of which show Kd values similar to dsDNA. We speculate that the high Kd of the Z-shaped DNA origami is caused by the presence of DNA topologies different from the canonical B-form. Despite being based on the honeycomb lattice, such non-B topologies may occur transiently, that is, resulting from thermal fluctuations. In the Z-shaped DNA origami, all helices are aligned parallel to its long axis, including those in the two arms (see Figure 2). These arms are therefore comparatively floppy and subject to strong fluctuations. These fluctuations travel from helix to helix via the crossovers, thereby inducing an oscillatory over- and underwinding of the helices in the vicinity of the crossovers (see CanDo70,71 simulations in the Supporting Information). Because groove binding requires a tight fit, both over- and underwinding will result in reduced binding and in sum to an increased Kd. The bow-tie design also features only parallel helices and could therefore show a similar behavior as the Z shape. However, because of the intrinsic underwinding of the helices in the square lattice, fluctuations in the bow tie may lead even to slightly improved groove binding because of the occurrence of transient B-form topologies. Note that in the case of intercalation, such fluctuations will not lead to different Kd values because here overwinding and underwinding result in lower and higher binding affinities, respectively, and thus compensate each other. Conclusions In conclusion, we have investigated the interaction of the drug MB with different DNA origami nanostructures under conditions that result in binding to the minor groove. The DNA origami superstructure is found to have a noticeable influence on the binding efficiency of MB. In particular, non-B topologies as for instance found in designs using the square lattice with 10.67 bp/turn may result in reduced binding affinity. Furthermore, also flexible DNA origami shapes that are prone to structural fluctuations may exhibit reduced groove binding because of the induction of transient over- and underwound DNA topologies. These issues should be considered when rationally designing DNA origami nanostructures for drug delivery applications employing groove-binding drugs. Methods Preparation of dsDNA Samples For the initial experiments addressing the binding mode of MB, lyophilized genomic dsDNA from salmon testes (Alfa Aesar) was dissolved at the desired concentration either in high-performance liquid chromatography (HPLC)-grade water (Carl Roth) or in 1× TAE (Calbiochem) with 10 mM MgCl2 (Sigma-Aldrich). MB, spermidine, and netropsin (all Sigma-Aldrich) were added to achieve the desired concentrations. For comparison with the DNA origami nanostructures, synthetic dsDNA was prepared by hybridization of two complementary oligonucleotides (Metabion) with the sequences 5′-TTG GAA CAG CAT TGA-3′ and 5′-TCA ATG CTG TTC CAA-3′. To this end, the readily mixed sample (100 μM of each strand in 1× TAE with 10 mM MgCl2) was heated to 94 °C in a thermocycler Primus 25 advanced (PEQLAB), kept at this temperature for 5 min, and cooled down to 20 °C with a cooling rate of 1.5 °C per minute. DNA Origami Synthesis and Purification All DNA origami nanostructures were fabricated as previously reported (see below). All structures are based on the 7249 nt long M13mp18 scaffold (purchased from Tilibit Nanosystems).Triangle: the staple sequences (purchased from Metabion) are taken from the article by Rothemund.4 DNA origami assembly was performed as previously described.80 6HB: the staple sequences (purchased from Metabion) are taken from the article by Bui et al.65 DNA origami assembly was performed as previously described.81 60HB: the staple sequences and the exact fabrication protocol (20 nM reactions) can be found in the article by Linko et al.66 Staple strands were purchased from IDT. Z shape and bow tie: the staple sequences and the exact fabrication protocol (20 nM reactions) can be found in the article by Shen et al.69 Staple strands were purchased from IDT. After assembly, the DNA origami nanostructures were purified as described previously80 by spin-filtering (Amicon Ultra filters from Millipore with 100 kDa molecular weight cutoff) and washing with 1× TAE supplemented with 10 mM MgCl2. Determination of DNA Origami Concentrations The concentrations of the DNA origami after purification were determined by UV–vis absorbance measurements using an Implen Nanophotometer P330 operated in dsDNA mode. Because different DNA origami nanostructures feature different numbers of base pairs and unpaired nucleotides, the so-determined concentration values had to be corrected. The measured concentration cm is a result of the combined absorption the single-stranded DNA (ssDNA) and the dsDNA fractions in the DNA origami. Because the extinction of ssDNA is about 1.5 times higher than that of dsDNA,82 the real concentration creal is given by Here, x is the fraction of paired bases relative to the total number of nucleotides (paired and unpaired) in the DNA origami. The numbers of base pairs and nucleotides of the DNA origami used in this work are given in Table S1. UV–Vis Spectroscopy UV–vis absorbance spectra with a spectral range from 200 to 800 nm were recorded with an Implen Nanophotometer P330 equipped with a sub-microliter cell. The sample volume for each measurement was 2 μL. After each measurement, the cell was washed at least twice with HPLC-grade water. To record the UV–vis spectra of samples with different DNA concentrations, a 20 μM MB solution in 1× TAE with 10 mM MgCl2 was measured first. Then, a small aliquot of a DNA-containing solution (80–300 μM in phosphates depending on the DNA structure, 1× TAE, 10 mM MgCl2, 20 μM MB) was added to the initial MB solution and mixed well. After recording the UV–vis spectra of the resulting solution, the addition and measurement cycle was repeated several times to record a complete concentration series. Determination of Kd Values For the determination of the dissociation constants, the measured absorption at 668 nm was averaged over at least five individual measurements at each DNA concentration in a concentration series, and the resulting standard deviations were treated as error limits. After transforming the data according to the protocol of Zhang and Tang,58 the Kd of each concentration series was determined from a linear fit to the data with instrumental weighting of the errors (see below). For each DNA origami, this determination of Kd has been performed for two to three independent concentration series. The so-obtained Kd values for each DNA origami were averaged, and the error limits of the individual linear fits were used for determining the standard error of the mean for each Kd value given in Figure 4 via propagation of error (see Table S3). The binding constant Kd follows from the relation Here, [Pho] refers to the molar concentration of phosphates, which has been used instead of the concentration of base pairs in order to account for the different fractions of paired and unpaired nucleotides in the different DNA origami designs. Δεap is the difference between the measured extinction coefficient of the DNA–MB solution, εa, which features both DNA-bound and free MB and the extinction coefficient of free MB in a solution without DNA, εf, with Here, εa and εf are determined by dividing the measured absorbance values by the molar MB concentration, which was kept constant at 20 μM in all experiments. Δε represents the difference of the extinction coefficients of DNA-bound MB, εb, and free MB, εf, that is, The dissociation constant Kd is then determined by plotting versus [Pho] and fitting the data with a linear function. In this case, the linear fit has a slope of and intercepts the y-axis at . Therefore, dividing the intercept by the slope yields the binding constant Kd. AFM Imaging For AFM imaging, a 5 μL droplet of each DNA origami sample (10 nM in 1× TAE with 10 mM MgCl2) with or without 20 μM MB was deposited on a freshly cleaved mica substrate. After adding 100 μL of 1× TAE with 10 mM MgCl2 to spread the sample over the whole substrate surface, the sample was incubated for 5–15 min, subsequently dipped in HPLC-grade water, and dried in a stream of ultrapure air. AFM imaging was performed in air using Agilent 5100 AFM in intermittent contact mode and HQ:NSC18/Al BS cantilevers from MikroMasch. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00934.Individual UV–vis absorbance spectra of MB–DNA binding in water and buffer, paired and unpaired bases in the different DNA origami designs, netropsin competition assay for the different DNA origami nanostructures, additional independent concentration series and binding isotherms, loading efficiency of MB, and averaging of Kd values (PDF) CanDo simulations of the Z-shaped DNA origami - top view (AVI) (AVI) CanDo simulations of the Z-shaped DNA origami - side view 1 CanDo simulations of the Z-shaped DNA origami - side view 2 (AVI) Supplementary Material ao8b00934_si_001.pdf ao8b00934_si_002.avi ao8b00934_si_003.avi ao8b00934_si_004.avi Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. Acknowledgments We thank K. Fahmy for many valuable discussions. Financial support from the Academy of Finland (grant nos. 286845 and 308578), the Sigrid Jusélius Foundation, and the Jane and Aatos Erkko Foundation is gratefully acknowledged. ==== Refs References Jones M. R. ; Seeman N. C. ; Mirkin C. A. Programmable materials and the nature of the DNA bond . Science 2015 , 347 , 1260901 10.1126/science.1260901 .25700524 Linko V. ; Dietz H. The enabled state of DNA nanotechnology . Curr. Opin. Biotechnol. 2013 , 24 , 555 –561 . 10.1016/j.copbio.2013.02.001 .23566376 Nummelin S. ; Kommeri J. ; Kostiainen M. A. ; Linko V. Evolution of Structural DNA Nanotechnology . Adv. Mater. 2018 , 30 , 1703721 10.1002/adma.201703721 . Rothemund P. W. K. Folding DNA to create nanoscale shapes and patterns . Nature 2006 , 440 , 297 –302 . 10.1038/nature04586 .16541064 Douglas S. M. ; Dietz H. ; Liedl T. ; Högberg B. ; Graf F. ; Shih W. M. 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PMC006xxxxxx/PMC6644411.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145896010.1021/acsomega.8b01176ArticleHardwood Kraft Lignin-Based Hydrogels: Production and Performance Zerpa Alyssa Pakzad Leila Fatehi Pedram Chemical Engineering Department, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1 E-mail: pfatehi@lakeheadu.ca. Tel: 807-343-8697. Fax: 87-346-7943.24 07 2018 31 07 2018 3 7 8233 8242 29 05 2018 16 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. In this study, hydrogels were synthesized through the radical polymerization of hardwood kraft lignin, N-isopropylacrylamide, and N,N′-methylenebisacrylamide. Statistical analyses were employed to produce lignin-based hydrogels with the highest yield and swelling capacity. The success of the polymerization reactions was confirmed by NMR and Fourier infrared spectroscopy. The lignin-based hydrogel was more thermally and rheological stable, but exhibited less swelling affinity, than synthetic hydrogel. The rheological studies indicated that the swollen hydrogels were predominantly elastic and exhibited a critical solution temperature that was between 34 and 37 °C. Compared with the synthetic hydrogel, lignin-based hydrogel behaved less elastic as temperature increased. In addition to inducing a green hydrogel, the results confirmed that hardwood lignin-based hydrogel would have different properties than synthetic-based hydrogels, which could be beneficial for some applications. document-id-old-9ao8b01176document-id-new-14ao-2018-011764ccc-price ==== Body Introduction Hydrogels are often described as three-dimensional polymeric networks formed from cross-linked hydrophilic homopolymers, copolymers, or macromers.1−4 They are insoluble polymer matrices capable of retaining a large amount of water in their swollen state; in some cases, up to a thousand times of their dry weight. Their swelling capability allows them to obtain the shape of their surroundings when confined.2,5,6 Depending on the source of polymers, hydrogels may be synthetic, natural, or hybrid.2 Hydrogels may be degradable in aqueous environments, making them biocompatible in most cases and a good carrier for nutrients to cells and their metabolic products.5 They have been deemed to be efficient in the protection of cells and fragile drugs, such as peptides and proteins.5 Some hydrogels have exhibited stimuli-induced swelling and deswelling capabilities without disintegration.7−9 The advantage of tunable properties has given hydrogels attention for biomedical and environmental applications.10,11 Poly (N-isopropylacrylamide) (PNIPAAm) is a thermoresponsive hydrogel.12−14 However, the applications of PNIPAAm hydrogels are limited by their fragility.15,16 These applications can be extended via enhancing their rigidity. Recently, lignin (LGN) has been incorporated into the production of flocculants, dispersants, and hydrogels because lignin is biocompatible and biodegradable with low toxicity.17−20 At a relatively low production cost,21 lignin presents the greatest available aromatic renewable resource worldwide, as well as a primary supplier of soil’s organic matter.6,22 Lignin provides the structural strength of plants by nature, which makes it a potential candidate for PNIPAAm hydrogel modifications. Previous studies have been conducted on synthesizing hydrogels using different types of lignin through free radical polymerization. Acetic acid lignin was incorporated into N-isopropylacrylamide (NIPAAm) for hydrogel production.23 The kinetics studies indicated that the lignin-containing hydrogel retained 7.2% more water than the lignin-free hydrogel over 10 min of swelling.23 In another work, wheat straw alkali lignin was cross-linked with acrylic acid and N,N′-methylenebisacrylamide (MBAAm) via free radical polymerization.24 The addition of acid hydrolysis lignin was found to greatly improve the surface morphology and swelling affinity of the hydrogel.25 Yu et al. developed lignosulfonate-g-acrylic acid hydrogels by grafting acrylic acid onto lignosulfonate with MBAAm as a cross-linker and lacase/tert-butyl hydroperoxide as the initiator.26 It is well known that the type of lignin (i.e., hardwood vs softwood vs nonwood) and its production process (i.e., enzymatic hydrolysis vs kraft vs sulfite treatment) affect its structure and chemical properties, which, in turn, affect its polymerization performance and end-use applications.27−32 There is currently limited research on the use of hardwood kraft lignin for hydrogel production following free radical polymerization. As hardwood is vastly available for end-use applications, it is of great importance to investigate the performance of hardwood lignin for hydrogel productions.17−21 The first objective of this study was to evaluate the cross-linking of hardwood-based kraft lignin with NIPAAm as the monomer and MBAAm as the cross-linker. The second objective of this study was to investigate the thermal and rheological properties of kraft lignin-based hydrogels, as well as their swelling performance. The main novelty of this work was the investigations on the use of hardwood kraft lignin as a commercially available resource in hydrogel production and on the properties and performance of the induced hydrogels under different scenarios. Results and Discussion Polymerization The free radical polymerization for producing lignin-based hydrogels is demonstrated in Figure 1. Azobisisobutyronitrile (AIBN) first undergoes thermal decomposition to generate radicals, which initiates the polymerization. Figure 1a demonstrates the thermal decomposition of the azo compounds with heating, which produces two 2-cyanoprop-2-yl radicals and nitrogen gas.33 In this case, 2-cyanoprop-2-yl radicals can then be transferred to lignin, NIPAAm, or MBAAm present in the reaction mixture. When the free radical is transferred to lignin, it will abstract hydrogen from the hydroxyl group located on lignin’s aromatic ring generating a phenoxy radical (I in Figure 1b) along with the corresponding resonance structures (II in Figure 1b).33 The phenoxy radicals will then attack the carbon double bonds of the NIPAAm monomers and the cross-linker MBAAm to form the initial propagating chain, which can then continue further to form the cross-linked structure (Figure 1c). The radical polymerization is most commonly terminated through the combination of two active chains.34 In addition, termination may occur via adjusting pH (i.e., sulfuric acid and sodium hydroxide). Figure 1 Radical polymerization reaction for lignin-based hydrogel production: (a) decomposition of AIBN initiator, (b) formation of phenoxy radicals, and (c) cross-linking reaction. Effect of Reaction Conditions Taguchi L9 orthogonal design is presented in Table 1. Typically, NIPAAm and MBAAm are used together to form hydrogels, and only using one of them cannot facilitate hydrogel production. Generally, the ratio of reactants affects the properties of the induced products in reactions. Compared with other reactants in this polymerization reaction system, lignin is more difficult to handle in terms of its solubility, reactivity, and pH adjustment in solutions. Therefore, to have a better control over the reactions, we decided to keep the amount of lignin constant in the reactions but to change the quantity of other components of the reaction. The range of analysis was optimized by evaluating the signal-to-noise (SN) ratio to maximize the responses of the model. The hydrogel sample of 2L was selected as the best hydrogel sample based on the results achieved. The control sample (2C) was produced following reaction conditions of 2L but without lignin. Table 1 Taguchi L9 Orthogonal Design Parameters run temperature (°C) time (h) NIPAAm content (g) pH yield (wt %) maximum swelling ratio (g/g) S/NLB 1L 65 3 1.2 2.0 76.09 15.41 24.62 2L 65 4 1.8 2.5 77.86 32.59 26.30 3L 65 5 2.4 3.0 80.7 20.19 25.41 4L 75 3 1.8 3.0 49.33 24.08 25.66 5L 75 4 2.4 2.0 84.57 14.00 24.67 6L 75 5 1.2 2.5 91.93 19.03 25.25 7L 85 3 2.4 2.5 65.49 21.76 25.58 8L 85 4 1.2 3.0 83.63 34.39 25.61 9L 85 5 1.8 2.0 90.67 20.91 25.74 Multivariate analysis (MANOVA) was applied to determine whether there are differences among the levels and factors. A 95% confidence interval was selected, implying that the response is deemed significant if the P-value is smaller than 0.05.35 Furthermore, post hoc analysis was applied to determine where these significant differences exist when considering multiple levels.36 Tukey’s test was selected as an effective post hoc model to compare all the possible pairs of means and determine where they differ within the data.36−38Table 2 demonstrates the results of the MANOVA for the responses of lignin-based hydrogels. It is observable that temperature presents a significant effect on yield (F = 7.84, p = 0.011, η2 = 0.635) and maximum swelling ratio (F = 14.07, p = 0.002, η2 = 0.076). The cleavage of the azo compounds may occur more rapidly at higher temperatures, increasing the concentration of radicals present in the solution and facilitating the reaction.39 This table also shows that the reaction time presented a large effect with respect to yield (F = 222.24, p < 0.001, η2 = 0.980). This is because the time extension allows for additional cross-linking to occur in the reaction, resulting in a higher product yield. A significantly large decrease in yield (F = 43.00, p < 0.001, η2 = 0.905) and swelling capacity (F = 16.67, p = 0.001, η2 = 0.787) with increasing NIPAAm content was determined between 18 and 24 g/g of lignin. The higher incorporation of monomer into the hydrogels was found to improve the hydrogel production. Furthermore, the yield (F = 50.89, p < 0.001, η2 = 0.919) and the maximum swelling ratio (F = 32.00, p < 0.001, η2 = 0.877) exhibit a significant effect between pH 2.0 and pH 2.5. This is most likely because concentration of sulfate ions in the reaction mixture leads to the abstraction of the terminated polymer chains, which, in turn, reinitiates the propagation step.40 Table 2 Multivariate Analysis of the Taguchi L9 Orthogonal Model for the Lignin-Based Hydrogels source dependent variable sum of squares degrees of freedom mean square F-value P-value η2 observed power model yield 2852.78 8 356.60 80.10 0.000 0.986 1.000 swelling ratio 777.82 8 97.23 20.53 0.000 0.948 1.000 temperature (°C) yield 69.07 2 34.54 7.84 0.011 0.635 0.852 swelling ratio 133.27 2 66.63 14.07 0.002 0.758 0.983 time (h) yield 1956.94 2 978.47 222.24 0.000 0.980 1.000 swelling ratio 183.67 2 91.83 19.39 0.001 0.812 0.998 NIPAAm content (g) yield 378.66 2 189.33 43.00 0.000 0.905 1.000 swelling ratio 157.85 2 78.92 16.67 0.001 0.787 0.993 pH yield 448.11 2 224.05 50.89 0.000 0.919 1.000 swelling ratio 303.03 2 151.52 32.00 0.000 0.877 1.000 pure error yield 39.62 9 4.40 swelling ratio 42.61 9 4.73 corrected total yield 2892.41 17 swelling ratio 820.43 17 Table 3 compares the means and standard deviations for both responses, as well as the associated coefficients. The responses exhibited a low coefficient of variation, implying that the obtained data are considered as precise and repeatable.41 Table 3 Descriptive Statistics 95% confidence interval dependent variable standard deviation mean lower bound upper bound coefficient of variation (%) yield 2.10 77.77 76.65 78.89 2.70 swelling ratio 2.18 22.48 21.32 23.64 9.68 To determine which lignin-based hydrogel sample exhibited the best responses, the signal-to-noise (SN) ratio was determined in the Taguchi analysis. Because the goal for this experiment was to maximize the response values, the larger-the-better (LB) SN ratio equation, eq 1, was selected42 1 where n is the number of experiments and yi is the collected experimental data. The optimal level can then be determined by selecting the largest SN ratio from each of the performance parameters.42Table 1 lists the signal to noise ratios for selected lignin-based hydrogel samples, along with their responses (yield and maximum swelling ratio). The SN values, which maximized both of these responses, were found to be the highest for 2L. Thus, this sample was selected as the optimum sample based on the results. NMR Evaluation Figure 2 illustrates the 1H NMR spectrum for the lignin-based hydrogels. The peak at 1.15 ppm corresponds to two methyl protons of the N-isopropyl group (A). The proton of the N-isopropyl group (E) is present at 4.1 ppm. These large peaks are dominant over the others due to the large content of NIPAAm within the hydrogels.43,44 The functional groups for lignin are depicted by a cluster of small peaks from 5 to 8 ppm, which may be due to the aromatic rings present in kraft lignin. The peak of dimethyl sulfoxide (DMSO) is observable at 2.6 ppm, which belongs to dimethyl sulfoxide, DMSO, (D).43 Figure 2 1H NMR spectrum for lignin-based hydrogels (sample 2L). Fourier Transform Infrared Spectroscopy (FTIR) Evaluation The FTIR spectra of the selected hydrogels with lignin, 2L, and without lignin, 2C, are shown in Figure 3. The bands located between 1535 and 1639 cm–1 are attributed to the amide groups found in NIPAAm as well as in N,N′-methylenebiscrylamide.45,46 These compounds also exhibit a broad spectrum between 3400 and 3200 cm–1, indicating the stretching of the N–H bond.45,47,48 According to Konduri and Fatehi,49 the presence of kraft lignin’s aromatic compounds yields a broad peak between 1593 and 1510 cm–1, which is attributed to the benzene ring vibrations. There are also absorption peaks present at 1130 and 1171 cm–1, which correspond to the stretching of the C–O bond on the primary alcohol and ether of kraft lignin, respectively.45 In addition, the C=O and C=C stretching may be depicted by the bands located at approximately 1200 and 1500 cm–1, respectively. The CH stretching of methyl or methylene groups are also shown to be present in the peaks between 2300 and 2400 cm–1.48 The peaks at 1535 and 1639 cm–1 were intense for both samples, indicating the presence of NIPAAm. For this reason, the peaks for NIPAAm overshadow the peaks at 1593 and 1510 cm–1 for aromatic structure of lignin. Figure 3 FTIR analysis for hydrogels with lignin, 2L, and without lignin, 2C. TGA Evaluation The thermal decomposition behavior of hydrogels of 2L and 2C are shown in Figure 4. An initial weight loss of 10% is generally due to moisture removal. Afterward, lignin demonstrates a higher thermal stability in comparison with the other samples, a desirable property for additional end-use applications.49 Above 200 °C, lignin exhibits a gradual decrease in weight loss and levels off at a 55% weight loss above 600 °C. This is because the thermal breakdown of lignin occurs via two competing reaction paths of the intramolecular condensation and the thermal depolymerization.50−52 Figure 4 Weight loss and weight loss rates of control and lignin-based hydrogels: (a) kraft lignin, (b) 2C, and (c) 2L. Despite relatively similar trends, sample 2C is shown to be slightly less thermally stable than 2L. Sample 2C was found to exhibit a major weight loss at approximately 415 °C, whereas sample 2L exhibited a primary weight loss at around 420 °C. Zarzyka and co-workers described that the use of N,N′-methylenebiscrylamide allows for a higher cross-linking density, which, in turn, decreases the chain mobility within the gels.53 Because N,N′-methylenebiscrylamide contributes to cross-linking, it can be assumed that it was readily consumed during the thermal decomposition reaction. Surface Properties and Swelling Behavior The surface area properties of the selected samples are shown in Table 4. The hydrogel without lignin, 2C, was found to have a higher surface area, pore volume, and pore size compared to the lignin-based hydrogel, 2L. This indicates that 2C hydrogel has a more porous structure.23 Lignin-based hydrogel is expected to become less hydrophilic than its synthetic form due to the incorporation of a hydrophobic polymer (lignin). Therefore, this hydrogel (2C) exhibited a better swelling performance in water, as presumably water molecules could diffuse into its pores more effectively. Table 4 Surface and Swelling Properties of Hydrogels sample surface area (m2/g) total pore volume (cm3/g) average pore size (A°) maximum swelling ratio (g/g) 2C 49.62 0.058 24.9 36.22 2L 44.56 0.048 22.1 32.59 Previous studies showed that the swelling performance of lignin hydrogels varied in a wide range depending on the hydrogel components (e.g., lignin types and monomers).6 For example, lignin-based poly(acrylic acid) (PAA) hydrogels showed relatively high swelling ratios of 400, 600, and 700 g/g for acid hydrolysis lignin–PAA, alkali lignin–PAA, and lignosulfonate–PAA hydrogels, respectively.24,25 The specific surface area of alkali lignin–PAA hydrogel was reported to be as high as 122.7 ± 4.51 m2/g.24 On the other hand, hydrogels synthesized by acrylamide and poly(vinyl alcohol) with alkaline and kraft lignin had a swelling ratio of 8 and 1.5 g/g, respectively.29,52 When cross-linking with NIPAAm, the hardwood kraft lignin-based hydrogel obtained in this study exhibited a much higher swelling ratio compared to the acetic acid lignin-based hydrogel.23 Rheological Behavior The cross-linked structure of hydrogels can be further characterized by applying dynamic oscillatory experiment. A sinusoidal oscillation with a given deformation and frequency can be applied onto a material to obtain sinusoidal output for strain.53 Their viscoelastic properties may be characterized by storage modulus (G′), which describes the material’s elasticity, and loss modulus (G″), which is attributed to the viscosity of materials. The elastic component characterizes a material’s solid-like ability to store energy (its stiffness), whereas the viscous component is the liquid-like capability to dissipate energy.54,55 Figure 5 illustrates the effect of frequency on storage (G′) and loss (G″) modulus as well as on dynamic viscosity (η) for both the control and lignin-based hydrogel samples. Some loss modulus values were unable to be measured accurately at low frequencies and are thus analyzed from 6.30 rad/s for both samples.56 The storage modulus is greater than that of the loss modulus for both cases (Figure 5), indicating that the hydrogel samples exhibit more elastic properties. This behavior is typical for hydrogels as the solid-like mechanical properties of their cross-linked structure are more dominant than the viscous properties, which is attributed to the small amorphous part of the polymer network.53 Figure 5 Frequency sweep of the control and lignin-based hydrogels: (a) moduli of 2C and 2L and (b) dynamic viscosity of 2C and 2L. In addition, both storage and loss modulus were found to increase with increasing shear frequency, allowing for more energy to be dissipated. Although frequency is shown to influence the moduli curves, its dependence is largely insignificant, indicating that the hydrogels have a well-structured three-dimensional network.57 Furthermore, the dynamic viscosity is shown to linearly decrease with increasing frequency, an attribute typical to hydrogels.53 The amount of energy dissipated was found to be slightly greater for the control samples (2C) than for the lignin-based sample (2L). This may be due to the incorporation of lignin resulting in a less cross-linked structure. In other words, the network structure of the control samples is more tightly cross-linked and is therefore better able to dissipate energy. For amplitude sweep test, linear viscoelastic region (LVR) for both samples are shown in Figure 6. The strain applied does not exhibit a strong effect on the moduli, which serves as an indication of the hydrogels’ rigidity.58 In addition, the storage modulus is shown to exhibit a greater plateau, indicating that the samples’ viscoelastic behavior.59 Figure 6 Strain amplitude sweep of the control and lignin-based hydrogels: (a) 2C and (b) 2L. On the other hand, the storage modulus is shown to significantly decrease with increasing strain, indicating a disturbance within the network structure. The loss modulus slightly increases before rapidly decreasing to 100% strain. This maximum indicates the microscopic failure within the hydrogel’s network structure, which indicates breaking of the interaction present within the polymer matrix.60 At this point, the storage and loss moduli exhibit a crossover where the hydrogel exhibits a phase change from primarily elastic to primarily viscous.56 This indicates the irreversible deformation of the three-dimensional network structure, which provides the hydrogel its elasticity.54,61 The crossover for the hydrogel 2C was shown to occur at a lower strain rate compared to the hydrogel 2L. This increase in rheological stability with applied strain is most likely due to the incorporation of lignin.53 In addition, the LVR has a larger width for the lignin-based sample compared with the control sample, further supporting that the incorporation of lignin increases the samples’ rigidity. Figure 7 shows the effect of the stress on the modulus resulting from amplitude sweep tests. The initial plateau designates the LVR, for which the change in modulus is denoted by the yield point (τy). As the modulus exhibits a crossover from mainly elastic to mainly viscoelastic, this crossover is known as the flow point (τf). These results for both these points are summarized in Table 5. The flow point and yield point for the lignin-based hydrogel were found to be greater than those for the control sample, indicating that lignin improved the hydrogels’ rheological performance. Figure 7 Stress amplitude sweep of the hydrogels of (a) 2C and (b) 2L. Table 5 Flow Point and Yield Flow Point for Control and Lignin-Based Hydrogels (2L and 2C) sample yield point, τy (Pa) flow point, τf (Pa) 2C 95.1 175.2 2L 216.1 628.9 Figure 8 demonstrates the rheological properties for both elastic and viscous moduli of the hydrogel samples at different temperatures. At low temperatures, the moduli curves dropped with increasing temperature. At approximately 34–37 °C, there is a slight valley in the moduli curves, which is attributed to the lower critical solution temperature (LCST) of NIPAAm (32–34 °C). At this temperature, the hydrogels undergo a reversible phase transition from their swollen state to a shrunken dehydrated state.23,47 Afterward, the difference between the elastic and viscous moduli becomes smaller, but does not reach a crossover temperature. In other words, the hydrogel samples do not exhibit a phase transition, but rather undergo a plateau with increasing temperature, indicating their thermal stability.57 Figure 8 Temperature ramp of the control and lignin-based hydrogels: (a) 2C and (b) 2L. The control hydrogel (2C) exhibits a larger modulus than the lignin-based hydrogel (2L), indicating that the control hydrogel has more elastic properties even with increasing temperature. In addition, following the LCST, the gap between the elastic and viscous modulus decreases more significantly for the lignin-based hydrogels, which suggests that the lignin-based hydrogels approach the sol–gel temperature more readily than the control hydrogel.62 Conclusions Hardwood lignin-based hydrogels were successfully produced through radical polymerization of lignin, NIPAAm, and MBAAm, as confirmed by the NMR and FTIR analyses. Thermogravimetric analysis determined that the hydrogels exhibited two decomposition stages attributed to the breakdown of the aliphatic alkenes groups and the remaining carboxylic and amine groups. Although lignin-based hydrogel had less swelling affinity, as it possessed smaller surface area and more porous structure than synthetic one, it was more thermally stable. The slightly greater energy dissipated for the control hydrogel than for the lignin-based hydrogel implied that the incorporation of lignin generated less cross-linked hydrogel. Lignin tended to increase the rigidity and rheological stability of hydrogel. Compared with the control hydrogel, lignin-based hydrogel behaved less elastic as temperature increased. These results suggest that hardwood lignin-based hydrogel can be produced, but its properties are different from synthetic-based hydrogels. Methodology Materials Mixed hardwood kraft lignin (LGN) was supplied by FPInnovations’ pilot plant facility located in Thunder Bay, ON. NIPAAm (97%), MBAAm (99%), azobisisobutyronitrile (AIBN, 98%), acetone (97%), dimethyl sulfoxide-d6 (DMSO, 99.9% atom D), and tetramethylthionine chloride (methylene blue) were obtained from Sigma-Aldrich. Sulfuric acid (98%) and sodium hydroxide (97%) were also obtained from Sigma-Aldrich and diluted with deionized water to 20 and 10%, respectively. Reaction In this set of experiments, 0.1 g of kraft lignin, 0.06 g of MBAAm, and varying concentrations of NIPAAm (1.2–2.4 g) were dissolved in round-bottom glass flasks with deionized distilled water. The pH of the solutions was then adjusted with 20% sulfuric acid and 10% sodium hydroxide to pH 2.0–3.0 before being purging with nitrogen gas for 30 min. Water was added into the flasks to generate the total mass of reaction (40 g), which includes the weight of the reactants. The flask was placed in a water bath and heated to the desired temperature before adding 0.08 g of the AIBN initiator. The reaction was then allowed to proceed at the steady-state temperature (65–85 °C), with a constant flow of nitrogen gas at 220 rpm. This procedure was repeated for samples with and without lignin. After completion, the hydrogel samples were extracted from the flasks and rinsed with acetone to remove unreacted monomers. The hydrogels were then rinsed with water to prevent further degradation and freeze-dried at −50 °C for over 24 h in a Labconco FreeZone 1L freeze-dryer. The yield of the hydrogel production was calculated following eq 2 2 where WHydrogel is the total dry weight of the hydrogel (g) and WLGN, WNIPAAm, WMBAAm, WAIBN are the initial weights of kraft lignin, NIPAAm, MBAAm, and AIBN, respectively. The L9 Taguchi orthogonal design was performed with four factors (each containing three levels) to investigate the effect of reaction parameters on the responses (i.e., yield of reaction and swelling ratio) for producing hydrogels. 1H NMR Spectroscopy The freeze-dried hydrogel samples were ground to powder before being dissolved in 0.5 g of deuterated dimethyl sulfoxide (DMSO-d6), and placed into a 5 mm, 500 MHz glass NMR tube. The sample containing tubes were inserted into the Varian Unity INOVA 500 MHz NMR machine. The 1H NMR spectra of the samples were acquired at a 15° pulse flipping angle, a 4.6 μm pulse width, a 2.05 acquisition time, and 1 s relaxation delay time. Fourier Transform Infrared Spectroscopy Fourier Transform Infrared Spectroscopy (FTIR) analysis was conducted for the selected hydrogel samples (0.001 g) using a Bruker Tensor 37 (Germany, ATR accessory). The IR spectra was recorded in transmittance mode within the wave number range of 500–4000 cm–1 with a 4 cm–1 resolution. Brunauer–Emmett–Teller (BET) Surface Analysis The surface area of the hydrogel particles was determined by using a Quantachrome surface area analyzer, Nova 2200e, instrument. The freeze-dried hydrogels were first ground to powder and passed through multiple sieves. The particles with the size fraction between 150 and 300 μm were selected for analysis. For each test, 0.05 g of the powder samples were taken into account for specific surface area analysis according to Brunauer–Emmett–Teller (BET) method via adsorption–desorption isotherms using nitrogen gas at −180 °C with the relative pressure range of 0.01–0.99. Thermogravimetric Analysis (TGA) The dried powder hydrogel samples were placed in a desiccator overnight before undergoing thermal analysis using a thermogravimetric analyzer (TGA i-1000 series, Instrument Specialist Inc.). Approximately, 8 mg of sample was heated at a constant flow rate of nitrogen (35 mL/min) from room temperature to 700 °C at the heating rate of 10 °C/min. Swelling Performance The freeze-dried hydrogel samples were cut and divided into samples weighing approximately 0.2 g. The dried samples (with a known weight) were immersed into 200 mL of deionized water for 24 h. The swelling ratio was determined by considering the swollen weight of the hydrogel samples and the initial weight of dried hydrogels following eq 3 3 where Wswollen is the weight of the swollen hydrogel and Wdry is the initial weight of the dry hydrogel. Rheology A rheometer, TA Instruments, Discovery HR-2, with a Peltier temperature control system was used for analyzing the viscoelastic properties of hydrogel samples. The upper geometry was a 40 mm steel parallel plate with a gap of 1 mm and a loading gap of 60 mm. The dynamic oscillatory measurements were carried out at a constant temperature of 25 °C, unless stated otherwise. The hydrogels were prepared for the rheology test according to the method described elsewhere.53 The hydrogel samples were saturated in deionized water before testing and were loaded onto the Peltier plate to cover the surface area of the parallel plate. Attempts have been made so that the same amount of the hydrogel samples with similar thickness was loaded on the Peltier plate for each test. Three tests of frequency sweep, amplitude sweep, and temperature ramp have been applied on the samples in this set of experiments. The frequency test was carried out at a shear stress of 0.2 Pa throughout the frequency range of 0.2–20 Hz (1.267–125.7 rad/s). The amplitude sweep was obtained at a constant frequency mode of 10 rad/s over a strain rate range of 0.01–1000%. The temperature ramp was recorded at a constant strain rate of 2%, with a low frequency of 10 rad/s over a temperature range of 0–50 °C. The temperature ramp rate was 5 °C/min. The authors declare no competing financial interest. Acknowledgments The authors would like to thank NSERC, Canada Foundation for Innovation, Ontario Research Fund, Canada Research Chairs, Northern Ontario Heritage Fund Corporation for supporting this research. ==== Refs References Peppas N. A. ; Khare A. 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PMC006xxxxxx/PMC6644412.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145882210.1021/acsomega.8b01030ArticleExtent of Helical Induction Caused by Introducing α-Aminoisobutyric Acid into an Oligovaline Sequence Tsuji Genichiro †Misawa Takashi †Doi Mitsunobu ‡Demizu Yosuke †† Division of Organic Chemistry, National Institute of Health Sciences, Kanagawa 210-9501, Japan‡ Osaka University of Pharmaceutical Sciences, Osaka 569-1094, Japan E-mail: demizu@nihs.go.jp. Phone & Fax: +81-44-270-6578.14 06 2018 30 06 2018 3 6 6395 6399 17 05 2018 05 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. The preferred conformations of a dodecapeptide composed of l-valine (l-Val) and α-aminoisobutyric acid (Aib) residues, Boc-(l-Val-l-Val-Aib)4-OMe (3), were analyzed in solution and in the crystalline state. Peptide 3 predominantly folded into a mixture of α- and 310-(P) helical structures in solution and a (P) α helix in the crystalline state. document-id-old-9ao8b01030document-id-new-14ao-2018-01030hccc-price ==== Body 1 Introduction In proteins, helices are abundant and important secondary structures, which recognize macromolecules, such as other proteins and DNA. Helical peptides that mimic proteins are capable of inhibiting protein–protein interactions, and a variety of helix-stabilizing methods have been developed to aid the production of such peptides. As representative techniques, the introduction of α,α-disubstituted α-amino acids (dAA)1 or cyclic β-amino acids2 into short oligopeptides and side-chain stapling3 can all help to stabilize helical structures. In particular, α-aminoisobutyric acid (Aib) is the simplest dAA, and it is commonly used as a helical promoter.4 We have previously reported that the introduction of Aib residues into natural amino acid sequences stabilized helical structures. For example, the oligopeptides Boc-(l-Leu-l-Leu-Aib)n-OMe (n = 3 or 4) preferentially form stable right-handed (P) helical structures.5,6 These peptides are able to act as organocatalysts for asymmetric reaction, such as enantioselective epoxidation catalysts of α,β-unsaturated ketones6 and Michael addition of a malonate.7 Furthermore, the amphipathic peptides R-(l-Xaa-l-Xaa-Aib)3-NH2 (R = FAM-β-Ala and Xaa = Arg or R = H and Xaa = Lys) were also folded into stable helical structures and were used as antimicrobial peptides8 and cell-penetrating peptides,9 respectively. In addition, we have recently reported that the azidolysine (Azl)-based peptide Boc-(l-Azl-l-Azl-Aib)3-OMe formed a stable helical structure, and the azide groups could be replaced with several functional groups via click reactions without influencing the peptide’s helical structure.10 Thus, the insertion of Aib residues into α-amino acid-based oligopeptides is useful for stabilizing helical structures and providing a variety of functions. However, there have not been any reports about the secondary structural changes that occur when Aib residues are introduced into oligopeptides that form extended β-sheet structures. In general, oligopeptides composed of β-branched amino acids, such as valine (Val) and isoleucine (Ile), form β-sheet structures with extended conformations. In particular, oligovalines have a strong tendency to form β-sheet conformations.11 In this study, we designed a dodecapeptide composed of l-Val and Aib residues, Boc-(l-Val-l-Val-Aib)4-OMe (3), and analyzed its preferred conformations in solution and in the crystalline state. 2 Results and Discussion The dodecapeptide Boc-(l-Val-l-Val-Aib)4-OMe (3) was synthesized using conventional solution-phase methods according to a fragment condensation strategy, in which 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) hydrochloride and 1-hydroxybenzotriazole (HOBt) hydrate were used as coupling reagents. Briefly, alkaline hydrolysis of the tripeptide Boc-l-Val-l-Val-Aib-OMe (1) afforded the acid 1-COOH, whereas Boc deprotection by trifluoroacetic acid furnished the amine 1-NH2. The amine 1-NH2 was coupled with 1-COOH to give the hexapeptide Boc-(l-Val-l-Val-Aib)2-OMe (2). The dodecapeptide 3 was prepared in a manner similar to that used to prepare the hexapeptide (Scheme 1). Scheme 1 Synthesis of Peptides 1–3 The dominant conformations of the synthesized peptides 1–3 in solution were analyzed based on their Fourier transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), and circular dichroism (CD) spectra. Figure 1 shows the IR spectra of the tri- (1), hexa- (2), and dodecapeptide (3) in the 3200–3500 cm–1 region (the amide A NH-stretching region) at a peptide concentration of 5.0 mM in CDCl3 solution. In the spectra, the weak bands in the 3425–3438 cm–1 region were assigned to free (solvated) peptide NH groups, and the strong bands in the 3325–3340 cm–1 region were assigned to peptide NH groups with N–H···O=C intramolecular hydrogen bonds. These IR spectra are similar to those of helical peptides containing Aib residues.12 Figure 1 IR spectra of peptides 1 (green), 2 (blue), and 3 (red) in CDCl3 solution (peptide concentration: 5.0 mM). In the 1H NMR spectra of the dodecapeptide 3, the N-terminal urethane-type N(1)H proton signal was unambiguously determined by the high-field position but the remaining eleven peptide NH protons could not be assigned. Figure 2 shows a solvent perturbation experiment involving the addition of the strong H-bond acceptor solvent dimethyl sulfoxide (DMSO-d6) [0–10% (v/v)]. Two NH chemical shifts in the high-field positions were sensitive to the addition of DMSO-d6. These results are indicative of a 310- or α-helical structure in solution.13 Figure 2 Plots of chemical shift values of the NH protons of peptide 3 as a function of the concentration of DMSO-d6 (v/v) in CDCl3 solution (peptide concentration: 5.0 mM). The CD spectra of the dodecapeptide 3 in 2,2,2-trifluoroethanol (TFE) showed negative maxima at 207 and 222 nm indicating that 3 formed a right-handed (P) helical structure. Judging from the R([θ]222/[θ]208) value,14 the secondary structure of 3 (R = 0.64) was a mixture of α- and 310-helical structures (Figure 3). This spectrum is similar to that of Boc-(l-Leu-l-Leu-Aib)4-OMe (R = 0.51).15 Figure 3 CD spectra of the dodecapeptide 3 (red) and Boc-(l-Leu-l-Leu-Aib)4-OMe (black) in TFE solution (peptide concentration: 0.1 mM). Peptide 3 formed good crystals for X-ray crystallographic analysis after the slow evaporation of methanol/water at room temperature. Its crystal and diffraction parameters, selected backbone and side-chain torsion angles, and intra- and intermolecular hydrogen-bond parameters are listed in the Supporting Information.16−19 The asymmetric unit in 3 contained two (P) α-helical structures with a flipped C-terminal Aib(12) residue (Figure 4a). The conformations of molecules A and B were well-matched, except for small differences in their side-chain conformations (Figure 4b). The mean ϕ and ψ torsion angles of the residues (2–11) were −63.1° and −39.9° for A and −62.6° and −40.7° for B, which are close to those of an ideal (P) α-helix (−60° and −45°, respectively). Regarding the intramolecular hydrogen bonds in molecules A and B, eight i ← i + 4 type hydrogen bonds were observed, respectively. In packing mode, molecules A and B were connected by intermolecular hydrogen bonds via methanol molecules, forming chains with head-to-tail alignments (···A···A···A··· and ···B···B···B···). Figure 4 (a) X-ray diffraction structure of 3. The methanol molecules have been omitted. (b) Superimposed structures of molecules A (green) and B (blue). 3 Conclusions We designed and synthesized a dodecapeptide-containing l-Val and Aib residues, Boc-(l-Val-l-Val-Aib)4-OMe (3), to investigate the influence of the helical promoter Aib on β-sheet structures. The conformation of 3 was analyzed based on its FT-IR, 1H NMR, and CD spectra in solution and X-ray diffraction analysis in the crystalline state. Peptide 3 predominantly folded into a mixture of α- and 310-(P) helical structures in solution and a (P) α helix in the crystalline state. Although oligopeptides composed of β-branched amino acids form β-sheet structures with extended conformations, the insertion of Aib residues into β-sheet-forming peptide sequences could change the conformations of helical structures. Thus, we revealed that the insertion of Aib residues into oligopeptides not only stabilized their helical structures but also markedly altered their secondary structures (from βsheets to helical structures). Not only helical but also unique secondary structures will be created by the combination of natural l- and/or d-amino acids and Aib residues,20 and these findings will be invaluable for the de novo design of peptide-based organic and bioorganic molecules. 4 Experimental Section 4.1 General 1H and 13C NMR spectra were recorded at 400 and 100 MHz in CDCl3 (tetramethylsilane as an internal standard). FT-IR spectra were recorded at 1 cm–1 resolution, with an average of 256 scans used for the CDCl3 solution method (0.1 mm path length for NaCl cell). High-resolution mass spectra were recorded with LCMS-IT-TOF spectrometer. CD spectra were recorded using a 1.0 mm path length cell in TFE. 4.2 Synthesis of Tripeptide 1 The tripeptide 1 was prepared by conventional solution-phase peptide synthesis strategy. Colorless crystals; mp 177–179 °C; [α]D24 = −95.7 (c 0.25, CHCl3); IR (CDCl3, cm–1): 3437, 2969, 2934, 2875, 1738, 1705, 1671; 1H NMR (400 MHz, CDCl3): δ 6.66 (s, 1H), 6.43 (d, J = 8.0 Hz, 1H), 4.99 (d, J = 7.2 Hz, 1H), 4.21–4.18 (m, 1H), 3.91 (dd, J = 6.8 Hz, 1H), 3.70 (s, 3H), 2.23–2.17 (m, 2H), 1.53 (s, 3H), 1.51 (s, 3H), 1.45 (s, 9H), 0.97 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 174.6, 171.6, 167.0, 156.1, 80.4, 60.5, 58.3, 56.4, 52.5, 50.2, 30.3, 30.2, 28.3, 24.8, 24.7, 19.3, 19.2, 17.7, 17.5; [HR-ESI(+)-TOF] m/z: calcd for C20H37N3O6Na [M + Na]+, 438.2575; found, 438.2591. 4.3 Synthesis of Hexapeptide 2 A solution of the tripeptide Boc-l-Val-l-Val-Aib-OMe (1) (415 mg, 1.0 mmol) and 1 M aqueous NaOH (2.0 mL, 2.0 mmol) in MeOH (10 mL) was stirred at room temperature for 24 h. The solution was neutralized with 1 M aqueous HCl and was extracted with AcOEt. Being dried over Na2SO4 and removing the solvent afforded the tripeptide-carboxylic acid 1-COOH, which was used for the next reaction without further purification. Trifluoroacetic acid (1 mL) was added to a solution of 1 (415 mg, 1.0 mmol) in CH2Cl2 (5 mL), and then the mixture was stirred at room temperature for 5 h. Removing the solvent afforded the crude N-terminal free tripeptide 1-NH2, which was used without further purification. A mixture of EDC (230 mg, 1.2 mmol), HOBt (162 mg, 1.2 mmol), N,N-diisopropylethylamine (418 μL, 2.4 mmol), the above 1-COOH (1.0 mmol), and the above 1-NH2 (1.0 mmol) in CH2Cl2 (10 mL) was stirred at room temperature for 3 days. The solution was washed with 3% aqueous HCl, saturated aqueous NaHCO3, and brine, before being dried over Na2SO4. After removing the solvent, the residue was purified by column chromatography on silica gel (n-hexane/AcOEt = 1:5) to give the hexapeptide 2 in 46% yield. Colorless crystals; mp 200–203 °C; [α]D24 = −27.4 (c 0.5, CHCl3); IR (CDCl3, cm–1): 3437, 3340, 2968, 2935, 2875, 1736, 1703, 1665; 1H NMR (400 MHz, CDCl3): δ 7.64 (s, 1H), 7.30 (d, J = 9.2 Hz, 1H), 7.17 (s, 1H), 6.93 (d, J = 6.8 Hz, 1H), 6.44 (d, J = 5.2 Hz, 1H), 5.01 (d, J = 2.6 Hz, 1H), 4.42 (dd, J = 8.8, 5.2 Hz, 1H), 4.18 (dd, J = 6.4, 4.4 Hz, 1H), 3.95 (dd, J = 4.4 Hz, 1H), 3.82 (dd, J = 4.4, 2.6 Hz, 1H), 3.68 (3H, s), 2.50–2.44 (m, 2H), 2.30–2.20 (m, 2H), 1.53 (3H, s), 1.52 (3H, s), 1.50 (9H, s), 1.50 (3H, s), 1.48 (3H, s), 1.06 (d, J = 6.8 Hz, 6H), 1.05–1.04 (m, 3H), 1.01 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.6, 175.2, 172.1, 171.9, 170.9, 170.5, 157.0, 81.8, 62.1, 60.8, 60.0, 58.6, 57.1, 55.8, 52.0, 29.4, 29.2, 28.9, 28.2, 27.5, 25.2, 24.7, 23.7, 19.6, 19.3, 19.2, 18.0, 17.5, 17.4, 17.2; [HR-ESI(+)-TOF] m/z: calcd for C34H62N6O9Na [M + Na]+, 721.4470; found, 721.4502. 4.4 Synthesis of Dodecapeptide 3 The dodecapeptide 3 was prepared using a method similar to that described for the preparation of 2. Yield 35%; colorless crystals; mp 302–304 °C; [α]D24 = −16.9 (c 0.5, CHCl3); IR (CDCl3, cm–1): 3425, 3325, 2967, 2936, 2876, 1734, 1703, 1656; 1H NMR (400 MHz, CDCl3): δ 7.80 (d, J = 4.8 Hz, 1H), 7.77 (s, 1H), 7.73 (s, 1H), 7.67 (d, J = 4.8 Hz, 1H), 7.53–7.51 (m, 3H), 7.21 (d, J = 5.6 Hz, 1H), 7.10 (d, J = 6.0 Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.72 (br s, 1H), 5.39 (br s, 1H), 4.41 (dd, J = 9.0, 5.8 Hz, 1H), 4.25 (dd, J = 7.2, 5.6 Hz, 1H), 3.89–3.84 (m, 3H), 3.82–3.79 (m, 1H), 3.71–3.62 (m, 2H), 3.67 (s, 3H), 2.47–2.36 (m, 2H), 2.29–2.15 (m, 6H), 1.52–1.48 (m, 33H), 1.12–0.97 (m, 48H); 13C NMR (100 MHz, CDCl3): δ 175.9, 175.9, 175.5, 173.8, 173.8, 173.0, 172.7, 172.6, 172.3, 171.5, 171.0, 157.2, 81.7, 62.9, 62.7, 62.5, 62.3, 60.9, 60.7, 59.2, 57.0, 56.8, 56.6, 55.8, 51.9, 29.8, 29.6, 29.5, 29.2, 29.2, 28.9, 28.3, 27.5, 27.4, 25.2, 24.6, 23.4, 23.3, 23.0, 19.9, 19.7, 19.5, 19.4, 19.4, 19.3, 19.2, 19.1, 19.1, 19.0, 18.9, 18.5, 18.0, 18.0, 17.8; [HR-ESI(+)-TOF] m/z: calcd for C62H112N12O15Na [M + Na]+, 1287.8262; found, 1287.8333. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01030.Crystallographic data and copies of the 1H NMR and 13C NMR spectra of the peptides (PDF) Crystallographic data of peptide 3 (CIF) Supplementary Material ao8b01030_si_001.pdf ao8b01030_si_002.cif The authors declare no competing financial interest. Acknowledgments This study was supported, in part, by JSPS KAKENHI grant number 17k08385 (Y.D.), by a grant from the Takeda Science Foundation (Y.D.), a grant from the Terumo Life Science Foundation (Y.D.), a grant from the Suzuken Memorial Foundation (Y.D.), and a grant from the Naito Foundation (Y.D.). ==== Refs References a Crisma M. ; Moretto A. ; Peggion C. ; Panella L. ; Kaptein B. ; Broxterman Q. B. ; Formaggio F. ; Toniolo C. Chiral, fully extended helical peptides . Amino Acids 2011 , 41 , 629 –641 . 10.1007/s00726-011-0839-9 .21293888 b Tomsett M. ; Maffucci I. ; Le Bailly B. A. 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PMC006xxxxxx/PMC6644413.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145907110.1021/acsomega.8b00825ArticleGraphene/Nickel Oxide-Based Nanocomposite of Polyaniline with Special Reference to Ammonia Sensing Ahmad Sharique †Ali khan Mohammad Mujahid ‡Mohammad Faiz ††Department of Applied Chemistry, Faculty of Engineering and Technology and ‡Applied Science and Humanities Section, University Polytechnic, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh 202002, India E-mail: faizmohammad54@rediffmail.com (F.M.).17 08 2018 31 08 2018 3 8 9378 9387 26 04 2018 20 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Polyaniline@graphene/nickel oxide (Pani@GN/NiO), polyaniline/graphene (Pani/GN), and polyaniline/nickel oxide (Pani/NiO) nanocomposites and polyaniline (Pani) were successfully synthesized and tested for ammonia sensing. Pani@GN/NiO, Pani/NiO, Pani/GN, and Pani were characterized using X-ray diffraction, UV–vis spectroscopy, Raman spectroscopy, scanning electron microscopy, and transmission electron microscopy. The as-prepared materials were studied for comparative dc electrical conductivity and the change in their electrical conductivity on exposure to ammonia vapors followed by ambient air at room temperature. It was observed that the incorporation of GN/NiO in Pani showed about 99 times greater amplitude of conductivity change than pure Pani on exposure to ammonia vapors followed by ambient air. The fast response and excellent recovery time could probably be ascribed to the relatively high surface area of the Pani@GN/NiO nanocomposite, proper sensing channels, and efficaciously available active sites. Pani@GN/NiO was observed to show better selectivity toward ammonia because of the comparatively high basic nature of ammonia than other volatile organic compounds tested. The sensing mechanism was explained on the basis of the simple acid–base chemistry of Pani. document-id-old-9ao8b00825document-id-new-14ao-2018-00825xccc-price ==== Body Introduction A variety of materials such as carbon nanomaterials, inorganic semiconductors, and conjugated polymers have been explored to fabricate gas/vapor sensors.1−7 Conducting polymer-based sensors have high sensitivity because of the large surface area, fast response, lower power consumption, small size, and lightweight. Among them, polyaniline (Pani) is considered to be the most promising and widely applied sensing material because of its low cost, easy preparation, high environmental stability, tuneable electrical properties, and unique functions by controlled charge-transfer processes.8−10 However, its low sensitivity and inadequate thermal stability still restrict its application in operable sensors. In order to improve the sensing performance of Pani, many efforts have been made by loading the noble metals, semiconductors, and other components to develop composite materials.11−13 Recently, nickel oxide (NiO) nanoparticles have drawn considerable attention because of their low price, acceptable sensing properties, and good environmental stability. Researchers developed gas sensors based on NiO films which have demonstrated significant sensing properties.14 Because of its high resistivity, there is still drawback in using NiO in composite materials for practical sensing applications. To overcome this problem, many carbonaceous materials with high electrical conductivity have been introduced to prepare carbon/NiO nanocomposites.15 A number of composites of Pani have been reported with metal oxides, metal nanoparticles, graphene (GN), and carbon nanotubes widely used in gas sensing applications.16 Also, many other nanocomposites have also been reported as ammonia sensors.17−21 We have decorated NiO nanoparticles on GN sheets, which increases the resultant surface area. The material obtained GN/NiO nanocomposite is also conductive with enhanced surface area, which may be a suitable material for making conducting nanocomposites with Pani for the purpose of gas/vapor sensing studies. In this work, the polyaniline@graphene/nickel oxide (Pani@GN/NiO) nanocomposite was selected and studied as an ammonia sensor by examining the dynamic response of electrical conductivity using a simple four-in-line-probe dc electrical conductivity measurement setup. Results and Discussion X-ray Diffraction Studies X-ray diffraction (XRD) spectra of Pani, polyaniline/nickel oxide (Pani/NiO), polyaniline/graphene (Pani/GN), and Pani@GN/NiO nanocomposites are shown in Figure 1. Figure 1a exhibits the XRD spectrum of Pani, which has two characteristic broad peaks at 2θ = 20.31° and 25.23°. The XRD spectrum of Pani/GN has broad peaks similar to those for Pani slightly shifted to 2θ = 20.45° and 25.27°, which is attributed to the presence of GN in the Pani/GN nanocomposite, as shown in Figure 1b. In Figure 1c, there are five extra sharp peaks along with characteristic broad peaks of Pani (at 2θ = 20.25° and 25.34°), which are observed at 2θ = 37.42°, 43.35°, 62.86°, 75.26°, and 79.42°, respectively, attributed to the presence of NiO nanoparticles in the Pani/NiO nanocomposite. The XRD spectrum of the Pani@GN/NiO nanocomposite is similar to that of the Pani/NiO nanocomposite, as shown in Figure 1d. The peaks due to Pani at 2θ = 20.05° and 25.69°, respectively, shifted from 2θ = 20.31° and 25.23°, which may be attributed to the interaction of Pani with NiO embedded on GN sheets. The five intense peaks observed in the Pani@GN/NiO nanocomposite at 2θ = 37.13°, 43.48°, 63.12°, 75.39°, and 79.56° may be ascribed to the (111), (200), (322), (311), and (222) planes, respectively, due to the reflection of NiO deposited on GN sheets with a slight shift compared to those in the Pani/NiO nanocomposite, indicating the successful preparation of the Pani@GN/NiO nanocomposite.22,23 Figure 1 XRD patterns of (a) Pani, (b) Pani/GN, (c) Pani/NiO, and (d) Pani@GN/NiO. Scanning Electron Microscopy Studies Figure 2 presents the scanning electron microscopy (SEM) images of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites. The SEM images of the Pani@GN/NiO nanocomposite demonstrated the sheet-type morphology due to the presence of GN/NiO, as shown in Figure 2a,b. However, the NiO nanoparticles deposited on GN sheets are not visible here, which indicates that the NiO nanoparticles deposited on GN sheets have a nucleating effect on the polymerization of aniline and are completely covered by the Pani shell. Figure 2c,d presents the SEM images of Pani, which seems to possess a nanotubelike morphology. Figure 2e presents the SEM image of the Pani/NiO nanocomposite, in which the polymer matrix is well deposited on NiO nanoparticles and the Pani is agglomerated by several nanoparticles. In Figure 2f, it may clearly be seen that the Pani is well deposited on GN, giving some sheetlike morphology in the Pani/GN nanocomposite. Figure 2 SEM micrographs of (a,b) Pani@GN/NiO nanocomposite, (c,d) Pani, (e) Pani/NiO, and (f) Pani/GN. Transmission Electron Microscopy Studies The morphologies of Pani@GN/NiO, Pani/NiO, Pani/GN, and Pani were further analyzed by transmission electron microscopy (TEM), as shown in Figure 3. In this synthesis, we deposited NiO nanoparticles on GN sheet and then aniline monomers were polymerized on the surface of GN/NiO. Figure 3a shows that the NiO nanoparticles were deposited on GN sheets upon which Pani nanotubes are deposited, signifying the successful incorporation of NiO nanoparticles onto GN sheets for a nanocomposite with Pani nanotubes. A lattice spacing of 0.818 nm can be attributed to the lattice spacing of the (200) plane of the NiO nanoparticles in the Pani@GN/NiO nanocomposite (see the magnified image of Figure 3a). Figure 3b shows that the NiO particles are uniformly distributed in the outer shell of the polymer matrix. In Figure 3c, it can be observed that the short tubes of Pani are precisely deposited on the GN sheets. We also synthesized Pani using the same procedure in the absence of the GN and NiO; short tubes of Pani were obtained, as shown in Figure 3d. In the light of the above observations of TEM analysis, it can be inferred that the NiO nanoparticles were precisely deposited on GN sheets and covered by Pani nanotubes. Furthermore, to evaluate the Brunauer–Emmett–Teller (BET) surface area, the N2 adsorption–desorption isotherms of the Pani@GN/NiO nanocomposite were measured. The BET surface area of the Pani@GN/NiO nanocomposite was found to be 275 m2 g–1 (Figure S1). Figure 3 TEM micrographs of (a) Pani@GN/NiO nanocomposite, (b) Pani/NiO nanocomposite, (c) Pani/GN nanocomposite, and (d) Pani. Optical Studies The UV–vis absorption spectra of Pani@GN/NiO, Pani/NiO, Pani/GN, and Pani are given in Figure 4. In Figure 4a, the band observed at 327 nm for Pani may be attributed to the π–π* electronic transition of benzenoid rings.24 In the case of the Pani/GN nanocomposite, the band red-shifted to 332 nm from 327 nm may be attributed to the interaction of π-electrons of benzenoid rings with π-bonds of GN, as shown in Figure 4b.25 In Figure 4c, the Pani/NiO nanocomposite illustrates the two bands at 281 and 323 nm, which may be related to the electronic transition from the valence band to the conduction band in the NiO and due to the π–π* transition of the benzenoid rings, respectively.26,27 In the case of Pani/NiO, a blue shift is observed in contrast to that in Pani, which may be attributed to the decrease in the conjugation of Pani by loading of NiO in Pani, reducing the path of polarons in Pani. From Figure 4d, it is illustrated that the Pani@GN/NiO nanocomposite reflected the two maxima at 276 nm, blue-shifted in comparison with Pani/NiO, and the other at 375 nm, red-shifted in comparison with Pani from 327 nm, which may be attributed to the increase in the extent of conjugation of Pani by forming an efficient network by electronic transition from NiO to GN.28 The significant red shift in the Pani@GN/NiO nanocomposite supports the enhanced dc electrical conductivity because of the ease in the movement of polarons by extended conjugation in Pani@GN/NiO. Figure 4 UV–vis spectra of (a) Pani, (b) Pani/GN nanocomposite, (c) Pani/NiO nanocomposite, and (d) Pani@GN/NiO nanocomposite. Raman Spectroscopy Figure 5 shows the Raman spectra of Pani@GN/NiO, Pani/NiO, Pani/GN, and Pani. In Figure 5a, the different modes of vibration are observed. The sharp peak at 1410 cm–1 may be associated with the presence of C–N+ polarons.29 The peaks observed at 1330 and 1570 cm–1 may be due to the presence of GN sheet bands (D and G), and the bands at 1370 and 1592 cm–1 may be due to two-magnon (2M) and two-phonon (2P) bands of NiO. In Figure 5b, the Raman spectrum of Pani/NiO displays two vibrational bands at 1363 and 1574 cm–1, which may be due to the presence of NiO nanoparticles that correspond to the 2M and 2P models.30−32Figure 5c displays the Raman spectrum of the Pani/GN nanocomposite, showing the two bands at 1345(D) cm–1 due to disordered structures in the GN layers and 1576(G) cm–1 due to sp2 electronic configuration of graphitic carbon phase in the Pani/GN nanocomposite.33 In the Raman spectra of Pani, the bands at 1350 and 1565 cm–1 may be attributed to C–N+ and C=C modes of vibration, respectively, as shown in Figure 5d. Significant shifts of Raman bands of Pani@GN/NiO are observed in comparison with the Pani/GN and Pani/NiO nanocomposites. The D and G bands of GN in the Pani/GN nanocomposite are blue-shifted from 1345 and 1576 to 1330 and 1570 cm–1, respectively, in the Pani@GN/NiO nanocomposite, whereas the 2M and 2P bands of NiO in Pani/NiO are red-shifted from 1363 and 1574 to 1370 and 1592 cm–1, respectively. The substantial band shifts of GN and nickel oxide constituent in the Pani/GN and Pani/NiO nanocomposites indicate a strong interaction between NiO and GN sheets, favoring the charge transfer from NiO to GN sheets28 and forming more holes in Pani. Thus, an increment in the electrical conductivity of Pani@GN/NiO also strongly supports the interaction and charge transportation between NiO and GN. Figure 5 Raman spectra of (a) Pani@GN/NiO nanocomposite, (b) Pani/NiO nanocomposite, (c) Pani/GN nanocomposite, and (d) Pani. Electrical Conductivity The initial dc electrical conductivities of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites were measured by a standard four-in-line-probe method, as shown in Figure 6a. The electrical conductivity of Pani was observed to be 1.832 S/cm. The electrical conductivity of the Pani/NiO nanocomposite prepared by the method mentioned above was observed to be 0.853 S/cm, which is slightly less than that of Pani. It may be proposed that the movement of polarons of Pani get retarded because of the Coulombic interaction between the polarons of Pani and lone pairs of oxygen of NiO, as shown in Figure 6b. Although, the d-orbitals of Ni may increase the number of polarons in the Pani chains by accommodating electrons from Pani, the Coulombic interaction seems to be a dominating factor. Shambharkar and Umare23 also reported that the electrical conductivity of the Pani/NiO nanocomposite decreases with a low NiO content in the nanocomposite. Figure 6 (a) Initial dc electrical conductivities of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites and the mechanism of conductivity behavior of (b) Pani/NiO, (c) Pani/GN, and (d) Pani@GN/NiO. The initial electrical conductivity at room temperature of the as-prepared Pani/GN nanocomposite was observed to be 9.2 S/cm, which was much higher than that of Pani (1.2 S/cm). It may be proposed that the charge carriers hop from Pani to GN, where they gain high mobility along the π-conjugated system of GN, as shown in Figure 6c. The overall mobility of charge carriers thus enhanced because of unobstructed movement along the GN nanosheets, leading to an increase in the electrical conductivity of the Pani/GN nanocomposite. Ansari et al.34 also reported that a sulfonated-Pani/GN nanocomposite exhibited a high electrical conductivity because of π–π interaction between sulfonated-Pani and GN. In the case of Pani@GN/NiO, the electrical conductivity was observed to be 11.34 S/cm, which was the highest among all samples. It seems that the NiO acts as a bridge between Pani and GN, which helps in the movement of charge carriers from Pani to GN and thus increases electrical conductivity because of a greater mobility of charge carriers along the π-conjugated system of GN nanosheets, as shown in Figure 6d. Thus, NiO bridges additionally facilitate the hopping movement of charge carriers between Pani and GN in the Pani@GN/NiO nanocomposites. Stability under Isothermal Ageing Conditions The isothermal ageing stabilities of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites were calculated, as shown in Figure 7, by using the following equation:13 1 where σt is the electrical conductivity at time t and σ0 is the electrical conductivity at time zero at experimental temperature. The as-prepared materials were examined for stability in terms of dc electrical conductivity retention with respect to time at different temperatures. The electrical conductivity of each of the samples was measured at the temperatures 50, 70, 90, 110, and 130 °C versus time at an interval of 5 up to 20 min. From Figure 7a,b, it can be observed that Pani and Pani/NiO are fairly stable up to 90 °C, whereas the Pani/GN and Pani@GN/NiO nanocomposites are stable up to 110 °C in terms dc electrical conductivity, as shown in Figure 7c,d. It may be inferred that the relative electrical conductivity of Pani@GN/NiO and Pani/GN nanocomposites showed greater stability than the Pani and Pani/NiO nanocomposite under isothermal ageing conditions. Figure 7 Relative electrical conductivity of (a) Pani, (b) Pani/NiO, (c) Pani/GN, and (d) Pani@GN/NiO under isothermal ageing conditions. Stability under Cyclic Ageing Conditions The cyclic ageing stabilities of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites were studied, as shown in Figure 8.13 The relative electrical conductivity (σr) was calculated using the following equation: 2 where σT is the electrical conductivity (S/cm) at temperature T (°C) and σ50 is the electrical conductivity (S/cm) at 50 °C, that is, at the beginning of each cycle. Figure 8 Relative electrical conductivity of (a) Pani, (b) Pani/NiO, (c) Pani/GN, and (d) Pani@GN/NiO under cyclic ageing conditions. The electrical conductivity was recorded for successive cycles and observed to be increased gradually for each of the cycle with a regular trend in all of these cases, which may be due to the increment of the number of charge carriers as polarons or bipolarons at elevated temperatures. Figure 8a shows the relative electrical conductivity of Pani following the same trend for each of the cycle with gain in the dc electrical conductivity as the temperature increases. Also, the relative electrical conductivities of Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites increased as the temperature increases from 50 to 130 °C, as shown in Figure 8b–d. Among all of these nanocomposites, the Pani@GN/NiO nanocomposite showed the lowest gain in conductivity with less deviation. Thus, it may be inferred that the Pani@GN/NiO nanocomposite is a more stable semiconductor than Pani and among all of these nanocomposites under cyclic ageing conditions. The difference observed may be attributed to the removal of moisture, excess HCl, or low-molecular-weight oligomers of aniline.35 Sensing The dc electrical conductivity responses of the sensors based on Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO were measured on exposure to ammonia (NH3) at room temperature (∼25 °C). A four-in-line-probe PID-200 (Scientific Equipment, Roorkee, India) dc electrical conductivity measuring instrument was used to study ammonia sensing properties. The pellet fabricated was in contact with the four probes and placed in a closed chamber of ammonia vapors (Figure 9). Figure 9 Instrumental setup of the ammonia sensor and the schematic presentation of the four-in-line probe. The electrical conductivity immediately decreases on exposure to ammonia for 60 s and rapidly reverts back on exposure to ambient air in next 60 s in all of the as-prepared materials, as shown in Figure 10a. However, the greatest change in electrical conductivity was observed in the case of the Pani@GN/NiO nanocomposite. Therefore, the Pani@GN/NiO nanocomposite was also tested for different concentrations of ammonia, as shown in Figure 10b. The highest change in electrical conductivity was observed for 1703 ppm of ammonia. As the concentration of ammonia increases from 170 to 1703 ppm, the more polarons of Pani get neutralized by the lone pair of electrons of ammonia molecules, leading to a decrease of electrical conductivity. The electrical conductivity change of the Pani@GN/NiO nanocomposite could be observed on exposure to ammonia as low as 170 ppm concentration. Figure 10 (a) Effect on the dc electrical conductivity of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO nanocomposites on exposure to (1703 ppm) ammonia vapors followed by exposure to ambient air with respect to time, (b) effect on the dc electrical conductivity of the Pani@GN/NiO nanocomposite on exposure to ammonia vapors at different concentrations, and (c) steady-state response of dc electrical conductivity of the Pani@GN/NiO nanocomposite on exposure to 1703 ppm ammonia followed by exposure to ambient air with respect to time. The steady-state response of Pani@GN/NiO was determined by first keeping the sample in 1703 ppm of ammonia environment for 180 s followed by 120 s in air for a total duration of 300 s (Figure 10c). It can be observed that the electrical conductivity immediately decreases on exposure to ammonia for about 65 s and attains saturation for further exposures until exposed to ambient air. The conductivity reverted in ambient air after 180 s of exposure to ammonia and became saturated at about 240 s. Reversibility The sensitivity and reversibility are the two important parameters for a gas sensor. Sensitivity may be defined as the time taken to reach the final value after exposure to vapors and to reach the initial value on exposure to ambient air, whereas the reversibility may be considered as the cycling between the analyte and ambient air without any loss in its sensing ability. The reversibility of Pani and Pani@GN/NiO nanocomposite was measured in terms of the dc electrical conductivity. The reversibility of both the samples was determined by first keeping the sample in 1703 ppm ammonia vapors for 30 s followed by 30 s in air for a total duration of 150 s. Figure 11a represents the reversibility response of Pani in terms of the dc electrical conductivity with a variation range from 1.849 to 1.812 S/cm in ambient air and on exposure to 1703 ppm of ammonia vapors, respectively. The change in conductivity observed was 0.037 S/cm, whereas in the case of the Pani@GN/NiO nanocomposite, the reversibility was observed with an excellent variation range compared to Pani, which is from 11.146 to 7.489 S/cm in ambient air and on exposure to 1703 ppm of ammonia vapors, respectively, as shown in Figure 11b. In the case of Pani@GN/NiO, the observed decrease in conductivity by 3.657 S/cm is indicative of much higher efficiency than that of Pani in terms of reversibility. There was around about 99 times greater variation observed in the conductivity of the Pani@GN/NiO nanocomposite than that of Pani. Figure 11 Variation in the electrical conductivity of (a) Pani and (b) Pani@GN/NiO on alternate exposure to (1703 ppm) ammonia vapors and ambient air. Sensing under Dry and Wet Atmospheres The NH3 response properties under completely dry and wet atmospheres were also studied. The effect of relative humidity (RH) on ammonia sensing properties was studied by mixing of humidity and NH3 vapors. It was seen that the dc electrical conductivity of the Pani@GN/NiO nanocomposite sharply decreases and reaches its saturation level within 50 s when exposed to 1700 ppm of dry ammonia prepared by heating of ammonium chloride and slaked lime mixture. Also, the sample Pani@GN/NiO was tested for pure water vapors, and it was observed that the decrease in electrical conductivity was not rapid as that it was in the case of completely dry ammonia, as shown in Figure 12. To confirm the effect of water vapors on the ammonia sensing properties of the Pani@GN/NiO nanocomposite, the sample was also exposed to an NH3 atmosphere at 1703 ppm at different humidity levels viz. 20, 30, 40, and 60% RH. The electrical conductivity of the sample continuously decreases with time at different RHs. The variations of humidity levels for the same concentration keep the conductivity stable. The conductivity variations of Pani@GN/NiO are not much effected at any RH, and the four curves were almost similar, from which we may conclude that humidity has only small effect on NH3 sensing properties. Figure 12 dc electrical conductivity response as a function of time of Pani@GN/NiO nanocomposite exposed to 1700 ppm dry ammonia, 20% (1703 ppm ammonia), 30% (1703 ppm ammonia), 40% (1703 ppm ammonia), 60% (1703 ppm ammonia) RH, and pure water vapors. Selectivity For a worthy and applicative gas sensor, high selectivity is also an important requirement. The conductivity responses of the Pani@GN/NiO nanocomposite to ammonia and volatile organic compounds (VOCs) viz. isopropanol, methanol, ethanol, acetone, acetaldehyde, and formaldehyde at room temperature (25 °C) are shown in Figure 13. It was observed that the conductivity response of the Pani@GN/NiO nanocomposite to 1703 ppm ammonia was 9.8, 6.2, 6.6, 7.9, 6.5, and 6.1 times higher than those for isopropanol, methanol, ethanol, acetone, acetaldehyde, and formaldehyde, respectively. Such a selectivity results from the difference of their sensing mechanisms, that is, the neutralization of polarons of Pani that plays an important role in changing electrical conductivity on exposure to VOCs. The greater the availability of lone pairs in a vapor/gas, the greater the observed electrical conductivity change. Ammonia has more electron-donating nature and thus neutralizes more number of polarons of Pani. On the other hand, in VOCs, there are interactions of lone pairs of electrons of oxygen of greater −I effect than that of nitrogen of Pani. Therefore, the lower electron-donating tendency of VOCs leads to a lower conductivity change. This also infers the higher selectivity of the Pani@GN/NiO nanocomposite toward NH3. Figure 13 Selectivity of the Pani@GN/NiO nanocomposite to 1703 ppm of ammonia and different VOCs (1 M) in water. The efficient gas sensing properties of Pani@GN/NiO may be attributed to the large surface area of Pani which anchored more adsorption and desorption of gaseous molecules as well as high polarons mobility due to the presence of GN, essential requirement for rapid response of any electrical conductivity-based sensor.13 It is expected that the NiO embedded on GN sheets interacts with lone pairs of nitrogen atoms of Pani, thus increasing the number of polarons in Pani and creating a more number of active sites at a large surface area for the interaction of lone pairs of ammonia molecules to polarons of Pani. However, the detailed understanding on the role of NiO and GN sheets in the sensing mechanism of the Pani@GN/NiO nanocomposite requires further investigations on this aspect. Proposed Mechanism for Sensing Sensing by the Pani@GN/NiO nanocomposite is based on the decrease in electrical conductivity on exposure to analyte vapors and to revert on exposure to ambient air. Therefore, the different aspects of emergence of high electrical conductivity in the nanocomposite have been discussed above in order to understand the sensing behaviour of the Pani@GN/NiO nanocomposite. The sensing mechanism of the Pani@GN/NiO nanocomposite was explained through dc electrical conductivity response by simple adsorption and desorption mechanism of ammonia vapors at room temperature, as shown in Scheme 1. In the Pani@GN/NiO nanocomposite, NiO nanoparticles embedded on GN sheets interact with lone pairs of nitrogen atoms of Pani; thus, the numbers of polarons and bipolarons increase in Pani chains. In the presence of ammonia vapors, the lone pairs of ammonia molecules bind with polarons of the Pani@GN/NiO nanocomposite, which impedes the mobility of polarons, leading to a decrease in electrical conductivity. When Pani@GN/NiO is exposed to ambient air, the ammonia molecules are desorbed from the surface and thus the electrical conductivity reverts to its initial value. Thus, the simple adsorption and desorption of ammonia vapors on the Pani@GN/NiO nanocomposite govern the mobility of polarons. Scheme 1 Proposed Mechanism of Interaction of Ammonia with the Pani@GN/NiO Nanocomposite However, the humidity present in atmosphere may also interfere in ammonia adsorption on the Pani@GN/NiO nanocomposite, but the conductivity changes of Pani@GN/NiO are not much affected at any RH so that humidity has only small effect on NH3 sensing properties. Therefore, in competition between ammonia and water molecules, it may be said that the change in conductivity of Pani@GN/NiO may be mainly due to ammonia vapors because ammonia has more available electrons than water. The selective response of the Pani@GN/NiO nanocomposite toward ammonia may be due to the basic nature of ammonia, which readily interacts with polarons of Pani, whereas all other VOCs tested are much less basic than ammonia because they contain an oxygen atom which is more electronegative than nitrogen, so the electronic interaction with Pani in these compounds is comparatively poorer. Conclusions Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO were successfully prepared and characterized by XRD, SEM, TEM, UV–vis, and XRD analysis. The Pani@GN/NiO nanocomposite showed the higher thermal stability in terms of dc electrical conductivity retention under isothermal and cyclic ageing conditions than pristine Pani. The results highlighted that the Pani@GN/NiO nanocomposite was found to be an excellent material for ammonia sensing with excellent selectivity against VOCs and rapid response. There was about 99 times greater variation in the electrical conductivity of Pani@GN/NiO nanocomposite than that of Pani during sensing. The enhancement of sensing properties in the Pani@GN/NiO nanocomposite may be attributed to the synergistic effect between NiO, GN, and Pani. Therefore, the sensor based on the nanocomposite of GN/NiO with Pani may be useful and efficient while the material is promising for ammonia vapor sensing. Experimental Section Materials Aniline (99%, E. Merck, India) and commercially available natural graphite powder (Sigma-Aldrich, USA) were used to prepare graphene oxide (GO). Sodium dodecylsulfate, ammonium persulfate (APS), sulfuric acid (H2SO4), hydrated nickel nitrate (Ni(NO3)26H2O), sodium hydroxide (NaOH), and hydrazine hydrate (85%) were obtained from the local suppliers and used as received. Preparation of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO Pani nanofibers were prepared through the interfacial polymerization reaction in which aniline monomers (10 mmol) were dissolved in hexane (10 mL) under stirring for 30 min. APS (30 mmol) was gently added to 10 mL of 1 M sulfuric acid solution under constant stirring. The solution of APS was added dropwise into aniline solution, and the mixture was allowed to react for 1 h. The final greenish reaction product was then filtered, washed with double distilled water, and then dried in an air oven at 70 °C for 10 h. In a typical synthesis procedure of the Pani/NiO nanocomposite, 2 mL of aniline was dissolved in the solution of 2 mL of 1 M H2SO4 in 90 mL of distilled water. A solution of 0.5 g of nickel oxide (NiO) was prepared in accordance with the existing literature36 and was then added to aniline solution. APS (5 g) dissolved in 10 mL of 1 M H2SO4 was added to the aniline solution, and the reaction mixture was stirred for 24 h at room temperature. The final reaction product was then filtered and washed thoroughly with distilled water and acetone until the filtrate became colorless. The resulting product was dried at 70 °C for 10 h. To prepare the Pani/GN nanocomposite, GO (20 mg) was prepared in accordance with the existing literature37 and was then dissolved in 10 mL of 1 M H2SO4 with constant stirring for 1 h. Subsequently, aniline solution (10 mmol) prepared in 1 M H2SO4 was mixed to the GO solution and stirred for another 1 h. The APS (30 mmol) solution prepared in 1 M H2SO4 was then added to the above solution. The final reaction mixture was kept under constant stirring, followed by a conventional microwave treatment at 300 W for 30 min. The resulting suspension was separated by centrifugation and washed with double distilled water and ethanol. The final product was dried at 70 °C under vacuum for 12 h. In the synthesis of GN/NiO, the as-prepared GO (25 mg) was ultrasonically dispersed in 10 mL of double distilled water for 1 h and 0.5 g of Ni(NO3)2·6H2O in 25 mL of distilled water was added into the above dispersion. After constant stirring for 6 h, 10 mL of 1 M NaOH aqueous solution was added into the above reaction mixture drop by drop. The pH value was adjusted to ∼10 by 10% aqueous ammonia. After that, 10 mL of hydrazine hydrate (85%) was added to it. The reaction mixture was stirred for several minutes before being treated with microwave at 300 W for 30 min. The product was separated by centrifuging and washed with double distilled water followed by ethanol. The product was dried in a vacuum oven at 50 °C for 24 h. The dried sample was heated in a N2 atmosphere at 300 °C for 3 h to obtain GN/NiO. The Pani@GN/NiO nanocomposite was prepared by mixing GN/NiO to aniline monomer in a weight ratio of 5:1. GN/NiO was dispersed in ethanol and water (1:1 weight ratio) by stirring for 60 min and then added to the aniline solution prepared in 1 M H2SO4. An aqueous solution of APS (10 mL) prepared in 1 M H2SO4 was added dropwise to the mixture of aniline and GN/NiO at 5–10 °C and kept under constant stirring for 24 h. The resultant product was washed several times by vacuum filtration using ethanol and double distilled water. The final product was dried at 50 °C for 12 h. Characterization The morphology, structure, and chemical composition of Pani, Pani/NiO, Pani/GN, and Pani@GN/NiO were investigated by a variety of methods. XRD pattern were recorded by a Bruker D8 diffractometer with Cu Kα radiation at 1.5418 Å. SEM studies were carried out by JEOL, JSM, 6510-LV (Japan). TEM studies were carried out by using JEM 2100, JEOL (Japan). The Raman spectra were taken using a Varian FT-Raman spectrometer. UV–vis spectra were recorded at room temperature using a Shimadzu UV–vis spectrophotometer (model 1601). dc electrical conductivity measurements were performed by using a four-in-line probe electrical conductivity retention measuring instrument with a PID controlled oven (Scientific Equipment, Roorkee, India). The thermal stability of all as-prepared materials in terms of dc electrical conductivity under isothermal and cyclic ageing conditions was also studied. The equation used in the calculation of dc electrical conductivity was 3 where I, V, W, and S are the current (A), voltage (V), thickness of the pellet (cm), and probe spacing (cm), respectively, and σ is the conductivity (S/cm).11,38 In thee testing of isothermal stability, the pellets were heated at 50, 70, 90, 110, and 130 °C in an air oven, and the dc electrical conductivity was measured at particular temperatures at an interval of 5 min in the accelerated ageing experiments. 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PMC006xxxxxx/PMC6644414.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145893910.1021/acsomega.8b00962ArticleStatistics of the Auger Recombination of Electrons and Holes via Defect Levels in the Band Gap—Application to Lead-Halide Perovskites Staub Florian †Rau Uwe †Kirchartz Thomas †‡† IEK5-Photovoltaik, Forschungszentrum Jülich, 52425 Jülich, Germany‡ Faculty of Engineering and CENIDE, University of Duisburg-Essen, Carl-Benz-Str. 199, 47057 Duisburg, Germany E-mail: t.kirchartz@fz-juelich.de.18 07 2018 31 07 2018 3 7 8009 8016 10 05 2018 05 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. Recent evidence for bimolecular nonradiative recombination in lead-halide perovskites poses the question for a mechanistic origin of such a recombination term. A possible mechanism is Auger recombination involving two free charge carriers and a trapped charge-carrier. To study the influence of trap-assisted Auger recombination on bimolecular recombination in lead-halide perovskites, we combine estimates of the transition rates with a detailed balance compatible approach of calculating the occupation statistics of defect levels using a similar approach as for the well-known Shockley–Read–Hall recombination statistics. We find that the kinetics resulting from trap-assisted Auger recombination encompasses three different regimes: low injection, high injection, and saturation. Although the saturation regime with a recombination rate proportional to the square of free carrier concentration might explain the nonradiative bimolecular recombination in general, we show that the necessary trap density is higher than reported. Thus, we conclude that Auger recombination via traps is most likely not the explanation for the observed nonradiative bimolecular recombination in CH3NH3PbI3 and related materials. document-id-old-9ao8b00962document-id-new-14ao-2018-009626ccc-price ==== Body Introduction One of the key prerequisites for optoelectronic materials are long nonradiative lifetimes1−7 for recombination via defects as compared to the recombination coefficients for direct radiative band-to-band transitions.8−13 Recombination via defects14,15 is usually assumed to be mediated by the emission of multiple phonons,16−21 whereas band-to-band recombination via multiphonon emission is thought to be extremely unlikely in inorganic semiconductors.22 Because the transition rates are strongly reduced for an increasing number of phonons17,22 involved in a single transition at low-to-moderate strength of electron–phonon coupling, direct band-to-band recombination is typically assumed to be entirely radiative in inorganic semiconductors. This is different in organic semiconductors due to the higher energy associated with molecular vibrations in organic molecules relative to the energy of phonons in inorganic semiconductors.23 Given that lead-halide perovskites due to the high atomic mass of Pb and I have particularly low phonon energies,24,25 it is initially rather surprising that there is evidence26,27 for recombination terms that are quadratic in charge-carrier density (like radiative recombination in high-level injection) and are nonradiative. In addition to multiphonon recombination, Auger recombination is the second archetypal nonradiative recombination mechanism.13,28−30 Auger recombination involving two free electrons and one hole or two free holes and one electron, respectively, should be cubic in charge-carrier density in the high-level injection and would therefore not be able to explain the observed features. However, Auger recombination involving trapped charge carriers could in principle explain the observed quadratic behavior.26 Thus far, trap-assisted Auger recombination has been mainly discussed for the case of highly doped semiconductors29−31 where this mechanism is most efficient because of the high density of free charge carriers. This implies that only the limiting linear case of low injection (with respect to the doping level) is usually considered. In addition, it has been discussed in the context of determining limiting efficiencies for Si solar cells, but recently also for perovskite solar cells.13,28,30,32 To study the potential effect of trap-assisted Auger recombination on bimolecular recombination in lead-halide perovskites close to the radiative limit, a model for the full recombination statistics is required. Therefore, we develop a detailed balance compatible rate equation model in analogy to the Shockley–Read–Hall (SRH) recombination statistics.14,15 Subsequently, we use known material properties of the lead-halide perovskites and previously derived equations33 for the transition rates to estimate the Auger coefficients for trap-assisted Auger recombination in perovskites. Finally, we derive the necessary trap density for trap-assisted Auger recombination to explain the observed nonradiative contribution to bimolecular recombination. The necessary defect density is at least on the order of 1017 cm–3 for midgap defects and increases for more shallow defects. This defect density is about 1 order of magnitude higher than the defect densities that have so far been observed in the experiment.34,35 Thus, we conclude that the trap-assisted Auger recombination is unlikely to be the reason for the observed nonradiative bimolecular recombination and probably does not pose a fundamental limitation to the efficiency of the lead-halide perovskites. Results and Discussion Recombination Statistics The complete picture of the Auger processes involving traps as outlined in ref (33) is illustrated in Figure 1. In total, four processes can be summarized by four transition coefficients T1...T4:(1) two electrons e and an empty trap ht transform into an electron e* (at nonthermal energy) and a trapped electron et according to the reaction scheme: 2e + ht ↔ e* + et. (2) an electron, a hole h, and an empty trap transform into a hole h* (at nonthermal energy) and a trapped electron: e + h + ht ↔ h* + et. (3) an electron, a hole, and a trapped electron transform into an electron (at nonthermal energy) and an empty trap: e + h + et ↔ e* + ht. (4) two holes and a trapped electron transform into a hole (at nonthermal energy) and an empty trap: 2h + et ↔ h* + ht. Figure 1 Illustration of various interactions between free charge carriers and defect states resulting in electron capture (with coefficients T1, T2) and hole capture (T3, T4), respectively. Please note that the processes 2 and 3 consist of two possible interactions each. The energy levels of the valence and conduction bands are here denoted as EV and EC, respectively. Furthermore, Et marks the imperfection level (trap depth). Thus, processes 1 and 2 result in electron capture and processes 3 and 4 in hole capture such that a net recombination of an electron–hole pair requires a combination of steps 1 or 2 with 3 or 4. To fulfill the requirements of detailed balance, we also have to consider the back reactions, i.e., impact ionization of trapped electrons or holes via hot electrons or holes. The rates R1...4,b for the back reactions are determined by the Auger coefficients C1...4* such that we have, e.g., for back 1R1,b = C1nN, where n* denotes the concentration of hot electrons and N the concentration of filled traps. If we assume that the thermalization of charge carriers is faster than the Auger processes or their inverses, the ratio between n* and the overall concentration n of electrons is constant and corresponds to the ratio n0/n0 between both concentrations at thermal equilibrium. Thus, we define the coefficient C1 = C1n0/n0 and, analogously, C2 = C2p0/p0, C3 = C3n0/n0, and C4 = C4p0/p0 using p0, p0, p*, and p as the analogous variables for the holes. Then, the rates for the four transitions can be written as 1 2 3 and 4 Here, we use the abbreviations 5 and 6 where n0 and p0 are the equilibrium concentrations of the electrons and the holes. The concentrations n1 and p1 correspond to the values of n and p, when the Fermi level lies at the trap depth ET. We eliminate the parameters C1,...,C4 in eqs 1–4 by using the principle of detailed balance36 and expressing them as a product of T1,...,T4 multiplied with either n1 or p1 in analogy to the derivation of Shockley–Read–Hall statistics. By assuming steady-state conditions dN/dt = 0, we may eliminate the concentration N of filled traps by writing 7 We then obtain for the (normalized) density of occupied trap states 8 The final recombination rate RTA is subsequently given by inserting eq 8 into RTA = R1 + R2 = R3 + R4 and is given by 9 Simulation Results Figure 2a illustrates eq 9 as a function of the excess charge-carrier concentration Δn = n – n0 using the parameters given in Table 1. The rate of Auger recombination involving interactions with defect states exhibits three regimes with different dependencies of RTA on the excess charge-carrier concentration Δn. We assume the semiconductor to be p-type (n0 ≪ p0) in this example (doping density NA = 3 × 1015 cm–3),37 thus, for Δn ≪ NA, we are in low-level injection conditions. In addition, we assume the defect to be close to the conduction band, i.e., n1 ≫ p ≫ p1. In this case, eq 9 simplifies to 10 i.e., the recombination rate scales linearly with Δn = n. This linear scaling is independent of the position of the trap and would also happen for a midgap trap, in which case, the rate would be 11 For higher excess charge densities, Δn > NA, we enter high-level injection conditions, where we may simplify eq 9 using the conditions n1 ≫ n = p = Δn ≫ p1 (for a trap close to the conduction band edge). Then, we obtain 12 i.e., a cubic relation between the rate and the excess carrier concentration. Only in the saturation regime, where n = p = Δn ≫ n1 ≫ p1, the recombination rate 13 starts to scale quadratically with Δn. Thus, the three regimes visible in Figure 2a differ from the situation encountered for Auger recombination of free charge carriers. Here, also three regimes are visible, but the order for the Auger recombination rate RA (for free carriers) is RA ∼ Δn (for low-level injection), RA ∼ Δn2 (for n ≈ p), and RA ∼ Δn3 for high-level injection. In contrast, the Auger recombination via traps features an intermediate cubic scaling law as long as the doping concentration is smaller than either n1 or p1. To estimate the magnitude of trap-assisted Auger recombination for the specific case of lead-halide perovskites, we use equations for the transition coefficients T1,...,T4 derived by Landsberg et al.,33 which are given by 14 where Table 2 provides the values for the abbreviations Ni, di, and bi for i = 1,...,4. Figure 2 (a, c) Recombination rate and (b, d) effective lifetime as a function of charge-carrier concentration. (a) The situation of a shallow trap (ET = 0.1 eV) with the solid gray line indicating the total trap-assisted Auger recombination and the dotted lines illustrate the approximations given by eqs 10, 12, and 13 valid in three different injection regimes. (c) The equivalent plot for the case of a midgap trap, which only shows a linear regime at low Δn and a quadratic regime at high Δn. (b) The effective lifetime τeff for Auger recombination via traps assuming a trap density NT = 1018 cm–3 and the radiative lifetime τrad = Δn/Rrad assuming Rrad = kradnp, with krad = 10–10 cm3/s. (d) The effective lifetime for a deep trap. For comparison, we also added the values for SRH recombination using capture coefficients determined as discussed in ref (22) and using different trap densities that are lower than the one for trap-assisted Auger recombination. Table 1 Input Parameters Used for the Calculations Presented in Figures 2 and 3 if Not Otherwise Stateda band gap Eg 1.6 eV effective mass of electrons me 0.2m0 effective mass of holes mh 0.2m0 relative permittivity εr 33.524 a There are various reported values for the effective mass of electrons and holes in the literature that are ranging from ∼0.1 to ∼0.3.37,49−53 Here, we use a value of 0.2m0 for simplicity, where m0 is the electron rest mass. Table 2 Definition of the Abbreviations Used To Determine the Trap-Assisted Auger Recombination Coefficients Ti Using Equation 15a i Ni di1 di2 bi 1 13 –260 2 0.5(33 – 15σL) 0.5(33 – 15σL) 3 4 a Here, Eg is the band gap, Et is the trap depth with respect to the conduction band, σ = mh/me is the ratio of the hole and electron effective mass, and σL = 1/σ. In this form, the notation is valid for free electrons in the conduction band interacting with defect states. For free holes in the valence band, Et, σ, and σL have to be adjusted accordingly. Figure 2b shows the resulting effective lifetime τeff for the trap-assisted Auger recombination assuming a high trap density of 1018 cm–3. We define the effective lifetime for a given process as τeff = Δn/R, where R is the recombination rate for a certain recombination mechanism. The effective lifetime τTA for the trap-assisted Auger recombination is therefore defined as τTA = Δn/RTA, whereas the effective radiative lifetime is τrad = Δn/Rrad. Here, Rrad is the radiative recombination rate. We note that for low-excess charge-carrier concentrations Δn, both the trap-assisted Auger recombination and radiative recombination lead to a constant effective lifetime, consistent with the recombination rate increasing linearly with Δn in both cases. Once Δn exceeds the doping concentration assumed to be NA = 3 × 1015 cm–3, the Auger lifetime drops drastically until Δn ≈ n1, at which point the slope gets flatter again. This is the logical consequence of the three regimes for RTA seen in Figure 2a. We also note that even for such a high trap density of NT = 1018 cm–3, the radiative lifetime is lower for all values of Δn. Figure 2c,d show the situation of a deep trap first for the recombination rate (c) and subsequently for the effective lifetime (d). For deep traps, n1 is very small (n1 ≪ NA), thus there exist only two regimes (low injection and high injection), but no saturation regime. In consequence, the effective lifetimes for radiative and trap-assisted Auger recombination have the same shape. For the assumed values of krad and NT, the values for τTA and τrad are quite similar. In Figure 2d, we also show for comparison the effective Shockley–Read–Hall lifetime via a deep trap. We do these calculations based on the theory of multiphonon recombination discussed in ref (22) and based on refs (17) and (18). Here, we observe that for trap densities already much lower than used for Auger recombination via traps, the SRH lifetime dominates over a wide range of Δn values. Having established the general features of the recombination statistics of Auger recombination involving traps, we now want to investigate more closely under which circumstances the recombination rate is comparable to the radiative recombination rate. In particular, we are interested in the case described by eq 13, where the scaling is quadratic. In this scenario, we may define the total bimolecular recombination rate Rbm as 15 i.e., the sum of radiative recombination (kradn2), minus the amount of light that is reabsorbed and contributes to the internal generation (prkradn2, with pr being the probability of reabsorption37,38) plus the trap-assisted Auger recombination. Figure 3a illustrates the effective Auger coefficient CT ≡ kTA/NT as a function of trap depth ET. As it is the case for Shockley–Read–Hall statistics, also the Auger recombination rate via defects shows its maximum for midgap traps, when detrapping of captured charge carries is least likely. The vertical dashed lines represent intrinsic defect levels according to the density functional theory calculations as reported in ref (39). In the next step, we evaluate the defect density NT, which has to be present in the samples to cause a certain nonradiative bimolecular recombination coefficient knon. Table 3 shows the experimental data from various groups on the total, radiative, and nonradiative bimolecular recombination coefficient. The values for the nonradiative recombination coefficient vary quite strongly. Therefore, we vary knon in the range between 10–12 and 10–10 cm3/s and show the necessary trap density as a function of trap position in Figure 3b. In comparison, we show the necessary trap depth to achieve a monomolecular Shockley–Read–Hall type lifetime τSRH = 1 μs assuming the multiphonon transitions as discussed in ref (22). We judge from Figure 3b that deep trap densities >1017 cm–3 are needed for midgap traps and higher for shallower traps. Although such trap densities at midgap would lead to very short SRH lifetimes that are not consistent with experiment, such trap densities may explain the experimental data if the trap is not midgap but at a trap depth of around 0.5–0.6 eV away from either the conduction or valence band. To put these densities into context, Table 4 compares the trap densities that have been measured on CH3NH3PbI3 in the literature. Table 4 suggests that so far most experimentally observed trap densities are in the range of 1015–1016 cm–3, i.e., in a range that would not lead to substantial trap-assisted Auger recombination. Figure 3 (a) Coefficient CT of trap-assisted Auger recombination as a function of the trap depth ET with respect to the conduction band edge EC. Vertical dashed lines represent trap depths of various intrinsic defects according to ref (39). (b) Trap density NT as a function of trap depths ET that would be needed to cause a nonradiative bimolecular recombination coefficient of 10–11 and 10–10 cm3/s (light blue and dark blue curves, respectively). In addition, we added the trap density needed to achieve a 1 μs monomolecular lifetime as calculated in ref (22) based on the theory of multiphonon Shockley–Read–Hall recombination. Table 3 Values for the Bimolecular Recombination Coefficients in Units of cm3/s of CH3NH3PbI3 from Literature references kext (cm3/s) krad (cm3/s) knon (cm3/s) (37) 4.78 × 10–11 8.7 × 10–10 neglected (27, 37)a 4.78 × 10–11 8.4 × 10–11 4.4 × 10–11 (54)b 1.4 × 10–10 to 2 × 10–11 6.8 × 10–10 0 (26)c 8.1 × 10–11 7.1 × 10–11 7.2 × 10–11 (26)d 7.9 × 10–11 1.8 × 10–10 5.6 × 10–11 a Data from ref (37) corrected using the evidence from ref (27) that only 66% of the bimolecular recombination is radiative. b Measurements done at different thicknesses. c Samples with PbI2 precursor. d Samples made from PbCl2 precursor. Table 4 Trap Densities Reported for CH3NH3PbI3 Thin Films in the Literature reference method trap density (cm–3) trap depth ET (eV) (34) thermally stimulated current (TSC) >1015 ∼0.5 (55) noise spectroscopy 4 × 1015 ∼0.8 (56) deep level transient spectroscopy (DLTS) ∼1015 0.62 ∼1015 0.75 (57) steady state photocarrier grating >1016 recombination center (58) TSC 9 × 1016 0.18 5 × 1016 0.49 (59) admittance spectroscopy ∼1016 cm–3 0.16 (60) admittance spectroscopy ∼1016 cm–3 a 0.27 ∼1017 cm–3 b 0.28 (35) DLTS 9 × 1013 to 5 × 1014 cm–3 0.78 a Dimethylformamide as solvent with hydroiodic acid as additive. b Dimethyl sulfoxide as solvent. Discussion and Outlook Having established that Auger recombination via traps is not likely to explain the observed nonradiative contributions to bimolecular recombination, it is useful to discuss the implications of this result and consider alternative explanations for the observed trends. First, we want to state that most experimental approaches to study bimolecular recombination would not be sensitive to whether bimolecular recombination is radiative or nonradiative. In most cases, the transient decay is fitted with a model that accounts for bimolecular recombination. In the case of transient photoluminescence experiments, it is clear that some of this recombination has to be radiative to generate a signal, but the determination of how much of this bimolecular recombination is radiative would require additional information. This information can either be another experiment that determines the total recombination as done by Richter et al.26 or some experimental circumstances that would distinguish radiative from nonradiative such as the sensitivity of radiative recombination to parasitic absorption via the modulation of the photon recycling probability as done previously by us.27 Certainly, two sets of experimental evidence we are aware of are not very much and future will tell whether these results are reproduced by others or are a peculiar feature of the samples or the data analysis in these articles. However, assuming that the data are representative, we want to briefly explore what other explanations there might be. In a simple zero-dimensional picture, the authors are not aware of any recombination mechanism that would be quadratic in charge-carrier density and still be nonradiative other than the one discussed here. Thus, it is logical to explore effects requiring more dimensions. There are essentially two options in our opinion: (i) diffusion of carriers at high-level injection inside the films leads to a decrease in signal that could be interpreted as a recombination but is not or (ii) lateral inhomogeneous lifetimes lead to a distribution of decay times that creates a decay that appears quadratic in the charge-carrier density but is not related to locally quadratic recombination mechanisms. Let us briefly look into the two options in more detail. After the excitation of a sample with a laser pulse, electrons and holes are created based on the generation profile of the sample. Disregarding interferences, it is clear that more electrons and holes would be created close to the front surface of the sample and less toward the back. As long as the electron and hole concentrations are roughly equal, the luminescence is higher than after equilibration of the charge-carrier distribution. This leads to a decay in the luminescence that requires no recombination to happen. Diffusion of charge carriers during a transient photoluminescence experiment has been taken into account in the classical theory articles on transient photoluminescence experiments,40,41 in first experiments on perovskite films with contact layers,42 and in the case of transient experiments on perovskite crystals.43,44 However, given the range of mobilities that is commonly reported,45,46 diffusion and equilibration of charge carriers in a film of few hundreds of nanometer thickness should happen in the sub-nanosecond range (e.g., ∼300 ps for a 300 nm film and a mobility of 20 cm2/(V s)). Thus, for this explanation to affect the experiments, there have to be at least some charge carriers with a substantially lower mobility to see an effect on the time scale where bimolecular recombination is typically observed. The second option is laterally inhomogeneous lifetimes that have been reported and studied in a range of publications.47,48 If by averaging over a certain area, the macroscopic decay curve does not capture one lifetime but a broad distribution of lifetimes, the tail of this distribution toward shorter lifetimes could influence the shorter time scales of a transient photoluminescence experiment and thereby affect the way we interpret the data. Future work of modeling the photoluminescence in two or three dimensions will have to show whether this is a likely explanation for the observed data in refs (26, 27). Both of these alternative approaches to explain the experimental data would not be a unique feature of every MAPI sample, but instead could vary from sample to sample. They would therefore not contradict reports6,7 of extremely high photoluminescence quantum efficiencies that leave little room for additional nonradiative pathways. Conclusions In summary, we have developed a rate model for the full recombination statistic of electrons and holes via trap-assisted Auger recombination. The model covers the linear low injection case with a recombination rate RTA ∝ Δn proportional to the density of excess charge carriers Δn, as well as two nonlinear situations, namely, high-level injection with RTA ∝ Δn3 and the saturation situation with RTA ∝ Δn2. The latter case represents the bimolecular recombination that directly competes with radiative recombination. As an example, our calculations following the theory of ref (33) yield the actual rates for the case of CH3NH3PbI3. The coefficients for this specific case are however relatively small. Therefore, we conclude that this mechanism is not likely to be the origin of the experimentally measured nonradiative bimolecular recombination coefficients. The authors declare no competing financial interest. Acknowledgments T.K. and U.R. acknowledge support from the DFG (Grant Nos. KI-1571/2-1 and RA 473/7-1) and from the Helmholtz Association via the PEROSEED project. T.K. thanks Igal Levine (Weizmann, Rehovot) for sharing most of the references for Table 4. ==== Refs References Correa-Baena J. P. ; Saliba M. ; Buonassisi T. ; Grätzel M. ; Abate A. ; Tress W. ; Hagfeldt A. Promises and Challenges of Perovskite Solar Cells . Science 2017 , 358 , 739 10.1126/science.aam6323 .29123060 Tress W. Perovskite Solar Cells on the Way to Their Radiative Efficiency Limit – Insights Into a Success Story of High Open-Circuit Voltage and Low Recombination . Adv. Energy Mater. 2017 , 7 , 160235810.1002/aenm.201602358 . Ahrenkiel R. K. Minority-Carrier Lifetime in III–V Semiconductors . In Minority Carriers in III–V Semiconductors: Physics and Applications ; Elsevier : 1993 ; Vol. 39 , pp 39 –150 . Sinton R. A. ; Cuevas A. 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PMC006xxxxxx/PMC6644415.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145782310.1021/acsomega.7b00928ArticleDesign of Polyproline-Based Catalysts for Ester Hydrolysis Hung Pei-Yu †Chen Yu-Han †Huang Kuei-Yen †Yu Chi-Ching †Horng Jia-Cherng †‡†Department of Chemistry and ‡Frontier Research Center on Fundamental and Applied Science of Matters, National Tsing Hua University, 101 Sec. 2 Kuang-Fu Rd., Hsinchu, Taiwan 30013, ROC E-mail: jchorng@mx.nthu.edu.tw. Phone: +886-3-5715131 ext. 35635. Fax: +886-3-5711082 (J.-C.H.).07 09 2017 30 09 2017 2 9 5574 5581 05 07 2017 24 08 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A number of simple oligopeptides have been recently developed as minimalistic catalysts for mimicking the activity and selectivity of natural proteases. Although the arrangement of amino acid residues in natural enzymes provides a strategy for designing artificial enzymes, creating catalysts with efficient binding and catalytic activity is still challenging. In this study, we used the polyproline scaffold and designed a series of 13-residue peptides with a catalytic dyad or triad incorporated to serve as artificial enzymes. Their catalytic efficiency on ester hydrolysis was evaluated by ultraviolet–visible spectroscopy using the p-nitrophenyl acetate assay, and their secondary structures were also characterized by circular dichroism spectroscopy. The results indicate that a well-formed polyproline II structure may result in a much higher catalytic efficiency. This is the first report to show that a functional dyad or triad engineered into a polyproline helix framework can enhance the catalytic activity on ester hydrolysis. Our study has also revealed the necessity of maintaining an ordered structure and a well-organized catalytic site for effective biocatalysts. document-id-old-9ao7b00928document-id-new-14ao-2017-009288ccc-price ==== Body Introduction Natural enzymes, exhibiting powerful efficiency and remarkable selectivity because of their unique folded structures, are usually involved in biochemical reactions. They play a major part in maintaining the functions in organisms. For instance, proteases are capable of accelerating the digestion of proteins into shorter fragments by hydrolyzing the peptide bonds. However, restricted to their length and complexity, most enzymes are difficult to acquire and vulnerable to environmental conditions, such as pH and temperature. Thus, artificial enzymes have received much attention in recent years because of their great stability and easy accessibility. Baker et al. and Korendovych et al. explored the efficient catalysts with rigid protein scaffolds by computational design.1,2 Ulijn et al. conducted phage display as a discovery technique for selecting active sequences.3 To demonstrate more details on the importance of three-dimensional structures, many artificial enzymes using various scaffolds, such as α-helical-coiled coils and barrels,4−6 β3-peptide bundles,7 short helices,8 and β-hairpins,9,10 were reported. Moreover, nanomaterials11,12 and self-assembled structures including membranes,13 amyloid fibers,14−17 and hydrogels18 were implemented for mimicking similar protein structures; yet, the activity of these supramolecular frameworks is still limited compared to natural enzymes. In natural enzymes, their active site is frequently composed of several residues, taking responsibility for specific binding and chemical catalysis. For example, chymotrypsin, a serine protease, possesses a hydrophobic pocket for binding to aromatic substrates and a catalytic site composed of His57, Asp102, and Ser195 residues.19 It is generally accepted that more than two amino acid residues that operate together at the active site of enzymes are regarded as a catalytic dyad or triad. Mechanically, nucleophilic residues (cysteine and serine) play a role of attacking the substrate; histidine acts as a general base to deprotonate the nucleophile; and acidic residues (aspartic acid and glutamine acid) function to polarize and align the base (Figure 1A).20,21 Additionally, it is well-documented that the guanidyl group in the arginine residue22 and the backbone NH group1,23,24 can aid in stabilizing the transition state of the substrate. These functional residues were initially applied to the primary sequence design25 and subsequently considered in the coassembly system.15,16,22 Figure 1 (A) Schematic diagram of the catalytic triad in a protease. (B) Cartoon representation of a PPII helix with His incorporated into the i position. Inspired by the previous reports, here we chose polyproline peptides as the scaffold and introduced a catalytic dyad or triad into the peptides. It was found that proline-rich sequences in proteins often form a polyproline II (PPII) structure, a left-handed helix with all trans peptide bonds, and three residues in each turn. On account of their structural stability and rigidity, oligoproline peptides were widely used as rulers and scaffolds for various studies.26,27 Hence, we designed and synthesized a series of 13-residue polyproline peptides containing a histidine residue at the i position and other residues with a functional side chain, such as Asp, Ser, and Cys, at the i + 3 or i – 3 position, as minimalistic biocatalysts (Figure 1B). According to the structure of a PPII helix, these positions would be located on the same side, and we predicted that the imidazole side chain of His could place an appropriate orientation and form strong interactions with the functional groups at position i – 3 or i + 3 to form a dyad or triad active site. Additionally, (2S,4R)-hydroxyproline (Hyp) and (2S,4R)-methoxyproline (Mop), 4-substituted proline derivatives, were found to have a high propensity to form PPII conformation because of the stereoelectronic effects.28,29 Therefore, they were also used to replace the regular proline residues for understanding the correlation between PPII structure and enzyme activity. The results showed that the interactions between different functional groups significantly contributed to the catalytic ability and that a well-formed PPII structure could lead to a higher catalytic efficiency. Results and Discussion To explore the activity of polyproline-based catalysts on ester hydrolysis, we chose (Pro)11 as the control peptide (free P11) and designed a series of peptides in which the specific position was substituted with His and the residues frequently found in the active sites of natural enzymes. For a single His-incorporated peptide (H6), the His residue was placed in the middle (position 6) of the peptide because the conformation of His might be fixed by the proline-rich sequence. In the multiple substitution peptides, His residues were incorporated into position 6, whereas nucleophilic (Cys, Ser, and His), neutral (Trp), and acidic (Asp) residues were placed at positions 3 and 9 to mimic the triad or dyad in natural enzymes. These peptides were designated as H3H6, W3H6, S3H6, S3H6D9, and C3H6D9. In addition, the peptides with Pro replaced by Hyp (Hyp-H3H6 and Hyp-C3H6D9) or Mop (Mop-H3H6) were applied to investigate the relationship between PPII-forming propensity and catalytic efficiency. A Gly–Tyr dipeptide was attached to the C-terminus of each peptide for concentration determination. Furthermore, all peptides except for free P11 have capped ends because a previous study found that the removal of the capping groups would reduce the catalytic activity.14 The sequences of designed peptides are shown in Table 1. Table 1 Peptide Sequences Used in This Study peptide sequencea free P11 NH-PPPPPPPPPPPGY-OH H6 Ac-PPPPPHPPPPPGY-NH2 W3H6 Ac-PPWPPHPPPPPGY-NH2 H3H6 Ac-PPHPPHPPPPPGY-NH2 S3H6 Ac-PPSPPHPPPPPGY-NH2 S3H6D9 Ac-PPSPPHPPDPPGY-NH2 C3H6D9 Ac-PPCPPHPPDPPGY-NH2 Hyp-H3H6 Ac-OOHOOHOOOOOGY-NH2 Hyp-C3H6D9 Ac-OOCOOHOODOOGY-NH2 Mop-H3H6 Ac-O′O′HO′O′HO′O′O′O′O′GY-NH2 a Ac indicates an acetylated N-terminus and NH2 indicates an amidated C-terminus; Hyp and O are the abbreviations for (2S,4R)-hydroxyproline; and Mop and O′ are the abbreviations for (2S,4R)-methoxyproline. After synthesis of the peptides, circular dichroism (CD) spectroscopy was utilized to characterize their secondary structures. As shown in Figure 2, all peptides exhibit a similar far-UV spectrum with a positive band between 220 and 230 nm and a negative peak near 205 nm, indicating the presence of PPII conformation. We used the maximal molar ellipticity at the positive band to evaluate their PPII-forming propensity and content. As shown in Figure 2 and Table 2, the replacement of proline to nonproline residues causes the decrease in the PPII structure. Compared to free P11, all peptides with Pro replaced by other residues display a much weaker positive band, and the multiple substitutions impose an even greater effect on PPII conformation than does the single substitution. Although the peptides with double- and triple-substituted nonproline residues show a much weaker characteristic CD signal of the PPII structure, W3H6 displays a stronger positive band around 230 nm due to the electronic absorption of aromatic residues.30,31 Therefore, the PPII propensity of W3H6 could not be evaluated solely by its ellipticity at the positive band. As expected, Hyp-H3H6, Hyp-C3H6D9, and Mop-H3H6 form a relatively intense PPII structure, and their molar ellipticity in the positive band is close to or even higher than that of free P11 (Figure 2B and Table 2), which can be attributed to that Hyp and Mop prefer a Cγ-exo pucker and a trans peptide bond to enhance PPII-forming propensity.28,29 Figure 2 Far-UV CD spectra for the designed peptides: (A) H6, W3H6, S3H6, and S3H6D9 and (B) H3H6, C3H6D9, Hyp-H3H6, Hyp-C3H6D9, and Mop-H3H6 at pH 7.4 and 25 °C. The spectrum of free P11 is included for comparison. Table 2 CD Parameters for the Peptides at 25 °C peptide λmax (nm) [θ]max (103 deg cm2 dmol–1) free P11 228 3.37 H6 228 1.66 W3H6 229 2.75 H3H6 229 0.86 S3H6 229 0.75 S3H6D9 231 0.43 C3H6D9 231 –0.18 Hyp-H3H6 226 6.00 Hyp-C3H6D9 226 4.62 Mop-H3H6 226 3.12 To evaluate the catalytic efficiency of our designed peptides and compare the results with the previous hydrolase designs, we chose p-nitrophenyl acetate (p-NPA) as a simple chromogenic substrate to monitor the catalytic reaction. The catalytic reaction is as follows: The initial hydrolytic rate (V0) was determined by monitoring the production of 4-nitrophenol at 405 nm and pH 7.4 with ultraviolet–visible (UV–vis) spectroscopy. As shown in Figure 3A,B, the dependence of the initial hydrolytic rate on the substrate concentration for each peptide is consistent with the Michaelis–Menten model, and the kinetic parameters can be obtained and calculated by fitting the curves to the model. The determined parameters are listed in Table 3. The reaction in the presence of free histidine residues was also monitored and used as a control. Figure S3 in the Supporting Information indicates that the rate of hydrolysis of p-NPA in the presence of free P11 was extremely low and close to that without any peptides or amino acids present in solution, suggesting that the polyproline backbone was unable to catalyze the reaction. This is likely attributed to the fact that free P11 does not contain nucleophiles to accelerate the reaction, and the proline residues lack NH groups on the backbone to stabilize the oxyanion intermediate of ester hydrolysis as suggested by previous studies.1,23,24 Figure 3 (A–C) Esterase activity for the designed peptides at pH 7.4 and 25 °C, with solid lines showing the fits to the Michaelis–Menten equation. (D) Esterase activity for the selected peptides under different pH conditions, with solid lines showing the fits to the modified Henderson–Hasselbalch equation (eq 1). Table 3 Kinetic Parametersa for p-NPA Hydrolysis by Designed Peptides at 25 °C and pH 7.4 peptide kcat (10–3 s–1) KM (mM) kcat/KM (M–1 s–1) free His (H-His-OH) 0.78 ± 0.16 1.73 ± 0.49 0.45 ± 0.16 free P11 0.28 ± 0.06 0.88 ± 0.32 0.31 ± 0.13 H6 0.70 ± 0.05 1.81 ± 0.17 0.39 ± 0.05 W3H6 1.15 ± 0.38 1.92 ± 0.85 0.60 ± 0.33 H3H6 2.44 ± 0.73 4.38 ± 1.50 0.56 ± 0.25 S3H6 0.86 ± 0.33 1.52 ± 0.82 0.56 ± 0.37 S3H6D9 0.97 ± 0.24 2.12 ± 0.69 0.46 ± 0.19 C3H6D9 1.05 ± 0.27 2.44 ± 0.78 0.43 ± 0.18 Hyp-H3H6 1.76 ± 0.16 1.42 ± 0.19 1.23 ± 0.20 Hyp-C3H6D9 1.73 ± 0.15 1.75 ± 0.20 0.99 ± 0.14 Mop-H3H6 1.88 ± 0.67 3.02 ± 1.30 0.62 ± 0.35 a Deviations of kcat and KM were the standard errors of fitting and were used to calculate the deviation of kcat/KM by the equation . To prevent the electrostatic repulsions and extra charges from affecting the catalytic efficiency, we introduced end caps by acetylating the N-terminus and amidating the C-terminus on the following peptides: H6, W3H6, H3H6, S3H6, S3H6D9, and C3H6D9. Of the designed peptides, His, Trp, Ser, Asp, or Cys was incorporated in the multiple substitution peptides for the purpose of forming catalytic dyads or triads. As shown in Table 3, the catalytic efficiency of the H6 peptide (kcat/KM is 0.39 M–1 s–1) is lower than that of free His (kcat/KM is 0.45 M–1 s–1), indicating that the single His incorporation in a polyproline peptide could not induce an effective electron transfer to catalyze the reaction. By contrast, the polyproline peptides with double substitutions (H3H6, S3H6, and W3H6) are 1.5-fold more active in comparison with H6, suggesting that the residues (His, Ser, and Trp) at position 3 of H3H6, S3H6, and W3H6 could interact with His at position 6 to facilitate the electron transfer and enhance the catalytic properties. Therefore, it could be inferred that all peptides with double substitutions may form a catalytic dyad in their active site. As detailed in Table 3, W3H6 is more efficient than H3H6 and S3H6 and has a kcat/KM value of 0.60 M–1 s–1. It could be explained by an increased nucleophilicity of His because of the histidine–aromatic interaction provided by Trp at position 3, which was also observed in barnase, a natural hydrolase.32 To validate our assumption that the PPII structure can facilitate a proper orientation between the side chains of His and Trp and generate strong interactions to enhance the catalytic activity, we prepared a dipeptide Trp–His (WH) and measured its catalytic efficiency. As shown in Figure S4 of the Supporting Information, its catalytic efficiency (kcat/KM is 0.52 M–1 s–1) is lower than that of W3H6, providing a piece of evidence that the PPII framework does play a positive role to increase its catalytic activity. Surprisingly, the catalytic activity of the peptides with triple substitutions slightly decreased compared with those with double substitutions. Their catalytic activity of the hydrolyzing p-NPA is around 0.45 M–1 s–1. It seems that introducing more residues into the PPII structure could not always benefit their catalytic abilities. As shown in Table 2, the peptides with triple substitutions (S3H6D9 and C3H6D9) dramatically reduce the propensity of forming a PPII structure, which may lead to an increased distance between the i and i + 3/i – 3 positions. Thus, it could be assumed that the catalytic triad may not be successfully formed in S3H6D9 and C3H6D9, and Asp cannot play a role as orienting the His residue and neutralizing the intermediates. To demonstrate more structure–activity relationships, we replaced the proline residues of H3H6 and C3H6D9 with Hyp and used Mop to substitute for the proline of H3H6 as well to form a more robust PPII structure. Interestingly, as shown in Figure 3B, we found that both Hyp-H3H6 and Hyp-C3H6D9 remarkably accelerated the hydrolysis of p-NPA. An approximately threefold increase in catalytic efficiency was observed for Hyp-H3H6 compared with free His residue. Both of the Hyp-H3H6 (kcat/KM is 1.23 M–1 s–1) and Hyp-C3H6D9 (kcat/KM is 0.99 M–1 s–1) exhibited a twofold higher efficiency than that of their corresponding peptides H3H6 (kcat/KM is 0.56 M–1 s–1) and C3H6D9 (kcat/KM is 0.43 M–1 s–1), respectively. As shown in Table 3, the peptides with Hyp-rich sequences show a relatively stronger binding affinity (lower KM) for the substrate than do their Pro-rich counterparts, leading to a much higher catalytic efficiency. It is likely that Hyp residues provide extra interactions and result in a stronger binding to the substrates. As reported in a few natural active sites, an oxyanion hole could stabilize the transition state of the substrate.1,23,24 Therefore, we believe that the OH group on the Hyp side chain could potentially serve as a hydrogen bond donor to stabilize the reaction intermediate and increase the reaction rate. This argument may also be supported by the observation that Mop-H3H6 exhibits a slightly higher activity (kcat/KM is 0.62) than H3H6 but a much lower activity than Hyp-H3H6 (Figure 3C). Because the OMe group on the Mop side chain cannot be a hydrogen bond donor, Mop cannot form similar hydrogen bonding interactions as Hyp does during the reaction, leading to a lower catalytic efficiency. In addition, the fact that Hyp-H3H6, Hyp-C3H6D9, and Mop-H3H6 have a higher catalytic efficiency than their corresponding peptides (H3H6 and C3H6D9) demonstrates the important role of a PPII structure. As aforementioned, the peptides containing Hyp or Mop residues favor the formation of a stable PPII structure, which may bring about a closer distance between the i and i + 3/i – 3 positions. As a result, it could be speculated that the PPII helix-forming propensity of our designed peptides also has a great impact on their catalytic activity. Although the efficiency of our designed peptides is not comparable to that of natural enzymes in catalyzing such an ester hydrolysis reaction, most of our designed polyproline-based peptides still exhibited a greater catalytic activity than some reported peptide-based nanostructures whose kcat/KM ranges from 0.09 to 0.62 M–1 s–1 at pH 7.5.12,15,16 Most notably, even as a short peptide, the activity of Hyp-H3H6 is better or comparable to the previously reported metal-based esterases (kcat/KM is 0.33–1.38 M–1 s–1 at pH 7.5).5 Because the protonated and deprotonated state of an active site would affect its catalytic activity, we investigated the hydrolysis reaction under different pH conditions for the selected peptides: W3H6, H3H6, S3H6D9, and Hyp-H3H6. As illustrated in Figure 3D, while the efficiency of these peptides remained low below pH 8.0, a noticeable increase could be observed as the pH values rose beyond 8.0. The catalytic efficiency (kcat/KM) of each selected peptide at pH 10.0 is in the range of 23–30 M–1 s–1, which is 30-fold greater than that at pH 7.4 (Table S3 in the Supporting Information). Using eq 1, a pair of kinetic pKa could be determined for each selected peptide (Table 4). As the typical pKa of a His imidazole ring is about 6, it is likely that the obtained pKa values around 6.5 represented deprotonation of a His side chain. Besides, we would suggest that the higher pKa reflected deprotonation of the Tyr side chain because its pKa is approximately 10.3 in the free form. We further used 1H NMR spectroscopy to measure the pKa of a His side chain in S3H6D9. A series of high-resolution one-dimensional (1D) 1H NMR spectra for S3H6D9 were acquired at pH values ranging from 4 to 8 (Figures 4A and S5 in the Supporting Information), whereas a titration curve was obtained by plotting the chemical shifts of His C2H versus pH (Figure 4B). By fitting the titration curve into the Henderson–Hasselbalch equation (eq 2), a pKa value of 6.64 and a Hill coefficient of 1.6 could be determined for S3H6D9. The pKa value measured by NMR is in good agreement with the pKa value derived by the kinetic hydrolysis assay, reflecting that the deprotonation of the His imidazole group could significantly increase the catalytic efficiency. Figure 4 (A) Representative high-resolution 1D 1H NMR spectra of peptide S3H6D9, with the C2H peak of His labeled with an “*”. (B) Titration curve for the peptide S3H6D9 produced from 1D 1H NMR titration experiments, with a solid line showing the fit to the Henderson–Hasselbalch equation (eq 2). Table 4 Kinetic pKa and Maximal Efficiency for p-NPA Hydrolysis by Selected Peptides peptide pKa1 pKa2 kcat/KM(max)1 (M–1 s–1) kcat/KM(max)2 (M–1 s–1) W3H6 6.73 ± 0.18 9.83 ± 0.01 0.50 ± 0.06 47.93 ± 0.49 H3H6 6.30 ± 0.43 9.74 ± 0.01 0.32 ± 0.07 42.36 ± 0.49 S3H6D9 6.27 ± 0.44 9.91 ± 0.03 0.29 ± 0.09 42.46 ± 1.39 Hyp-H3H6 6.74 ± 0.93 9.92 ± 0.11 0.81 ± 0.46 53.78 ± 5.93 Furthermore, to have more insights into the structure of designed peptides, we conducted Hartree–Fock (HF) calculations on the conformation of oligopeptides. As it was found that Ac-(Pro)5-OMe is the simplest peptide model to form a stable PPII structure,33 we used the seven-residue oligopeptide models, Ac-(Pro)2Ser(Pro)2HisPro-OMe, Ac-Cys(Pro)2His(Pro)2Asp-OMe, and Ac-(Pro)7-OMe, to simplify the computational studies, in which Ac-(Pro)2Ser(Pro)2HisPro-OMe was used to mimic S3H6, whereas Ac-Cys(Pro)2His(Pro)2Asp-OMe was utilized as a model to mimic C3H6D9. In the energy-minimized structure, the dihedral angles of Ac-(Pro)2Ser(Pro)2HisPro-OMe and Ac-Cys(Pro)2His(Pro)2Asp-OMe are similar to those of Ac-(Pro)7-OMe and an idealized PPII helix, indicating that this peptide could form a PPII-like helix (Table S4 in the Supporting Information). For an ideal PPII helix, every third residue is about 9 Å apart, suggesting that the distance between the two side chains at positions i and i + 3 should be less than 9 Å to form possible interactions. As shown in Figure S6 of the Supporting Information, the distance between the side chains of Ser and His in the energy-minimized structure of Ac-(Pro)2Ser(Pro)2HisPro-OMe is 5.4 Å, which is significantly shorter than 9 Å, suggesting that the functional groups of Ser and His might interact and form a reaction dyad to catalyze ester hydrolysis. This finding may in part explain why S3H6 exhibits a better activity than H6. For the optimized structure of Ac-Cys(Pro)2His(Pro)2Asp-OMe (Figure 5), the distance between Cys and His is 2.90 Å, whereas the distance between His and Asp is up to 6.7 Å. In comparison, the distances within the catalytic triad of chymotrypsin, a serine protease, are shorter than 3 Å,19 which facilitates the proton transfer between them. It could be inferred that the functional groups in the triple substitution peptide, C3H6D9, might not form a catalytic triad because the long distance between His and Asp could hinder them from forming an appropriate orientation between the functional groups. Therefore, the observation in the computational structure may rationalize why the triple substitution peptides exhibited a less catalytic efficiency than did the double substitution peptides. Figure 5 Energy-minimized structure model for Ac-Cys(Pro)2His(Pro)2Asp-OMe representing the peptide C3H6D9. The distances between the side chains of Cys, His, and Asp are indicated in the diagram. Conclusions In the current work, a series of polyproline peptides were designed and prepared to form PPII helices and serve as a scaffold to catalyze ester hydrolysis. We found that introducing a pair of residues containing His into the PPII structure could successfully imitate a catalytic dyad and improve the catalytic efficiency. However, introducing more than two nonproline residues could dramatically perturb the PPII structure and failed to form a catalytic triad in the polyproline-based scaffold. Surprisingly, the replacement of Pro to Hyp in the peptide not only favors forming a stable PPII helix but also provides additional interactions to accelerate the hydrolysis of ester. In addition, the deprotonation of histidine side chains could enhance the catalytic activity of peptides. Our results provide compelling lines of evidence that the secondary structure of the peptide-based enzymes could have substantial impacts on their catalytic activity for ester hydrolysis and demonstrate new insights into the structure–activity relationship of polyproline-based catalysts. This work also reveals valuable information for the design of potentially useful peptide-based enzymes. With a view to develop remarkable artificial enzymes, the studies of organizing suitable secondary and tertiary structures to improve their catalytic activity are currently on the way in our laboratory. Materials and Methods General Chemical reagents and [(9-fluorenylmethyl)oxy]carbonyl (Fmoc) amino acids were purchased from Advanced ChemTech, Alfa Aesar, ECHO, JT Baker, and Sigma-Aldrich and used without further purification. Fmoc-(2S,4R)-Mop was synthesized by the method previously described.29 Preloaded Tyr resin was prepared by attaching Fmoc-Tyr-OH to 2-chlorotrityl resin using the procedure previously described.31 The loading capacity of Fmoc-Tyr resin was determined according to the previously reported method (Applied Biosystems Technical Note 123485, Rev. 2, http://www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/generaldocuments/cms_040640.pdf). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectra were obtained using an Autoflex III SmartBeam LRF200-CID spectrometer (Bruker Daltonics). Peptide Synthesis and Purification All peptides were synthesized on a 0.05 mmol scale by standard solid-phase methods and Fmoc chemistry protocols. HBTU-mediated coupling reactions were carried out on a Discover SPS microwave peptide synthesizer (CEM Corp.). Preloaded 2-chlorotrityl resin was used to produce a free C-terminus, whereas the use of the Rink amide resin generated an amidated C-terminus upon cleavage. Cleavage of the peptides from the resin and side-chain deprotection was performed using a solution of 95% trifluoroacetic acid (TFA)/2.5% triisopropylsilane (TIS)/2.5% H2O (v/v) or 94% TFA/1% TIS/2.5% H2O/2.5% 1,2-ethanedithiol (v/v) to treat the peptide-resin product for 3 h at ambient temperature. The filtrate was then precipitated and washed with ice-cold methyl t-butyl ether and purified by reverse-phase HPLC with a semipreparative C18 column. H2O/acetonitrile gradients with 0.1% (w/v) TFA were used as the eluting solvent system to purify the peptides. The purified products were identified by MALDI-TOF-mass spectrometry (Table S1 and Figure S2 in the Supporting Information). All peptides were more than 90% pure according to the HPLC analysis as shown in Figure S1 of the Supporting Information. Preparation of Peptide Stock Solutions Purified and lyophilized peptides were dissolved in 25 mM buffer solution [MES (pH 6.0–6.5), HEPES (pH 7.0), Tris (pH 7.4–9.0), and CAPS (pH 10.0)] to make a 0.5 mM peptide stock solution. The concentration of peptides was determined by the UV absorbance at 280 nm in 6 M guanidine hydrochloride at pH 6.5, using an extinction coefficient of 1420 M–1 cm–1 for Tyr and 5500 M–1 cm–1 for Trp. CD Spectroscopy CD spectra were recorded on an Aviv model 410 CD spectrometer with a 1 mm path-length-quartz cuvette. The measurements were performed in pH 7.4 and 25 mM Tris buffer with a peptide concentration of 100 μM. The far-UV CD spectra were taken from 190 to 260 nm with an averaging time of 10.0 s and a wavelength step of 1 nm at 25 °C. Kinetic Assays The catalytic activities of the peptides were determined using p-NPA as the substrate. The kinetic measurements were performed on a JASCO V-630 spectrophotometer associated with an STR-773 water thermostatic cell holder and stirrer by monitoring the absorbance of the hydrolysis product (p-nitrophenol) at 348 nm or 405 nm at 25 °C. A volume of 100 μL of the freshly prepared peptide stock solution was added to 1900 – x μL of buffer solution. After having recorded the initial absorbance (blank), a volume of x μL of 20 mM p-NPA stock in acetonitrile was subsequently added to obtain a final p-NPA concentration of 100–1600 μM (the final acetonitrile content was less than 5% in all reaction mixtures). The reaction mixture was stirred for 60 s before collecting the data, and the absorbance was recorded every 10 s for 150–1200 s (as shown in Table S2 of the Supporting Information). Initial rates were determined from linear fits of the plots of absorbance versus time, and the values were from averaging duplicate measurements. The extinction coefficients of the hydrolysis product at different pH values were experimentally determined by measuring the absorbance of p-nitrophenol in the specified buffers and by taking the average of at least three repeats. The reported kinetic parameters (kcat and KM) were derived by fitting the data to the Michaelis–Menten equation, and the pH-dependent curves were fit to the following modified Henderson–Hasselbalch equation 1 Determination of pKa by NMR A series of S3H6D9 (1.2 mM) peptide samples were prepared in 90% H2O/10% D2O-containing sodium 3-(trimethylsilyl)-1-propanesulfonate in solution as the chemical shift reference. Their pH values in the range of 4–8 were adjusted by adding NaOD or DCl directly into the samples. High-resolution 1D 1H NMR spectra were acquired at 25 °C using a Varian VNMRS-700 NMR spectrometer (Varian, Inc.) at National Tsing Hua University Instrumentation Center. The spectra were analyzed using Bruker Topspin (v.2.1) software, and the pKa values were determined by fitting a plot of the chemical shift (δ) of His C2H versus pH into the modified Henderson–Hasselbalch equation 2 where δ is the observed chemical shift, δd is the chemical shift for the fully deprotonated species, δp is the chemical shift for the fully protonated species, and n is the Hill coefficient, which is 1 for a residue that follows ideal Henderson–Hasselbalch behaviors. Structure Modeling Geometry optimization for the model peptide in the aqueous phase was carried out using the HF/6-31+g(d) and CPCM-SCRF solvation method, conducted by the Gaussian 09 software.34 The initial structure was generated with idealized torsion angles for the PPII backbones based on the reported structural data.35 Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00928.Measured molecular weights of the peptides, detailed experimental conditions for pH-dependent kinetic assays, pH-dependent kinetic parameters for ester hydrolysis, dihedral angles of energy-minimized structural models, HPLC chromatograms, MALDI-TOF spectra, UV–vis-monitored ester hydrolysis reaction curves, additional esterase activity plots, additional pH-dependent 1H NMR spectra, and energy-minimized structure model for Ac-(Pro)2Ser(Pro)2HisPro-OMe (PDF) Supplementary Material ao7b00928_si_001.pdf The authors declare no competing financial interest. Acknowledgments We are thankful to the financial support from the Ministry of Science and Technology of Taiwan (MOST 105-2113-M-007-018 & MOST 106-2113-M-007-016) and National Tsing Hua University (106N501CE1). We are also grateful to the National Center for High-Performance Computing (NCHC) for computer time and facilities. ==== Refs References Richter F. ; Blomberg R. ; Khare S. D. ; Kiss G. ; Kuzin A. P. ; Smith A. J. T. ; Gallaher J. ; Pianowski Z. ; Helgeson R. C. ; Grjasnow A. ; Xiao R. ; Seetharaman J. ; Su M. ; Vorobiev S. ; Lew S. ; Forouhar F. ; Kornhaber G. J. ; Hunt J. F. ; Montelione G. T. ; Tong L. ; Houk K. N. ; Hilvert D. ; Baker D. Computational design of catalytic dyads and oxyanion holes for ester hydrolysis . J. Am. Chem. Soc. 2012 , 134 , 16197 –16206 . 10.1021/ja3037367 .22871159 Moroz Y. S. ; Dunston T. T. ; Makhlynets O. V. ; Moroz O. V. ; Wu Y. ; Yoon J. H. ; Olsen A. B. ; McLaughlin J. M. ; Mack K. L. ; Gosavi P. M. ; van Nuland N. A. J. ; Korendovych I. V. 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PMC006xxxxxx/PMC6644416.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145805410.1021/acsomega.8b02002ArticleFabrication of Ordered 2D Colloidal Crystals on Flat and Patterned Substrates by Spin Coating Banik Meneka Mukherjee Rabibrata Instability and Soft Patterning Laboratory, Department of Chemical Engineering, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India E-mail: rabibrata@che.iitkgp.ac.in (R.M.).17 10 2018 31 10 2018 3 10 13422 13432 12 08 2018 05 10 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Spin coating is a simple and rapid method for fabricating ordered monolayer colloidal crystals on flat as well as patterned substrates. In this article, we show how a combination of factors, particularly concentration of the dispensed colloidal solution (Cn) and spin-coating speed, influences the ordering process. We have performed systematic experiments on different types of substrates with two types of colloidal particles (polystyrene and silica). We also show that even when perfect ordering is achieved at some locations, there might be a significant spatial variation in the deposit morphology over different areas of the sample. Our experiments reveal that higher Cn is required for obtaining perfect arrays, as the diameter of the colloids (dD) increases. Interestingly, a combination of higher Cn and rotational speed (expressed as revolutions per minute) is required to achieve perfect ordering on a topographically patterned substrate, as compared to that on a flat surface, because of loss of inertia of the particles during outward flow because of impact on the substrate features. Finally, we also identify the relation between the particle diameter and the height of the pattern features to achieve topography-mediated particle ordering. document-id-old-9ao8b02002document-id-new-14ao-2018-02002eccc-price ==== Body Introduction It is well-known that monodispersed colloidal particles self-assemble into hexagonal close-packed (HCP) structures, under appropriate conditions, which are also known as two-dimensional (2D) colloidal crystals, and find wide applications in fabrication of optical chips,1 photonic band gap materials and photonic crystals,2−4 data storage,5 chromatography,6 sensors,7 masks for nanosphere lithography,8 etc. Various interfacial self-assembly techniques, such as sedimentation,9 confined convective self-assembly,10 dip coating,11 drop casting and evaporative drying,12 deposition during retraction of liquid meniscus within a microfluidic chamber,13 electrophoretic deposition,14,15 self-assembly at the gas–liquid interface,16,17 Langmuir–Blodgett (LB) technique,18 inkjet printing,19 spray coating,20 spin coating,21−41 and so forth, have been utilized to fabricate monolayer colloidal crystals with HCP ordering. Almost all methods rely on the convective assembly of particles engendered by lateral capillary forces, which cause an attractive interaction between the particles that push them together, favoring the nucleation of a close-packed ordered monolayer. Surface stabilization of the particles is important to prevent uncontrolled aggregation in the early stage of self-assembly.11 Methods such as sedimentation or electrophoretic deposition are well suited for three-dimensional assembly, as they easily form multilayers. Although dip coating and LB-based techniques are capable of producing high-quality 2D crystals, they are inherently slow, require large volume of colloidal solution, and are difficult to scale up. It turns out that spin coating, which is widely used for coating polymer thin films over large areas, is also well suited for creating 2D colloidal crystals. Major advantages of spin coating include rapid formation (∼few minutes), high-throughput, extremely low amount of colloidal solution requirement (∼microliter), high degree of reproducibility, scalability, and direct integration with standard microfabrication approaches. The possibility of creating a 2D monolayer array of latex particles [polystyrene (PS) beads] on a rigid substrate by spin coating was first demonstrated by Deckman and Dunsmuir in early 80s.21,22 Subsequently, on the basis of this concept, Van Duyne pioneered the concept of nanosphere lithography, which is widely used as a physical mask for subsequent additive deposition of various types of materials.8,23 Monolayer arrays of many other types of colloids such as silica, titania, core–shell particles, hollow titania spheres, and nanoparticles of gold, silver, cobalt, etc. have been fabricated by spin coating.24−26 Jiang and McFarland spin-coated a colloidal sol comprising a mixture of a tri-acrylate monomer and a photoinitiator to fabricate a well-ordered nonclose-packed hexagonal array. The monomer, which acts as a spacer, is subsequently photopolymerized to form either a polymer nanocomposite36 or a macroporous polymer with reverse opal structure (in conjugation with etching),37 spanning over large areas.38 However, it is important to point out that as the spin-coating process is radially symmetric about the center of rotation, it is not possible to arrange all the particles in the form of a single defect-free crystal spanning over the entire substrate.29 It has been shown by Yethiraj and co-workers that under idealized conditions, it becomes possible to obtain crack-free orientationally correlated polycrystal (OCP) structures,29,31 which comprise defect-free single-crystal domains radially arranged with respect to the center of the film. While each single crystalline domain laterally spans about 10 μm (represented with the correlation length, lR), their orientation undergoes continuous macroscopic rotation on length scales much larger than the diameter of the colloidal particles.31 Application of an external magnetic field has been shown to be a promising approach that enhances the uniformity of the deposited colloidal particles, provided the colloids are magnetic in nature.39−41 Apart from monolayer arrays with HCP ordering, colloidal particles arranged in a nonhexagonal manner also find applications in many exotic areas such as optics and photonics and as sensing materials.42,43 Such arrays are typically created by depositing the colloids on a chemically or a topographically patterned substrate, which acts as a template. The possible formation of a topography-directed non-HCP colloidal array was first reported by van Blaaderen et al. based on gravity settling of silica particles, which was referred to as colloidal epitaxy.44,45 Subsequently, several other techniques such as micromoulding,46,47 electrostatic assembly,48 flow inside the microfluidic cell,49 dip coating,50 LB technique,51 spin coating,52−57 and so forth have been used to obtain pattern-directed particle arrays with a nonhexagonal geometry. A spin-coated pattern-directed particle array was first demonstrated by Ozin and Yang where they obtained non-HCP arrays of silica microspheres into anisotropically etched square pyramid pits,52 based on a combination of gravity-driven sedimentation and evaporation-induced capillary forces, which lead to rapid settling, self-assembly, and crystallization within the pits. Brueck and co-workers reported the self-assembled array formation of sub-100 nm silica particles inside line gratings and circular holes. They highlighted the critical role of pH of the casting solution and showed that good ordering was possible only when pH was 7.53 The same group showed the possible fabrication of aligned multilayer particle arrays inside deeper trenches, where the layer thickness could be controlled by multiple spin-coating steps. Varghese et al. obtained size selective array of particles within the pattern grooves from a mixture of particles of different sizes.56 From the above discussion, it is clear that spin coating is a simple method, which can be used to fabricate monolayer colloidal crystals with both HCP and non-HCP ordering on flat and patterned substrates, respectively. However, in spin coating, particle array formation depends on a combination of capillary, gravitational, centrifugal, and electrostatic forces. The situation gets further complicated on a topographically patterned substrate where the solution layer undergoes topography-mediated rupture during spin coating.57−59 Further, because of symmetry issues discussed already,29 as well as nonplanarization of colloidal suspensions during spin coating from a volatile medium,40 it becomes impossible to achieve perfect ordering with the particles over a large area, and at best crack-free OCP structures can be obtained.31 Colson et al. approached this complex parameter optimization problem from the concept of Experimental Design and predicted the optimum condition for obtaining a defect-free array over ASA ≈ 200 μm2 area.60 Most experimental papers only report the optimized condition that offers a near perfect array, and there are not too many systematic experimental studies that show how the morphology varies with change in various input parameters. Lack of such a study often renders it difficult to identify the conditions for perfect ordering, particularly over large areas. In this article, we show the conditions under which perfect colloidal arrays can form on flat as well as patterned surfaces of a variety of materials based on systematic experimental investigations. Our results show that a uniform array is formed within a narrow parameter window spanning over large areas. The best structures we obtained exhibit correlation length lR ≈ 15 ± 2.5 μm and ASA ≈ 210 ± 12 μm2 in each single crystalline domain comprising HCP monolayer arrays of the colloidal particles, with no crack between adjacent domains over the entire sample surface (15 mm × 15 mm). Such a structure will be referred to as “perfectly ordered structure” in the subsequent section of the paper. We show that such perfect ordering is achieved within a very narrow parameter window that depends strongly on the diameter of the colloidal particles (dD). We also show that there can be a significant spatial variation in the morphology of the structures and extent of ordering primarily because of nonplanarization of the colloidal suspension,30 and therefore, care must be taken to examine the structure at different locations of the sample. We hope the reported results will act as a guide, particularly for beginners to identify the actual optimized condition based on a first trial that may lead to nonuniform deposition. Further, on a patterned surface comprising grating geometry, we show how the morphology of the ordered particles within the grooves depend on relative commensuration between the particle diameter (dD) and the groove width (lP), in addition to the concentration of the particles in the colloidal solution (Cn). Finally, by coating the colloidal solution on grating patterned substrates with the same periodicity (λP) and lP, but a different groove depth (hP), we identify the minimum height of the features necessary to successfully align the particles. Results and Discussion Self-Assembly of PS Colloids on the Sylgard 184 Substrate As discussed in the Introduction section, the objective of the work is to identify the condition at which perfect monolayer arrays of colloidal particles can be achieved. We performed systematic experiments for each size and types of colloids on different substrates by casting the sols with different colloidal concentrations (Cn) at different revolutions per minute (rpm), to find out the condition that leads to perfect ordering. Figure 1 highlights one such optimization attempt, showing various degrees of ordering with PS colloids having dD = 600 nm, as a function of Cn and rpm on the UVO-exposed Sylgard 184 substrate. A UVO-exposed poly(dimethyl siloxane) (PDMS) substrate is deliberately chosen as it is hydrophobic in nature (θE-W ≈ 109.5°). We show that it becomes possible to create a perfect array even on a hydrophobic surface using methanol as the solvent. However, it must be highlighted that because of UVO exposure, the PDMS surface becomes almost completely wetted by methanol, and therefore, ordered array formation gets favored. Figure 1 Morphology of the colloidal deposit at different Cn–rpm combinations, when PS colloids with dD = 600 nm are spin-coated on a flat UVO-exposed PDMS substrate. The location is 4 mm away from the center of the substrate. It may be noted that in all cases, the surfactant used is sodium dodecyl sulfate (SDS) and its concentration is 0.025 wt %. The presence of surfactant molecules in the colloidal suspension in appropriate quantity is essential for achieving perfect ordering, as they get adsorbed on the particle surface and control the interaction between the particles and surrounding. Self-assembly of colloidal particles into monolayer colloidal crystals require repulsive interactions between the particles, as strong attraction leads to the formation of highly disordered structures.61 Though a detailed analysis on how ordering is influenced by the surfactant concentration is beyond the scope of the present study (and will be taken up separately), we observed that disordered structures form both when the surfactant concentration is very low (or absent) or higher than 0.1 wt %, when micelles start to form. Apart from SDS, we could also obtain perfect ordering using a nonionic surfactant (Triton-X). However, no ordering could be achieved when a cationic surfactant [hexadecyltrimethylammonium bromide (HTAB)] was used. This is attributed to the Coulombic binding of the positively charged head group of HTAB onto PS and silica particles, both of which have negative surface charge, as verified by dynamic light scattering measurements (data shown in Figure S1 of online Supporting Information). As the head group of the cationic surfactant molecules adsorbs on the particle surface, their hydrophobic tail orients outward and adjacent surfactant-covered particles experience strong attraction in the presence of alcohol because of hydration pressure. This leads to agglomeration and consequent suppression of ordered crystal formation. Before presenting the experimental results, we feel it will be appropriate to highlight the key theoretical results related to spin coating,62−64 particularly some of the recent theoretical papers which are specific to spin coating of colloidal dispersions.39−41 This will help us in qualitatively explaining some of the experimental findings reported in this paper. It is well-known that in the initial stage of spin coating, the dispensed solution flows radially outward because of centripetal forces and the advancing solution meniscus reaches the edge of the sample. The excess solution (including the particles) is subsequently thrown out, which is known as splash drainage. Spin coating of simple fluids has been modeled based on lubrication approximation ever since the pioneering work of Emslie et al.,62 where the primary focus was to find out how film thickness (h) varies as a function of spinning time (t) and radial distance from the center of spinning (r). However, the model neglected the role of evaporation, and thinning was considered only because of centrifugal forces. Meyerhofer subsequently improved the model by incorporating the effect of evaporation by considering spin coating to be a two-step process comprising two different stages: (a) initial phase dominated by the radially outward flow and (b) evaporation-dominated later stage where there is virtually no flow. The model considered rate of evaporation (e) to depend on rpm as e ∝ (rpm)1/2.63 Subsequent improvement on spin-coating modeling was proposed by Cregan and O’Brien,64 based on the argument that solvent evaporation starts almost instantaneously with deposition of the solution and therefore cannot be neglected even in the initial phase. Although the model is based on lubrication approximation and utilizes matched asymptotic expansion techniques, it considers a constant rate of evaporation and predicts the deposited layer thickness of the solute (not colloids) h∞(s) rather accurately and is given as64 1 where C0 is the initial solute concentration, γ is the kinetic viscosity, ω is the angular velocity, and E is the rate of evaporation of the solvent. Equation 1 can be simplified as 2 where is constant for a specific experimental condition and β = 2/3. The Cregan model was extended specifically for colloids by González-Viñas and co-workers, who argued that for a particulate system the film thickness h∞(s) should be replaced by a term “compact-equivalent height”, which depends on the kind of deposited structure and can be calculated as the product of packing fraction and Vornoi cell volume.65 Aslam et al. proposed that the term h∞(s) in eq 1 for a simple solute should be replaced by compact-equivalent height (CEH) for colloidal deposition, which gives 3 where the contact A1 can be obtained by simply replacing the solute concentration C0 with the initial colloid concentration (Cn) in the expression of A mentioned above. Further, for the submonolayer deposit with a hexagonal structure, the expression of CEH is given as41 4 where ϵ2 is the local occupation factor, which is defined as the area occupied by the colloidal clusters relative to the total area. In this paper, the term fractional surface coverage (Fs) can be considered identical to the occupation factor (ϵ2) used by Pichumani and González-Viñas.39 It may also be noted that we have written eq 4 in terms of dD, the diameter of the colloidal particles, rather than the particle radius as in refs (40) and (41). Figure 1 shows the morphology of the colloidal deposit obtained for various Cn–rpm combinations. At low rpm, the centripetal force remains weak and therefore fails to spread the particles uniformly. Thus, the evolution is dominated by viscous shear forces, which in turn results in the particles getting arranged in a disordered fashion (series A, Figure 1). As the rpm increases, the centripetal force becomes stronger, resulting in uniform spreading. This also enhances the evaporation rate up to a point where the forces acting on the colloidal particles get balanced. Under this condition, the particles move on the substrate surface easily and self-assemble into a monolayer with HCP ordering that spans over a large area (frames B22, C33, and D44 of Figure 1). It also becomes clear that the balance between the shear force and the drying rate can only be maintained when Cn is increased along with an increase in rpm. However, increase of Cn to much higher values leads to a sharp enhancement in the viscosity of the remnant colloidal suspension during the late stage of spinning. This hinders the mobility of the particles on the substrate surface, and HCP ordering gets suppressed in favor of random disorganized structures (frames B25 and C35, Figure 1). When the speed of rotation is increased further, the radial outflow of the particles increases and consequently, a larger amount of particles are thrown out of the substrate because of splash drainage. Consequently, the remnant suspension rotating on the substrate becomes very dilute. Consequently, the remaining PS particles fail to form monolayer coverage on the substrate, though they may show localized HCP ordering (series E, Figure 1). The gradual morphology transition along each column and row of Figure 1 highlights the influence of enhanced rpm and Cn on the ordering process, respectively. It can be clearly seen that the fractional coverage of the surface (Fs) reduces with an increase of rpm, for a given Cn, as the proportion of splash drainage increases at higher rpm. On the other hand, Fs gradually increases with an increase in Cn for a constant rpm, as more number of particles are initially dispensed on the substrate. In fact, in most cases, the morphology gradually transforms from monolayer with partial coverage to scattered multilayers with an increase in Cn, where the drying front fails to drag the particles over the initially assembled particles. Important observation in Figure 1 is the formation of a perfectly ordered monolayer with HCP ordering in frames B22, C33, and D44 (Fs ≈ 1.0). This means that a perfect monolayer coverage is possible for different combinations of Cn and rpm. At this point, it is worth highlighting that all the images in Figure 1 are captured at a location which is approximately 4 mm away from the center of the substrate. However, in order to claim that a specific coating condition is optimum for achieving perfect crystalline ordering, it is important to check the structure uniformity over the entire sample substrate. We checked this by performing careful atomic force microscopy (AFM) scan of the samples at different radial distances (rD) from the center of the sample at 1 mm intervals. The samples chosen for this study include the ones which are seen to give perfect ordering in Figure 1. In addition, few samples corresponding to different Cn–rpm combinations were also explored, the details of which are mentioned in the legend of Figure 2A. We note that at an rpm of 200 as well as at a higher rpm (=2500), perfect ordering is not achieved anywhere on the sample surface. For the low rpm case, the morphology comprises a scattered multilayer over major portion of the substrate (rD > 2 mm), indicating weak action of the centripetal force, which fails to drag the particles. On the other hand, at higher rpm, the centripetal force is much higher than the shear force and a large amount of particles are lost because of splashing. Consequently, Fs never exceeds 50% over the entire substrate. For the sample with Cn = 0.52% rotated at 400 rpm, we observe a slight undercoverage till rD ≈ 2 mm but a very high degree of multilayer formation beyond rD > 5 mm. An opposite trend is observed in the sample with Cn ≈ 1.2%, rotated at rpm = 1000. Here, a near perfect array with Fs ≈ 1.0 is obtained in the outer section of the sample (rD ≥ 4 mm). However, the areas close to the center show a significant undercoverage (Fs down to ≈0.75%). As can be seen in Figure 2A, the most uniform coverage is observed in the sample with Cn = 0.67%, rotated at rpm = 600. Thus, for PS colloids with dD ≈ 600 nm, we identify a combination of Cn = 0.67% and rpm = 600 to be optimum. Figure 2 Variation of fractional coverage Fs as a function of distance from center (rD) for different Cn–rpm combinations. AFM images in insets (A1–A3) show perfectly ordered HCP morphology at rD = 2, 5, and 7 mm. In all cases, PS colloids with dD = 600 nm have been used. The trend in Figure 2A highlights the critical role of solvent evaporation time on the array formation. Unlike water, which has a vapor pressure = 3.17 kPa, methanol has a much higher vapor pressure (13.02 kPa), which means methanol evaporates much faster. Therefore, particle spreading can take place only till the adequate amount of solvent is present. This means at low rpm (200 and 400), even before major part of the particles can reach the sample periphery, the solution starts to dry up, which is also associated with the enhancement of viscosity, and therefore, multilayers are formed toward the outer areas of the sample. Thus, for an organic solvent, the balance of shear force and centripetal force must be established within a very narrow time window, before major part of the solvent evaporates away. This in turn allows perfect array formation at Cn which is typically much lower than that reported in the literature with an aqueous colloidal sol, which also implies much reduced splash drainage of the colloidal particles. Further, Figures 1 and 2 clearly highlight the success of our work in obtaining a neat and perfect array on a hydrophobic surface (θE-W ≈ 109°) of the cross-linked PDMS substrate, UVO-exposed for 30 min (Table 1). In fact, Choi et al. have clearly mentioned that it is nearly impossible to obtain a perfect array on a hydrophobic surface.32 We show that this limitation can be circumvented by using methanol as the solvent. The only requirement is methanol must wet the surface, which is achieved by 30 min UVO exposure, despite the substrate remaining hydrophobic. Table 1 Details of the Flat Substrates Used sl. no. substrate RMS roughness (nm) water contact angle (θE-W°) methanol contact angle (θE-M°) surface energy (mJ/m2) 1 glass 0.429 ± 0.043 17.3 8.2 56.4 2 silicon wafer 0.305 ± 0.039 11.1 2.3 59.17 3 PS film coated on glass 0.486 ± 0.044 91.5 5.1 38.3 4 PMMA film coated on glass 0.493 ± 0.038 73.2 3.2 41.8 5 cross-linked PDMS film on glass 0.505 ± 0.042 114.5 35.8 24.2 5A UVO-exposed PDMS film on glass 0.514 ± 0.023 109.5 2.4 28.3 Self-Assembly of PS and Silica Colloids on Different Substrates In Table 2, we show the optimized conditions for obtaining HCP arrays on different substrates with colloids of different sizes and of different materials. The optimized Cn–rpm combination is obtained by performing experiments in line with Figures 1 and 2 for particles of each diameter on each type of substrate. The corresponding images of the ordered arrays are shown in Figures S2–S5 of online Supporting Information Table 2 Optimized Conditions for Perfect Ordering on Flat Substrates material of particle dD (nm) substrate Cn (wt/vol %) rpm figure number in online Supporting Information PS 300 glass 0.25 wt % 1000 rpm, 120 s S2A silicon wafer 0.2 wt % 1000 rpm, 120 s S2B PS film 0.3 wt % 800 rpm, 120 s S2C PMMA film 0.25 wt % 800 rpm, 120 s S2D UVO-exposed PDMS film 0.4 wt % 800 rpm, 120 s S2E PS 600 glass 0.5 wt % 800 rpm, 120 s S3A silicon wafer 0.4 wt % 800 rpm, 120 s S3B PS film 0.6 wt % 600 rpm, 120 s S3C PMMA film 0.5 wt % 600 rpm, 120 s S3D UVO-exposed PDMS film 0.67 wt % 600 rpm, 120 s S3E PS 800 glass 0.8 wt % 600 rpm, 120 s S4A silicon wafer 0.75 wt % 800 rpm, 120 s S4B PS film 0.85 wt % 500 rpm, 120 s S4C PMMA film 0.8 wt % 500 rpm, 120 s S4D UVO-exposed PDMS film 0.9 wt % 500 rpm, 120 s S4E silica 350 glass 0.4 wt % 800 rpm, 120 s S5A silicon wafer 0.3 wt % 1000 rpm, 120 s S5B PS film 0.47 wt % 600 rpm, 120 s S5C PMMA film 0.4 wt % 600 rpm, 120 s S5D UVO-exposed PDMS film 0.5 wt % 600 rpm, 120 s S5E In order to highlight the dependence of the optimum condition on simultaneous variation of several parameters, we construct morphology phase diagrams. Two types of morphology phase diagram can be constructed, which are shown in Figure 3. The phase diagram shown in Figure 3A can be constructed for each particle with a specific dD—substrate combination to identify the optimum condition. The diagram shown in Figure 3B is obtained by plotting each of the optimum points obtained for different dD—substrate combinations. To generate a plot like the one shown in Figure 3A, which is specific to PS particles with dD = 600 nm on UVO-exposed PDMS substrates at a location that is 4 mm away from the center of the substrate, we classify the morphology obtained at different Cn–rpm combinations into three subcategories, which are perfectly ordered, underfilled, and overfilled, as has been discussed in the context of Figure 1. The three distinct morphologies are represented with different symbols. The information in Figure 2 is further used to identify the Cn = 0.67% and rpm = 600 as the optimum condition which is marked with a square. It can further be seen that the Cn at which transition from underfilled to overfilled structures take places varies linearly with rpm. Figure 3 Morphology phase diagram (A) for PS colloids having dD = 600 nm on a UVO-exposed flat PDMS substrate and (B) for colloids of different sizes on different types of flat substrates. While each color represents colloid of a specific type and dD, each symbol represents a type of substrate, as per legend provided in the figure. Figure 3B represents the global morphology phase diagram which is constructed by using the data reported in Table 2, each one of which has been obtained by constructing a particle and substrate specific morphology phase diagram similar to that shown in Figure 4A. Representing data with simultaneous variation of four parameters (Cn, rpm, dD, and substrate type) on a single plot was itself challenging, and therefore, the logic adopted for representation needs to be mentioned. In the plot, a particular color of the symbols represents particles of a specific dD. In contrast, different types of substrates have been represented with different shapes of the symbols. The figure highlights that for a particular type of particle, perfect ordering is achieved on three polymeric substrates [PS, polymethylmethacrylate (PMMA), and UVO-exposed PDMS] at nearly identical conditions. In contrast, it emerges out from Figure 3B that a higher rpm is required to obtain perfect ordering on glass and silicon substrates. This probably is a signature of slippage of the solvent on a polymeric substrate and needs to be explored in greater detail. Also, it becomes evident from both Figure 3B and Table 2 that for obtaining perfect ordering, Cn increases with an increase in dD, a trend that is counterintuitive, as lesser number of larger particles are required to cover the same surface area, when dD is larger. It may however be explained rather easily from eq 4, which suggests that to achieve constant surface coverage (Fs) or occupation factor (ϵ2), is proportional to dD.41 As Cn is very dilute, one may further argue that 1 – Cn ≈ 1, and hence, Cn exhibits direct proportionality with dD. Figure 4 Morphology of the colloidal deposit at different Cn–rpm combinations, when PS colloids with dD = 600 nm are spin coated on the patterned UVO-exposed PDMS substrate with type 2 geometry (λP = 1.5 μm). Table 2 also reveals that for a particular dD, the optimum Cn increases slightly, with lowering of the substrate surface energy. This can be attributed to the reduced strength of the particle–substrate interaction with lowering of γ. However, how exactly the surfactant molecules adsorb on the substrate and thus influence the ordering process is not fully clear and will be analyzed separately. It can be seen that there is no monotonic correlation of Cn and rpm with the substrate surface energy for the formation of a perfect array, though the parameters are quite close on various substrates. This observation is an indirect evidence that the surface has a much lower role on the assembly, apart from its wettability. We argue this happens because of the presence of the surfactant molecules, which favor wetting of solution on the substrate, which is the necessary condition for achieving ordering. Template-Directed Assembly of Colloids Particle Diameter Approximately Equal to Pattern Line Width (dD ≈ lP) It is well-known that physical confinement provided by the patterned substrate effectively traps the colloidal particles during spin coating.51 The grooves act as the preferred location for the deposition of the particles, as the solvent layer ruptures over the substrate pattern protrusions.57 Consequently, the remaining liquid flows in the grooves, localizing the colloids there. Subsequently, the lateral capillary forces between the particles drive them to form closely packed structures aligned along the grooves. It has also been recently shown by Aslam et al. that a patterned substrate also eliminates axial symmetry by spin coating and therefore suppresses the formation of OCP structures.57 In this section, we show how the morphology of the particle deposit varies as a combination of Cn and rpm. As a test case, we examine the ordering of dD = 600 nm PS particles on the type 2 grating patterned substrate of the UVO-exposed cross-linked PDMS. For a type 2 substrate, the groove width is lP ≈ 750 nm, the groove depth is hP ≈ 250 nm, and the periodicity is λP ≈ 1.5 μm. Using a substrate of the same material as that in Figure 1 allows direct comparison between the conditions that leads to a perfect ordering on a flat substrate and a patterned substrate. As dD and lP are comparable, we consider that the particle and the patterned substrate are commensurate. Figure 4 shows various deposition morphologies of dD = 600 nm PS particles on type 2 substrate, obtained at different Cn–rpm combinations. The trend is similar to that observed on a flat surface; for a particular Cn, Fs-P (surface coverage or fill fraction on a patterned substrate) gradually reduces with an increase in rpm because of higher splash drainage. On the other hand, increase of Cn at a constant rpm leads to the deposition of more particles, leading to disorder, as particles get deposited on top of the stripes, after filling the grooves (frames A14, A15, B24, and B25 of Figure 4). Among all the frames of Figure 4, perfect ordering is observed in frame B23 for a parameter combination of Cn = 0.75% and rpm = 1000. It is interesting to note that perfect ordering on a patterned substrate requires higher Cn as well as higher rpm for particles with the same dD as compared to the flat substrate of the same material. This means that on a patterned substrate, more number of particles are required for perfect ordering. This is counterintuitive as perfect ordering on a grating patterned substrate implies that particles align only inside the grooves, and therefore, only about 50% of the number of particles is required to achieve the same, in comparison to a perfect HCP structure on a flat surface (Fs ≈ 1.0). The observation can be explained well with the help of eq 4 (modified Cregan model).41 As Fs (or ϵ2) is almost half on a grating patterned substrate, as compared to that on a flat substrate, CEH on a patterned substrate is also approximately half than that on a flat substrate. From eq 3, we note that CEH = A1ω–β. Figure 4 shows that Cn = 0.75% for perfect ordering on a patterned substrate, which is compared to a value of Cn = 0.67% on a flat surface (Figure 2). As the values of Cn are rather close to each other, the values of are also rather close on a flat and a patterned substrate. Thus, from eq 4, lower CEH becomes possible on a patterned substrate only when ω is higher as compared to that on a flat substrate. We summarize the optimized conditions for obtaining perfectly ordered structures (Fs-P ≈ 1.0) on type 1 and type 2 substrates of different materials, with both silica and PS colloids of different sizes, as presented in Table 3, by performing experiments in line with Figure 4 for particles of each dD on each type of patterned substrate. Figure 5A shows the perfectly ordered structure obtained with silica colloids having dD = 350 nm on a patterned type 1 PMMA substrate, under conditions mentioned in Table 3. Interestingly, on patterned substrates, we find that the rpm at which perfect ordering is obtained for a particular colloid is independent of the substrate material, though Cn differs slightly from substrate to substrate. This clearly highlights the critical role of colloidal spreading on the formation of perfect structures, rather than the particle–substrate interaction, which seems to influence the ordering process weakly. The results imply that the evolution during spinning up to the stage of topography-mediated rupture of the solvent layer is not much influenced by the substrate. Also, as already discussed, in all cases higher rpm is required for obtaining a perfect array, as compared to that on a flat substrate of the same material. Figure 5 (A) Perfectly ordered structure with Fs-P ≈ 1.0 obtained by spin coating silica colloids having dD = 350 nm on type 1 patterned PMMA substrate. (B) Unique ordered structures with PS colloids having dD = 600 nm on type 2 patterned PMMA substrate, where the particles fully cover the patterned substrate. Inset (B1) shows the cross-sectional AFM line profile and (B2) represents a schematic highlighting the particle arrangement. The overall pattern is HCP, but particles rest at two different elevations. Table 3 Optimized Conditions for Perfect Ordering on Patterned Substrates material of particle dD (nm) substrate substrate type Cn (wt %) rpm PS 300 patterned PS film type 1 0.45 1400 rpm, 120 s patterned PMMA film type 1 0.45 1400 rpm, 120 s UVO-exposed patterned PDMS film type 1 0.5 1400 rpm, 120 s PS 600 patterned PS film type 2 0.67 1000 rpm, 120 s patterned PMMA film type 2 0.65 1000 rpm, 120 s UVO-exposed patterned PDMS film type 2 0.75 1000 rpm, 120 s silica 350 patterned PS film type 1 0.55 1200 rpm, 120 s patterned PMMA film type 1 0.5 1200 rpm, 120 s UVO-exposed patterned PDMS film type 1 0.6 1200 rpm, 120 s Until this point, we have considered a perfectly ordered structure to be one where only the grooves are completely filled up by the colloids in an uninterrupted threadlike manner (Fs-P ≈ 1.0). However, Figure 5B shows another interesting morphology where the entire patterned substrate is covered in a near HCP manner by PS colloids (dD = 600 nm). The section B1—B1 in the inset of Figure 5B shows that some particles are at a lower level as compared to others. This happens when Cn is adequately high and they start depositing covering the entire patterned substrate. The particles which lead to within the grooves arrange themselves in a zigzag threadlike manner. The remaining particles, which deposit over the substrate stripes, align themselves in an interesting arrangement of alternate single and double particles along the length of the stripes, alongside the particle thread formed within the groove. The arrangement is schematically shown in inset (B2) of Figure 5B. This structure forms when Cn ≈ 2.5% and rpm = 1000. Because lP is higher than dD, the particles align in a zigzag fashion inside the grooves, allowing more particles to pack themselves side by side over the stripes. As larger number of particles are dispensed (Cn for optimum coverage = 0.75%, as per Table 3) on the surface, the applied centripetal force fails to splash out significant fraction of the excess particles that are available after filling of the grooves. The morphology is novel and highlights that the hexagonal arrangement still leads to minimum energy configuration even if the particles remain at multiple levels. Effect of Template Height on Pattern-Directed Assembly In this section, we show how the feature height (hP) of the substrate patterns influences the pattern-directed ordering. For this purpose, type 2 substrates of different hP were used. The experiments were performed with dD = 600 nm PS colloids so that the dD and λP remain commensurate. The initial Cn–rpm combination chosen was the same as that which is seen to give perfect ordering in Figure 4, frame B23. It can be seen that perfect pattern-directed ordering is obtained till hP ≈ 150 nm, when the Cn–rpm combination was kept unaltered. It can be seen in Figure 6B that the ordering is lost when hP ≈ 125 nm. The uniqueness of Figure 6B can be understood by comparing it with frames A13–A15 and B24–B25 of Figure 4 which also shows distorted arrays on patterned substrates. However, in all those frames of Figure 4, the grooves are totally filled and the excess particles are seen to get localized over the stripes, resulting in disorder. In contrast, in Figure 6B, the particles are seen to accumulate over the stripe tops, despite some grooves remaining vacant. This means that during the radial outward flow, the hP of the features is too low to intercept the flow and guide the particles within the grooves. When we performed similar experiments with dD = 300 nm on type 1 substrates of different hP, we observed that ordering gets lost below hP ≈ 75 nm. The trend, including the critical hP below which topography-guided ordering is lost, is nearly similar for all substrates having identical pattern topography. It can be seen that the limiting value of hP/dD lies between 0.2 and 0.25 below which the topography of the substrate patterns fails to provide confinement to the particle organization process. On the basis of the first results reported in this article, a detailed investigation on this problem will be taken up separately. Similar transition from order to disordered structures with gradual reduction of hP has been observed earlier in the context of pattern-directed dewetting of thin polymer films.66 A detailed investigation on this topic is underway. It will be particularly interesting to explore if there is a critical hP around which there will be a transition from OCP structures to purely template-guided ordered structures, which is likely to depend on dD of the colloids as well. Figure 6 Morphological variation in template-guided assembly of PS colloids (dD = 600 nm) on type 2 gratings of height (A) 150 and (B) 125 nm. Particle Diameter Smaller Than Pattern Line Width (dD < lP) In Figure 7, we show how the morphology of the deposit depends when dD is much smaller than the groove width of the patterned substrate. Unlike all the earlier cases, where perfect ordering means a single thread of particles aligned along the substrate groove, more complex ordering is possible when dD and lP are noncommensurate. We produce one such example with dD = 300 nm PS particles coated on type 2 PMMA substrate, which has lP = 750 nm. While we obtained a variety of morphologies depending on the combination of Cn and rpm, we report the morphology which can be considered as the perfectly ordered structure and can be considered analogous to Figure 4, frame B23. In Figure 7, the laterally coexisting particles are seen to arrange in a hexagonal manner, though they are aligned along the groove. Figure 7 Particle doublet obtained with PS colloids having dD = 300 nm on type 1 substrate, obtained with Cn = 05% and rpm = 1000. Conclusions In this article, we have systematically demonstrated the conditions under which perfect arrays of monodispersed colloids can be obtained on both defect-free flat and topographically patterned substrates of different materials, using spin coating. On the flat surface, we obtained structures that exhibit correlation length lR ≈ 15 ± 2.5 μm and ASA ≈ 210 ± 12 μm2 in each single crystalline domain comprising HCP monolayer array of the colloidal particles, with no crack between adjacent domains over the entire sample surface (15 mm × 15 mm). We have shown in details the optimization process, and the role of individual parameters, particularly Cn and rpm on the extent of ordering process and constructed a morphology phase diagram to highlight the collective role of Cn and rpm on perfect ordering with particles of different sizes on different surfaces. We have also highlighted the need to check the spatial variation of the as-cast morphology, as there can be a significant variation in the extent of ordering and Fs over the surface. Our results also highlight the critical role of surfactants added on the formation of uniform particle arrays, which might eventually open up a new route to overcome the nonplanarization effect in spin coating, particularly from a volatile organic solvent. Our intention is that the report will significantly help beginners in the area to quickly optimize the parameters by identifying the nature of the defect in the structure and appropriately modulating either rpm or Cn. To facilitate this, we have highlighted the likely types of defects that may result when Cn and/or rpm is low or high. We also show that higher Cn is required for obtaining perfect arrays with larger dD particles, which is in line with the recent theoretical predictions on the spin coating of colloidal particles by Aslam et al.41 On a patterned surface, we note that both higher rpm and Cn are required for obtaining perfect ordering, which can be attributed to lower Fs as compared to that on a flat surface and has been explained well from the theory. We also explored how the ratio of dD and hP can influence the ordering process. If the features are shallower than 20% of the particle diameter, then the patterns fail to confine the particles. Or in other words, a grating much shallower than the particle diameter is sufficient for confining the particles. We also report the formation of novel multielevation structures under certain conditions when the topographically patterned substrate is fully covered with particles at high Cn, as well as interesting structure comprising an array of particle doublets when dD is nearly 50% of lP. As a final summary, we understand that 2D monolayer colloidal array formation on both flat and patterned substrates by spin coating is a complex problem because of its multiparameter nature. While we have carefully examined the dependence of Cn and rpm on the ordering process, various other parameters such as effect of surfactant type and concentration, relative commensuration between particle size and pattern geometry, and so forth also influence the ordering process, which needs detailed investigation. Materials and Methods Substrates Glass slides, silicon wafer, films of cross-linked PDMS, PS (molecular weight: 280 kDa, Sigma-Aldrich USA), and PMMA (molecular weight: 120 kDa, Sigma-Aldrich USA) were used as different substrates. The size of the substrates was 15 mm × 15 mm. The glass and silicon substrates were cut from glass slides and silicon wafer (test grade, Wafer World Inc.) respectively. They were subsequently cleaned following a standard procedure. All polymer films were spin-coated on cleaned glass pieces. The thickness of the PDMS film was ∼10 μm, which was obtained by spin-coating a degassed mixture of part A to part B (1:10 wt/wt) of Sylgard 184 (a thermo curable, PDMS-based elastomer, Dow Corning, USA) in n-hexane (SRL, India) at 2500 rpm for 1 min. The coated film was cured at 120 °C for 12 h in an air oven for complete cross-linking. The thickness of the PS and PMMA films was ∼500 nm, which was obtained by coating dilute solutions [10% (w/v) PS and PMMA] in toluene on the glass slides at 2500 rpm for 1 min. After coating, the PS and PMA films were annealed at 60 °C for 3 h in a vacuum oven to remove the remnant solvent. All the flat substrates were characterized using an atomic force microscope. Contact angle goniometry (ramé-hart Instrument Co.) was used to measure the water and methanol contact angle on each of the substrates, which along with the substrates surface energy are listed in Table 1. All the substrates were used as it is, except cross-linked PDMS substrates which were not wetted by the solvent (methanol). To make it wettable, the cross-linked PDMS substrates were exposed to UVO (PSD Pro UV–O, NovaScan, USA). UV irradiation at 185 nm wavelength leads to the production of ozone from atmospheric oxygen. The ozone molecules, in turn, dissociate into oxygen at 254 nm irradiation.67 The reactive oxygen radicals, thus created, attack the methyl groups present in Sylgard 184 (Si–CH3) and replace them with silanol groups (Si–O–H), hence generating a superficial oxide layer of higher surface energy and leads to enhanced wetting of the samples. The drastic drop in methanol contact angle on the UVO-cured PDMS substrate can be observed in Table 1. Patterned Substrates Some PS, PMMA, and PDMS films were patterned using topographically patterned stamps, which were obtained from peeling the foils of commercially available optical data storage disks such as DVD and CD.68 The PS and the PMMA films were patterned using capillary force lithography.69,70 The cross-linked PDMS films were patterned using a UVO-mediated soft embossing technique reported elsewhere.68 The morphology of all three films patterned using the same stamps was identical, implying the formation of a perfect negative replica of the stamp pattern, which was verified using an atomic force microscope. While the grating patterns obtained with a DVD foil has the periodicity λP ≈ 750 nm, groove width lP ≈ 350 nm, and groove depth hP ≈ 100 nm (Figure S6, online Supporting Information, marked as type 1 patterns), the films patterned with a CD foil has λP ≈ 1.5 μm, groove width lP ≈ 750 nm, and groove depth hP ≈ 250 nm (Figure S7, online Supporting Information, marked as type 2 patterns). Grating patterns with the same λP but different hP was obtained following stress relaxation-associated imprinting of partially precured PDMS films, which is reported in detail elsewhere.71,72 In short, PDMS films of different viscoelasticities were created by precuring the films for different durations before embossing. These films, once imprinted with flexible foils, underwent stress relaxation, which resulted in films with different hP. Figure 8A shows the hP of different PDMS substrates used in our study, created as a function of precuring time (tP) of the PDMS film. Figure 8B1,B2 shows the cross-sectional line profiles of the type 1 and type 2 patterns having different hP. Figure 8 (A) Variation of pattern feature height (hP) with precuring time (tP). (B) AFM cross-sectional profile of patterns with different features hP, as a function of tP. Colloidal Particles Monodispersed colloids of PS with diameters dD ≈ 300, 600, and 800 nm were purchased from Sigma, UK. Monodisperse silica particles of diameter (dP) ≈ 350 nm were synthesized following Stöber’s method by the hydrolysis of tetraethyl orthosilicate (99.99%, Sigma-Aldrich) in ethanol (99.99%, Sigma-Aldrich) medium in the presence of ammonium hydroxide (28%, Sigma-Aldrich) as a catalyst.73 The details about the reaction can be found in the online Supporting Information (Section S4.0). Prior to spin coating, dilute solution of PS and silica colloids in methanol was mixed with 0.025 wt % (unit of Cn is in terms of wt/vol %) solution of SDS (purest grade purchased from Merck, India) in methanol to stabilize the colloidal dispersion and was sonicated in a water bath for 1 h. All the results reported in this paper are with SDS. However, in some cases, a nonionic surfactant (Triton-X) or a cationic surfactant (HTAB) was also used. It is quite clear that the presence of surfactants favors better ordering, and therefore, surfactant concentration is going to be an important factor influencing the morphology of the deposits. A detailed analysis on how the nature and concentration of the surfactant influence the ordering process will be analyzed separately. Spin-Coated Array Fabrication For spin coating, the bare substrate (mounted onto the chuck) was first started to rotate and allowed to attain the final rotational velocity. Subsequently, 100 μL of the stabilized colloidal dispersion was dispensed on the rotating substrate using a micropipette. This protocol of dispensing the colloidal suspension was adopted to minimize the effects of acceleration on the sample. Coating over all the samples was performed at a room temperature of 25 °C and a relative humidity of 30%. The rpm and the colloid concentration (Cn) vary from sample to sample and are mentioned in the appropriate sections. The morphology of the colloidal arrays was investigated using an atomic force microscope (Agilent Technologies, AFM 5100) in intermittent contact mode using a silicon nitride cantilever (PPP-NCL, Nanosensors Inc., USA) and a field emission scanning electron microscope (JSM7610F, JEOL, Japan). For every AFM image, the fractional coverage (Fs) was calculated using the “slice” feature of Pico Image Basic (Version 5.1), an integrated AFM imaging and analysis software package. Details about the calculation procedure of Fs can be found in Section S5.0 of the online Supporting Information Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02002.Surface charge of PS and silica colloids; HCP array formation by PS and silica colloids on different surfaces; details of the different patterns used; synthesis of silica colloids; and fractional coverage calculation procedure (PDF) Supplementary Material ao8b02002_si_001.pdf The authors declare no competing financial interest. Acknowledgments R.M. acknowledges DST, Government of India, for funding under its SERI initiative, grant number: DST/TM/SERI/DSS/361(G). ==== Refs References Kamenetzky E. A. ; Magliocco L. G. ; Panzer H. P. Structure of Solidified Colloidal Array Laser Filters Studied by Cryogenic Transmission Electron Microscopy . 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PMC006xxxxxx/PMC6644419.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145886910.1021/acsomega.8b01061ArticleEfficient Perovskite Solar Cells with Reduced Photocurrent Hysteresis through Tuned Crystallinity of Hybrid Perovskite Thin Films Qi Jun †§Yao Xiang †§Xu Wenzhan †Wu Xiao †Jiang Xiaofang †Gong Xiong †‡Cao Yong †† Institute of Polymer Optoelectronic Materials and Devices, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, P. R. China‡ Department of Polymer Engineering, College of Polymer Science and Polymer Engineering, The University of Akron, Akron, Ohio 44325, United States E-mail: xgong@uakron.edu. Fax: (330) 972 3406.28 06 2018 30 06 2018 3 6 7069 7076 19 05 2018 19 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Hybrid perovskite materials used for realization of efficient perovskite solar cells have drawn great attention in both academic and industrial sectors. It was reported that the crystallinity of hybrid thin-film perovskite materials plays an important role in device performance. In this study, we report a novel and simple method to tune the crystallinity of CH3NH3PbI3 thin film for device performance of perovskite solar cells. By employing tetraphenylphosphonium chloride on the top of PbI2 thin layer in the two-step perovskite deposition processes, the crystallinity of the resultant CH3NH3PbI3 thin film was tuned. As a result, perovskite solar cells by the CH3NH3PbI3 thin film with tuned crystallinity exhibit an enlarged open-circuit voltage and enhanced short-circuit current, thus boosted efficiency as well as reduced photocurrent hysteresis compared to pristine CH3NH3PbI3 thin film. These results indicate that our study provides a new simple way to boost device performance of perovskite solar cells through tuning the crystallinity of CH3NH3PbI3 thin film. document-id-old-9ao8b01061document-id-new-14ao-2018-01061pccc-price ==== Body 1 Introduction In the past years, hybrid perovskite materials used for realization of efficient perovskite solar cells (PSCs) have drawn great attention in both academic and industrial sectors due to their advanced features, such as large absorption coefficient,1,2 high charge-carrier mobility,3,4 and long charge-carrier diffusion lengths.5,6 Power conversion efficiencies (PCEs) of more than 22% have been reported from PSCs fabricated by CH3NH3PbI3 with large crystal and generic interfacial engineering in PSCs device structure.7−10 However, there is still a gap to realize 31% PCE, a theoretical value, from CH3NH3PbI3-based PSCs.11 Toward the end, device performance parameters, short-circuit current (JSC), open-circuit voltage (VOC), and fill factors (FFs) are required to be enhanced for boosting PCE. In the case of PSCs fabricated by CH3NH3PbI3 thin film, VOC is estimated to be 1.3 V.11 But the reported VOC always exhibited unavoidable optical and electrical losses,12,13 which were induced by interfacial states.14−16 Toward the end, passivating the charge defects and improving the energy disorder at the electron extraction layer have been utilized to realize large VOC.16−22 On the other hand, many effects have been also devoted to enhance JSC PSCs.11,23−25 Han et al. reported a solvent-annealing process to control the crystal orientation transformation to improve charge-carrier collection and prolong charge-carrier lifetime, thus boosting JSC.26 Recently, Leblebici et al. have found that different crystal facets of individual grains have a direct impact on both VOC and JSC.27 Thus, optimization of microscopic facet orientation of CH3NH3PbI3 crystals in polycrystalline perovskite thin film could boost both VOC and JSC.26−29 In this study, we report a novel and simple method to tune the crystallinity of CH3NH3PbI3 thin film for boosting PSCs device performance. By employing tetraphenylphosphonium chloride (TPPCl) on the top of PbI2 thin layer in the two-step perovskite deposition processes, the crystallinity of the resultant CH3NH3PbI3 thin film was tuned. As a result, the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer exhibit a VOC of 1.12 V, a JSC of 21.71 mA/cm2, an FF of 74.43, and a corresponding PCE of 18.04%, with dramatically reduced hysteresis, compared to the PSCs fabricated by pristine CH3NH3PbI3 thin film, which exhibit a VOC of 1.04 V, a JSC of 21.08 mA/cm2, an FF of 74.41, and a corresponding PCE of 16.34%, with serious hysteresis. 2 Results and Discussion TPPCl is selected to tune the crystallinity of CH3NH3PbI3 thin film through modification of PbI2 thin-layer surface originated from the Cl– anion, which probably chelated with the Pb2+ cations and altered the kinetics of thin-film formation.29 The molecular structure of TPPCl is shown in Scheme 1a. Figure 1a–e presents the scanning electron microscopy (SEM) images of pristine PbI2 thin film and PbI2 thin films treated with the TPPCl ultrathin layers. It is found that pristine PbI2 thin film possesses layered crystals with sizes of tens of nanometers and many voids. Such observation is in good agreement with the morphologies of PbI2 thin film reported by others.30,31 After the PbI2 thin films are treated with different TPPCl ultrathin layers cast from different concentrations of TPPCl solutions, PbI2 thin films still possess layered crystals with sizes of tens of nanometers and many voids, but a mass of interlaced particles like strip is adhered to the surfaces of PbI2 thin films. As the TPPCl solution is at 1 mg/mL, several well-distributed large TPPCl particles are formed on the surface of PbI2 thin film. As the TPPCl solution is further increased to 1.5 mg/mL, large TPPCl nanoparticles are aggregated on the surface of PbI2 thin film. Figure 1 Top view of SEM images of pristine PbI2 thin film and PbI2 thin film treated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of (a) 0 mg/mL, (b) 0.25 mg/mL, (c) 0.5 mg/mL, (d) 1 mg/mL, and (e) 1.5 mg/mL. Top view of SEM images of pristine CH3NH3PbI3 thin film and CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of (f) 0 mg/mL, (g) 0.25 mg/mL, (h) 0.50 mg/mL, (i) 1 mg/mL, and (j) 1.5 mg/mL. Scheme 1 (a) Molecular Structure of TPPCl and (b) the Device Structure of Perovskite Solar Cells Figure 1f–j presents the SEM images of pristine CH3NH3PbI3 thin film and CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers. Compared to pristine CH3NH3PbI3 thin film, no significant change in the film morphology is observed in the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from TPPCl solution with a concentration of 0.25 mg/mL. However, the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from TPPCl solution with a concentration of 0.5 mg/mL, displays larger and more inerratic grains. The CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from TPPCl solution with a concentration of 1.0 mg/mL, shows larger gains and higher gains continuity with distinctly less grain boundaries. The CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from TPPCl solution with a concentration of 1.5 mg/mL, possesses very fine grain with flatness feature of grain boundary. Thus, these results demonstrate that the TPPCl ultrathin layers affect the qualities of resultant CH3NH3PbI3 thin films. Figure 2 presents the X-ray diffraction (XRD) patterns of pristine CH3NH3PbI3 thin film and CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers. No XRD patterns assigned to TPPCl ultrathin layer are found in all CH3NH3PbI3 thin films, indicating that TPPCl is evaporated during the thermal annealing for the formation of CH3NH3PbI3 thin films. All CH3NH3PbI3 thin films exhibit the same diffraction peaks located at 14.1, 28.5, and 31.9°, corresponding to the (110), (220), and (114) crystal planes. No XRD patterns assigned to TPPCl ultrathin layer are found in all. These results indicate that all CH3NH3PbI3 thin films possess the tetragonal crystal phase.29,32 However, the peak intensities are different. The highest peak intensity (the (110) plane) is observed from the CH3NH3PbI3 film pretreated with the TPPCl ultrathin layer cast from a 1.0 mg/mL concentration of TPPCl solution, indicating that the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer possesses the best crystallinity, which is in good agreement with the SEM results. All of these results imply that the CH3NH3PbI3 film pretreated with the TPPCl ultrathin layer cast from a 1.0 mg/mL concentration of TPPCl solution, possesses optimal photovoltaic properties. Figure 2 XRD patterns of pristine CH3NH3PbI3 film and CH3NH3PbI3 thin films pretreated with the TPPCl ultralayers cast from TPPCl solutions with concentrations of 0, 0.25, 0.50, 1, and 1.5 mg/mL. Figure 3 depicts the UV–vis absorption spectra of pristine CH3NH3PbI3 thin film and CH3NH3PbI3 film pretreated with the TPPCl ultrathin layers. All CH3NH3PbI3 thin films have identical absorption spectrum with the same onset of absorption, indicating that the TPPCl ultrathin layers do not have any influence on the band gap of the CH3NH3PbI3 thin film. However, the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layers possesses gradually enhanced absorbance ranging from 380 to 770 nm along with the TPPCl ultrathin layers cast from increased concentrations of TPPCl solutions. The CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1.0 mg/mL concentration of TPPCl solution possesses the highest absorbance. Such high absorbance indicates that more light will be absorbed by the CH3NH3PbI3 thin film, implying that more photocurrent could be generated. Figure 3 UV–vis spectra of pristine CH3NH3PbI3 film and CH3NH3PbI3 films pretreated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of 0, 0.25, 0.50, 1, and 1.5 mg/mL. The photovoltaic properties of pristine CH3NH3PbI3 thin film and CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers are evaluated through investigation of device performance of PSCs with a device structure of ITO/PTAA/CH3NH3PbI3/PC61BM/BCP/Ag, as shown in Scheme 1b, where ITO is indium tin oxide, which acts as the anode; PTAA is poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine], which acts as the hole extraction layer; PC61BM is 6,6-phenyl-C61-butyric acid methyl ester, which acts as the electron extraction layer; BCP is bathocuproine, which acts as the hole blocking layer; and Ag is sliver, which acts as the cathode. The current density versus voltage (J–V) characteristics of PSCs are shown in Figure 4a. The device performance parameters are summarized in Table 1. The PSCs fabricated by the pristine CH3NH3PbI3 thin film exhibit a VOC of 1.04 V, a JSC of 21.08 mA/cm2, an FF of 74.41, and a corresponding PCE of 16.34%. These device performance parameters are consistent with reported values from PSCs with the same device structure.7−9 The PSCs fabricated by the CH3NH3PbI3 thin film pretreated with TPPCl ultrathin layers exhibit enlarged VOC, enhanced JSC, and thus boosted PCEs. The best device performance (VOC of 1.12 V, JSC of 21.71 mA/cm2, FF of 74.43, and the corresponding PCE of 18.04%), is observed from the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1.0 mg/mL concentration of TPPCl solution. Figure 4 (a) J–V characteristics and (b) external quantum efficiency (EQE) spectra of PSCs fabricated by pristine CH3NH3PbI3 film and CH3NH3PbI3 films pretreated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of 0, 0.25, 0.50, 1, and 1.5 mg/mL. Table 1 Device Performance Parameters of PSCs solution concentrations for deposition of the TPPCl ultrathin layer (mg/mL) VOC (V) JSC (mA/cm2) FF (%) PCE (%) HI (%) 0 1.04 21.08 74.41 16.34 3.74 0.25 1.06 21.32 75.17 17.04 1.73 0.5 1.08 21.77 74.84 17.51 1.11 1 1.12 21.71 74.43 18.04 1.12 1.5 1.08 21.38 65.75 15.26 –3.11 Figure 4b presents the external quantum efficiencies (EQE) of PSCs fabricated by either pristine CH3NH3PbI3 thin film or CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers. Enhanced EQE values are observed from the PSCs fabricated by the CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers compared to those by pristine CH3NH3PbI3 thin film. The integrated JSC values are 20.16 mA/cm2 for the PSCs fabricated by pristine CH3NH3PbI3 thin film, and 20.42, 20.83, 20.80, and 20.46 mA/cm2 for the PSCs fabricated by CH3NH3PbI3 thin films pretreated with the TPPCl ultrathin layers cast from the TPPCl solutions with concentrations of 0.25, 0.5, 1.0, and 1.5 mg/mL, respectively. These JSC values are consistent with the JSC extracted from J–V characteristics, as shown in Figure 4a. The increased JSC is attributed to the enlarged grain size (Figure 2) and stronger light-harvesting ability of CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers (Figure 3).33,34 To further understand enhanced JSC, transient photocurrent (TPC) and transient photovoltage (TPV) measurements are conducted to assess the intrinsic properties of charge-carrier extraction and charge-carriers recombination in PSCs.35,36Figure 5a presents the normalized TPC of the PSCs fabricated by either pristine CH3NH3PbI3 thin film or CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers. The PSCs fabricated by CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers possess slightly shorter charge extraction lifetimes (0.17, 0.24, and 0.23 μs) compared to that (0.29 μs) by the PSCs fabricated the pristine CH3NH3PbI3 thin film, indicating that reduced trap-assisted charge recombination and better charge extraction efficiencies have taken place in these PSCs. However, the longest charge extraction lifetime (0.43 μs) is observed in the PSCs fabricated by CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layers cast from a 1.5 mg/mL concentration of the TPPCl solution, indicating that PSCs fabricated by such thin film exhibit the poorest device performance. Figure 5 (a) Transient photocurrent and (b) transient photovoltage decays of PSCs fabricated by pristine CH3NH3PbI3 thin film and CH3NH3PbI3 thin films pretreated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of 0, 0.25, 0.50, 1, and 1.5 mg/mL. Figure 5b shows the normalized TPV of the PSCs fabricated by either pristine CH3NH3PbI3 thin film or CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers. The PSCs fabricated by CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers possess longer charge recombination lifetimes to reach the same TPV compared to those fabricated by pristine CH3NH3PbI3 thin film. The longest charge recombination lifetime is observed in the PSCs fabricated by CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1 mg/mL concentration of the TPPCl solution. The longer charge recombination lifetime demonstrates the more suppressed charge recombination in PSCs. Thus, the PSCs fabricated by CH3NH3PbI3 thin film pretreated with TPPCl ultrathin layers possess larger JSC values compared to those fabricated by pristine CH3NH3PbI3 thin film. The light intensity dependence on VOC is further investigated to illustrate the charge-carrier recombination in PSCs. Figure 6a displays the light intensity dependence on VOC for the PSCs fabricated by either pristine CH3NH3PbI3 thin film or CH3NH3PbI3 thin films pretreated with TPPCl ultrathin layers. According to VOC ∝ S In(I)37 (where S is the slope and I is the light intensity), the smallest S (0.038) is observed in the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1 mg/mL concentration of the TPPCl solution, indicating that suppressed trap-assisted charge recombination occurred in PSCs.38 Therefore, the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1 mg/mL concentration of the TPPCl solution exhibit the highest JSC value among all PSCs. Figure 6 (a) VOC dependence on different light intensities and (b) J–V characteristics, in the dark, of the PSCs fabricated by pristine CH3NH3PbI3 thin film and CH3NH3PbI3 thin films pretreated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of 0, 0.25, 0.50, 1, and 1.5 mg/mL. To understand the underlying physics of enlarged VOC, the J–V characteristics of PSCs are measured in the dark, and the results are shown in Figure 6b. According to the Shockley–Queisser model,39 the relationship between J and VOC is described as where J0 is the reverse dark current density, q is the electron charge, n is the diode ideality factor, k is the Boltzmann constant, and T is the temperature. Thus, it concludes that a large VOC is anticipated from the PSCs with a low value of J0. As shown in Figure 6b, the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with TPPCl ultrathin layers possess low J0 values compared to those fabricated by the pristine CH3NH3PbI3 thin film. In particular, the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1 mg/mL concentration of the TPPCl solution possess the lowest J0. Thus, the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer cast from a 1 mg/mL concentration of the TPPCl solution exhibit the largest VOC value. The hysteresis behaviors of PSCs are further investigated. Figure 7a–e presents the J–V characteristics of the PSCs fabricated by either pristine CH3NH3PbI3 thin film or the CH3NH3PbI3 thin film pretreated with TPPCl ultrathin layers under either forward scan direction (from the negative voltage to the positive voltage) or reserved scan direction (from the positive voltage to the negative voltage). The hysteresis index (HI) is defined as .40Table 1 summarizes PCEs and the corresponding HI values of PSCs under different scan directions. Clearly, the PSCs fabricated by pristine CH3NH3PbI3 thin film exhibit PCEs of 17.00 and 17.66% under forward and reversed scan directions, respectively, which reveal an HI value of 3.74%. A stable PCE and the smallest HI are observed from the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with TPPCl ultrathin layer cast from a 1 mg/mL concentration of the TPPCl solution. Thus, the PSCs fabricated by the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layers possess reduced photocurrent hysteresis. Such reduced photocurrent hysteresis behavior probably originated from suppressed charge recombination due to the improved crystallinity of CH3NH3PbI3 thin films. Figure 7 J–V characteristics of the PSCs fabricated by pristine CH3NH3PbI3 thin film and the CH3NH3PbI3 thin films pretreated with the TPPCl ultrathin layers cast from TPPCl solutions with concentrations of 0, 0.25, 0.50, 1, and 1.5 mg/mL and measured under forward and reverse scan directions. 3 Conclusions In summary, we reported a simple method to tune the crystallinity of CH3NH3PbI3 thin film in a two-step process. Compared to pristine CH3NH3PbI3 thin film, large and inerratic grains have been observed in the CH3NH3PbI3 thin film pretreated with the TPPCl ultrathin layer. As a result, the PSCs fabricated by CH3NH3PbI3 thin film pretreated with TPPCl ultrathin layer exhibit a VOC of 1.12 V, a JSC of 21.71 mA/cm2, an FF of 74.43, and a corresponding PCE of 18.04%, with dramatically reduced hysteresis compared to the PSCs fabricated by pristine CH3NH3PbI3 thin film, which exhibit a VOC of 1.04 V, a JSC of 21.08 mA/cm2, an FF of 74.41, and a corresponding PCE of 16.34%, with serious hysteresis. Our results provide a new strategy to boost device performance through tuning the crystallinity of perovskite thin films. 4 Experimental Section 4.1 Materials Bathocuproine (BCP), tetraphenylphosphonium chloride (TPPCl), n-butyl alcohol, anhydrous N,N-dimethylformamide (DMF), and ethanol were purchased from Sigma-Aldrich. Lead(II) iodide (PbI2) was purchased from Alfa Aesar. Phenyl-C61-butyric acid methyl ester (PC61BM) was purchased from 1-Material Inc. Poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine] (PTAA) and methylammonium iodide (MAI) were purchased from Xi’an Polymer Light Technology Corp. All materials were used as received without further purification. 4.2 Solution Preparation PbI2 was first dissolved in DMF at a concentration of 400 mg/mL and then stirred at 60 °C for 12 h to form a PbI2 solution. Afterward, the PbI2 solution was filtered using a filter of size 0.45 mm to obtain a transparent yellow solution. The MAI was dissolved in ethanol to form an MAI solution with a concentration of 35 mg/mL. TPPCl was dissolved in n-butyl alcohol with concentrations of 0.25, 0.5, 1, and 1.5 mg/mL. 4.3 Preparation of CH3NH3PbI3 Thin Film The CH3NH3PbI3 thin films were prepared by a two-step deposition method. PbI2 solution was first spin-cast on the top of the PTAA layer, which was cast from the corresponding solution, and thermally annealed at 80 °C for 5 min. After PbI2 thin film was cooled to room temperature, the TPPCl ultrathin layer was coated on the top of PbI2 thin film from TPPCl solution at 4000 rpm for 30 s. Afterward, the MAI thin layer was spin-coated on the top of either pristine PbI2 thin film or the PbI2/TPPCl thin film and thermally annealed at 100 °C for 2 h for converting PbI2 and CH3NH3I into CH3NH3PbI3 thin film. 4.4 Characterization of CH3NH3PbI3 Thin Film The surface profilometer (Tencor, Alpha-500) was used to measure the film thickness. UV–vis absorption spectra of CH3NH3PbI3 thin films were recorded on an HP 8453 spectrophotometer. Top-view images of CH3NH3PbI3 thin films were obtained using a field-emission scanning electron microscope (Zeiss Merlin). 4.5 Fabrication of Perovskite Solar Cells Precleaned indium tin oxide (ITO) glass substrates were first treated by oxygen plasma for 3 min. Afterward, ∼10 nm of the PTAA film was spin-cast on the top of the ITO substrates from PTAA solution and then thermally annealed at 100 °C for 10 min. The CH3NH3PbI3 thin films were prepared by the two-step deposition method described above. Then, an ∼40 nm thick PC61BM layer was spin-cast on the top of the CH3NH3PbI3 thin film. Afterward, an ∼10 nm BCP thin film was spin-cast on the top of the PC61BM layer. Finally, an ∼100 nm silver (Ag) electrode was thermally evaporated on the top of the BCP layer in vacuum with a base pressure of 2 × 10–6 mbar. The device area was measured to be 5.7 mm2. 4.6 Characterization of Perovskite Solar Cells The current density–voltage (J–V) characteristics of PSCs were measured under 1 sun from an AM 1.5 G solar simulator (Japan, SAN-EI, XES-40S1), where light intensity was calibrated using a standard silicon solar cell with a KG5 visible filter. The photocurrent hysteresis properties of PSCs were assessed by testing the PSCs at both forward (from −1.2 to 0.2 V) and reverse (from 0.2 to −1.2 V) directions at a scan rate of 3 V/s. The external quantum efficiency (EQE) spectra of PSCs were recorded on a DSR100UV-B spectrometer with an SR830 lock-in amplifier, a bromine tungsten light source, and a calibrated Si detector. 4.6.1 Transient Photovoltage and Transient Photocurrent Measurements A digital oscilloscope (Tektronix TDS 3052C) with input impedances of 1 MΩ and 50 Ω was used to monitor the charge density decay and charge extraction time of PSCs. The transient photovoltage of PSCs was measured under 0.03 sun illumination with a small perturbation by an attenuated laser pulse (500 nm). The laser-pulse-induced photovoltage variation (ΔV) was smaller than 5% of the VOC produced by the background illumination. The transient photocurrent of PSCs was measured by applying laser pulses to the short-circuited devices in the dark with an excitation wavelength of 500 nm, a pulse width of 120 fs, and a repetition rate of 1 kHz. Author Contributions § J.Q. and X.Y. contributed to this work equally. The authors declare no competing financial interest. Acknowledgments This study was financially supported by NSFC (51329301). ==== Refs References Stoumpos C. C. ; Malliakas C. D. ; Kanatzidis M. G. Semiconducting Tin and Lead Iodide Perovskites with Organic Cations: Phase Transitions, High Mobilities, and Near-Infrared Photoluminescent Properties . Inorg. Chem. 2013 , 52 , 9019 –9038 . 10.1021/ic401215x .23834108 Green M. 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PMC006xxxxxx/PMC6644420.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145892110.1021/acsomega.8b01188ArticleRationalizing the Formation of Activity Cliffs in Different Compound Data Sets Hu Huabin Stumpfe Dagmar Bajorath Jürgen Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Endenicher Allee 19c, D-53115 Bonn, Germany E-mail: bajorath@bit.uni-bonn.de. Phone: 49-228-73-69100.11 07 2018 31 07 2018 3 7 7736 7744 30 05 2018 26 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Activity cliffs are formed by structurally analogous compounds with large potency variations and are highly relevant for the exploration of discontinuous structure–activity relationships and compound optimization. So far, activity cliffs have mostly been studied on a case-by-case basis or assessed by global statistical analysis. Different from previous investigations, we report a large-scale analysis of activity cliff formation with a strong focus on individual compound activity classes (target sets). Compound potency distributions were systematically analyzed and categorized, and structural relationships were dissected and visualized on a per-set basis. Our study uncovered target set-dependent interplay of potency distributions and structural relationships and revealed the presence of activity cliffs and origins of cliff formation in different structure–activity relationship environments. document-id-old-9ao8b01188document-id-new-14ao-2018-01188zccc-price ==== Body 1 Introduction Activity cliffs are formed by structurally similar (analogous) active compounds with large differences in potency.1−4 Because activity cliffs represent small chemical changes having large biological activity effects, they embody the pinnacle of structure–activity relationship (SAR) discontinuity,3 which is detrimental for quantitative SAR predictions.2 However, discontinuous SARs and activity cliffs often reveal SAR determinants, especially when encountered during early stages of compound optimization, and thus provide viable information for medicinal chemistry.3,4 For a consistent assessment of activity cliffs, similarity and potency difference criteria must be clearly defined.3 On the basis of globally assessed potency range distributions of pairs of active analogues, an at least 100-fold difference in potency (on the basis of equilibrium constants, if available) has been proposed and frequently been used as an activity cliff criterion.4,5 The definition of activity cliffs also depends on the molecular representations and similarity measures that are used.4,6 Compound similarity for activity cliff definition can be quantified in different ways, for example, by calculating Tanimoto similarity on the basis of molecular fingerprint representations or by applying substructure-based similarity criteria.3,4 Numerical similarity measures, such as the Tanimoto coefficient, yield a continuum of values, and a threshold must be set for defining activity cliffs. By contrast, substructure-based methods produce a binary readout, for example, two compounds share the same core structure—and are classified as similar—or they do not. In addition to comparing molecular graph-based (two-dimensional) representations, activity cliffs have also been determined in three dimensions by calculating the similarity of experimental compound binding modes taken from complex X-ray structures.7 For graph-based activity cliff definition, substructure similarity assessment is—in our experience—generally more consistent than numerical similarity calculations and often easier to interpret from a chemical perspective.4 Among substructure-based approaches, the matched molecular pair (MMP) concept8,9 is particularly attractive for activity cliff definition. An MMP is defined as a pair of compounds that are only distinguished by a chemical modification at a single site.8 This modification corresponds to the exchange of a pair of substructures,8,9 which is termed a chemical transformation.9 By introducing appropriate transformation size restrictions, the formation of MMPs can be limited to structural analogues typically generated during compound optimization.10 Applying this similarity criterion yields a structurally conservative and chemically intuitive definition of activity cliffs.4,10 Moreover, transformation size-restricted MMPs can be efficiently generated algorithmically,9,10 hence enabling large-scale analysis of activity cliff populations. In light of these considerations, our preferred activity cliff definition encompasses the formation of a transformation size-restricted MMP by two compounds sharing the same biological activity that have an at least 100-fold difference in potency.4,10 Whenever possible, potency differences are determined on the basis of (assay-independent) equilibrium constants. The so-defined activity cliffs have been termed MMP-cliffs.10 The definition of activity cliffs is focused on compound pairs and hence accounts for pairwise relationships. However, activity cliffs in compound data sets are mostly not formed by isolated compound pairs (i.e., pairs without structural neighbors forming additional activity cliffs). Rather, the vast majority of activity cliffs are formed in a coordinated manner by groups of structurally related compounds with large potency variations, meaning that individual compounds are involved in the formation of multiple activity cliffs with different analogues.11,12 In activity cliff networks where nodes represent compounds and edges pairwise activity cliffs, compound subsets forming coordinated cliffs give rise to the formation of disjoint clusters.12 These activity cliff clusters are a rich source of SAR information and much more informative than cliffs considered as isolated.13 More than 95% of MMP-cliffs detected across different data sets were formed in a coordinated manner.14 In activity cliff networks, clusters often include “hubs,” that are, nodes representing molecules that are centers of local activity cliff formation with multiple partner compounds. Such molecules have also been termed “activity cliff generators.”15,16 In addition to activity cliff coordination, the frequency with which activity cliffs occur across different data sets has been determined.5,14 There has been substantial growth in activity cliff information over time. For example, from June 2011 until January 2015, the number of MMP-cliffs originating from the ChEMBL database,17 the major public repository of compounds and activity data from medicinal chemistry sources, nearly doubled; with a total of more than 17 000 MMP-cliffs available at the beginning of 2015.14 In addition, the target coverage of MMP-cliffs increased from about 200 to 300 individual target proteins over this period of time. However, despite this strong growth, the proportion of bioactive compounds involved in the formation of MMP-cliffs across different compound data sets remained essentially constant at close to 23%.14 So far, activity cliffs have been studied in exemplary compound sets on a case-by-case basis or surveyed by global statistical analysis.5,14 In addition, cliff populations have been organized and visualized in network representations.12,13 However, what has not been attempted thus far is systematically exploring and comparing activity cliff formation in different compound activity classes (also called target sets). To these ends, we have analyzed in detail potency distributions and structural relationships between compounds in many different target sets, studied how activity cliffs were formed, and determined the differences between sets. Hence, the focus of our current study has been on details of activity cliff arrangements in individual compound sets rather than on global statistical exploration. Our analysis revealed many characteristic differences in activity cliff formation between target sets. 2 Materials and Methods 2.1 Activity Cliff Definition For our current analysis, we introduced a modification of our preferred MMP-cliff definition stated above.4,10 For MMP generation, standard random fragmentation of exocyclic single bonds9 was replaced by fragmentation according to retrosynthetic (RECAP) rules,18 yielding (transformation size-restricted) RECAP-MMPs (RMMPs).19 Retrosynthetic MMPs were generated to further increase the chemical relevance (synthetic accessibility) of compound pairs, forming cliffs. Accordingly, the formation of an RMMP was used as a similarity criterion for activity cliffs, and an at least 100-fold difference in potency between RMMP compounds was required, as before. The so-defined activity cliffs are referred to as RMMP-cliffs. 2.2 Compounds and Activity Data Bioactive compounds with high-confidence activity data were assembled from ChEMBL version 23.17 The following selection criteria were applied: First, only compounds involved in direct interactions (type “D”) with human targets at the highest confidence level (assay confidence score 9) were selected. Second, only numerically specified equilibrium constants (Ki values) were considered as potency measurements. Equilibrium constants were reported as pKi values. On the basis of these selection criteria, a total of 71 967 unique compounds were obtained with activity against a total of 904 targets. Accordingly, these compounds were organized into 904 target sets. 2.3 RMMP Analysis RMMPs were systematically generated for all target sets, yielding 354 094 target set-based RMMPs (243 110 unique RMMPs) that were formed by 46 977 compounds from 574 target sets. For the subsequent analysis, only target sets that contained at least 100 RMMPs were retained, which resulted in 237 sets yielding a total of 347 025 target-based RMMPs (238 795 unique RMMPs) formed by 44 451 compounds. For each target set, an RMMP network was generated in which nodes represented compounds and edges pairwise RMMP relationships. In this network, each separate RMMP cluster represented a unique series of analogues. RMMP networks were also used to represent RMMP-cliffs by highlighting edges that represented both RMMP and activity cliff relationships. All network representations were drawn with Cytoscape.20 2.4 Potency Distributions For the 237 qualifying target sets, compound potency distributions were monitored in boxplots. On the basis of the interquartile range (IQR), that is, the range between quartile 1 (Q1) and 3 (Q3), target sets were assigned to three different categories, as shown in Figure 1: category 1 (CAT 1), IQR was smaller than 1 order of magnitude (<10-fold difference in potency); CAT 2, IQR fell between 1 and less than 2 orders of magnitude (10- to 100-fold difference); and CAT 3, IQR no smaller than 2 orders of magnitude (≥100-fold difference in potency). By definition, the IQR represented the potency range of ∼50% of the compounds in each target set. Figure 1 Potency distribution in target sets and categorization. The compound potency distributions of all 237 target sets were analyzed in a boxplot and the IQR, that is, the difference between quartile 3 and 1, was determined. On the basis of the IQR, target sets were divided into three different categories (CAT 1: IQR < 1; CAT 2: 1 ≤ IQR < 2; CAT 3: IQR ≥ 2). 3 Results and Discussion 3.1 Study Concept Activity cliffs have so far mostly been studied on the basis of individual compound series or by global statistical analysis.3−5 Our current study was designed to systematically investigate, for the first time, the differences in activity cliff formation and frequency between different target sets by relating compound potency distributions and structural relationships to each other. Therefore, potency distributions were determined for many different target sets, categorized, compared, and related to intra-set analogue relationships, which were systematically determined. Primary goals of the analysis included the assessment of differences in activity cliff formation and frequency between different target sets and the rationalization of such differences on the basis of potency and structural criteria, as defined in the following. To better understand target set-dependent activity cliff distributions, they were visualized in network representations. Taken together, these features set our current analysis apart from previous studies of activity cliffs in computational and medicinal chemistry.3,4 3.2 Structural Relationships Close structural relationships between active compounds are one of the two major determinants of activity cliffs, in addition to potency differences. RMMP (or MMP) calculations reveal close structural relationships and identify pairs of analogues. Importantly, however, the number of RMMPs produced by a given target set cannot be reliably used as an indicator of structural homogeneity. Rather, the presence or absence of multiple subsets of analogues comprising different series strongly influences structural heterogeneity or homogeneity, which is reflected by the cluster structure of RMMP networks, as illustrated in Figure 2. Here, two target sets with similar numbers of RMMP-forming compounds are compared. The target set on the left was dominated by a large cluster of analogues and was thus structurally homogeneous, whereas the set on the right contained 20 different small clusters and 1 larger cluster and was structurally heterogeneous. It follows that the cluster structure of RMMP networks must be carefully considered as a prerequisite for RMMP-cliff formation. Figure 2 Structural similarity in target sets. For two exemplary target sets, RMMP networks are shown in which blue nodes represent compounds and edges pairwise RMMP relationships. Separate clusters represent a unique series of analogues. Although the number of RMMP-forming compounds (CPDs) was similar for both target sets, the number of clusters differed significantly. 3.3 Potency Distributions and Profiles The likelihood of large potency differences between similar compounds can be estimated by monitoring the potency distributions of target sets. For our analysis, we assigned potency distributions to three different categories (CAT 1–3) on the basis of boxplot-derived IQR values, as shown in Figure 1. CAT 1, 2, and 3 comprised 25, 169, and 43 target sets, respectively. Hence, the majority of target sets fell into CAT 2 whose IQR spanned 1 to 2 orders of magnitude in potency and thus delineated an activity cliff-relevant range, which was further expanded by CAT 3. These observations supported our categorization of potency distributions. Accordingly, potency distributions became increasingly variable from CAT 1 to 3, as revealed by the potency distribution profiles in Figure 3. The CAT 1 profiles in Figure 3a reflect narrow potency distributions on the basis of which activity cliff formation is unlikely. By contrast, the CAT 2 profiles in Figure 3b and, especially, CAT 3 profiles in Figure 3c reveal large potency variations between structural analogues, resulting in a principally high propensity of activity cliffs. Figure 3 Potency distribution profiles. Shown are exemplary potency distribution profiles for target sets belonging to different categories [(a), CAT 1; (b), CAT 2; (c), CAT 3] according to Figure 1. Black dots represent RMMP compounds and red dots singletons not participating in RMMPs. 3.4 RMMP-Cliffs In 207 of the 237 qualifying targets sets, RMMP-cliffs were identified, amounting to a total of 11 834 cliffs. Table 1 reports that the number of RMMP-cliffs increased over target sets of CAT 1, 2, and 3, with on average 2, 52, and 69 cliffs per set, respectively. Thus, there was a general trend of increasing number of RMMP-cliffs with increasing variability of potency distributions. The very small number of RMMP-cliffs for CAT 1 sets was directly attributable to the narrow potency distributions characterizing this category. Table 2 reports that the 48 target sets containing 50 to a maximum of 820 RMMP-cliffs exclusively belonged to CAT 2 and CAT 3 that had activity cliff-relevant IQR values. By contrast, target sets with less than 50 RMMP-cliffs were found in all 3 categories. Figure 4 shows that the majority of target sets with large number of 100 or more RMMP-cliffs belonged to CAT 2, which was due to the large number of 169 target sets in this category compared to only 43 sets in CAT 3. A systematic increase in the number of activity cliffs with increasing IQR values was not observed. Figure 4 RMMP-cliffs vs IQR values. For each of the 237 target sets, the number of RMMP-cliffs (y-axis) is plotted against increasing IQR values (x-axis). Red vertical lines separate target sets belonging to CAT 1, 2, and 3. Table 1 Target Set Statisticsa CAT # target sets # clusters (mean) # RMMP-cliffs (mean) 1 25 10 2 2 169 54 52 3 43 37 69 a For each target set category (CAT), the number (#) of target sets, mean number of RMMP clusters per set, [# clusters (mean)], and mean number of RMMP-cliffs are reported. Table 2 RMMP-Cliff Distributiona # RMMP-cliffs (range) # target sets CATs 0 30 1, 2, 3 [1, 10) 77 1, 2, 3 [10, 20) 33 1, 2, 3 [20, 50) 49 1, 2, 3 [50, 100) 20 2, 3 [100, 500) 25 2, 3 [500, 820] 3 2, 3 a For different ranges of RMMP-cliffs, the number of target sets (# targets) and categories (CATs) they belong to are reported. However, despite these general trends, the propensity to form RMMP-cliffs could not solely be attributed to the variability and spread of potency distributions. Rather, as further discussed below, potency distributions in target sets must be viewed in combination with RMMP networks and their cluster structure. Table 1 also reports that target sets in CAT 1, 2, and 3 contained on average 10, 54, and 37 RMMP clusters, respectively. Thus, CAT 2 and CAT 3 sets contained large number of clusters (analogue series) whose local potency distributions strongly influenced RMMP-cliff formation. 3.5 Interplay of Potency Patterns and Structural Relationships The 207 target sets containing RMMP-cliffs were individually examined to evaluate potency distribution profiles and RMMP networks in context and rationalize why RMMP-cliffs were formed with different frequencies. The analysis revealed a number of characteristic features determining cliff formation that are summarized in Figure 5 by comparing exemplary target sets. Figure 5a (top) shows a set of phosphodiesterase 3A inhibitors with a flat CAT 1 potency distribution profile, which prohibited RMMP-cliff formation, despite the presence of two analogue series with in-part extensive RMMP relationships. In addition, Figure 5a (bottom) displays somatostatin receptor 5 ligands with a variable CAT 2 distribution and more than 100 RMMP-forming compounds. Although cliff formation was more likely in this case, the target set did not contain any RMMP-cliffs either. This was a direct consequence of a heterogeneous cluster structure and local potency distributions over different subsets of analogues forming 16 clusters, as revealed by the RMMP network of this set. Figure 5 Differences in RMMP-cliff formation. In (a–c), exemplary target sets with characteristic differences in activity cliff formation are compared, as described in the text. For each set, its potency distribution profile and RMMP network are shown and RMMP statistics are reported. Network nodes are colored by potency using a continuous color spectrum from red (lowest potency in the target set) over yellow (intermediate) to green (highest potency). If available, compounds forming exemplary RMMP-cliffs are shown and consistently labeled in all display items. Figure 5b shows two different sets of kinase inhibitors with similar CAT 2 potency distributions but different RMMP cluster structures that yielded 40 (top) and 27 (bottom) RMMP-cliffs, respectively. Exemplary RMMP-cliffs are displayed. In both instances, the target sets were structurally heterogeneous but RMMP-cliffs were formed across different clusters, revealing high degrees of SAR discontinuity. In Figure 5c, sets of anandamide amidohydrolase (top) and Bcl-X (bottom) inhibitors are compared having CAT 2 (top) and CAT 3 (bottom) distributions, respectively. The anandamide amidohydrolase inhibitors contained only 49 RMMP-forming compounds. The RMMP network was dominated by a densely connected cluster of 19 analogues that formed 79 coordinated RMMP-cliffs (exemplary cliffs are shown). Thus, in this case, the number of RMMP-cliffs was much larger than the number of participating analogues because of extensive coordination of cliffs. Hence, this cluster represented an SAR hotspot. By contrast, the Bcl-X inhibitors contained a much larger number of 119 RMMP-forming compounds that were distributed over 20 clusters. Although the CAT 3 potency distribution of this target set was highly variable, the majority of compounds in individual clusters had comparable potency, whereas the potency levels of clusters significantly differed, giving rise to the presence of only three RMMP-cliffs. Taken together, the results in Figure 5 were representative of many target sets we studied. Analyzing the potency distribution profiles and in combination with RMMP networks revealed the characteristic features of target sets and clearly rationalized differences in RMMP-cliff frequency across target sets. 4 Conclusions Herein, we have reported a systematic analysis of RMMP-cliffs in more than 200 target sets to investigate and better understand the origins of cliff formation and differences in the frequency of cliffs. Our study was strongly focused on individual target sets and their comparison. Potency distributions were determined and categorized, and structural relationships were analyzed at the level of RMMPs and organized in networks. Structural homogeneity of target sets and potency distributions of increasing variability generally supported the formation of RMMP-cliffs. However, the interplay of structural and potency relationships determined the frequency with which RMMP-cliffs were formed, as revealed by relating potency profiles and RMMP networks to each other and studying local potency distributions across different RMMP clusters. The analysis scheme introduced herein reveals target set-dependent formation of activity cliffs, provides immediate visual access to characteristic activity cliff-relevant features of target sets, and rationalizes differences in the frequency of cliffs across sets. Author Contributions The study was carried out by all authors, and the manuscript was written with contributions of all authors. All authors have approved the final version of the manuscript. The authors declare no competing financial interest. Acknowledgments H.H. is supported by the China Scholarship Council (CSC). ==== Refs References Lajiness M. Evaluation of the Performance of Dissimilarity Selection Methodology . 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PMC006xxxxxx/PMC6644421.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145808010.1021/acsomega.8b01942ArticleTailoring the Emission of Fluorinated Bipyridine-Chelated Iridium Complexes Zhang Guang †§∥Baumgarten Martin †Schollmeyer Dieter ‡Müllen Klaus †† Max Planck Institute for Polymer Research, Ackermannweg 10, Mainz D-55128, Germany‡ Institute für Organische Chemie, Johannes-Gutenberg Universität, Mainz D-55128, Germany§ Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University, Suzhou 215123, China* E-mail: baumgart@mpip-mainz.mpg.de (M.B.).* E-mail: muellen@mpip-mainz.mpg.de (K.M.).22 10 2018 31 10 2018 3 10 13808 13816 08 08 2018 25 09 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. New functionalized tris(2′,6′-difluoro-2,3′-bipyridinato-N,C4′)iridium(III) ((dfpypy)3Irs) complexes, including small molecules and their dendrimer embedded analogoues, were synthesized and characterized. It is demonstrated that both the fac-(dfpypy)3Ir-based polyphenylene dendrimers and (triisopropylsilyl)ethynyl (TIPSE)-substituted (dfpypy)3Ir complexes induce large bathochromic shifts (∼50 nm) of emission bands compared with fac-(dfpypy)3Ir. This is due to the pronounced 3π–π* character of emissive excited states and the extended conjugation. A further remarkable feature is the small bathochromic shift of the emissions of fac-tris(2-phenylpyridine)iridium (fac-(ppy)3Ir)-based polyphenylene dendrimers when compared to those of the iridium (Ir) complex core. Obviously, the triplet metal-to-ligand charge transfer makes emission less sensitive to extended conjugation than the 3π–π* transition. This finding suggests new concepts for designing blue phosphorescent dendrimer emitters. Both the dendrimers and the TIPSE-substituted (dfpypy)3Ir complexes represent new green and the trimethylsilyl-functionalized (dfpypy)3Ir new blue phosphorescent emitters. Incorporation of TIPSE moieties into the ligands of iridium complex gives rise to enhanced phosphorescence. document-id-old-9ao8b01942document-id-new-14ao-2018-019422ccc-price ==== Body Introduction Phosphorescent emitters (PEs) such as iridium (Ir) and platinum (Pt) complexes have been intensively investigated since the first report on PE-based organic light emitting diodes (OLED) by Baldo et al. in 1998.1−5 PEs could provide much higher efficiencies than conventional organic singlet fluorophores because the former generates both singlet and triplet excitons in devices (corresponding to 100% internal quantum efficiency and around 20% external quantum efficiency (EQE) theoretically).1−8 Significant improvements of red,9 green,10 blue,11−13 and white14 OLEDs based on small-molecule PEs have been achieved so far and all have reached or passed 20% EQEs. PEs have also received attention in applications such as photocatalysis,15−17 chemical sensors,18,19 solar cells,20,21 and biolabeling,22 due to their intriguing photophysical properties. The neat films of PEs are prone to undergo severe self-quenching.23−27 Therefore, the emitters (also called dopants) are usually incorporated into a matrix to ensure high photoluminescence quantum yields (PLQYs). The matrix composed of host materials, e.g., 4,4′-bis(N-carbazolyl)-1,1′-biphenyl, serves as medium for charge transport and energy transfer to the dopants.6,28−30 However, these devices based on small molecules still suffer from expensive ultra high vacuum fabrication and inhomogeneous distribution of the dopants in the matrix. Dendrimers can overcome these problems because of their good film formation and generation-by-generation site-specific functionalization.31−35 In a rigid and structurally well-defined dendritic molecule, the core can be a phosphorescent emitter while the surface is functionalized with host moieties. Thus, the ratio between the hosts and dopants and their positions are accurately controlled in a dendritic structure.32,33 There have been many studies on dendrimer-based phosphorescent emitters, especially green and red ones, since the pioneering work of Samuel and Burn et al. in 2001.35−43 So far, very few blue PEs were reported and they all suffered from poor color purity and/or low efficiencies.44−48 Accordingly, it is worthwhile to develop high-performance, dendrimer-based blue PEs. Shape-persistent polyphenylene dendrimers (PPDs) as emitters have been widely studied.49−54 Core and surface-functionalized, first-generation PPDs were shown to improve OLED performance through the shielding effect and appropriate choice of surface moieties. The latter were thought to bring about efficient surface-to-core energy transfer and charge transport.50,51 PPDs were also employed as phosphorescent green and red emitters.49,53 Interestingly, the peak emission of fac-(ppy)3Ir-based PPDs (G1–G4) (Figure 1) in solution was located at 516 nm, i.e., very close to that of fac-(ppy)3Ir (λem: ∼508 nm).55 It follows that the attachment of polyphenylene dendrons on fac-(ppy)3Ir does not change the emission wavelength notably. Figure 1 Molecular structures of fac-(ppy)3Ir, G1, fac-(dfpypy)3Ir, and the targeted iridium complexes. fac-Tris(2′,6′-difluoro-2,3′-bipyridinato-N,C4′)iridium(III) (fac-(dfpypy)3Ir) (Figure 1) was described as a pure blue PE (λmax: 438 and 463 nm) with high PLQY (∼0.71) and thermal stability (5% loss at 452 °C from thermogravimetric analysis).56 We thus chose this chromophore as the core of a PPD, en route to unprecedented blue phosphorescent dendrimer emitters. In this contribution, as depicted in Figure 1, several (dfpypy)3Ir-based small molecules and a first-generation PPD with carbazoles in the periphery (1) are introduced, whereby carbazoles act as charge transport and energy transfer moieties. Results and Discussion Synthesis A trimethylsilyl (TMS)-substituted Ir complex 4 was considered as the initial candidate because TMS groups could be transformed into iodo substituents later.57 As depicted in Scheme 1, 2-bromo-5-trimethylsilylpyridine (8) was synthesized from 2,5-dibromo-pyridine (10) and trimethylsilylchloride (TMSCl) in high yield (82%). Then, compound 8 was reacted with 2,6-difluoropyridinyl-3-boronic acid (9) obtained from commercial 2,6-difluoropyridine (11) to furnish the functionalized bipyridine ligand (7) by Suzuki coupling in moderate yield (50%). Subsequently, the one-step reaction between bipyridine 7 and iridium(III) acetylacetonate (Ir(acac)3) to the iridium complex (4 and 5) failed.56,58−60 A two-step method was therefore applied by refluxing the bipyridine ligand 7 with IrCl3, providing a dichloro-bridged dimer (6) as a yellow solid in moderate yield (48%). Then, the dimer (6) and the ligand (7) were heated under basic conditions,47 leading to a yellow solid (5). Attempted substitution of the TMS group with iodine remained unsuccessful. Scheme 1 Synthetic Routes for TMS-Functionalized (dfpypy)3Ir (a) (1) n-BuLi, Et2O, −78 °C, 1 h, (2) TMSCl, room temperature (rt), 12 h, 82%; (b) (2,6-difluoro-3-pyridinyl)boronic acid 9, Pd(PPh3)4, K2CO3, tetrahydrofuran (THF), water, 85 °C, 24 h, 50%; (c) IrCl3·nH2O, 2-ethoxyethanol, 140 °C, 24 h, 48%; (d) compound 7, AgSO3CF3, K2CO3, mesitylene, 170 °C, 24 h, 53%; (e) UV light, THF, 12 h, 90%; (f) (1) isopropylamine, THF, n-BuLi, 0 °C, 30 min for making fresh lithiumdiisopropylamide (LDA), (2) LDA, THF, −78 °C, 1 h, (3) triisopropylborate, rt, 12 h, 1 M HCl (aq), 100%; (g) ICl, CCl4, 80 °C, 12 h; (h) Ir(acac)3, ethylene glycol, 200 °C. Tri-cyclometalated iridium(III) complexes usually exist in two configurations, i.e., a facial and meridional, with C3 and C1 symmetry, respectively.60−62 The obtained TMS-functionalized Ir complex 5 possesses meridional configuration, as demonstrated by its 1H and 19F NMR spectra (Supporting Information). The facial isomer 4 was easily obtained by stirring a THF solution of 5 under UV light for 12 h in high yield (90%) (Scheme 1e).60−62 For Ir complex 4, only 4 proton signals are present in the low-field region of its 1H NMR spectra, corresponding to the 4 protons of the bipyridine ligand; in addition, two peaks in the 19F NMR spectra represent the two fluorine atoms in one ligand, thus demonstrating a C3 symmetry of complex 4. On the other hand, the number of corresponding NMR signals for molecule 5 was tripled in comparison. Single crystals of both complexes 4 and 5, suitable for X-ray diffraction, were obtained by slow addition of methanol to a dichloromethane (DCM) solution. As shown in Table S2 and Figure 2, the facial configuration possesses nearly identical Ir–N bonds (∼2.12 Å) and Ir–C bonds (∼2.00Å), similar to those in fac-(dfpypy)3Ir56 and consistent with its C3 symmetry. For the meridional one, however, the Ir–N2 bond has a notably longer length (∼0.1 Å) than that of others. This suggests that these bonds can break and reorganize to form the facial isomer 4, which is in accord with the successful meridional-to-facial transformation upon photochemical treatment.60−62 Moreover, several short intermolecular contacts exist for both materials (e.g., F···H–C, F···C, C···C, N···H–C, and face-to-face π···π interactions) (Supporting Information), similar to those in fac-(dfpypy)3Ir.56 Figure 2 Crystal structure views of Ir complexes 4 and 5 with ellipsoids at 50% probability (proton atoms were hidden for clarity). With the successful synthesis of the TMS-substituted Ir complexes, the preparation of (dfpypy)3Ir-based PPDs became possible. In view of the Diels–Alder reaction with carbazole-functionalized tetraphenylcyclopentadienone (21), ethynyl units had to be introduced (Scheme 2), yielding TIPSE-functionalized ligands (14 and 15). The first step involved generating the bromo-functionalized bipyridines (16 and 17) from 2,6-difluoro-pyridine-5-boronic acid (9) and 2-iodo-4-bromopyridine (19) or 2-iodo-5-bromopyridine (18) by Suzuki coupling in which the iodo reacted much faster than the bromo groups. Then, (triisoprypylsilyl)acetylene was allowed to react with compounds 16 and 17 by Sonogashira coupling to afford the TIPSE-functionalized bipyridine ligands 14 or 15 in high yields (85 and 96%). Scheme 2 Synthetic Routes for TIPSE-Functionalized Ir Complexes (a) Pd(PPh3)4, K2CO3, THF, water, 80 °C, 24 h, 39% for 16, 34% for 17; (b) Pd(PPh3)2Cl2, PPh3, CuI, triethylamine, (triisopropylsilyl)acetylene, 80 °C, 24 h, 85% for 14 and 96% for 15; (c) IrCl3·nH2O, 2-ethoxylethanol, 140 °C, 24 h, 57% for 12, 24% for 13; (d) TIPSE-substituted bipyridine 14 or 15, K2CO3, AgSO3CF3, 1,3,5-trimethylbenzene, 170 °C, 24 h, 25% for 2, 20% for 3. For iridium complex formation, the functionalized bipyridines 14 or 15 were treated with IrCl3·nH2O to furnish the dichloro-bridged dimers 12 and 13 in moderate yields (57 and 24%) (Scheme 2). Thereafter, TIPSE-functionalized Ir complexes 2 and 3 were obtained by a reaction between the dimer and TIPSE-substituted bipyridine 14 or 15 under basic conditions. The low yields (25 and 20%) are similar to those commonly reported for Ir complexes.47 The collected iridium complexes 2 and 3 were in meridional configurations, as confirmed by 1H and 19F NMR measurements (Supporting Information). Compound 2 could not be transformed into its facial isomer by photochemical treatment, probably due to decomposition upon prolonged UV light irradiation. Finally, dendrimers 20 and 1 were obtained starting from the TIPSE-substituted molecule 2. As depicted in Scheme 3, first, the TIPS fragments were removed by treatment with tetrabutylammonium fluoride (TBAF).12 With the ethynyl-substituted Ir complex 22 available, dendrimer 20 was obtained by a Diels–Alder reaction between 22 and molecule 21 in moderate yield (55%). The resulting product is meridional, as confirmed by the 1H NMR and 19F NMR spectra (Supporting Information). As the facial isomer usually displays a higher PLQY than that of the meridional one,60,63 the former was successfully prepared by photolysis in 83% yield. The facial dendrimer 1 was comprehensively characterized by 1H and 19F NMR spectroscopy, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass, and high-resolution mass spectrometry (HRMS). Scheme 3 Synthetic Routes for Iridium Complex-Based Dendrimers (a) TBAF, THF, rt, 46%; (b) o-xylene, 150 °C, 48 h, 55%; (c) UV light, THF, 12 h, rt, 83%. In the 1H NMR spectra of dendrimer 1 (Figure 3) proton Ha ortho to the fluorine atom appears as a singlet peak at around 5.90 ppm. Protons Hb and Hc occur at 8.13 and 7.52 ppm, respectively. The carbazole protons at 4,5-positions (Hf) are found at 8.05 ppm, and the tert-butyl group protons at 1.33 ppm are clearly assigned. Moreover, there are only two peaks present in the 19F NMR spectra, corresponding to the two fluorine atoms in one ligand of the molecule. Figure 3 1H NMR and 19F NMR (inset) spectra of dendrimer 1 (solvent: CD2Cl2). The MALDI-TOF mass spectra of dendrimer 1 exhibits a single peak of the molecular ion, in good agreement with the molecular mass of the dendrimer (Figure S3). The HRMS spectra show clear isotope patterns of the molecular ion, which is consistent with the calculated result (Figure S3). Photophysical Characterization As depicted in Figure 4a, both TMS-substituted Ir complexes 4 and 5 exhibit strong absorptions between 250 and 300 nm due to the ligand-based π–π* transitions.56 The relatively weak bands between 350–400 nm are attributed to the singlet metal-to-ligand charge transfer (1MLCT).56,60 In addition, as shown in the inset of Figure 4a, a shoulder between 400 and 450 nm is tentatively assigned to a mixture of spin–orbital coupling-enhanced ligand (L)-centered 3π–π* (3LC) and triplet MLCT (3MLCT) transitions, which is based on a comparison with fac-(dfpypy)3Ir.56 This is consistent with the two absorption bands of molecule 4, in which one centered at 412 nm is presumably ascribed to 3LC and the other (λmax: 438 nm) to 3MLCT according to the proposed orbital diagram of the Ir complexes (Figure 6, top left). The facial one appears as a stronger and deeper blue emitter than the meridional one (Figure 4b), which is similar to the situation prevailing in other reported Ir complexes,60,63 such as fac-(ppy)3Ir and mer-(ppy)3Ir.60 Both emission spectra are structured, resembling fac-(dfpypy)3Ir.56 The small bathochromic shift of the emission of compound 4 against that of fac-(dfpypy)3Ir56 is due to the electron-donating effect of the TMS groups (Table 1). In line with that, the emission is essentially attributed to ligand-based 3π–π* transitions rather than 3MLCT because electron-donating moieties lead to a blue-shifted emission for a 3MLCT-type phosphorescence.64 As indicated in Figure 6, TMS-substituted bipyridine (7) has a higher highest occupied molecular orbital (HOMO) but about the same lowest unoccupied molecular orbital (LUMO) level compared to those of difluoro-substituted bipyridine 26. With the new orbital formation between Ir and ligand, it shows that the transition energy of MLCT and LC of material 4 increases and decreases respectively, compared with those of fac-(dfpypy)3Ir and consequently causes blue- and red-shifted emissions individually. This is consistent with the conclusion from Lee et al., that the emission of fac-(dfpypy)3Ir is mainly attributed to a 3LC nature of the emissive excited states, even though their calculated results support 3MLCT.56 Figure 4 UV–vis absorption (a) and photoluminescence spectra (b) of compound 4 and 5 in 10–4 M DCM solution (excited at 330 nm). Figure 5 UV–vis absorption (a) and emission spectra (b) of compounds 3, 2, 20, and 1 (10–5 M in DCM, ex: 385 nm for 3 and 2 and 348 nm for 20 and 1, measured at room temperature). Figure 6 Molecular orbital distributions and energy levels of Frontier molecular orbitals (FMOs) of the ligands (calculated using DFT, B3LYP, 6.31G method). Table 1 Photophysical and Electrochemical Properties of Ir Complexes λab (nm) λem (nm) Sol (rt) Sol (rt) Sol (77 K) ETb PLQYc HOMO (ev)d LUMO (ev)d Ega fac-(dfpypy)3Ir 375e 438, 463e 0.71e 5 259, 370, 436, 441 448, 475 446, 475 2.78 –6.03 –2.60 3.43 4 262, 334, 370, 411, 438 444, 472 440, 469 2.82 0.58 –6.01 –2.57 3.44 3 270, 329, 376 508 479, 509 2.59 0.73 2 289, 349, 383 489, 525 486, 521 2.55 0.30 –5.68 –2.83 2.85 1 297, 334, 348 515 486, 517 2.55 0.31 –5.55 –2.11 a Calculated from the energy difference between HOMO and LUMO. b Calculated from the highest energy peak of emission spectra (77 K) (Figure S5), which is 1240/λem. c Measured in toluene solution with quinine sulfate in 0.5 M H2SO4 solution as the standard. d Calculated from cyclic voltammetry (CV) by comparing the first redox onset of the compounds and the oxidation onset of ferrocene. e Obtained from ref (56). For the absorption of Ir complexes 2 and 3 (Figure 5), the first two bands between 270 and 350 nm are due to the π–π* transitions of the ligands. The weaker one (370–400 nm) is likely attributed to the 1MLCTs.56,60 The little shoulders above 400 nm are assigned to a mixed state of spin–orbital coupling-enhanced 3π–π* and 3MLCT, referring to fac-(dfpypy)3Ir.56 Both are green emitters with pronounced bathochromic shifts (∼45 nm for compound 3) in contrast to fac-(dfpypy)3Ir (Figure 5b), which should relate to the extended conjugations. This also suggests that the emissive excited states exhibit dominant 3π–π* rather than 3MLCT nature because the former induces bigger bathochromic shift than the latter when the conjugation is extended. As shown in Figure 6, for Ir complex 2, due to the smaller energy gap of ligand 14 than that of difluoro-substituted bipyridine (26), the reduction of transition energy is considerable for LL but is much less notable for MLCT, compared to that for fac-(dfpypy)3Ir. This is consistent with the 3LC nature of the emissive excited states of materials 4 and 5. Additionally, both the bathochromically shifted absorption and emission of Ir complex 2 compared with molecule 3 is ascribed to the bigger band gap of 4-TIPSE-substituted bipyridine 15 than that of ligand 14, which indicates that conjugation at 5 position is more effective (Figure 6). Moreover, Ir complex 3 appears as a stronger emitter than phosphore 2 (Figure 5b), owing to very high extinction coefficient between 400 and 500 nm (ε ≤ 6000 M/cm) of lumiphore 3, which corresponds to 3LC and 3MLCT transitions. This is comparable to that of highly emissive fac-(ppy)3Ir (ε ≤ 6000 M/cm) in the same region.65 This is also consistent with the higher PLQY of Ir complex 3 than that of 2 (Table 1). The Ir complex 3 is a rare case of a strongly emitting cyclometalated Ir complex with alkynyl groups in the ligand.49,53 As to the absorption of dendrimers, a strong band at around 297 nm is characteristic for carbazole absorptions.66 The band between 320 and 350 nm and the one ranging from 370 to 400 nm are due to electronic transitions of the ligands50 and 1MLCTs, respectively. The less notable shoulders (400–450 nm) stem from a mixed state of spin–orbit coupling-enhanced 3π–π* and 3MLCT.56 Both dendrimers are green emitters (Table 1), similar to materials 2 and 3. Consequently, dendrimer 1 exhibits a bathochromically shifted emission of 50 nm compared to that of fac-(dfpypy)3Ir. This strong shift is correlated to 3π–π* rather than 3MLCT, similar to complex 2. This feature is quite different from fac-(ppy)3Ir-based PPDs bearing very small redshifts (∼8 nm) in emissions as compared with parent fac-(ppy)3Ir.53 Lo et al. argued that this is due to the lack of conjugation between dendron and ligand.67 We rather propose that this is due to the dominant 3MLCT nature of emissions of fac-(ppy)3Ir-based PPDs (Figure S6). As shown in Figure 6, for molecule 25, polyphenylene dendrons indeed significantly decrease the band gap of the ligand compared with bipyridine 26 (4.25 vs 5.05 eV calculated). The close band gaps between TIPSE-substituted bipyridine 14 and ligand 25 and the similar emission colors of Ir complexes 2 and 1 further manifest that their luminescence has primary 3LC nature. The facial dendrimer displays slightly stronger emission than that of the meridional one, similar to other Ir complexes, e.g., materials 5 and 4, but the emission intensity of both are much lower than that of compound 3. This is consistent with the much weaker absorptions of 3LC and 3MLCT of the dendrimers than those of compound 3. Extending the conjugation of the ligand is an effective way to tune the color of emission of metal–organic complexes,68 which has been reported for dendrimer-based blue PEs as well.44 Electrochemistry The redox properties of some of the prepared Ir complexes were studied by cyclic voltammetry (CV). Compound 4 and 5 exhibit two oxidation processes, starting above 1.5 V (Figure S7), which indicates their relatively low HOMOs (∼−6.00 eV) (Table 1), similar to that in fac-(dfpypy)3Ir.56 Both of them present three reversible reduction couples (LUMO: ∼−2.60 eV). Going to Ir complex 2, due to the extended conjugations, the HOMO (−5.68 eV) is raised and the LUMO (−2.83 eV) is decreased compared with molecules 4 and 5. As to dendrimer 1, the HOMO and LUMO were calculated to be −5.55 and −2.11 eV, respectively. This is consistent with the FMOs of the peripheral carbazoles,66 probably owing to the big ratio between the number of carbazole groups and Ir complex segment within one molecule (6:1). Conclusions We find that the TMS-functionalized and TIPSE-substituted (dfpypy)3Irs and (dfpypy)3Ir-based polyphenylene dendrimers exhibit bathochromically shifted emissions, when compared with parent fac-(dfpypy)3Irs due to the dominant 3LC nature of their emissive excited states. This demonstrates that strong red-shifted emission occurs when both extended conjugations and significant 3LC character are involved in a phosphore. It is also found that when both extended conjugations and primary 3MLCT nature are present in an Ir complex, the bathochromic shift is less notable or much weaker, as seen, for example, in fac-(ppy)3Ir-based polyphenylene dendrimers. Therefore, this work provides useful insights into the design of new blue phosphorescent dendrimer emitters besides the most commonly utilized method of breaking the conjugation between the PE core and the dendron with alkyl segments.45 Appropriate steps are: (i) to select the core of a dendrimer with a blue PE with dominant 3MLCT nature of its emissive excited state or (ii) to utilize a UV PE with major 3LC nature of its emissive excited states as the core of the dendrimer to push the emission of the dendrimer into pure blue region by extended conjugations. Experimental Section Materials and Methods All chemicals and solvents were purchased from commercial sources and used as received except where noted. Reactions were all conducted under argon atmosphere. 1H NMR and 13C NMR spectra were recorded on Bruker AMX 250, AC 300, and AMX 500 NMR spectrometers. The solvents for NMRs were CD2Cl2 with the reference peak at 5.32 ppm (1H) and 53.84 ppm (13C) and DMSO-d6 with the reference peak at 2.50 ppm (1H). Field desorption mass spectra were recorded on a VG-Instruments ZAB 2-SE-FDP using 8 kV accelerating voltage. MALDI-TOF mass spectra were recorded by a Bruker Reflex II spectrometer with fullerene as the reference and dithranol as the matrix. HR-MALDI-TOF mass spectra were recorded by a SYNAPT G2-Si time-of-flight instrument equipped with a MALDI source (Waters Corp. Manchester, U.K.). UV–vis absorption spectra were measured by a Varian Cary 4000 UV–vis spectrometer (Varian Inc. Palo Alto) using quartz cells with path length of 1 cm. Fluorescence spectra were recorded with a SPEX Fluorolog 2 spectrometer. Cyclic voltammetry (CV) measurements were conducted on a computer-controlled GSTAT12 workstation using a three-electrode system in which a Pt wire, a silver wire (or a saturated calomel electrode), and a glassy carbon electrode were used as counter, reference, and working electrode, respectively. The measurements were conducted in 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) solution under argon environment with a scan rate of 100 mV/s at room temperature. The solvents were DCM for the oxidation part and acetonitrile for the reduction part. Ferrocene/ferrocenium (Fc/Fc+) worked as the internal reference throughout the measurements. The calculations of HOMO and LUMO using CV were based on the following equations, HOMO (eV) = −Eoxonset + EFconset – 4.80 and LUMO (eV) = −Eredonset + EFconset – 4.80, where Eoxonset, EFc/Fc+onset, and Eredonset mean the onset oxidation potential of the targeted molecule, the onset oxidation potential of ferrocene, and the onset reduction potential of the targeted molecule compared with the reference electrode. The calculated isotope distributions of the molecular mass of the dendrimer were conducted with mMass software. The X-ray intensity data for 4 was measured on a STOE IPDS-2T X-ray diffractometer system equipped with a Mo-target X-ray tube. The data frames were collected using the program X-Area and processed using the program Integrate routine within X-Area. The reflection intensities were corrected for absorption on the basis of the crystal faces using XRED-32. The X-ray intensity data for 5 was measured on a Bruker SMART APEX2 CCD-based X-ray diffractometer system equipped with a Mo-target X-ray tube. The data frames were collected using the program APEX2 and processed using the program SAINT routine within APEX2. The data were corrected for absorption on the basis of the multiscan technique, as implemented in SADABS. Structure solution and refinement were performed using SHELT-2014. Crystal of compound 4 contains one molecule of CH2Cl2. The density functional theory calculations for ground-state geometry optimizations were performed using PC controlled Gaussian software. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01942.Synthetic procedures and characterization data of the targeted molecules: 1H NMR, 13C NMR, X-ray single-crystal diffraction, low-temperature photoluminescence, and cyclic voltammetry (PDF) Crystallographic data (CIF) Supplementary Material ao8b01942_si_001.cif ao8b01942_si_002.pdf Author Present Address ∥ Institute of Functional Nano & Soft Materials, Soochow University, Suzhou, 215123, China (G.Z.). The authors declare no competing financial interest. Acknowledgments The authors gratefully thank the Deutsche Forschungsgemeinschaft (SFB625) for providing financial support for this research. The authors also gratefully thank Dr Wen Zhang for MALDI-TOF mass characterizations. 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PMC006xxxxxx/PMC6644422.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145880710.1021/acsomega.8b00574ArticlePorous Organic Polymer-Derived Carbon Composite as a Bimodal Catalyst for Oxygen Evolution Reaction and Nitrophenol Reduction Gopi Sivalingam †Giribabu Krishnan ‡Kathiresan Murugavel ††Electro Organic Division and ‡Electrodics and Electrocatalysis Division, CSIR-Central Electrochemical Research Institute, Karaikudi 630003, TamilNadu, India E-mail: kathiresan@cecri.res.in. Tel: 04565-241312.11 06 2018 30 06 2018 3 6 6251 6258 26 03 2018 31 05 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Ethylene diamine-based porous organic polymer (EPOP) was synthesized, carbonized at different temperatures, and characterized. The successful formation of the triazine polymer was confirmed by Fourier-transform infrared spectroscopy, 13C, and 15N cross-polarization magic angle spinning solid-state NMR. The two-dimensional layered architecture and graphitic nature of the samples resembled that of nitrogen-doped amorphous carbon, as confirmed by Raman, powder X-ray diffraction, and transmission electron microscopy measurements. The catalytic activity of these materials toward nitrophenol reduction and electrocatalytic activity toward oxygen evolution reaction (OER) were systematically evaluated in detail. Electrocatalytic activity toward oxygen evolution reaction was systematically evaluated by chronoamperometry and linear sweep voltammetry. Results clearly demonstrate that all of these catalysts exhibit good OER activity and excellent stability. Among all catalysts, EPOP-700 showed better OER activity, as reflected by its onset potential and current density, comparable with that of the metal-based OER catalysts and better than that of metal-free catalysts. Further, their catalytic activity toward the reduction of 4-nitrophenol to 4-aminophenol was tested with NaBH4; although all of these catalysts showed good catalytic activity; EPOP-800 displayed better catalytic activity. document-id-old-9ao8b00574document-id-new-14ao-2018-005743ccc-price ==== Body Introduction Porous organic polymers (POPs), a subclass of organic polymers/organic materials, show great advantage over conventional polymers in the area of catalysis due to their high surface area.1,2 They are highly cross-linked and are amorphous in nature.1,3 Proper tuning of the porosity in these materials can be achieved with desired functional groups or linking units. It is anticipated that porous organic polymers with high nitrogen content are desirable for catalytic applications.4 It is also advantageous that the “N” atom can coordinate to metal atoms;5 hence, their surface can be modified with desired metals to further improve their catalytic properties.6 Among the porous organic polymers, covalent triazine frameworks constitute an important class because of their high nitrogen content, high surface area, high thermal and chemical stability, easy preparation on a large scale, and use in catalytic applications.6−9 Thomas and co-workers developed a novel ionothermal process for the synthesis of covalent triazine frameworks from aromatic nitriles using ZnCl2 at elevated temperatures (≥400 °C) in molten state.10 Such a high-temperature procedure usually yields highly porous framework due to the partial decomposition of organic moiety, although it was reported that nitrile trimers are stable up to 400 °C.6,10,11 POPs can also be synthesized from melamine3 and cyanuric chloride (C-Cl) under mild conditions;8,12,13 however, POPs prepared under mild conditions exhibit very low surface area.14,15 It is further shown that these syntheses are solvent dependent. The condensation reaction in dimethylacetamide and N-methyl pyrrolidine yielded 4 times high surface area material than the one obtained using dioxane as a solvent, indicating the impact of solvent on porosity in such reactions. Fifteen POPs are electron rich and they exhibit good π-electron mobility along the triazine ring and if conjugated with an aromatic linker, they exhibit good electronic conductivity, which enhances their chemical, electrochemical, and photocatalytic activity.16 Altogether, POPs find application in energy storage devices, gas storage/separation, photocatalysis, etc. wherein the triazine nitrogen is shown to play a dominant role.16−21 Recent applications of POPs focus on the heteroatom doping, multilayer assembling, and metal cocatalysts aimed toward further enhancement of their catalytic properties.22−25 Oxygen evolution reaction (OER) is one of the most promising sustainable systems to generate clean and effective energy. OER is a complex multistep reaction as it involves several surface-adsorbed intermediates, and this reaction usually requires large overpotential for the actual process, which distinctly reduces the process efficiency even if the benchmark catalysts are applied.26 Numerous electrocatalysts have been developed till date to improve the efficiency of oxygen evolution.27 Ru- and Ir-based noble metal catalysts were frequently employed as benchmark catalysts due to their efficiency in oxygen evolution reaction. Further, nanoscale catalysts with reduced noble metal content and high surface area were developed to reduce the cost. Later, metal nanocatalysts on carbon matrices were developed. In this scenario, metal-free catalysts have attracted wide attention owing to their analogous performance with noble metal catalysts. Frequently studied metal-free catalysts include graphene-based materials, carbon nanotubes (CNTs), and so on, which are usually expensive. Metal-free catalysts were also developed from inexpensive carbon-based materials by high-temperature treatment under inert conditions. On the other hand, 4-nitrophenol (4-NP) is an important organic pollutant obtained from agricultural and industrial sources.28 Metal-catalyzed reduction of 4-NP with excess NaBH4 yields 4-aminophenol (4-AP), a key intermediate in the production of paracetamol and dyeing industry. Hence, development of an efficient catalyst for the reduction of 4-NP to 4-AP will be of broad interest owing to its industrial value. In general, metal or metal-based nanocatalysts were employed for the catalytic reduction of 4-NP to 4-AP with excess NaBH4. Till date, very few metal-free catalysts based on graphene materials are known for the reduction of 4-NP to 4-AP, which are usually expensive.28,29 In this context, development of a catalyst with dual performance in OER and nitrophenol reduction would be interesting, provided they display excellent catalytic activities in both the reactions, as they follow a different pathway. Recently, we reported on the synthesis of phenylenediamine-based POP network and its electrocatalytic activity toward the nitrobenzene reduction and oxygen evolution reaction (OER).14 Further, the carbonized sample was tested for its energy storage performance.30 There are very few reports available on OER and nitrophenol reduction using metal-free catalysts derived from POPs. Herein, we report the synthesis, carbonization, and characterization of ethylene diamine-based porous organic polymer (EPOP) and their catalytic activity toward oxygen evolution reaction and nitrophenol reduction. Results and Discussion The reported procedure was followed to obtain EPOP (Scheme 1).14 EPOP-600, EPOP-700, and EPOP-800 were obtained by the carbonization of EPOP sample at 600, 700, and 800 °C under inert atmosphere (3 h) in a tubular furnace, respectively. Scheme 1 Synthesis of EPOP and Its Carbon Composites The successful formation of the porous organic polymer is confirmed by Fourier-transform infrared spectroscopy (FT-IR) analysis. The absence of cyanuric chloride (C-Cl) stretching vibration at 850 cm–1 and a broad peak at 3400 cm–1 corresponding to “NH2” of ethylene diamine confirms the absence of both the starting materials (Figure S1). Apart from this, key peaks such as the stretching frequencies of triazine rings were observed at 1347 and 1571 cm–1, bending and stretching vibration of sp3 “CH2” moiety were observed at 1447 and 2940 cm–1 respectively. The peak at 804 cm–1 corresponds to the breathing mode of vibration of triazine unit (Figure S2). All of these characteristic peaks indicate the successful formation of triazine polymer, which is further confirmed by solid-state 13C and 15N cross-polarization magic angle spinning (CP-MAS) spectroscopy. The FT-IR of the carbonized sample (EPOP-800) showed key features of the triazine ring to some extent; however, the characteristic peaks corresponding to CH2 group were absent and the pattern looked very similar to that of the nitrogen-doped graphene.31 The successful formation and the nature of the formed porous organic polymers can be confirmed by cross-polarization magic angle spinning (CP-MAS) solid-state 13C and 15N NMR spectroscopy. 13C CP-MAS measurement of EPOP sample (Figure S3a) confirmed the coexistence of triazine and ethylene diamine moieties in the polymer; the aromatic sp2 carbon of triazine was observed at δ 166 ppm, and the aliphatic sp3 carbon of ethylene diamine was observed at δ 41 ppm, indicating that these two individual moieties are covalently linked during the synthesis [starting materials are soluble in organic solvents; upon completion of the reaction, the product EPOP was washed several times with organic solvents to remove unreacted starting materials]. Similarly, 15N CP-MAS measurement of EPOP sample (Figure S3b) showed two broad peaks at δ −172 and −90 ppm, corresponding to the triazine N and linker ethylene diamine N, respectively. This confirms the presence of two different N (triazine N and ethylene diamine N) atoms in the sample. Raman spectroscopy is an important tool that specifies the defects and disorderly nature of carbon materials. Figure 1 shows the Raman spectra of carbon composites of EPOP, and the Raman spectra of EPOP is given in Figure S4a. It is clear that after carbonization at different temperatures, we observe only two peaks at 1330 (D-band) and 1600 cm–1 (G-band). This clearly proves that EPOP has undergone chemical and structural changes. The observed peaks lie in the D- and G-band region; usually, D-band arises due to the disorders and imperfection present in the carbon lattice. G-band is assigned to one of the two E2g modes corresponding to stretching vibrations in the basal-plane (sp2 domains) of carbon lattice. The D- and G-band peaks were observed at 1346, 1358, and 1342 cm–1 and 1542, 1550, and 1567 cm–1, EPOP-600, 700, 800 respectively. The G peaks of all samples shift to higher frequencies as the pyrolyzing temperature increased. The quantitative amount of blueshift for EPOP-700 and EPOP-800 is 8 and 25 cm–1, respectively, when compared to that of EPOP-600. It is obvious that the shift in Raman peaks may be attributed to the effect of doping and strain. Usually, the doping of nitrogen in carbon-based materials, such as graphene and CNTs, the blueshift of G peak is apparent, which we observed in our case. Typically, it is known that the compressive/tensile strain in carbon materials (graphene and CNTs) may induce a blue/redshift of Raman peaks. Doping of nitrogen atoms in carbon lattice may lead to the defect pinning and distortion of lattice. The doping may be associated with bond formation and this produces deformation and stress fields. The formation of pyrrolic nitrogen by doping of nitrogen will have a C–N bond length of 1.37 Å, which shortens compared to that of C–C bond (1.42 Å). The extent of structural disorder present in the carbonized POP can be considered from the ID/IG ratio.32 The ID/IG ratios of all three carbonized samples were found to be 1.47 (EPOP-600), 1.41 (EPOP-700), and 0.87 (EPOP-800). The increase in ID/IG ratio indicates more structural disorder; if the value is high, it indicates a low degree of graphitization. The order of graphitization for all three carbonized samples can be given as EPOP-800 > EPOP-700 > EPOP-600. Figure 1 Raman spectrum of carbon composites of EPOP. Figure 2a–d shows the powder X-ray diffraction (PXRD) pattern of EPOP and its carbon composites. All samples displayed a broad peak, and hence deduction of any structural information of the POP becomes feeble. EPOP showed a broad peak around the 2θ value of 23.7°, ascribed to the graphitic carbon like structure, with a d-spacing of 3.72 Å,32 whereas EPOP-600 and EPOP-800 showed a broad peak with a spacing of 3.68 and 3.59 Å, respectively, suggesting the presence of graphitic structure.32,33 EPOP-700 showed a broad peak around the 2θ value of 26.1° close to that of graphitic carbon with d-spacing of 3.47 Å.33 From the PXRD, we infer that all samples were found to resemble graphitic carbon. The reason for the resemblance to graphitic carbon could be attributed to the carbonization process; a similar kind of observation was made for room-temperature analogues.6 Further, these results were supported by Transmission electron microscopy (TEM) analysis. Figure 2 XRD profile of (a) EPOP, (b) EPOP-600, (c) EPOP-700, and (d) EPOP-800. TEM image of the EPOP showed stacked sheetlike network, whereas the high-resolution transmission electron microscopy (HR-TEM) images of the carbonized samples EPOP-600 and EPOP-700 showed exfoliated sheetlike structure. EPOP-800 displayed carbon sheetlike morphology, which was crumpled to spherelike morphology on the topographical view34 (Figure 3). Selected area electron diffraction patterns of all of these samples showed amorphous nature of the material (Figures S5–S8). These results are highly consistent with XRD analyses. Figure 3 (a) TEM image of EPOP, HR-TEM images of (b) EPOP-600, (c) EPOP-700, and (d) EPOP-800. Field emission scanning electron microscopy (FE-SEM) images of EPOP and the carbonized samples were shown in Figures S9–S12. EPOP sample exhibited irregularly agglomerated particle-like morphology. In the case of carbonized samples, change in morphology was observed. As the carbonization temperature increased from 600 to 800 °C, the morphology changed from irregularly agglomerated network to highly agglomerated network, which could be attributed to the high pyrolysis temperature.4 The oxygen evolution activities of the prepared carbon sample (EPOP, EPOP-600, EPOP-700, and EPOP-800) were studied in 1 M KOH using linear sweep voltammetry (LSV). The prepared samples were coated on carbon paper and used as working electrode. Figure 4 shows the polarization curves of the prepared carbon sample. Among these samples, EPOP-700 showed an onset potential of 1.527 V, which is lower than that of the other carbon samples (EPOP: 1.673 V, EPOP-600: 1.70 V, EPOP-800: 1.65 V). EPOP-700 sample exhibited overpotential of 297 mV to attain a current density of 10 mA/cm2; however, other carbon samples showed relatively high overpotential, as shown in Table 1. Also, at high current density of 300 mA/cm2, the EPOP-700 sample showed a low overpotential of 580 mV, whereas other samples exhibited high overpotential. The obtained results with high current density are found to be better than the metal-based OER catalysts.35 The influence of pH on the OER activity has been studied for EPOP-700 sample (Figure S21). Among the four catalysts used in this study, EPOP-700 showed high electrocatalytic activity, which could be ascribed to the presence of nitrogen atoms present on the surface/edges of the sample. It is evident from the elemental analysis that the N content in the sample EPOP-700 has relatively high nitrogen content than other samples as a result of high nitrogen atom doping during carbonization process (Supporting Information (SI), Table 1). From N %, it may possibly be concluded that 700 °C is the ideal temperature to attain high nitrogen doping with respect to EPOP samples. The presence of nitrogen atoms on the surface impacts the polarity and hydrophilicity of the sample, which is known to improve mass transfer properties at the electrode–electrolyte interface. The OER kinetics of the samples EPOP, EPOP-600, EPOP-700, and EPOP-800 were examined using a Tafel plot. From figure 4b, it is apparent that favorable reaction kinetics and a small Tafel slope of 76 mV/dec was found for EPOP-700 among all studied catalysts (SI, Table 2). The Tafel slope value implies that a lower electrochemical polarization occurred at the interface. The enhanced OER activity of EPOP-700 sample can be explained on the basis of N % (SI, Table 1). A high N % of 16.87 was obtained for EPOP-700 in comparison to other samples. The morphology analysis of EPOP-700 reveals the high stability of the carbon after prolonged OER for 10 h (Figure S19). EPOP-700 sample displayed better electrocatalytic performance among the studied catalysts (EPOP, EPOP-600, and EPOP-800), and this improved performance might be related to the increased electron transfer at the electrode interface accompanied by high ionic conductivity. To elucidate this, electrochemical impedance spectroscopy (EIS) measurements for all four catalysts were carried out (Figure S17). Nyquist plots obtained from the EIS measurement showed a lower resistance of 90 Ω for EPOP-700 compared to that of other catalysts (EPOP = 293 Ω, EPOP-600 = 238 Ω, EPOP-800 = 102 Ω). This lower charge-transfer resistance arises from the efficient interfacial contact of porous carbon network with electrolyte. EPOP-700 catalyst holds a smaller Rct, which facilitates the faster charge-transfer rate and hence better OER performance. Also, we anticipate that such OER performance might be concomitantly related to the intrinsic activity of the catalyst, high electrochemical surface area (ECSA), and efficient charge-transfer kinetics prevailing at the electrode surface. The mechanistic aspect of the EPOP-700 can be explained as follows: the nitrogen atom present in the network of EPOP-700 might be negatively charged because of the electron withdrawing nature, and hence the adjacent carbon atoms might become positively charged (Figure S20). The positively charged carbon adsorbs OH– from the electrolyte, which leads to the accumulation of OH– on the surface of the catalyst, and this may have positive influence on the catalytic reaction. For the rate-determining step of OER, the adjacent positively charged carbon atoms should undergo facile recombination of the two adsorbed species. The electron density localized on the nitrogen atoms in the carbon network may reside near the Fermi level, and so they can participate in the electrocatalytic reaction.36 Figure 4 Electrochemical performance of EPOP and its carbon composites toward OER. (a) LSV; (b) Tafel plots in 0.1 M KOH solution at 2 mV/s. Table 1 Electrochemical Data of the Catalysts s. no catalyst onset (V) current density (mA) Tafel slope (mV/dec) overpotential (mV) active surface area ECSA (cm–2) 1 EPOP 1.673 53 169 440 0.0568 2 EPOP-600 1.70 22 177 470 0.0016 3 EPOP-700 1.527 322 76 297 0.233 4 EPOP-800 1.65 37 106 420 0.0598 To evaluate the catalytic performance of EPOP and its carbon composites toward the reduction of nitro group to amino group, the catalytic reduction of 4-NP was carried out in excess NaBH4 in aqueous medium. The decrease and increase in absorption intensity of 4-NP or 4-AP is measured in the presence of different catalysts as a function of time, as shown in Figure 5a–d. As the time increases, absorbance at 400 nm corresponding to 4-nitrophenolate anion decreases and the absorbance at 298 nm corresponding to 4-aminophenolate anion increases, indicating the catalytic activity of all four catalysts. An identical experiment was carried out with excess NaBH4 without catalyst. It is remarkable that even after a day, no change in intensity of 4-nitrophenolate peak was observed, indicating that the catalyst is necessary for this conversion. The difference in the degree of catalytic activity can be correlated with the complete disappearance of the peak at 400 nm vs function of time. Although all catalysts displayed good catalytic activity toward the reduction of 4-NP to 4-AP, the time taken for the complete reduction of 4-NP to 4-AP varied. It is evident that EPOP-800 sample took 35 min for the complete conversion of 4-NP to 4-AP, displaying the best catalytic activity among all. The order of catalytic activity can be derived as EPOP-800 > EPOP-700 > EPOP-600 > EPOP. From these observations, it can be concluded that the catalyst with a high degree of graphitization showed better catalytic activity in 4-NP reduction, whereas the same is not in the case of OER catalysis that follows a different mechanism. The reaction followed pseudo first-order kinetics. The improved catalytic performance of EPOP-800 can be ascribed to the facile adsorption of 4-NP on the catalyst’s surface. The concept of facile adsorption of 4-NP on the carbon surface has been reported using density functional theory calculation in the literature.37 The rate constant for 4-NP reduction of all catalysts was tabulated (SI, Table 3), and the activity factors for all catalysts were found to be 63.3, 90, 113, 150 min–1/g, respectively. The observed rate constant and activity factor values are better than those of the reported nonmetallic catalysts.29,37 Figure 5 UV–vis absorption spectra for the catalytic reduction of 4-nitrophenol by NaBH4 over (a) EPOP, (b) EPOP-600, (c) EPOP-700, and (d) EPOP-800. Conclusions In conclusion, porous organic polymers based on ethylene diamine and triazine were successfully synthesized, carbonized at different temperatures, and characterized. The as-synthesized materials were studied for the catalytic activity toward OER and nitrophenol reduction. Among the catalysts employed in this study, EPOP-700 sample displayed excellent OER activity with an overpotential of 580 mV at a current density of 300 mA/cm2, with a prolonged stability over 10 h. These results were further complimented by Tafel slope and EIS measurements. A small Tafel slope of 76 mV/dec and lower resistance of 90 Ω were obtained for EPOP-700 compared to that of other catalysts employed in this study. The excellent activity of EPOP-700 could be attributed to high N doping, as apparent from elemental analysis. The obtained results were found to be better than those of the metal-free catalysts and are comparable with those of metal-based OER catalysts. In addition, these catalysts showed excellent catalytic activity toward the reduction of 4-NP to 4-AP. EPOP-800 sample displayed excellent catalytic activity by reducing 4-NP to 4-AP in 35 min, whereas other catalysts took relatively longer times. The observed rate constant and activity factor values are better than those of the reported nonmetallic catalysts. Experimental Section Materials and Methods All reagents and solvents were purchased from Sigma-Aldrich/Alfa Aesar and used without further purification. 13C and 15N CP-MAS measurements were carried out on a Bruker Avance 400 spectrometer, operating at 100.6 MHz for 13C and 40.53 MHz for 15N using a Bruker 4 mm double resonance probe-head at a spinning rate of 10 kHz. The X-ray diffraction (XRD) patterns were measured at room temperature (RT) using a Bruker D8 ADVANCE instrument using Cu Kα radiation with a wavelength of 1.5418 Å. The powder diffraction covered the angle ranges from 5–65°, with a step angle of 0.02°/min. The morphological structures of the prepared samples were captured using a scanning electron microscope (SEM) of TESCAN, VEGA 3 with Bruker detector. Thermogravimetric analysis (TGA) was used to study the thermal degradation of synthesized materials using a TGA/SDT Q600, TA instruments at a scanning rate of 5 °C/min, from room temperature to 1000 °C under nitrogen atmosphere. FT-IR analysis was carried out on a Bruker Tensor 27 (Optik GmbH) using RT DLaTGS (Varian) detector. Transmission electron microscope (Tecnai 20 G2 (FEI make), Netherlands) was used to analyze the surface morphology. Brunauer–Emmett–Teller surface area and porosity analysis was carried out on an Accelerated Surface Area and Porosimetry system (ASAP-2020 V4.03 (V4.03 H)) at 77 K. Raman spectra were recorded using a high-resolution Renishaw Raman microscope employing a He–Ne laser of 18 mW at 633 nm. Cyclic voltammetry was carried on an autolab PGSTAT 302N workstation at room temperature in a standard three-electrode cell. The working electrode was a carbon paper (area = 1 cm2, mass loading of 2 mg/cm2) and modified glassy carbon, and the counter electrode was a Pt wire. The reference electrode was Ag/AgCl/KCl (3 M) electrode. The oxygen evolution reaction was studied by using linear sweep voltammetry carried out at room temperature using 1 M KOH as the electrolyte at a scan rate of 1 mV/s. For the ease of comparison of results, the potentials were converted to the reversible hydrogen electrode scale. Ink Preparation The ink was prepared by equimolar preparation 1:1:1 (water/isopropyl alcohol/Nafion), and the solution was sonicated for 3 h. A small volume (100 μL) of this solution was added to 5 mg of the catalyst to prepare the slurry and was sonicated for 30 min. The catalyst ink was coated on the surface of tory carbon by manual brush coating. The modified tory carbon was allowed to dry at room temperature. Nitrophenol Reduction Reduction of 4-NP to 4-AP was carried out in the presence of excess NaBH4, which is an important organic catalytic reaction. This reaction was carried out to evaluate the catalytic performance of EPOP catalyst. Two milligrams of 4-NP was dissolved in 40 mL of deionized water and sonicated for half an hour; the solution was light yellow. Then, NaBH4 (120 mg) was added to the 4-NP solution. In the absence of EPOP catalysts, a small amount of bubbles was observed because of the hydrogen generation by the reduction reaction between NaBH4 and water. However, in the presence of EPOP (3 mg) catalysts, a large amount of bubbles was observed and the gas release rate became much faster. Synthesis of EPOP Under inert conditions, ethylene diamine (1.4 g, 24.4 mmol) was dissolved in anhydrous 1,4-dioxane (100 mL) under constant stirring at RT. To this solution, K2CO3 (4.5 g, 32.5 mmol) was added and stirred for 30 min. Then, the solution was cooled to 10 °C and cyanuric chloride (3 g, 16.27 mmol) dissolved in 100 mL anhydrous 1,4-dioxane was added dropwise over 8 h. The mixture was allowed to warm to RT and refluxed for 3 days (during the course of the reaction, color changed from dark brown to pale brown). The solution was cooled to RT; the mixture was filtered, washed with 1,4-dioxane and methanol to remove unreacted SM, and dried under vacuum to yield EPOP (yield = 90%). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00574.FT-IR, 13C and 15N NMR, HR-TEM, FE-SEM, TGA, electrochemical active surface area calculation, SEM image of EPOP-700 modified electrode after OER measurements, EIS, chronoamperometry, XPS (PDF) Supplementary Material ao8b00574_si_001.pdf The authors declare no competing financial interest. Acknowledgments Dr. M.K. thanks DST-INSPIRE for the faculty award and DST-SERB Start-up Research grant for research funding. Director and support staffs of CSIR-CECRI are gratefully acknowledged for their constant encouragement and support. ==== Refs References Zhang Y. ; Riduan S. N. Functional porous organic polymers for heterogeneous catalysis . Chem. Soc. Rev. 2012 , 41 , 2083 –2094 . 10.1039/c1cs15227k .22134621 Patra B. C. ; Khilari S. ; Manna R. N. ; Mondal S. ; Pradhan D. ; Pradhan A. ; Bhaumik A. 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PMC006xxxxxx/PMC6644423.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145902610.1021/acsomega.8b01137ArticleUnusual Nucleophilic Addition of Grignard Reagents in the Synthesis of 4-Amino-pyrimidines Tinson Ryan A. J. Hughes David L. Ward Leanne Stephenson G. Richard School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, Norfolk NR4 7TJ, U.K. E-mail: g.r.stephenson@uea.ac.uk.10 08 2018 31 08 2018 3 8 8937 8944 25 05 2018 18 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Pyrimidines have always received considerable attention because of their importance in synthesis and elucidation of biochemical roles, in particular that of vitamin B1. Herein, we describe a reaction pathway in a Grignard reagent-based synthesis of substituted pyrimidines. A general synthesis of α-keto-2-methyl-4-amino pyrimidines and their C6-substituted analogues from 4-amino-5-cyano-2-methylpyrimidine is reported. The presence of the nitrile substituent in the starting material also results in an unusual reaction pathway leading to C6-substituted 1,2-dihydropyrimidines. Grignard reagents that give normal pyrimidine products under standard reaction conditions can be switched to give dihydropyrimidines by holding the reaction at 0 °C before quenching. document-id-old-9ao8b01137document-id-new-14ao-2018-011378ccc-price ==== Body Introduction The synthesis of pyrimidines has always been a priority topic for investigation because of their widespread use as scaffolds in medicinal, pharmaceutical, and academic chemistries. Recently, there has been a marked return to prominence of these structures, particularly in the functionalized amino pyrimidine series,1 as synthetic targets. Although a majority of these examples are 2-amino pyrimidines, examples of 4-2 and 6-aminopyrimidines3 are also notable features. These vital building blocks of life are found in many biologically significant molecules, including structures like DNA 1, drugs such as zidovudine 2,4,5 and barbiturate sodium thiopental 3 (Figure 1). Pyrimidines are also investigated for the treatment of the neurological disorders Beriberi and Korsakoff syndrome and as a recognition motif for binding in thiamine diphosphate-dependent enzymes (compound 4, Figure 1).6,7 Figure 1 (a) DNA fragment, HIV drug zidovudine, sodium thiopental, and vitamin B1. Because of their versatile hydrogen-bonding interactions, these small fragments have been intensively investigated by medicinal chemists during the past decade. Their applications in GSK’s antimalaria drug trimethoprim 5,8 antibiotic bacimethrin 6,9 and in the treatments of thiamine deficiency,10−12 are well-documented (Figure 2). Figure 2 Aminopyrimidines in GSK’s trimethoprim and antibiotic bacimethrin. Commonly, the synthesis of pyrimidine rings utilizes a sodium alkoxide-catalyzed condensation reaction between urea, an aldehyde, and a malonic ester. Most methodologies to form these rings utilize this process, now called the Biginelli reaction.13−15 Assembly of the core aromatic structure permits further functionalization with a range of reactive groups. The initial synthetic strategies to access the valuable 4-amino-2-methyl pyrimidine substitution pattern were originally developed by Williams16 (Scheme 1). The reported procedure is outlined below and was subsequently modified by others for the development and production scale-up of vitamin B1 synthesis. Scheme 1 Williams’ Synthesis of 4-Amino-2-methyl Pyrimidine Although the 4-amino-2-methyl pyrimidines have been investigated with many differing structural modifications,17−19 there are very few examples for the synthesis of α-keto-4-amino-2-methyl pyrimidines and their analogues.20 As the core pyrimidine ring is observed widely throughout nature, it was surprising to find such a lack of examples because these α-keto structural analogues could be good candidates for inhibitor mimics and for potential structural manipulations in structure–activity relationship studies. To address this issue, we have devised a new synthetic route, which uses cheap and readily available starting materials, to construct α-keto-4-amino-2-methyl pyrimidines. Results and Discussion Our approach to α-keto-4-amino-2-methyl pyrimidines is based on the inclusion of a nitrile substituent at the 5-position to enable the introduction of a wide variety of R groups. This allows us to exploit the easy availability of 2-methyl-4-amino-5-cyano-pyrimidines (Scheme 2).17 Scheme 2 Synthetic Approach to Access α-Keto-4-amino-2-methyl Pyrimidines with a Wide Variety of R Groups Thus, our synthesis of a range of differently substituted α-keto-2-methyl-4-amino pyrimidines began by employing the established condensation reaction of cheap and accessible reagents, acetamidine hydrochloride 14 and ethoxymethylenemalonitrile 15, which after recrystallization of the crude product from ethanol gave the required nitrile 13 in a 78% yield (Scheme 3, step 1).17 Scheme 3 New Synthesis of the Pyrimidyl Ketones We now turned to a series of preliminary experiments to assess the optimum temperature for addition of the Grignard reagent to the nitrile. Addition of the Grignard reagent at 0 °C and warming to 40 °C gave total consumption of the starting material after 16 h. Lower temperatures resulted in incomplete consumption and required longer reaction times. Addition of the appropriate Grignard reagent produced the desired keto products 12 with various R groups after simple column chromatography in yields of 16–68% (see Scheme 4 and Table 1, product type A). The infrared spectra of the products showed an unusually low position for the C=O stretching vibration of the ketones at about 1650 cm–1. In the 1H nuclear magnetic resonance (NMR) spectra, the signals for the NH2 protons came at two separate chemical shifts, presumably because of H-bonding to the adjacent carbonyl oxygen by one of the protons of the amino substituent. This strong intramolecular hydrogen bonding also accounts for the unusual position of the C=O stretching vibration for these compounds and is consistent with the data reported for 1-(2-aminophenyl)ethanone.21 During the purification of our ketone products, it became apparent that other byproducts had been formed during the Grignard reaction. Characterization of these byproducts allowed us to identify some unexpected structures, which reveal an unusual alternative pathway for the addition of the Grignard reagent at the C-6 position of the pyrimidine ring (Scheme 4, product types B and C). Scheme 4 Surprising Range of Products Isolated from the Addition of Grignard Reagents to the Nitrile-Substituted Aminopyrimidine Table 1 Product Structures and Yields of Grignard Addition Products A, B, or C entry R temp, °C % yield (type A) % yield (type B) % yield (type C) 1 Me 0–40a 18 (50%)b 2 Me 40c 17 (85%) 3 Et 0–40a 19 (68%)b 20 (27%)d 4 Et 40c 21 (80%) 5 Pr 0–40a 22 (16%)b 23 (20%)d 6 Pr 0–25e 24 (67%) 7 Pr 40c 22 (22%) 8 Bu 0–40a 25 (18%)d 9 Bu 0–25e 26 (87%) 10 i-Pr 0–25e 27 (56%) 11 t-Bu 40c 28 (42%) 12 phenyl 0–40a 29 (25%)b 30 (40%)d 13 phenyl 0–25e 31 (81%) 14 vinyl 40c 32 (76%) 15 HC≡C 40c 16 HC≡C 0–25e a Grignard reagent was added at 0 °C, and the reaction mixture was then warmed to 40 °C and then left to cool and stirred at rt overnight. b By procedure I. c By procedure III (Grignard reagent was added at 40 °C). d By procedure II (like procedure I but neutralized with aqueous NaHCO3, before extraction). e By procedure IV in which the reaction temperature was maintained at 0 °C for 3 h before being allowed to warm to rt overnight. The formation of these unexpected substitution patterns (see Scheme 4) was apparent from the lack of the C6 aromatic proton at ∼8.0 ppm in the 1H NMR spectrum and the retained strong C≡N stretching vibration at 2100 cm–1 in the infrared spectrum. After adjusting the reaction conditions, addition of the methyl and ethyl Grignard reagents at 40 °C instead of 0 °C gave the [1,2]-dihydropyrimidine analogues of type C in good yields (80–85%). These structures were identified by the presence of the methyl signal as a doublet with 3J coupling (6.0 Hz) to the CH proton (q, 3J = 6.0 Hz) located at 4.1 ppm (e.g., see Figure 3). The assignment of these spectral features was confirmed by the analysis of 1H correlation spectroscopy data. The [1,2]-dihydro products also retained the distinctive nitrile stretch at 2100 cm–1 in their infrared spectra. The key features discussed above allow the characterization data for the products of type B/C and the anticipated product of type A to be easily distinguished. Remarkably, these [1,2]-dihydro compounds remained stable at room temperature over a period of weeks, despite the loss of aromaticity following the addition of the Grignard reagent. Some aryl-22 and ferrocenyl-substituted23 examples appear to share the same stability shown by our nitrile-substituted structures, whereas Lyle24 has reported instability in the cyano-substituted dihydropyridine analogues. Figure 3 Structure of racemic 17 with the CHMe feature that gives a characteristic C6 quartet and C7 doublet in the 1H NMR spectrum. A plausible mechanism for the synthesis of side products of types B/C could arise as a consequence of the Schlenk equilibrium25 between the Grignard reagent RMgX, R2Mg, and MgX2. Coordination of the Lewis acidic MgX2 to the N1 nitrogen of the pyrimidine to form 16 would make the C6 position more electrophilic and hence promote attack by the Grignard reagent (Scheme 5). Moreover, an electron-withdrawing group adjacent to this position would further increase the electrophilicity of the C6 position providing an explanation for why, in the 5-cyano series, this side-reaction is far more favored. An alternative possibility, however, would be a radical mechanism for the transfer of the R group, followed by loss of H• by an oxidative step to form 17. Scheme 5 Proposed Mechanism of the formation of [1,2]-Dihydropyrimidine 17 Examples of reactions that are capable of forming stereogenic centers from an aromatic system using Grignard reagents are rare, but a notable precedent for this type of transformation has been described in the synthesis of antifungal agent Voriconazole by Pfizer.26 The methyl and ethyl C6-substituted analogues have been reported previously in the synthesis of vitamin B1 analogues, but were acquired by condensation chemistry as described by Todd et al.27,28 This condensation approach has not been applied to the synthesis of other analogues containing modifications at this position, so the new examples described here, which are obtained by the Grignard approach, will open up a general access to this class of structures. Interestingly, the Schlenk equilibrium is often controlled by changes to the Grignard reagent, solvent, or temperature. To assess the optimum conditions, a range of temperatures from (−78 to 40 °C) were tested, but this served only to increase the consumption of the starting nitrile 13 and did not affect the product outcome. It was noticeable that addition of the Grignard at 0 °C followed by warming to 40 °C gave a mixture of products, so some Grignard additions at 40 °C were attempted. As the Schlenk equilibrium should favor formation of the R1MgX species at higher temperatures and in a polar aprotic solvent, the involvement of Lewis acidic MgX2 could be reduced, and therefore C-6 addition should be decreased. This, however, with our initial choice of methyl and ethyl Grignard reagents, gave 17 and 21 as the sole products (Table 1, entries 2 and 4). A range of solvents were then examined to assess their affect upon product distribution. As tetrahydrofuran (THF) had already been used for the preliminary experiments, we sought to decrease the solvent polarity. Diethyl ether was tested under the same conditions, and from the analysis of the 1H NMR spectra, it was obvious that only methyl ketone and the unreacted starting material were present. Attempts using 1,4-dioxane resulted in the precipitation of the MgX2 salts. Removal of these MgX2 salts by filtration was expected to improve the selectivity for the formation of the ketone compound; however, this proved not to be the case, and the under these conditions, the attempted reaction gave only the recovered unreacted starting material. On the basis of the mechanism proposed in Scheme 5, it seemed probable that the products that retained the nitrile group originated by initial addition of the nucleophile to the heteroaromatic ring (producing the [1,2]-dihydro product of type C) and that oxidation under the reaction conditions later resulted in rearomatization to give 5-cyano-6-alkyl-2-methylpyrimidine products of type B. This hypothesis was tested for Grignard reagents (R1 = Et, Pr, Bu, and Ph; Table 1 entries 3, 5, 8, and 12) that tended to give low yields and only products of types A and B. By avoiding the high temperature of 40 °C and keeping the reaction mixture at 0 °C for 3 h, more time was allowed for the addition at C6 to go to completion before the reaction was quenched. Under these conditions (Table 1, entries 6, 9, 10, and 13), the [1,2]-dihydro products of type C were isolated at moderate to good yields. Bulky Grignard reagents (branched R1 groups such as i-Pr and t-Bu) give poorer results (56 and 42%, respectively). In the other cases, (R1 = Me, Et, Pr, and Bu), the yields of the [1,2]-dihydro products ranged from 67 to 87%. Extending the study to phenyl- and vinylmagnesium bromide, we were able to show that the low temperature conditions worked well to improve the yields with phenylmagnesium bromide, giving [1,2]-dihydro product 31 in an 81% yield (compare entries 12 and 13), and our original 40 °C reaction conditions were suitable with vinylmagnesium bromide, giving 32 in a 76% yield. On the basis of these trends, it appears that the unexpected formation of [1,2]-dihydropyrimidine is in fact the preferred addition pathway and proceeds efficiently, albeit slowly, at 0 °C. At higher temperatures, the addition at the nitrile begins to become significant, so that reactions that are allowed to warm up before all of the Grignard reagent has been consumed can produce substantial amounts of the ketones (type A products). Type B products in many cases arise by rearomatization of [1,2]-dihydropyrimidines, accounting for the failure to isolate type C products in these cases. In other cases, however, the addition of the Grignard reagent can be performed at 40 °C to give methyl- and ethyl-substituted [1,2]-dihydropyrimidines in a high yield. We proposed that this difference arises from the differences in stability of type C products under strong acid (HCl; entries 1, 3, 5, 8, and 12) conditions used in the quench, compared to the weak acid (ammonium chloride) used for entries 2, 4, 6, 7, 9, 10, 11, 13, and 14. Steric bulk in the nucleophile (e.g., entry 8) seems to block addition at the nitrile, and type A products were not formed with butylmagnesium bromide. Alkynyl Grignard reagents are less reactive and HC≡CMgBr failed to add to 13, even at 40 °C. Crystallization and X-ray analysis of the simple methyl substituted example 17 confirmed the [1,2]-dihydro structure and the presence of the stereogenic center at C6 (Figure 4). C6 is displaced 0.078(2) Å from the good mean plane of the rest of the heterocyclic ring, which supports the presence of extended π-overlap into the nitrile substituent. The shorter bond length of C1–C2 [1.379(2) Å] when compared to the C1–C6 bond of the C–CHMe [1.510(2) Å] suggests the presence of the alkene π bond in conjugation with the nitrile substituent within the aromatic π-system. Figure 4 diagram for the crystal structure of 4-amino-2-methyl-5-cyano-6-methyl-[1,2]-dihydropyrimid-ine 17. Thermal ellipsoids are drawn at the 50% probability level. Measurement of the CN bond length C11–N12 [1.158(2) Å] indicates a typical length for this group, although the C1–C11 C–CN [1.402(2) Å] bond length lies between a normal C–C bond (1.5 Å) and an sp2 C=C (1.3 Å), thereby suggesting the presence of a partial sp2 bond character between the aromatic π-system and the nitrile bond. At C6, the CHMe position adopts the classic tetrahedral geometry with a bond angle for the atom group N5–C6–H6 at 109.0(9)° and the bond length for C1–C6 at 1.510(2) Å, whereas the shorter bonds between the sp2 centers of C1–C2 and C4–N3 have lengths of 1.379(2) and 1.312(2) Å, respectively. Interestingly, these structures form a hydrogen-bonded dimer pair (racemic, with R and S configurations) around a center of symmetry, forming a binding pattern similar to that observed in DNA base pairs (Figure 4), which becomes extended through further pairs of hydrogen bonds (see the Supporting Information). This paper identifies three competing pathways for the addition of Grignard reagents to the readily available 4-amino-2-methyl-5-cyanopyrimidine starting material, giving access to either the sought after α-keto-4-amino-2-methyl pyrimidines or alternative products arising from substitution of the pyrimidine ring itself. Especially in the cases where the exclusive formation of a single product has been identified, the results presented here should open the way for the exploitation of these substitution patterns in future research. Furthermore, studies are now in progress to extend the range of examples that give exclusively the unusual chiral 1,2-dihydropyrimidines to introduce alternative reactive functional groups at the C6 position (e.g., alkynes) to prepare the way for the development of an enantioselective version of the procedure. Ideally, this research would facilitate a wider range of ongoing synthetic modifications such as Diels–Alder cycloaddition chemistry and metal-catalyzed cross-coupling reactions, which could utilize the vinyl adduct 32. Each in their own way, the intended α-keto products and the unusual ring-substituted products are ideal structures to access a wide range of pyrimidine-derived compounds that will be of value in medicinal and bioorganic chemistry. Our procedure avoids the need for extensive purification steps and so provides a potential “gateway compound” suited for easy functionalization in future studies. It also offers potential access to key intermediates in the substantial quantities needed for sustained biochemical/pharmaceutical research projects and scale-up processes.29 Furthermore, the ketones made available in this way would be suitable prochiral candidates for reductions to provide useful intermediates for the chiral synthetic pool and/or enzymatic studies.30,31 Experimental Section General Considerations All reactions were carried out in dry solvents, unless otherwise stated. Anhydrous THF, diethyl ether, and toluene were distilled over sodium following literature methods. All solvents and chemicals were purchased from appropriate suppliers including Sigma-Aldrich, Acros, TCI, Fluorochem, Alfa Aesar, and Fisher. Infrared spectra were obtained on a PerkinElmer spectrum 100 Fourier transform infrared spectrometer, with most compounds dissolved in dichloromethane and measured as a film on a NaCl disc. In cases where compounds were not soluble in this solvent, the infrared spectrum was obtained using an attenuation total reflection plate. 1H and 13C NMR spectra were obtained using a Bruker (Ascend) 500 MHz spectrometer and a sample express autosampler. Mass spectra were measured by the EPSRC UK National Mass Spectrometry Facility, Swansea, UK using an LTQ Orbitrap XL spectrometer. Melting points were recorded on a BUCHI melting point apparatus B545. X-ray diffraction data were recorded at the UEA on an Oxford Diffraction Xcalibur-3/Sapphire3 CCD diffractometer. The data were then processed with the CrysAlisPro-CCD and -RED programs.32,33 The structure was determined by the direct method routines in the SHELXS program34 and refined by full-matrix least-squares methods on F2’s in SHELXL34 using WinGX.35 Scattering factors for neutral atoms were taken from the International Tables for X-ray Crystallography.36 Preparation of 4-Amino-5-cyano-2-methylpyrimidine (13)17 Sodium (0.19 g) was added in small pieces to ethanol (4.0 mL) to produce a 2 M solution of sodium ethoxide. Then, acetamide hydrochloride (0.80 g, 8.50 mmol) was added. Filtration through celite gave a clear solution, which upon addition of ethoxymethylene malonitrile (0.50 g, 4.10 mmol) produced a yellow precipitate that was collected by filtration and recrystallized from ethanol to give the title compound as fine yellow needles (1.10 g, 70%). mp 245–247 °C [lit.17 mp 246–248 °C]. 1H NMR (500 MHz, DMSO-d6): δ 8.52 (s, 1H), 7.77 (s, 2H), 2.40 (s, 3H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 170.6, 162.8, 161.6, 116.2, 87.1, 26.4 ppm. IR νmax: 3378, 3334 (NH2), 2223 (sp CN), 1672, 1584, 1542 (C–H, sp2 stretch) cm–1. Preparation of tert-Butylmagnesium Bromide 2-Methyl-2-bromopropane (1.0 mL, 8.9 mmol, 1 equiv) in dry THF (9.0 mL) was added dropwise to a predried round-bottom flask containing magnesium turnings (427 mg, 17.8 mmol, 2.0 equiv) in anhydrous THF (20 mL). After activation with a small amount of iodine, addition was maintained to keep a gentle reflux. The reaction was then stirred at room temperature (rt) for 3 h. The Grignard reagent was titrated against 1,10-phenanthroline and isopropyl alcohol. Typical Procedure I for the Synthesis of α-Keto-4-amino-2-methylpyrimidines (Products of Type A) 5-(4-Amino-2-methylpyrimidinyl)propan-1-one (19) To 4-amino-5-cyano-2-methylpyrimidine 13 (200 mg, 1.5 mmol) in THF (5 mL) was added ethylmagnesium bromide in THF (3 M, 5.3 mmol, 1.74 mL, 3.5 equiv) dropwise at 0 °C. The reaction mixture was warmed to 40 °C and left to stir overnight. The reaction was then quenched with 1 M HCl (10 mL), stirred for a further 24 h, and then extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered, and evaporated under pressure to leave a fine powder. Column chromatography on silica eluting with EtOAc/hexanes (1:3 v/v) gave the product as a white solid (168 mg 68%). Rf = 0.6 EtOAc/hexanes 1:3 v/v. mp 160–162 °C. 1H NMR (500 MHz, CDCl3): δ 8.73 (s, 1H), 8.62 (s, 1H), 5.67 (s, 2H), 2.87 (q, 3J = 7.3 Hz, 2H, H-4), 2.47 (s, 3H, H-1), 1.15 (t, 3J = 7.3 Hz, 3H, H-5) ppm. 13C NMR (126 MHz, CDCl3): δ 201.4, 171.1, 161.9, 158.9, 109.2, 29.5, 26.4, 8.1 ppm. IR νmax: 3385, 3263, 3109, 2980 (sp3 C–H stretch), 1657, 1625, 1528 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C8H12N3O, 166.0975; found, 166.0972. Typical Procedure II for the Synthesis of 4-Amino-5-cyano-2-alkyl- or 2-Arylpyrimidines (Products of Type B) 4-Amino-5-cyano-2-methyl-6-phenylpyrimidine (30)32 To 4-amino-5-cyano-2-methylpyrimidine 13 (200 mg, 1.5 mmol) in THF (5 mL) was added phenylmagnesium bromide in THF (3 M, 5.2 mmol, 1.74 mL, 3.5 equiv) dropwise at 0 °C. The reaction was warmed to 40 °C and left to stir overnight, then quenched with 1 M HCl (10 mL), and stirred for a further 24 h. The reaction was then neutralized with aqueous NaHCO3 and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered, and evaporated under reduced pressure to leave a fine powder. Column chromatography on silica eluting with EtOAc/hexanes (gradient, 5:1 v/v to pure EtOAc) gave the product as a white powder (315 mg, 40%). Rf = 0.2 EtOAc/hexanes 5:1. v/v. 1H NMR (500 MHz, DMSO-d6): δ 7.87–7.82 (m, 2H), 7.63–7.51 (m, 3H), 2.47 (s, 3H) ppm (this compound would only dissolve in DMSO-d6; signals for the NH2 protons were not observed because of exchange with H2O in the solvent). 13C NMR (126 MHz, DMSO-d6): δ 169.7, 168.4, 164.7, 136.8, 131.3, 129.4, 129.0, 116.7, 84.2, 26.4 ppm. IR νmax: 3379, 3330 (NH2), 2168 (sp CN), 1680, 1626, 1561 cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C12H11N4, 211.0978; found, 211.0978. Typical Procedure III for the Synthesis of 4-Amino-5-cyano-2-methyl-6-alkyl- or 6-Alkenyl-[1,2]-dihydropyrimidines at 40 °C (Products of Type C) 4-Amino-5-cyano-2-methyl-6-vinyl-[1,2]-dihydropyrimidine (32) To 4-amino-5-cyano-2-methylpyrimidine 13 (2.5 g, 19 mmol, 1 equiv) in THF (50 mL) was added vinylmagnesium bromide in THF (3 M, 65 mmol, 21.8 mL, 3.5 equiv) dropwise at 40 °C. The reaction mixture was left to stir overnight and then quenched with saturated aqueous ammonium chloride (20 mL) at 0 °C and stirred for further 48 h and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered, and evaporated under reduced pressure to give the product as a fine yellow powder (2.24 g, 76%). mp 171–173 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.47 (br s, 1H), 5.88 (s, 2H), 5.76 (ddd, 3J = 16.9, 9.9, 6.9 Hz, 1H), 5.07–5.00 (m, 2H), 4.49 (d, 3J = 6.9 Hz, 1H), 1.88 (s, 3H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 160.8, 159.4, 139.9, 122.1, 114.3, 52.5, 51.4, 21.8 ppm. IR νmax: 3316, 3302 (NH2), 2171 (sp CN), 1734 (sp2 CO), 1683, 1638, 1601 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C8H11N4, 163.0978; found, 163.0975. Typical Procedure IV for the Synthesis of 4-Amino-5-cyano-2-methyl-6-alkyl- or 6-Alkenyl-[1,2]-dihydropyrim-idines from 0 °C to rt (Products of Type C) 4-Amino-5-cyano-2-methyl-6-propyl-[1,2]-dihydropyrimidine (24) To 4-amino-5-cyano-2-methylpyrimidine 13 (200 mg, 1.5 mmol) in anhydrous THF (5 mL) at 0 °C was added propylmagnesium chloride (2 M, 5.2 mmol, 2.6 mL, 3.5 equiv) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 3 h and then left to warm to rt overnight. The reaction mixture was cooled to 0 °C and quenched with saturated aqueous ammonium chloride (10 mL) and left to stand for 3 h. The product was extracted with EtOAc (3 × 10 mL), washed with brine (10 mL), dried over MgSO4, filtered, and evaporated to dryness to give the title compound as a fine yellow powder (180 mg, 67%). mp 163–165 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.21 (s, 1H), 5.74 (s, 2H), 4.04 (dd, J = 18.8, 4.6 Hz, 1H), 1.84 (s, 3H), 1.46–1.23 (m, 4H), 0.92–0.88 (m, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 161.4, 160.1, 122.6, 51.7, 49.3, 41.4, 22.2, 16.9, 14.2 ppm. IR νmax: 3310, 3252 (NH2), 2100 (CN), 1650, 1617, 1538 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C9H15N4, 179.1291; found, 179.1291. Synthesis of α-Keto-4-amino-2-methylpyrimidines 5-(4-Amino-2-methylpyrimidinyl)ethanone (18) Following procedure I, using methylmagnesium bromide followed by column chromatography on silica eluting with ethyl acetate/hexanes (1:3 v/v) gave the title compound as a white solid (470 mg, 50%). mp 170–172 °C. 1H NMR (500 MHz, CDCl3): δ 8.70 (s, 1H), 8.57 (s, 1H), 5.72 (s, 1H), 2.50 (s, 3H), 2.48 (s, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 198.2, 171.4, 161.8, 159.8, 109.8, 26.6, 26.1 ppm. IR νmax: 3372, 3114, 1651 (sp2 C=O), 1590, 1520 (sp2 C–H aromatic stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C7H10N3O, 152.0818; found, 152.0815. 5-(4-Amino-2-methylpyrimidinyl)butan-1-one (22) Following procedure I, using n-propylmagnesium bromide followed by column chromatography on silica eluting with EtOAc/hexanes (2:1 v/v) gave the title compound as a white solid (43 mg, 16%). Rf = 0.3 EtOAc/hexanes 2:1 v/v. mp 147–149 °C. 1H NMR (500 MHz, CDCl3): δ 8.73 (s, 1H), 2.81 (t, 3J = 7.4 Hz, 3H), 2.50 (s, 3H), 1.73–1.64 (m, 2H, H-5), 0.94 (t, 3J = 7.4 Hz, 3H, H-6) ppm. 13C NMR (126 MHz, CDCl3): δ 200.6, 170.5, 162.0, 158.2, 109.3, 40.3, 25.8, 17.9, 13.8 ppm. IR νmax: 3373, 3265 (NH2) 1653, 1629, 1534 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C9H14N3O, 180.1131; found, 180.1129. This product was also obtained following procedure III; using n-propylmagnesium bromide followed by column chromatography on silica eluting with EtOAc/hexanes (2:1 v/v) gave the title compound as a white solid (60 mg, 22%). 5-(4-Amino-2-methylpyrimidinyl)phenone (29) Following procedure I, using phenylmagnesium bromide followed by column chromatography on silica eluting with EtOAc/hexanes (gradient, 5:1 v/v to pure EtOAc) gave the title compound as a white powder (200 mg, 25%), Rf = 0.5 EtOAc/hexanes 5:1 v/v. mp 182–184 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.33 (s, 1H), 8.12 (s, 2H), 7.68–7.63 (m, 3H), 7.61–7.54 (m, 2H), 2.45 (s, 3H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 196.2, 170.63, 162.8, 161.8, 138.6, 132.5, 129.4, 129.0, 108.9, 26.3 ppm. IR νmax: 3381, 2925, 1627, 1577, 1598 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C12H12N3O, 214.0975; found, 214.0973. Synthesis of 4-Amino-5-cyano-2-alkyl- or 2-Arylpyrimidines 4-Amino-5-cyano-6-ethyl-2-methylpyrimidine (20)27 Following procedure II, using methylmagnesium bromide followed by column chromatography on silica eluting with EtOAc/hexanes (gradient, 1:1 v/v to pure EtOAc) gave the title compound as a white powder (66 mg, 27%). Rf = 0.7 EtOAc/hexanes 1:1 v/v. mp 197–199 °C [lit.27 mp 193–194 °C]. 1H NMR (500 MHz, CDCl3): δ 5.42 (s, 2H), 2.66 (q, 3J = 7.6 Hz, 2H), 2.38 (s, 3H), 1.16 (t, 3J = 7.6 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 175.5, 170.4, 163.3, 115.3, 86.3, 30.6, 26.4, 12.8 ppm. IR νmax: 3377, 3340 (NH2), 2223 (CN), 1682, 1553, 1577 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C8H11N4, 163.0978; found, 163.0974. 4-Amino-5-cyano-2-methyl-6-propylpyrimidine (23) Following procedure II, using n-propylmagnesium bromide followed by column chromatography on silica eluting with EtOAc/hexanes (2:1 v/v) gave the title compound as a yellow/white powder (53 mg, 20%). Rf = 0.6 EtOAc/hexanes 2:1 v/v. mp 176–177 °C. 1H NMR (500 MHz, CDCl3): δ 5.50 (s, 2H), 2.71 (t, 3J = 10 Hz, 2H), 2.47 (s, 3H), 1.75–1.66 (m, 2H), 0.94 (t, 3J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 173.0, 169.1, 162.1, 114.1, 85.6, 37.5, 25.1, 21.2, 12.4 ppm. IR νmax: 3368, 3338 (NH2) 1653, 1629, 224 (CN), 1678, 1553, 1417 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C9H13N4, 177.1135; found, 177.1133. 4-Amino-6-butyl-5-cyano-2-methylpyrimidine (25) Following procedure II, using n-butylmagnesium bromide followed by column chromatography on silica eluting with EtOAc/hexanes (gradient, 1:1 v/v to pure EtOAc) gave the title compound as a white solid (50 mg, 18%). Rf = 0.6 EtOAc/hexanes 1:1 v/v. mp 180–182 °C. 1H NMR (500 MHz, CDCl3): δ 5.69 (s, 2H), 2.72 (t, 3J = 10 Hz, 2H), 2.46 (s, 3H), 1.68–1.60 (m, 2H), 1.40–1.31 (m, 2H), 0.88 (t, 3J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 175.2, 170.4, 163.6, 116.0, 87.1, 37.0, 31.6, 26.8, 23.1, 14.2 ppm. IR νmax: 3339, 3379, 2227 (sp CN), 1687, 1559, 1461 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C10H15N4, 191.1291; found, 191.1289. Synthesis of 4-Amino-5-cyano-2-methyl-6-alkyl- or -6-Phenyl- or 6-Alkenyl-[1,2]-dihydropyrimdines 4-Amino-5-cyano-2,6-dimethyl-[1,2]-dihydropyrimidine (17) Following procedure III, using methylmagnesium bromide gave the title compound as a fine yellow powder (2.4 g, 85%). mp 221–222 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.27 (s, 1H), 5.77 (s, 2H), 4.17 (q, 3J = 6.0 Hz, 1H), 1.84 (s, 3H), 1.18 (d, 3J = 6.0 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 161.0, 159.7, 122.4, 53.6, 45.5, 25.5, 22.1 ppm. IR νmax: 3399, 3304, 2152 (sp CN), 1658, 1608, 1563 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C7H11N4, 151.0978; found, 151.0975. 4-Amino-5-cyano-6-ethyl-2-methyl-[1,2]-dihydropyrimidine (21) Following procedure III, using ethylmagnesium bromide gave the title compound as a fine yellow powder (2.1 g, 80%). mp 213–215 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.19 (s, 1H, H-6), 5.75 (s, 2H, H-2), 4.09 (td, 3J = 4.6, 2.3 Hz, 1H), 1.86 (s, 3H), 1.50–1.38 (m, 2H), 0.86 (t, 3J = 7.4 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 161.5, 160.3, 122.7, 51.0, 50.6, 31.2, 21.7, 8.1 ppm. IR νmax: 3372, 3326 (NH2), 2180, 2149 (sp CN), 1659, 1600, 1565 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C8H13N4, 165.1135; found, 165.1135. 4-Amino-6-butyl-5-cyano-2-methyl-[1,2]-dihydropyrimidine (26) Following procedure IV, using n-butylmagnesium bromide gave the title compound as a fine yellow powder (250 mg, 87%). 1H NMR (500 MHz, DMSO-d6): δ 8.22 (s, 1H), 5.74 (s, 2H), 4.08 (td, J = 4.8, 2.4 Hz, 1H), 1.86 (s, 3H), 1.45–1.40 (m, 2H), 1.34–1.25 (m, 4H), 0.90 (t, J = 6.9 Hz, 3H) ppm. 13C NMR (126 MHz, DMSO): δ 161.1, 160.2, 122.3, 51.3, 49.6, 38.4, 25.4, 22.8, 21.9, 14.5 ppm. IR νmax: 2159 (sp CN), 1631, 1588, 1520 cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C10H17N4, 193.1448; found, 193.1447. 4-Amino-5-cyano-2-methyl-6-iso-propyl-[1,2]-dihydropyrimidine (27) Following procedure IV, using iso-propylmagnesium bromide gave the title compound as a fine yellow powder (150 mg, 56%) mp 170–171 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.14 (s, 1H), 5.75 (s, 2H), 3.89 (d, J = 2.6 Hz, 1H), 1.87 (s, 3H), 1.57 (s, 1H), 0.86 (d, 3H), 0.82 (d, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 162.1, 160.8, 123.0, 55.4, 49.9, 36.7, 21.7, 17.3, 16.4 ppm. IR νmax: 3368, 3321 (NH2), 2175 (CN), 1652, 1601, 1565 (sp2 C–H stretch) cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C9H15N4, 179.1291; found, 179.1290. 4-Amino-5-cyano-2-methyl-6-tert-butyl-[1,2]-dihydropyrimidine (28) Following procedure III, using tert-butylmagnesium bromide gave the title compound as a fine yellow powder (120 mg, 42%). mp 217–219 °C. 1H NMR (500 MHz, DMSO-d6): δ 8.38 (s, 1H), 5.82 (s, 2H), 3.61 (s, 1H), 1.96 (s, 3H), 0.85 (s, 9H) ppm. 13C NMR (126 MHz, DMSO-d6): δ 161.9, 161.6, 124.2, 58.9, 48.9, 40.8, 25.0, 21.9 ppm. IR νmax: 2159 (sp CN), 1637, 1598, 1562 cm–1. HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C10H17N4, 193.1448; found, 193.1445. 4-Amino-5-cyano-2-methyl-6-phenyl-[1,2]-dihydropyrimidine (31) Following procedure IV, using phenylmagnesium bromide gave the title compound as a fine yellow powder (256 mg, 81%). 1H NMR (400 MHz, DMSO-d6): δ 8.79 (br s, 1H), 7.4–7.37 (m, 2H), 7.31–7.27 (m, 3H), 5.91 (s, 2H), 5.11 (s, 1H), 1.92 (s, 3H) ppm. 13C NMR (101 MHz, DMSO-d6): δ 145.9, 129.4, 129.0, 128.2, 127.9, 127.2, 127.0, 122.4, 53.8, 22.1. IR νmax: 3345, 3317 (NH2), 2123 (CN), 1646, 1601, 1550 (sp2 C–H stretch) cm–1 HRMS (ESI-LTQ Orbitrap XL) m/z: [M + H]+ calcd for C12H13N4, 213.1135; found, 213.1137. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01137.Copies of 1H and 13C NMR spectra for all new compounds and X-ray structure details for compound 17 (PDF) Crystallographic data for compound 17 (CIF) Supplementary Material ao8b01137_si_001.pdf ao8b01137_si_003.cif Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript, and all authors have contributed equally. The authors declare no competing financial interest. Acknowledgments We thank the EU Interreg Trans Manche/Channel cross-border project “Academy-Industry Chemistry Channel” (AIcc: ref 4196) for the financial support and the EPSRC mass spectrometry facility at the University of Swansea for HRMS data. ==== Refs References a Wang C. ; Cai J. ; Zhang M. ; Zhao X. 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PMC006xxxxxx/PMC6644424.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145879710.1021/acsomega.8b00818ArticleIn Situ Shape Change of Au Nanoparticles on TiO2 by CdS Photodeposition: Its Near-Field Enhancement Effect on Photoinduced Electron Injection from CdS to TiO2 Fujishima Musashi †Ikeda Takuya ‡Akashi Ryo ‡Tada Hiroaki ‡†Faculty of Science and Engineering and ‡Graduate School of Science and Engineering, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan E-mail: h-tada@apch.kindai.ac.jp.06 06 2018 30 06 2018 3 6 6104 6112 25 04 2018 25 05 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Hemisphere-like gold nanoparticles (NPs) were loaded on TiO2 (Au/TiO2) by the deposition–precipitation method. Subsequent photodeposition of CdS on the Au surface of Au/TiO2 at 50 °C yields Au(core)–CdS(shell) hybrid quantum dots with a heteroepitaxial (HEPI) junction on TiO2 (Au@#CdS/TiO2), whereas nonHEPI Au@CdS/TiO2 was formed by CdS photodeposition at 25 °C. In the HEPI system, the shape of the Au core changes to an angular shape, whereas it remains in a hemisphere-like shape in the nonHEPI system. The hot photodeposition technique was applied to the Au NP-loaded mesoporous TiO2 nanocrystalline film (Au/mp-TiO2). Using Au@CdS/mp-TiO2 and Au@#CdS/mp-TiO2 as the photoanodes, two-electrode quantum dot-sensitized photoelectrochemical cells were fabricated for hydrogen (H2) generation from water, and the performances of the cells were evaluated under illumination of simulated sunlight. In the photocurrent and the rate of H2 evolution, the Au@#CdS/mp-TiO2 photoanode cell surpasses the CdS/mp-TiO2 and Au@CdS/mp-TiO2 ones. Three-dimensional finite-difference time-domain calculations for the model systems indicated that the angular shape Au core generates an intense electric field at the corners and edges, extending the electric field distribution over the Au core–CdS shell interface. The striking shape effect on the cell performances can originate from the promotion of the CdS excitation and charge separation due to the near-field enhancement by the deformed Au core. document-id-old-9ao8b00818document-id-new-14ao-2018-00818pccc-price ==== Body 1 Introduction Plasmonic photocatalysts consisting of metals and semiconductors have recently attracted much interest as the key materials for solar-to-chemical transformations.1−3 The optoelectric properties of the plasmonic metals such as Au nanoparticles (NPs) strongly depend on the shape in addition to the size and loading amount.4,5 Periodic arrays of Au nanorods (NRs) were formed on a TiO2 single crystal electrode (Au NR/TiO2) by electron beam lithography, and the plasmonic activity for photoelectrochemical water splitting was studied under external biased conditions.6 Also, TiO2-capped Au NR array with redox catalysts electrochemically prepared using porous alumina templates shows photocatalytic activity for water splitting without external wiring.7 In both the systems, two peaks corresponding to the transverse and longitudinal modes of the localized surface plasmon resonance (LSPR) unique to the Au NRs were observed in the action spectrum of incident photon-to-current efficiency (IPCE), and the hot-electron transfer mechanism is proposed for the reaction.8 However, the study on the effect of the shape on the plasmonic photocatalytic activity is limited because the synthesis of such catalysts usually needs expensive experimental equipment and elaborated techniques. On the other hand, a large contact area and a high-quality interface between the metal and semiconductor are crucial for the efficient interfacial electron transfer enhancing photocatalytic activity of plasmonic photocatalysts.9,10 Colloidal solutions of Au and CdS NRs were separately synthesized, and Au-NR/CdS-NR hybrids were prepared by mixing them.11 The authors showed that the Au-NR/CdS-NR exhibits high visible-light activities for the oxidation of salicylic acid and reduction of p-nitrophenol due to the large contact area between Au- and CdS-NRs. This colloid-based method is convenient and versatile for the preparation of various metal–semiconductor nanohybrids; however, organic modifiers are necessary as the shape-controlling agents contaminating the interface between the components. Whereas postheating at high temperatures improves the interface quality by removing the organic modifiers, and may cause aggregation of metal NPs, and further, damage of the components. Consequently, if a low temperature technique can be developed for changing the shape of the components in metal–semiconductor nanohybrids with a large contact area and high-quality interfaces, it could provide basic and useful information about the shape effect on the activity of the plasmonic photocatalysts. Here, we report in situ shape change in the Au NP on TiO2 with the selective photodeposition of the CdS shell on the Au NP at 50 °C, and the mechanism on the low temperature formation of the heteroepitaxial (HEPI) junction between the Au(core) and CdS(shell) (Au@CdS). Furthermore, Au@CdS/TiO2 is applied as the photoanode of quantum dot-sensitized photoelectrochemical cells (QD-SPECs) for H2 generation from water, and the shape effect of the Au core on the cell performances is discussed on the basis of the results of three-dimensional finite-difference time-domain (3D-FDTD) calculations. 2 Results and Discussion 2.1 Hot Photodeposition of CdS on Au/TiO2 The morphology of the samples was checked by transmission electron microscopy (TEM). The loading amount of Au on TiO2 was determined to be 0.0190 ± 0.002 g TiO2 g–1 by inductively coupled plasma spectroscopy (ICPS). Au NPs with a hemisphere-like form and a mean diameter (dAu) of 8.0 nm (standard deviation = 1.8 nm) were deposited on TiO2 by the deposition–precipitation method.12Figure 1A shows the TEM image for the sample with CdS photodeposited on Au/TiO2 at 50 °C. A CdS shell is formed on the surface of every Au NP to yield Au@CdS/TiO2. Similar TEM images were observed for the samples prepared at 25 °C (Figure S1),13 where the mean shell thickness of 7.7 nm (standard deviation = 2.3 nm) was confirmed.12 The selective photodeposition of CdS on Au was explainable in terms of the electron pool effect of Au NP and the strong affinity of Au and sulfur.14 The amounts of CdS photodeposited on Au/TiO2 at 25 and 50 °C were comparable to 0.025 ± 0.003 g TiO2 g–1. Figure 1B shows X-ray diffraction (XRD) patterns for the samples prepared at 25 and 50 °C. The former has several diffraction peaks of anatase TiO2 with a shoulder at 27 < 2θ < 30°. In the pattern for the latter, two weak peaks are present at 2θ = 26.62 and 28.42° assignable to the diffraction from the (002) and (101) planes of hexagonal CdS. Figure 1C,D show Cd 3d- and Au 4f-X-ray photoelectron (XP) spectra for Au@CdS/mp-TiO2, prepared by varying photodeposition time (tPD), respectively. In spectrum C, two signals are observed at binding energy (EB) = 411.9 and 405.2 eV due to the emission from the Cd 3d3/2 and Cd 3d5/2 orbitals, respectively, intensifying with an increase in tPD. In spectrum D, Au/TiO2 has emissions from the Au 4f5/2 orbital at EB = 86.4 eV and the Au 4f7/2 orbital at EB = 82.8 eV, which disappear at tPD > 0.5 h. Evidently, the present photodeposition technique yields a hexagonal CdS shell completely covering the Au core, and the rise in the deposition temperature from 25 to 50 °C somewhat increases the CdS crystallinity. Figure 1 (A) TEM image of Au@CdS/mp-TiO2 prepared by photodeposition at 50 °C. (B) XRD patterns for Au@CdS/mp-TiO2 prepared by photodeposition at 25 and 50 °C. Cd 3d (C) and Au 4f (D)-XP spectra for Au@CdS/TiO2 prepared by photodeposition at 25 °C. To gain information about the junction between the Au core and the CdS shell, high-resolution (HR) TEM observation and analysis were carried out. Figure 2A,B compare the typical HR-TEM images for Au@CdS/TiO2 prepared at 25 and 50 °C. As shown in image A, the CdS photodeposition at 25 °C yields nonHEPI Au@CdS hybrids on TiO2 (Au@CdS/TiO2) and the shape of the Au core remained to be in a hemisphere-like shape. In image B, the CdS shell formation induces the Au(111) facets with the d-spacing of 0.236 nm. The d-spacing of 0.335 nm for the CdS shell agrees with the value for the hexagonal CdS(0002) plane. The CdS shell grows on the Au(111) surface with a HEPI relation of CdS{0001}/Au{111} (Au@#CdS/TiO2): the symbol # denotes the HEPI junction between the Au core and the CdS shell below. Figure 2C,D show the side view and top view of a model for the CdS/Au NP interface, respectively. The interface consists of the Au(111) plane and the S plane perpendicular to the c-axis of CdS. Importantly, the photodeposition of CdS on Au/TiO2 at 50 °C simultaneously changes the shape of the Au core from a hemisphere-like shape to an angular shape with edges and corners. A similar shape change in the Au core was also observed in the CdS photodeposition on Au/ZnO.10 Figure 2 HR-TEM images for Au@CdS/TiO2 prepared by photodeposition at 25 °C (A) and 50 °C (B). The side view (C) and top view (D) of a model for the CdS/Au NP interface of the sample prepared at 50 °C (d1 = 0.7172 nm, and d2 = 0.7493 nm, d3 = 0.8651 nm, d4 = 0.8274 nm). So far, several Au NP-metal chalcogenide heteronanostructures with the HEPI junction have been synthesized at temperatures in the range from 170 to 300 °C.15−18 The basic mechanism on the low temperature formation of Au@#CdS/TiO2 in the present system is discussed. The general requirements to form the HEPI junction are small lattice mismatch <15% for core–shell structures15 and slow crystal growth near the equilibrium conditions.19 Upon UV-light irradiation of Au/TiO2 in ethanol containing Cd2+ ions and S8, the excited electrons in the conduction band (CB) of TiO2 are transferred to the Au NPs. Simultaneous excitation of the Au NP-LSPR can lead to the hot-electron transfer from the Au NPs to the CB of TiO2,8 which is overwhelmed by the electron transfer in the opposite direction under these conditions.20 The accumulation of the electrons in Au NPs rises its Fermi energy (EF) to approach the CB minimum of TiO2. S8 is selectively adsorbed on Au NPs due to the strong S-Au bond (∼184 kJ mol–1),21 gradually reduced to S2– ions by the electrons accumulated in Au NPs with the concentration being maintained low near the Au surface to react with Cd2+ ions. Consequently, the CdS shell slowly grows on the surface of Au NP. The CdS deposition induces the Au(111) facet so as to generate the maximum S-Au interfacial bonds stabilizing the interface between the Au(111) with the largest surface Au-atom density and the S plane of CdS(0001). Both the Au(111) and CdS(0001) planes have hexagonal symmetry, and the three unit cells in the Au(111) plane (0.8651 nm) fit with the two unit cells in the CdS(0001) plane (0.8274 nm) with a lattice mismatch degree of −4.36%. The fact that the HEPI junction between the Au core and the CdS shell can be induced by an increase in the photodeposition temperature from 25 to 50 °C indicates that the activation of the mobility of the Au surface atoms is important for the formation of high-quality junction. The melting point of transition metals is known to dramatically decrease with increasing electron occupancy in the d-band above half filling or increasing EF. Recent full-multiple scattering calculations indicated that the bulk-state Au has 6.9% vacancy in the d-band due to the hybridization of the 5d orbitals with 6s and 6p orbitals.22 The EF upward shift in Au NPs by the photoinduced electron transfer from TiO2 may cause the surface phonon softening of Au NPs. The enhancement of the vibrational amplitude on the surface atoms can assist to reconstruct and relax the lattice mismatch at the interface. Thus, the present photodeposition technique, satisfying the requirements for the formation of HEPI, enables its low temperature formation. 2.2 Application to the QD-SPEC Cells Au@#CdS/TiO2 is very promising as the photoanode for the QD-SPEC cell for H2 generation from water,23−25 where the optical properties are fundamental to the application. Figure 3A compares UV–visible absorption spectra for Au/mp-TiO2, CdS/mp-TiO2, Au@CdS/mp-TiO2, and Au@#CdS/mp-TiO2. The absorption edge for the pristine mp-TiO2 composed of anatase TiO2 nanocrystals is located at ∼390 nm. In the spectrum for Au/mp-TiO2, the LSPR of Au NPs appears around 550 nm, and CdS/mp-TiO2 has absorption at λ < 510 nm due to the CdS interband transition. Although Au@CdS/mp-TiO2 possesses both the features of Au/mp-TiO2 and CdS/mp-TiO2, the CdS shell formation induces significant broadening and redshift in the LSPR peak from 550 to 640 nm. This remarkable redshift and broadening in the LSPR peak would be due to the large refractive index of the CdS shell (2.32)26 and the potent interaction between the Au core and the CdS shell. The absorption spectrum for Au@#CdS/mp-TiO2 is similar to that for Au@CdS/mp-TiO2, but the former has a significantly stronger LSPR than the latter at λ < 730 nm. Figure 3 (A) UV–visible absorption spectra for Au/mp-TiO2 (green line), CdS/mp-TiO2 (black line), Au@CdS/mp-TiO2 (blue line), and Au@#CdS/mp-TiO2 (red line), and IPCE action spectra for the QD-SPEC cells using CdS/mp-TiO2 (black circle), Au@CdS/mp-TiO2 (blue circle), and Au@#CdS/mp-TiO2 (red circle). (B) solar-to-current efficiency (STCE) as a function of applied voltage (Eapp) for the CdS/mp-TiO2 (black), Au@CdS/mp-TiO2 (blue), and Au@#CdS/mp-TiO2 (red) photoanode cells. Two-electrode QD-SPEC cells were fabricated using Au/mp-TiO2, CdS/mp-TiO2, Au@CdS/mp-TiO2, and Au@#CdS/mp-TiO2 as the photoanode and Pt cathode, and the photocurrents were measured under illumination of simulated sunlight (λ > 430 nm, AM 1.5, one sun). The STCE was calculated from the photocurrent (Jph) using 1 1 where Erev (V) and I are the standard state reversible potential for the overall cell reaction and the light intensity (100 mW cm–2), respectively. The Erev was calculated to be +0.23 V from the standard reaction Gibbs energy of +44.3 kJ mol–1 for the present overall cell reaction.26Figure 3B shows STCEs for the QD-SPEC cells as a function of applied bias (Eapp). The photocurrent for the Au/mp-TiO2 photoanode cell was very small. This is probably because of the poisoning of the Au NP surface by the oxidized S2– ions. The CdS/mp-TiO2 photoanode cell shows a volcano-shaped one with a maximum of 0.0095% at Eapp = 0.125 V. The presence of the Au core greatly increases the STCE, decreasing the Eapp, where the STCE reaches maximum. Strikingly, the Au@#CdS/mp-TiO2 photoanode cell provides a maximum STCE of 0.037% at Eapp = 0.025 V, which is substantially larger than the value of 0.028% at Eapp = 0 for Au@CdS/mp-TiO2. To elucidate the origin for the Au core effect, three-electrode PEC cells with the structure of a working electrode\|0.25 M Na2S, 0.35 M Na2SO3\|Ag/AgCl (reference electrode)\|counter electrode were constructed. A stable photocurrent was observed for each cell (Figure S2). Furthermore, the photocurrent was measured for the PEC cells with CdS/mp-TiO2, Au@CdS/mp-TiO2, and Au@#CdS/mp-TiO2 as the working electrodes under irradiation of monochromatic light by varying its wavelength. In Figure 3A, the IPCE action spectra for the cells are also shown. The onset wavelength for the photocurrent (λon) ranges from 560 to 600 nm, and the order of the IPCE value at 430 < λ < 600 nm is CdS/mp-TiO2 < Au@CdS/mp-TiO2 < Au@#CdS/mp-TiO2. The discrepancy between the λon and the CdS absorption edge (∼510 nm) indicates that the interfacial electron transfer from CdS to TiO2 occurs via the transition from the valence band (VB) of CdS to the CB of TiO2 (path 2)27 besides the injection from the CB of CdS to the CB of TiO2 (path 1).14 In the Au@CdS/mp-TiO2 and Au@#CdS/mp-TiO2 photoanodes, not the Au NP core but the CdS shell acts as the photosensitizer, because the photocurrent hardly flows around the LSPR peak of the Au core. Also, the Au core and further, the high-quality junction between the Au core and the CdS shell enhances the electron injection from CdS to TiO2. Figure 4A compares the rate of H2 evolution under simulated sunlight illumination (λ > 430 nm, AM 1.5, one sun) of the QD-SPEC cells with different photoanodes. In the Au@CdS/mp-TiO2 and Au@#CdS/mp-TiO2 photoanode cells, the amount of H2 increases proportionately to irradiation time, while good linearity in the plot has also recently been confirmed for the CdS/mp-TiO2 photoanode cell.28 These results indicate that every photoanode stably works under these conditions. The rate of H2 evolution is in the order of CdS/mp-TiO2 (0.03 mL h–1 cm–2) ≪ Au@CdS/mp-TiO2 (0.18 mL h–1 cm–2) < Au@#CdS/mp-TiO2 (0.20 mL h–1 cm–2). The mean lifetime of the electrons injected into the CB of TiO2 (τn) can be estimated by analyzing the potential decay curve in the three-electrode QD-SPEC cell by 2 2 where kB and q are the Boltzmann constant and the elementary charge of electron, respectively, and Voc is obtained from the subtraction of the electrode potential in the dark (Edark) from the electrode potential under light illumination (Eph).29Figure 4B shows plots of log(τn/s) vs Voc. Although the τn decreases with increasing Voc in every system, the value for the Au@CdS/mp-TiO2 cell is larger than that for CdS/mp-TiO2 at the same Voc. The lifetime for the Au@#CdS/mp-TiO2 cell is further longer than that for the Au@CdS/mp-TiO2 cell. Clearly, the Au core greatly enhances the charge separation to induce the remarkable increase in the STCE. In Figure 3A, no photocurrent is observed at λ > 600 nm, which excludes the hot-electron transfer mechanism in this system.8 Thus, the superior performance of Au@#CdS/mp-TiO2 over Au@CdS/mp-TiO2 could result from the shape change of the Au core rather than the high-quality interface.10 Figure 4 (A) Time courses for H2 generation for the QD-SPEC cells. (B) Plots of hot electron lifetime (τn) as a function of photovoltage under open-circuit conditions (Voc). Photoanodes used in the measurements were CdS/mp-TiO2 (black), Au@CdS/mp-TiO2 (blue), and Au@#CdS/mp-TiO2 (red), respectively. 2.3 FDTD Calculations Three-dimensional FDTD simulations were performed for a model of Au@CdS/TiO2 consisting of a CdS shell (thickness, lCdS = 7.7 nm)-coated Au hemisphere (dAu = 8 nm) placed on a TiO2 slab (Figure 5A). To clarify the shape effect of the Au core, we also modeled Au@#CdS/mp-TiO2 by a CdS shell-coated Au nanocube placed on a TiO2 slab (Figure 5B), where the volumes of the cube and the shell were set to be the same as those of Au@CdS/TiO2. The models were illuminated with incident visible light parallel with either the z-axis or x-axis toward the hemisphere. The excited localized electromagnetic fields were monitored at the cross-sectional planes of the models. To identify the location of the excited fields, polarized light sources were used in this simulation. Figure 5C shows wavelength dependence of maximum local electric field enhancement factors monitored on the x–y plane (EFxy) and the x–z plane (EFxz) under x-polarized light incident from the z-axis direction (k//z, E//x). The enhancement factor defined as the ratio of the local maximum electric field intensity to the incident electric field intensity is highly dependent on the excitation wavelength. The small EF in the off-resonant wavelength region below 500 nm indicates that the interband transitions of Au and CdS hardly contribute to the local electric field enhancement. The peak value of EFxy for Au@#CdS/TiO2 (red dotted line) is 9.62 × 103 at an incident light wavelength of 673 nm which is smaller than the value of 2.31 × 104 at 694 nm for Au@CdS/mp-TiO2 (black dotted line). On the other hand, EFxz for Au@#CdS/TiO2 (red solid line) surpasses EFxz for Au@CdS/TiO2 (black solid line) at a wavelength of over 510 nm, where a significant difference in LSPR absorption was observed (Figure 3A). The peak EFxz value of 9.62 × 103 for the former is about 1 order of magnitude larger than the value of 1.01 × 103 for the latter. Figure 5D shows the wavelength dependence of EFs monitored on the x–y plane (EFxy) and the x–z plane (EFxz) under z-polarized light incident from the x-axis direction (k//x, E//z). EFxy and EFxz for Au@#CdS/TiO2 (red dotted and solid lines) far exceed those for Au@CdS/TiO2 (black dotted and solid lines) in a wavelength over ca. 530 nm, respectively. It is noteworthy that EFxy and EFxz for Au@#CdS/TiO2 are almost comparable with each other within the simulation wavelength range both in Figure 5C,D, whereas EFs for Au@CdS/TiO2 are highly dependent on the location of the monitor planes. Figure 5 FDTD simulation models of (A) Au@CdS/TiO2 and (B) Au@#CdS/mp-TiO2. Inset image of (B) represents the structural notation for a nanocube. Wavelength dependence of a maximum local electric field enhancement factor (EF) under (C) x-polarized light incident from the z-axis direction (k//z, E//x) and (D) z-polarized light incident from the x-axis direction (k//x, E//z) for Au@CdS/TiO2 (black) and Au@#CdS/mp-TiO2 (red). Values of the EF were monitored on the x–y plane (EFxy, dotted line) and the x–z plane (EFxz solid line), respectively. Figure 6A,C show local electric field distribution in the x–y plane (upper row) and the x–z plane (lower row) for Au@CdS/TiO2 and Au@#CdS/mp-TiO2 excited by an x-polarized light incident from the z-axis direction (k//z, E//x), respectively. In all of these images, the LSPR is due to dipole resonance excitation because the core size of the models is sufficiently smaller than the incident light wavelength.30Figures 6Ai,ii and 6Ci,ii display an off-resonant electric field with the EFs of approximately unity. As shown in Figure 6Aiii–viii, an intense local electric field is formed on the equator of the Au hemisphere adjacent to the CdS shell and the TiO2 slab irrespective of the incident light wavelength. A similar local electric field distribution was also confirmed for Au@SiO2/ZnO.31 The simulated field distribution can be explained by the lightning rod effect (LRE), where the electric field at the curved surface is highly intensified due to the condensation of electric field lines.32 As clearly seen in Figure 6Ciii–viii, the local electric field for Au@#CdS/mp-TiO2 is confined at the edge and the corner of the Au nanocube due to LRE likewise. The dominant LSPR mode for each incident light wavelength was assigned by using structural notation of B, S, T, E, and C which represent bottom, side, top, edge, and corner, respectively (Figure 5B inset). The dominant modes are the BE–BC–SE–TC mode at 591 nm, the BE–BC mode at 673 nm, and the BC mode at 795 nm, respectively. Among these modes, the first and the second modes are the coupled mode denoted by the hyphened notation of more than two single modes. As for the first mode, SE–TC modes are the modes formed in the direction perpendicular to the TiO2 slab (parallel with the z-axis), whereas the BE–BC modes are the modes existing on the perimeter of the bottom square of the Au nanocube. Also, the presence of the TE mode that is out of the monitor planes was confirmed by using the polarized light inclined 45° from the x-axis. Thus, local field enhancement occurs at the sharpened edges and corners even in the position away from the TiO2 slab. In contrast, the field enhancement for Au@CdS/TiO2 is limited in the x–y plane and the mode component distant from the TiO2 slab surface is of negligible magnitude. To inspect SE–TC modes in more detail, we carried out the FDTD simulation under irradiation of z-polarized light incident from the x-axis direction. EFxz for Au@#CdS/TiO2 far exceeds that for Au@CdS/TiO2 in a wavelength over 535 nm (Figure 5D). Figure 6B exhibits electric field distribution for Au@CdS/TiO2 where intense local fields are formed on the periphery of the Au hemisphere similar to the local field excited by x-polarized light incident from the z-axis direction (Figure 6A). Electric fields are present at the zenith of the Au hemisphere due to the hemispherical structure resonating with the incident electric field parallel to the z-axis. Also, asymmetric electric field distribution on the periphery (Figure 6Bv–viii) is due to damping of electromagnetic energy toward the surrounding environment during propagation of the electromagnetic wave from the right side (+x) to the left side (−x) of the model. The periphery of the hemisphere plays an exclusive role of propagation pathway of electromagnetic energy, whereas the curved surface of the hemisphere hardly does. As for Au@#CdS/TiO2, intense electric fields excited due to resonance with z-polarized light are recognized at the top corner of the Au nanocube (Figure 6D). Interestingly, propagating electric fields do not weaken the strength even on the outgoing side. Similarly to LSPR excitation by z-incident light, LSPR excitation occurs at the corners (BC, TC) and edges (BE, TE, SE). From the analysis of the field distribution and the LSPR modes, we can attribute the enhancement of EFxz for Au@#CdS/TiO2 mainly to the excitation of SE and TC modes. Symmetric electric field distribution in the cubic model would result from the presence of complex propagation pathways consisting of BC, BE, SE, TC, and TE. Complex propagation pathways such as the one from the right-side SE to the left-side SE through TC and TE enable efficient energy transfer over the cubic model even if energy loss occurs during light propagation. Figure 6 Local electric field distribution for Au@CdS/TiO2 excited with (A) x-polarized light incident from the z-axis direction (k//z, E//x) at wavelengths of 401 (i, ii), 601 (iii, iv), 694 (v, vi), and 728 nm (vii, viii) and (B) z-polarized light incident from the x-axis direction (k//x, E//z) at wavelengths of 531 (i, ii), 608 (iii, iv), 694 (v, vi), and 723 nm (vii, viii). Local electric field distribution for Au@#CdS/TiO2 excited with (C) x-polarized light incident from the z-axis direction (k//z, E//x) at wavelengths of 435 (i, ii), 591 (iii, iv), 673 (v, vi), and 795 nm (vii, viii) and (D) z-polarized light incident from the x-axis direction (k//x, E//z) at wavelengths of 584 (i, ii), 651 (iii, iv), 708 (v, vi), and 791 nm (vii, viii). Upper and lower row images are local electric field distribution monitored on the x–y and x–z planes, respectively. Inset arrows represent the relation between k and E. White broken line is a guide to the eye for the CdS shell surface and the TiO2 slab surface. Field enhancement studies for hemispherical and cubic particles have been reported so far, but are focused on the particles with a geometrical length of over 10 nm.33−35 This is the first report comparing the electric field enhancement between hemispherical and cubic NPs with a size of sub-10 nm, i.e., 8.0 nm in equatorial diameter for the former and 5.1 nm in edge length for the latter. The cubic core structure is superior to the hemispherical structure with respect to the ability to extend the local electric field away from the TiO2 slab surface even on the side opposite to incident light. The results of FDTD simulation using polarized unidirectional light illuminated to ideal model systems give an insight to interpret the spectroscopic and photoelectrochemical data shown in Figure 3A,B which were obtained by the omnidirectional illumination of unpolarized light on samples. Thus, the presence of SE and TC modes in Au@#CdS/TiO2 suggests that the electric interaction between the Au core and the CdS shell is augmented by replacing the Au hemisphere with the Au nanocube. 2.4 Action Mechanism of Au@#CdS Hybrid QD-SPEC Cell The action mechanism of the Au@#CdS/mp-TiO2 system can be basically explained by the near-field enhancement mechanism (Scheme 1)36 in contrast to the hot-electron transfer mechanism for water splitting by the half-cut Au@#CdS plasmonic photocatalyst.10 Visible-light irradiation (λ > 430 nm) excites the electrons in the VB of the CdS shell to the CB. The CB electrons with a potential of ECB = −1.12 V (vs standard hydrogen electrode (SHE))37 are injected into the CB of TiO2 (ECB = −0.95 V at pH 14).38 Under irradiation conditions, the LSPR of the Au core is excited simultaneously. The intense near-field generated at the Au/CdS and Au/TiO2 interfaces can enhance the electron injection via both path 1 and path 2 because the transition rates are proportional to the square of the local electric field.39 For the effective operation of this enhancement mechanism, the spectral overlap between the LSPR of the Au core and the interband transition of the CdS shell or the electron transition from the VB of CdS to the CB of TiO2 is necessary.40 First, the spectral overlap and the EFs in the energy region increase with the deformation of the Au core from hemisphere to an angular shape (Figures 3A and 5). Second, the angular shape Au core extends the electric field distribution over the Au core–CdS shell (Figure 6C,D). The near field of the Au core can also enhance the charge separation.41 Third, the assumption that the Au core-induced local electric field in the direction normal to the TiO2 surface (z in Figure 5) mainly assists the electron injection from CdS to the CB of TiO2 explains the longer lifetime of the electrons or the more effective charge separation for Au@#CdS/mp-TiO2 than Au@CdS/mp-TiO2 (Figure 4B). Fourth, the higher crystallinity of the CdS shell in Au@#CdS/mp-TiO2 would further contribute to its excellent performance since the recombination through the defects can be minimized. On the other hand, the holes in the VB of CdS (EVB = +1.30 V) easily oxidize S2– ions (E0(S2–/S) = −0.45 V).42 These shape effects of the Au core rationalize the excellent performances of the Au@#CdS hybrid QD-SPEC cell for H2 generation from water. Scheme 1 Action Mechanism of the Au@#CdS/mp-TiO2 Photoanode in the QD-SPEC Cell for H2 Generation The potential is shown with respect to the standard hydrogen electrode (SHE). 3 Conclusions This study has shown that the CdS photodeposition on Au/TiO2 at as low as 50 °C leads to shape change in the Au NP from hemisphere-like shape to an angular shape with the formation of a HEPI junction between Au and CdS (Au@#CdS/TiO2). Striking shape effects are presented in the performance of the QD-SPEC cell using Au@#CdS/TiO2 as the photoanode for H2 generation from water under simulated sunlight. We anticipate that the versatility of the present hot photodeposition technique40 widely contributes to the improvement in the solar-to-chemical conversion efficiency of not only the QD-SPEC cells but also for heteronanostructured photocatalysts. 4 Experimental Section 4.1 Sample Preparation Mesoporous TiO2 thin films (mp-TiO2) were prepared using a previously reported procedure.27 An anatase TiO2 paste (20 nm in a mean particle size, PST-18NR, Nikki Syokubai Kasei) was coated on fluorine-doped tin oxide film-coated glass substrates (<10 Ω/square) by a squeegee method, and air calcination at 773 K was conducted for the as-coated samples to form a mesoporous structure in the film. The mp-TiO2 films thus prepared and anatase TiO2 particles (A-100, specific surface area = 8.1 m2 g–1, Ishihara Sangyo) were used as supports of Au NPs. Au NP-loaded TiO2 (Au/TiO2) was prepared by the deposition–precipitation method. An aqueous solution of HAuCl4 (0.243 mM, 100 mL) was neutralized by 1 M aq NaOH to be pH 6. To this solution, mp-TiO2 or TiO2 particles were added and stirred at 70 °C for 1 h. The resulting film or precipitate was harvested from the solution and rinsed with distilled water. The sample was dried in vacuo at room temperature and heated at 500 °C for 1 h to obtain Au/mp-TiO2 and Au/TiO2. Au@CdS/(mp-)TiO2 and Au@#CdS/(mp-)TiO2 were prepared by photodeposition of the CdS shell layer on the Au-loaded TiO2 film or particles. The Au-loaded sample was immersed in a solution of S8 (1.36 mM) and Cd(ClO4)2 (13.6 mM) in EtOH (200 mL), and deaerated by Ar bubbling for 0.5 h in the dark. UV irradiation (λ > 320 nm, I310–400 = 3.6 mW cm–2) was carried out with a high-pressure mercury lamp (H-400P, Toshiba) at 298 K for the preparation of Au@CdS/(mp-)TiO2 and at 323 K for the preparation of Au@#CdS/(mp-)TiO2 by varying the photodeposition time (tPD), respectively. After irradiation, the films were taken out from the solution and the particles were harvested by centrifugal separation from the solution. The obtained films and particles were rinsed with ethanol repeatedly to be stored at ambient temperature after vacuum drying. CdS-deposited mp-TiO2 (CdS/mp-TiO2) was prepared by the photodeposition method for the pristine mp-TiO2 film. 4.2 Characterization The loading amounts of Au and CdS were quantified by inductively coupled plasma spectroscopy (ICPS-7500, Shimadzu). X-ray diffraction (XRD) analysis was carried out with a Rigaku SmartLab X-ray diffractometer. Diffuse reflectance UV–vis spectra of the samples were recorded on a UV-2600 spectrometer (Shimadzu) with an integrating sphere unit (Shimadzu, ISR-2600Plus) at room temperature. BaSO4 was used as a reference material to evaluate the reflectance (R∞) of the samples. The reflectance was transformed to the Kubelka–Munk function [F(R∞)] presenting the relative absorption coefficient by the equation F(R∞) = (1 – R∞)2/2R∞.43 Transmission electron microscopy (TEM) measurements were performed using a JEOL JEM-2100F at an applied voltage of 200 kV. X-ray photoelectron (XP) spectroscopy measurements were performed using a Kratos Axis Nova X-ray photoelectron spectrometer with a monochromated Al Kα X-ray source (hν = 1486.6 eV) operated at 15 kV and 10 mA. The takeoff angle was 90°, and multiplex spectra were obtained for Cd 3d and Au 4f photopeaks. 4.3 Photoelectrochemical Measurements The solar-to-current efficiency (STCE) was measured for a two-electrode QD-SPEC cell consisting of CdS/mp-TiO2, Au@CdS/mp-TiO2, and Au@#CdS/mp-TiO2 (photoanode)\|0.25 M Na2S + 0.35 M Na2SO3 (aqueous electrolyte solution)\|Pt (cathode). Photocurrents (Jph) of the cell were evaluated under illumination by a solar simulator (PEC-L10, Peccell technologies, Inc.) at one sun (λ > 430 nm, AM 1.5, 100 mW cm–2). Upon photoelectrochemical measurement, a bias voltage (Eapp) in the range between 0 and 0.23 V was applied to the cell. Also, argon bubbling was carried out to remove the oxygen in the solution. A three-electrode PEC cells consisting of CdS/mp-TiO2, Au@CdS/mp-TiO2, and Au@#CdS/mp-TiO2 (working electrode)\|0.25 M Na2S + 0.35 M Na2SO3 (aqueous electrolyte solution)\|Ag/AgCl (reference electrode)\|Pt (counter electrode) were constructed to record the action spectra of IPCE. A 500 W-Xe short arc lamp (UXL-500D-O, Ushio Inc.) was used as a light source to illuminate one sun (λ > 430 nm, AM 1.5, 100 mW cm–2) onto the cell. J–E curves for the PEC cells under light illumination or in the dark were measured with a potentiostat/galvanostat (HZ-5000, Hokuto Denko). The action spectra of IPCE were recorded under illumination of monochromatic light from a xenon lamp through a monochromator (full width at half-maximum = 10 nm, HM-5, JASCO). The short-circuit current at the rest potential was measured as a function of excitation wavelength (λ/nm). Calculation formula for the incident photon-to-current efficiency (IPCE) is shown below (3) 3 where Jph(E), NA, I (W cm–2), F, h, and c are the photocurrent at an electrode potential of E, Avogadro constant, light intensity, Faraday constant, Planck constant, and speed of light, respectively. 4.4 Three-Dimensional FDTD Simulations Local electric field distribution and wavelength dependence of the enhancement factor (EF) for Au@CdS/mp-TiO2 and Au@#CdS/mp-TiO2 were simulated by the 3D FDTD-method Maxwell solver using FDTD Solutions (Lumerical Solutions, Inc.). Au@CdS/mp-TiO2 was modeled by a CdS hemispherical shell-covered Au hemispherical particle placed on a TiO2 slab (500 × 500 × 50 nm3). The diameter of the Au core particle and the thickness of the CdS shell were set to be the experimental values (8 and 7.7 nm), respectively. For Au@#CdS/mp-TiO2, the Au core particle was modeled by a nanocube incorporated into the CdS hemispherical shell. The volumes of the Au nanocube (5.1 × 5.1 × 5.1 nm3) and the surrounding CdS shell for Au@#CdS/mp-TiO2 were kept the same as the corresponding volumes for Au@CdS/mp-TiO2. The origin of the Cartesian coordinate (x = y = z = 0) was located at the midpoint of the cross-sectional plane of the Au hemisphere and the center of the bottom rectangle of the Au nanocube. The position of the top surface of the TiO2 slab was fixed at z = 0. The near-field enhancement for the models was simulated using the total-field scattered-field source. In the simulation, a polarized light source with the wavelength from 300 to 900 nm (f = 200–353 THz) was irradiated to the core–shell hemisphere from the normal direction (wave vector k//z) or the parallel direction (k//x) to the TiO2 slab. The light incidence of k//z and k//x was performed with polarization along the x-axis (E//x) and z-axis (E//z), respectively. For the purpose of improving the accuracy of simulation, the mesh override region with a finer mesh size than that in the previous study41 was set to the core–shell hemispheres. The simulation region was surrounded by a perfectly matched layer absorbing boundary to remove unwanted light reflection at the simulation boundary. To minimize the computational time, antisymmetric and symmetric boundary conditions were used in the x-axis and y-axis directions, respectively. The enhancement factor is defined by the following equation: EF = \|E\|2/\|E0\|2, where \|E\| and \|E0\| are the amplitude of the local maximum electric field and the amplitude of incident electric field, respectively. Optical constants for Au, CdS, and TiO2 used in the simulation were taken from refs (44−46). The refractive index of surrounding medium was set to be a value of air (n = 1.0). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00818.TEM image, photochronoamperometry curves (PDF) Supplementary Material ao8b00818_si_001.pdf The authors declare no competing financial interest. Acknowledgments The authors acknowledge Dr. S. Naya for helpful discussion. 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PMC006xxxxxx/PMC6644425.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145808710.1021/acsomega.8b01714ArticleLanthanum Hydroxide Nanorod-Templated Graphitic Hollow Carbon Nanorods for Supercapacitors Wang Zijie Perera Wijayantha A. Perananthan Sahila Ferraris John P. Balkus Kenneth J. Jr.Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 West Campbell Road, Richardson, Texas 75080-3021, United States E-mail: balkus@utdallas.edu.23 10 2018 31 10 2018 3 10 13913 13918 20 07 2018 11 10 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Lanthanum hydroxide nanorods were employed as both a template and catalyst for carbon synthesis by chemical vapor deposition. The resulting carbon possesses hollow nanorod shapes with graphitic walls. The hollow carbon nanorods were interconnected at some junctions forming a mazelike network, and the broken ends of the tubular carbon provide accessibility to the inner surface of the carbon, resulting in a surface area of 771 m2/g. The hollow carbon was tested as an electrode material for supercapacitors. A specific capacitance of 128 F/g, an energy density of 55 Wh/kg, and a power density of 1700 W/kg at 1 A/g were obtained using the ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, as the electrolyte. document-id-old-9ao8b01714document-id-new-14ao-2018-01714qccc-price ==== Body Introduction Graphene-based materials with designed nanoarchitectures have been studied for a wide range of applications.1,2 Many methodologies have been established to prepare three-dimensional or porous carbons with graphitic walls to achieve high surface area and high conductivity, which are of great importance for electrical double-layer capacitors.3−7 The hydrophobic nature and high surface area of carbon-based materials also provide advantages when employed as catalyst supports, which allows better adsorption and diffusion of reactions.8,9 Among these carbon materials, templated carbon has been prepared using hard templates, such as zeolites and mesoporous silica.10−16 Unfortunately, most of them have amorphous carbon framework, which limited their use in energy storage as well as catalysis.17 Graphene- and carbon nanotube (CNT)-based carbon electrodes with porous or three-dimensional structures have shown excellent performance in supercapacitors.18−22 Therefore, it is of great interest to make templated carbon with graphene-like walls for energy storage and other applications. Mesoporous silica has been used with various organic precursors to produce carbons with improved graphitic content.17 Recently, a lanthanum-exchanged zeolite was reported to be an excellent template for carbon synthesis.23 The resulting carbon replicated the zeolite pore structure with graphene-like walls and conductivity comparable to gold. It is assumed that the carbon source reacts with La first to form LaC2, which is then turned to La(OH)3 by reacting with steam leading to the growth of graphitic carbon (Figure 1). This work demonstrated the application of La as a catalyst for the synthesis of graphitic carbon with controlled morphology and structure. In the present study, pure lanthanum hydroxide nanorods (LaNR) were employed as templates to produce hollow carbon nanorods. Compared to the La-zeolite, the pure lanthanum hydroxide templates are removed by HCl, and the resulting solution can be used to make new nanorods. Figure 1 Schematic illustration of the carbon formation mechanism on La(OH)3 surface. Results and Discussion The LaNRs were hydrothermally synthesized using a solution of La(NO3)3·6H2O and NaOH at 110 °C for 24 h followed by centrifugation, drying, and annealing at 200 °C for 1 h. The lanthanum hydroxide nanorod-templated carbon (LaNRTC) was synthesized by chemical vapor deposition (CVD) using acetylene and steam at 600 °C. Washing with concentrated HCl removes the La(OH)3 template leaving hollow replicas of the nanorods. The LaNR shown in Figure 2a,d possesses a rod shape with an average diameter of 16 nm and an average length of 140 nm. The lattice fringes of LaNR (Figure 2b) have a d-spacing of 0.285 nm, corresponding to the (200) face of La(OH)3 (JCPDS 36-1481). Figure 1b,e shows the morphology of the templated carbon before removal of LaNR. The transmission electron microscopy (TEM) image shows that carbon covers the surface of the LaNR. The scanning electron microscopy (SEM) image (Figure 2b) shows better contrast compared to that of the pure LaNR since the carbon covered on the surface of LaNR is conductive. These images indicate that the carbon was grown only on the surface of LaNR without formation of amorphous carbon elsewhere. This demonstrates that the La(OH)3 serves as both a template and catalyst for carbon growth, which is consistent to our previous work that no carbon was formed under the same conditions if only mesoporous silica was placed in the reactor.24 And after La(OH)3 was deposited on mesoporous silica, carbon was formed along the surface of the templates. After removal of the LaNR by using acid, the resulting carbon retains the rod shape morphology with similar particle size (Figure 2c), and the inner space is empty as shown in the TEM image (Figure 2f). The acid may access LaNR through defects in the carbon coating, leading to openings in the tubular carbon network. The edge of the resulting carbon exhibits lattice fringes with a d-spacing of 0.40 nm attributed to the (002) planes of graphitic carbon, indicating the graphitic nature of the lanthanum hydroxide nanorod-templated carbon. The d-spacing is bigger than graphite (0.34 nm) due to less than five graphene layers on the edge of the carbon, where the d-spacing has been reported to increase as the number of the graphene layers decreases.25 Figure 2 SEM and TEM images of (a, d) lanthanum hydroxide nanorods (LaNR); lanthanum hydroxide nanorod-templated carbon (b, e) before (C@LaNR) and (c, f) after (LaNRTC) template removal. The LaNRTC after template removal shows a broad peak around 22° in the X-ray diffraction (XRD) and the corresponding d-spacing is calculated using Bragg equation, which is consistent to the d-spacing measured in the TEM image. This is attributed to the (002) planes of the carbon, whereas the LaNR and C@LaNR mainly exhibit XRD patterns for nanocrystalline La(OH)3 (JCPDS 36-1481) (Figure 3a). This is also consistent to the TEM images (Figure 2), where the LaNRTC shows a few layers of carbon. The Raman spectra exhibit a strong G band (∼1600 cm–1) with a IG/ID ratio of 1.03. Previously, a La-zeolite-templated carbon was reported to possess five- or seven-member rings, which contributed to the peaks for disordered carbon (D band), but they are still sp2 hybridized.23 In that case, the overall carbon material has graphitic framework with good conductivity. Combining the results from TEM and Raman, the LaNRTC possesses graphitic walls. Figure 3 (a) Powder X-ray diffraction patterns of LaNR, C@LaNR, and LaNRTC; (b) Raman spectrum of LaNRTC; (c) N2 adsorption–desorption isotherms of LaNR and LaNRTC; (d) density functional theory (DFT) pore size distributions of LaNR and LaNRTC. Figure 3c shows the N2 adsorption–desorption isotherms for the LaNR template and the resulting carbon, LaNRTC. The isotherm of the pure LaNR shows a type II isotherm with a low Brunauer–Emmett–Teller (BET) surface area of 54 m2/g, indicating no porosity but space between nanoparticles. In contrast, the LaNRTC shows a type IV isotherm corresponding to mesoporous features with a clear hysteresis between 0.4 and 1.0. The adsorption below 0.1 also indicates some microporosity. The BET surface area of the LaNRTC is much higher than that of the pure LaNR (771 vs 54 m2/g). The micropore volume and total pore volume of LaNRTC is much higher than LaNR, as shown in Table S1. Both LaNRTC and LaNR show similar DFT pore size distribution, which might due to the absence of uniform pore structure (Figure 3d). The large increase in surface area is due to the transformation of the morphology from rods to tubular network with accessibility to inner surface of the tubes. The TEM images in Figure 4 show both closed ends and open ends. The open ends result in pore openings as large as 30 nm (Figure 4). Micropores resulting from defects are not apparent in these images. Additionally, some of the tube junctions were fused together as interconnected L-shaped channels. It is assumed that the carbon grows from one rod to the other across the tightly packed junctions rather than just covering an individual rod. This arises because during the CVD process the carbon cannot completely coat the randomly stacked La(OH)3 nanorods. Thus, there are hollow nanorods and a complicated tubular carbon network. Overall, the material is composed of carbon tubes, tube stacks, and fused mazelike nano carbon tube networks. The large increase in the surface area from pure LaNR to LaNRTC is much higher than that of most CNTs (<500 m2/g).7 The TEM images in Figure 4 also represent the relationship between the nanorods template and the pore structure of resulting carbon. Since the synthesis of La(OH)3 nanorods with various sizes and shapes has not been well established, the pore structure is not controlled by different La(OH)3 templates here, but it is of great interest for future work. The hollow carbon tubes with different length and diameters are presented in Figure 4, and they are formed on La(OH)3 nanorods with various sizes. The size of La(OH)3 nanorods controls the resulting carbon’s dimensions and inner diameters, which affect the porosity of each carbon tube in the same batch. By using different La(OH)3 templates, it is possible to control the pore structure of the resulting carbon in bulk. Figure 4 Schematic illustration of the formation and TEM images of broken ends and connected junctions in the LaNRTC. The LaNRTC was tested as an electrode material for supercapacitors using 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIM-TFSI) as the electrolyte. Figure 5a shows the cyclic voltammograms (CVs) of the LaNRTC at different scan rates (10, 25, 50, 75, and 100 mV/s) in the range of −2.0–2.0 V.26−29 The shape of the CV is nearly rectangular indicating ideal double-layer capacitive behavior. The capacitances of the electrodes were calculated from CV using the following equation where I is the current as a function of time, υ is the scan rate. The specific capacitance in different scan rates was calculated using following equation where m is mass of both electrodes. The highest specific capacitance obtained for the LaNRTC was 128 ± 2 F/g at 10 mV/s. Table 1 summarizes the obtained specific capacitance values at different scan rates. To compare the LaNRTC with a mesoporous carbon templated by mesoporous silica, wrinkled mesoporous carbon was prepared according to reported procedure.24 The capacitance of LaNRTC is higher than the mesoporous carbon templated by La2O3 supported on wrinkled mesoporous silica (Figure S1), which has a capacitance of 70 F/g. The capacitance of LaNRTC is among the best results for mesoporous graphenes,20,30−36 and aligned CNTs18,37 as well as activated carbons38−41 using ionic liquids as the electrolyte. Unlike these carbons, the LaNRTC does not require complicated porous silica templates, lengthy preparation process, or additional activation steps. And the ionic liquids allow a higher operating voltage window, which is more practical for various applications. Figure 5 Electrochemical characterizations of LaNRTC: (a) cyclic voltammograms; (b) charge–discharge curves from 1 to 6 A/g; (c) Nyquist plots; (d) Ragone plot; (e) specific capacitance retention as a function of cycle number. Table 1 Specific Capacitance (F/g) in Different Scanned Rates scan rates (mV/s) specific capacitance (F/g) 10 128.1 25 126.2 50 125.0 75 122.6 100 120.2 Figure 5b shows the charge–discharge curves for the LaNRTC in different current densities (1–10 A/g). The JME cells were charged to 3.5 V and discharged completely at different current densities. Linear discharge curves show the ideal capacitive behavior. After charging up to 3.5 V, it is important to stabilize the coin cell before it starts to discharge. A plateau at 3.5 V is purposely introduced to investigate the stability of the coin cell. At 1 A/g, the IR drop is ∼0.2 V, which shows the good conductive behavior of the JME cell. Energy densities (Ed) and power densities (Pd) were calculated using following equations where t is the time taken to discharge to 0 V from the initial voltage (ΔV), subtracting IR drop at the beginning of discharge. The energy density and power density of the LaNRTC were found to be 55 Wh/kg and 1700 W/kg, respectively, at 1 A/g. In contrast, the mesoporous carbon templated by La2O3 supported on wrinkled mesoporous silica (Figure S1) has an energy density of 35.8 Wh/kg and a power density of 1499 W/kg at 1 A/g. Figure 5c shows the Nyquist plots for the LaNRTC in EMITFSI electrolyte. Electrochemical impedance spectroscopy (EIS) can provide information such kinetics in the double-layer charging–discharging processes. The almost vertical increase in the lower frequency region is indicative of an ideal capacitance behavior. In the Nyquist plot, the x axis intercepts at the highest frequency region indicating bulk electrolyte resistance, which mainly depends on the electrolyte solution. Figure 5d shows the Ragone plot of the LaNRTC, polyacrylonitrile (PAN) nanofibers,42 and carbon nanotubes (CNTs) at different current densities 1–10 A/g by comparing energy densities and power densities. Energy densities were determined by discharging from 3.5 to 0 V. When the current density increases, the energy density decreased, but retained 90% of its original energy density at discharge rate of 10 A/g, whereas PAN and CNTs retained only 75 and 80%, respectively. The energy density of the LaNRTC is much higher than PAN nanofibers and the pure CNTs, as shown in Figure 5d. Figure 5e shows the plot of specific capacitance retention against cycle number. After the 100th cycle, 98% of the specific capacitance was retained, whereas 97% of the capacitance was retained after 1000th cycle. Capacitance (89%) was retained after 5000th cycles, indicating the good cycling ability. This combined with the high energy density, and power density of the LaNRTC makes this carbon a good electrode material for supercapacitors. Conclusions In conclusion, the lanthanum hydroxide nanorods function as both catalyst and template for carbon growth, resulting in hollow carbon nanorods. The hollow carbon nanorods possess connected junctions and open ends, which result in a mazelike structure with high surface area and porosity. The LaNRTC shows excellent performance as an electrode material for supercapacitors taking advantage of its unique structure as well as low resistance. The templating method using lanthanum hydroxide nanoparticles also creates possibilities for the preparation of other graphitic carbons with controlled morphology. Experimental Section Synthesis of La(OH)3 Nanorods In a typical synthesis, 5.77 g of La(NO3)3·6H2O in 10 mL of deionized water was mixed with a solution of 14.7 g of NaOH in 20 mL of water and heated to 110 °C in a 45 mL Teflon-lined autoclave for 24 h. The solid product was collected by centrifugation and washed with water several times then dried overnight at 100 °C. The product was then annealed at 200 °C for 1 h. Synthesis of La(OH)3 Nanorod-Templated Carbon The La(OH)3 nanorods (∼1 g) were placed in a ceramic boat centered in a horizontal quartz tube reactor. The reactor was heated to 600 °C under N2 (200 mL/min). Then, acetylene (30 mL/min) and steam (water is pumped at 5 mL/h) were fed into the reactor for 1.5 h followed by carbonization under N2 at 850 °C for 2 h. The resulting carbon/La(OH)3 composite was washed with concentrated HCl and water to remove the La(OH)3 template. For comparison, a mesoporous carbon was prepared with the same method, but by using La(OH)3 supported on wrinkled mesoporous silica as the template following reported procedure.24 Fabrication of Coin Cell-Type Supercapacitors Symmetric JME cells were fabricated according to a previously reported procedure for JME packaging (Figure S2).43 Electrodes were prepared by mixing the La(OH)3 nanorod-templated carbon with 2% poly(tetrafluoroethylene) binder. The carbon electrodes were immersed in the electrolyte, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIM-TFSI), for 1 h at room temperature under vacuum prior for assembling the coin cell. The two electrodes were separated by a Teflon film (0.02 mm) and constructed under inert environment. Characterization Scanning electron microscope (SEM) images were recorded on a Zeiss-LEO model 1530 scanning electron microscope. Transmission electron microscope (TEM) images were recorded using a JEOL 2100 transmission electron microscope. Powder X-ray diffraction (XRD) patterns were acquired on a Rigaku Ultima IV diffractometer using Cu Kα radiation. Raman spectra was obtained on a Jobin Yvon Horiba high-resolution LabRam Raman microscope. The N2 adsorption–desorption isotherms and BET surface areas were measured using a Quantachrome AS1 Autosorb. Electrochemical measurements were made using a BT2000 Arbin battery testing system. Electrochemical impedance spectroscopy (EIS) was measured on a EG&G Princeton Applied Research potentiostat/galvanostat. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01714.Experimental details on the synthesis and characterizations of lanthanum hydroxide nanorods and lanthanum hydroxide nanorod-templated carbon as well as details of coin cell fabrication (PDF) Supplementary Material ao8b01714_si_001.pdf Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. Acknowledgments We thank the financial support from Robert A. Welch Foundation (AT-1153). ==== Refs References Han S. ; Wu D. ; Li S. ; Zhang F. ; Feng X. Porous Graphene Materials for Advanced Electrochemical Energy Storage and Conversion Devices . Adv. 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PMC006xxxxxx/PMC6644426.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145888610.1021/acsomega.8b00948ArticleKinetics of Catalytic Wet Peroxide Oxidation of Phenolics in Olive Oil Mill Wastewaters over Copper Catalysts Maduna Karolina †‡Kumar Narendra ‡Aho Atte ‡Wärnå Johan ‡Zrnčević Stanka †Murzin Dmitry Yu. ‡† Faculty of Chemical Engineering and Technology, Department of Reaction Engineering and Catalysis, University of Zagreb, Marulicev trg 19, 10000 Zagreb, Croatia‡ Faculty of Science and Engineering, Laboratory of Industrial Chemistry and Reaction Engineering, Åbo Akademi University, Biskopsgatan 8, FI 20500 Turku-Åbo, Finland E-mail: dmurzin@abo.fi.03 07 2018 31 07 2018 3 7 7247 7260 09 05 2018 21 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. During olive oil extraction, large amounts of phenolics are generated in the corresponding wastewaters (up to 10 g dm–3). This makes olive oil mill wastewater toxic and conventional biological treatment challenging. The catalytic wet peroxide oxidation process can reduce toxicity without significant energy consumption. Hydrogen peroxide oxidation of phenolics present in industrial wastewaters was studied in this work over copper catalysts focusing on understanding the impact of mass transfer and establishing the reaction kinetics. A range of physicochemical methods were used for catalyst characterization. The optimal reaction conditions were identified as 353 K and atmospheric pressure, giving complete conversion of total phenols and over 50% conversion of total organic carbon content. Influence of mass transfer on the observed reaction rate and kinetics was investigated, and parameters of the advanced kinetic model and activation energies for hydrogen peroxide decomposition and polyphenol oxidation were estimated. document-id-old-9ao8b00948document-id-new-14ao-2018-00948mccc-price ==== Body 1 Introduction Phenols are important industrial chemicals widely used as reactants and solvents in numerous commercial processes and therefore are often present in industrial effluents. The major anthropogenic sources of phenol-contaminated wastewaters are petrochemical, pharmaceutical, wood, pulp, and paper and food processing industries as well as landfill and agricultural area leachate waters.1 There are several environmental concerns regarding phenols; thus, they are considered to be hazardous in industrial wastewaters, harmful even at low concentration levels (ppm range). Wastewaters containing phenols should therefore undergo a special treatment. In EU, the current limits for wastewater emission of phenols are 0.5 mg dm–3 (0.5 ppm) for surface waters and 1 mg dm–3 (1 ppm) for sewage systems with maximum allowed concentration levels in potable and mineral waters of 0.5 μg dm–3 (0.5 ppb). Significant quantities of phenolics are generated in olive oil mill wastewater (OOMW) including organic contaminants such as lignin, tannins, and polyphenolic compounds. Significant amounts of olive mill wastewater exceeding several million tons are produced in Europe alone despite stringent legislation2 and are not properly treated. The properties of OOMWs depend on the method of extraction, feedstock properties, and region and climate conditions. In general, OOMW is a dark brown acidic effluent (pH = 4.0–5.5), with a distinctive odor and high conductivity, comprising besides water (80–83%) organic compounds (15–18%) and inorganic elements (2%, potassium salts and phosphates). The concentration of phenols and polyphenols in OOMW can be as high as 20 wt %.3 Although several studies were reported on removal of phenolics in OOMW,4 significant efforts are still needed. Often separation-based technologies are suggested as an alternative to biological processes, however, their effective application can be hindered by high operational costs and sustainability concerns such as generation of secondary toxic wastes because the toxic compounds are not destroyed but only separated.5 Therefore, in the current work, the focus was on catalytic approaches to diminish the content of phenolics in OOMW. In particular, the catalytic wet peroxide oxidation (CWPO) process is a suitable method6 generating hydroxyl radicals during hydrogen peroxide decomposition. Hydrogen peroxide is generally considered as a nontoxic and ecologically attractive oxidant. Application of heterogeneous catalysts, such as zeolites,7 in CWPO of organic compounds has been reported. Transition metal-exchanged (mostly iron and copper) zeolites of FAU or MFI morphology showed promising results; however, there are still some open issues such as resistance to leaching of the active metal during the reaction. Apart from few recent reports,8−11 most of the studies describe application of powdered catalysts for which mass transfer limitations can be neglected.7,12,13 It is apparently clear that scaling up of a commercial CWPO process requires detailed studies with the pelletized catalysts. In this case, external and internal mass transfers (i.e., diffusion processes in the boundary layer surrounding the catalyst pellet and in the pores of the catalyst) should be properly considered. In this work, the activity of copper-containing catalysts was tested in catalytic wet peroxide oxidation of OOMW with a special attention to the stability of copper during the reaction, namely, its resistance to leaching. The influence of interparticle and intraparticle diffusion was investigated, and the reaction kinetics parameters of the proposed pseudo-second-order kinetic model were estimated. 2 Experimental Section 2.1 Catalyst Preparation A series of copper-containing 13X zeolite catalysts were prepared by postsynthesis ion-exchange from the sodium form of the commercial 13X zeolite acquired from UOP Italy (13X-APG Molsiv, SiO2/Al2O3 = 3.2, wNa2O = 20 wt %, dB, range ≤ 2 mm). Depending on the bead size, between 2.5 and 10 g dm–3 zeolite was ion-exchanged with a 0.05 M copper acetate solution under agitation at 298 K for 0.5 to 3 h, followed by filtration of the samples and drying overnight at room temperature to obtain copper-bearing zeolites with a similar metal content. The detailed preparation method was described previously.14 After copper ion-exchange, postsynthesis thermal treatment was performed consisting of calcination at 1273 K for 5 h (ramp 2 K min–1) to achieve materials exhibiting a higher stability against the loss of the active metal component during the reaction. The list of prepared catalysts and their designated names is presented in Table 1. Table 1 Overview of Prepared Catalysts and Their Preparation Procedures and Designated Names sample bead size range mm preparation method copper content, wt % (UV–vis) Cu/13X-1 0.40–0.63 ion exchange (10 g dm–3, 2 h) 8.6 Cu/13X-K1273-1 ion exchange + calcination at 1273 K Cu/13X-2 0.315–0.40 ion exchange (10 g dm–3, 0.5 h) 7 Cu/13X-K1273-2 ion exchange + calcination at 1273 K Cu/13X-3 0.63–0.80 ion exchange (10 g dm–3, 3 h) 8 Cu/13X-K1273-3 ion exchange + calcination at 1273 K Cu/13X-4 0.10–1.00 ion exchange (10 g dm–3, 3 h) 7 Cu/13X-K1273-4 ion exchange + calcination at 1273 K Cu/13X-5 1.25–2.00 ion exchange (2.5 g dm–3, 1 h) 7 Cu/13X-K1273-5 ion exchange + calcination at 1273 K 2.2 Catalyst Characterization Textural characterization of the catalysts was performed by nitrogen physisorption at 77 K using a Sorptomatic 1900 Carlo Erba instrument. Prior to measurements, the samples were outgassed at 423 K for 3 h at reduced pressure below 0.1 mbar. The specific surface area and pore volume calculations were performed using Dubinin’s equation for microporous and Brunauer–Emmett–Teller equation for mesoporous samples. Pore size distributions were acquired using the Horvath–Kawazoe method. The crystalline structures of the parent zeolite- and prepared zeolite-based catalysts containing copper were evaluated by powder X-ray diffraction (XRD) analysis on a XRD 600, Shimadzu instrument. Cu Kα was used as the radiation source at the wavelength of 0.154 nm with 2θ from 5 to 60° with a 0.02° step size. The peak identification was performed using X’Pert HighScorePlus software. The morphology of the fresh- and spent-zeolite-based copper catalysts Cu/13X and Cu/13X-K1273 was studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM analysis was performed on carbon-coated samples using a LEO Gemini 1530 instrument equipped with a Thermo Scientific UltraDry Silicon Drift Detector. The transmission electron microphotographs were taken by a JEM-1400 Plus transmission electron microscope (TEM) operated at 120 kV acceleration voltage. The powdered samples were suspended in 100% ethanol under ultrasonic treatment for 10 min. For each sample, a drop of ethanol suspension was deposited on a Cu fiber carbon grid (200 mesh) and evaporated, after which the images were recorded. Copper loading was measured using a UV/vis spectrometer (UV1600PC, Shimadzu) at 270 nm for the parent solution of copper acetate applied during ion exchange and later confirmed by energy-dispersive X-ray microanalysis (EDXA) during SEM analysis and by inductively coupled plasma-optical emission spectrometry (ICP-OES) (PerkinElmer, Optima 5300 DV) after dissolution in HF. The basicity of the prepared catalysts was elucidated using temperature-programmed desorption (TPD) of CO2 on AutoChem 2010 (Micromeritics Instruments) in the temperature range of 373–1173 K according to the method described by Kumar et al.15 Infrared spectroscopy (ATI Mattson FTIR) was applied to elucidate the strength of Brønsted and Lewis acid sites using the KBr pellet technique working in the range of wavenumbers of 4000–400 cm–1 with pyridine as the probe molecule. A detailed description of the analytical procedure is available.16 2.3 Catalytic Experiments The catalytic experiments were carried out under atmospheric pressure in a 250 cm3 glass batch reactor equipped with a pH electrode and a temperature sensor. The stirring speed in the range between 50 and 800 min–1 and catalyst particle sizes from ca. 0.3 to 2.0 mm were varied to address the impact of mass transfer. For elucidation of reaction kinetics, the catalyst loading, reaction temperature, and hydrogen peroxide concentration were varied. OOMW was supplied by a private oilery (Dalmatia Region, Croatia) from a three-phase extraction process of olive oil production from green olive stock mixture (local sort Olea europaea var. oblica). Basic properties of the wastewater are presented in Table 2. Table 2 OOMW Properties at Source (293 K) pH γTOC, g dm–3 COD, g O dm–3 γTPh, g dm–3 total solids, g dm–3 4.79 10.7 36 1.8a 27 a Total phenols content constitutes 17 wt % total organic carbon (TOC) content of the OOMW. Prior to reactions, OOMW was filtered through a 100 μm nylon filter bag and diluted with distilled water (v/v = 50:50). UV–vis absorbance was applied to monitor the concentration of phenolics and hydrogen peroxide. The standard Folin–Ciocalteu method at 765 nm described in the literature17 was used to measure the total phenol concentration. A standard curve of gallic acid was used for quantification, and the results were expressed as gallic acid equivalent (GAE) concentrations. The ammonium metavanadate spectrophotometric method at 450 nm adopted from ref (18) was used for measuring hydrogen peroxide concentrations. The measured absorbances were recalibrated with reference to OOMW sample blanks not containing phenols or hydrogen peroxide to eliminate the potential error from the existing color or turbidity of the wastewater. Total organic carbon (TOC) was evaluated with a TOC-V CSN Shimadzu analyzer using diluted reaction mixtures, and chemical oxygen demand (COD) of the selected samples was measured by a UV/vis spectrometer using the dichromate colorimetric method at 605 nm (Hach-Lange cuvette tests). The copper content in the reaction mixture reflecting metal leaching was determined by atomic absorption spectrometry of diluted reaction mixture solutions on Shimadzu AAS 6300 using a Cu hollow cathode at λ = 324.9 nm. X-ray powder diffraction analysis and N2 physisorption measurements were conducted to reveal potential structural changes and coking. 3 Results and Discussion 3.1 Catalyst Characterization After testing all prepared catalysts having different sizes, it was concluded (see below) that Cu/13X-1 with the size range of 0.4–0.63 mm is the most appropriate for CWAO. Table 3 thus contains results obtained from N2 physisorption analysis of Cu/13X-1 and its thermally treated counterpart. The incorporation of copper in 13X zeolite did not have a significant effect on the measured surface area. Table 3 Specific Surface Areas, Pore Volumes, and Copper Loadings of Prepared-Zeolite-Based Catalysts copper content, wt % sample UV/vis EDXA ICP-OES specific surface area, m2 g–1 pore volume, cm3 g–1 average pore size,a nm 13X-APG Molsiv 59419 0.3119 2 Cu/13X-1 8.6 7.5 8.0 61819 0.3419 2 Cu/13X-K1273-1 11.5b 13.2 13.9 2619 0.0319 5 a Calculated using dPORE = 4VPORE/S. b Calculation based on a 25% reduction of the catalyst’s mass during postsynthesis thermic treatment. The thermal treatment resulted in a decrease of both specific surface area and pore volume with a shift of the pore size distributions (Figure 1) from the microporous (Cu/13X-1) to the mesoporous range (Cu/13X-K1273-1). Such pronounced differences in the physical properties for the catalyst calcined at 1273 K can be attributed to structural changes during thermal treatment. Figure 1 Pore size distributions in Cu/13X-1 and Cu/13X-K1273-1 catalysts. XRD diffractograms of Cu/13X-1 already presented in ref (19) confirm the FAU structure as no shifts in the peak positions and no significant diffraction lines assigned to any new or impurity phase were observed. XRD suggested19 high crystallinity of the copper-containing material as incorporation of copper into the zeolite framework via ion exchange does not influence the crystal structure. In agreement with the literature,20 the obtained results indicate that Cu2+ ions are well dispersed in the zeolite framework of 13X and that the size of copper particles is below the detection limit for the XRD measurement (<2–4 nm). In fact, from the TEM image of a Cu/13X-1 catalyst (Figure 2a), very small metal particles highly dispersed in single zeolite crystals can be observed. Their average size calculated using TEM analysis was 1.7 nm. Figure 2 TEM images of Cu/13X-1 (a) and Cu/13X-K1273-1 (b) catalysts. The copper-bearing zeolite calcined at 1273 K exhibited phase transformations from a zeolite to a silicate-based material upon heating. As previously reported,19 several phases were determined for Cu/13X-K1273-1, including magnesium silicate, copper oxide, anorthoclase (Na0.85K0.14AlSi3O8), and andesine (Na0.685Ca0.347Al1.46Si2.54O8). Changes in crystal phases upon thermal treatment were in line with a decrease of the surface area and pore volume (Table 3). The size of CuO in Cu/13X-K1273-1 according to the Debye–Scherrer equation was 26.0 and 25.1 nm for the respective peaks at 35.5 and 38.6°. An average metal particle size analysis using TEM was not applicable for the Cu/13X-K1273-1 catalyst because of a poor resolution between the dark metal particles and the dark surface of single catalyst crystals (Figure 2b). The increase in the size of the metal particles in the Cu/13X-K1273-1 catalyst is most probably a consequence of metal sintering and clustering of smaller metal particles into larger ones that occurs during thermal treatment.21 The morphology, shape, and size of crystals of Cu/13X-1 and Cu/13X-K1273-1 catalysts were additionally characterized by SEM. From the transmission electron micrograph (Figure 2a) and the scanning electron micrograph (Figure 3a) of the Cu/13X-1 catalyst, specific needle-shaped crystals were identified. Figure 3 SEM images of Cu/13X-1 (a) and Cu/13X-K1273-1 (b) catalysts. Although agglomerated, these can be associated with X zeolite morphology similar to that reported previously.22 Single crystals in Cu/13X-K1273-1 were observed to be larger in size and of irregular shapes and a broad crystal size range (Figures 2b and 3b) in agreement with XRD, showing the presence of several crystal phases. To evaluate metal dispersion across the surface, SEM imaging in a backscattering mode of the pellets and the cross sections of pellets was performed (Figure 4). Figure 4 Backscattering SEM images of pellets and cross sections of pellets: Cu/13X-1 (a, b) Cu/13X-K1273-1 (c, d). The brighter areas in the backscattering images are representative of the higher densities of the more heavy elements (copper). It can be noticed that copper is consistently spread over the surface of the Cu/13X-1 catalyst (Figure 4a,b), whereas in the case of the Cu/13X-K1273-1 catalyst (Figure 4c,d), copper is mainly located on the outer catalyst surface and in the narrow band close to the pellet surface several micrometers in width. Migration of copper from inside of the pellet to its outer surface is most probably a consequence of the structural changes during thermal postsynthesis treatment. XPS analysis was used for the identification of the oxidation state of copper cations in Cu/13X-1 and Cu/13X-K1273-1. From the XPS spectra presented in Figure 5, characteristic peaks were identified for Cu 2p, O 1s, Al 2p, and Si 2p for both catalysts. Differences in the high-resolution spectra of Cu 2p and O 1s indicate that the nature of copper species is different in Cu/13X-1 and thermally treated Cu/13X-K1273-1 catalysts. The first exhibits only two main peaks at 934 (Cu 2p3/2) and 953.3 eV (Cu 2p1/2), confirming the presence of Cu1+ as in Cu2O. In the high-resolution Cu 2p spectra of the latter, strong Cu2+ satellite peaks at 943.3 and 964.2 eV were present, contributing to the presence of the CuO phase, as previously identified by XRD.23 Differences in O 1s signals additionally confirm the distinction between copper oxides found on the surface of Cu/13X-1 and Cu/13X-K1273-1 catalysts. It should be noted that reduction of finely dispersed Cu2+ under exposure to the X-ray beam during XPS analysis in the case of the Cu/13X-1 catalyst cannot be excluded. Therefore, a difference between the catalysts can also be related to difficulties in reduction of larger CuO particles in the case of Cu/13X-K1273-1 during the XPS measurements. Figure 5 XPS survey and high-resolution spectra of Cu/13X-1 (a) and Cu/13X-K1273-1 (b). During catalyst preparation, the influence of metal incorporation into the zeolite support as well as the influence of postsynthesis thermal treatment on the acid–base properties of the parent and copper-bearing zeolites has been investigated. CO2-TPD profiles of the parent zeolite as such (13X), calcined form (13X-K1273), copper zeolite (Cu/13X-1), and the calcined material (Cu/13X-K1273-1) were presented previously.19 The calculated amounts of desorbed CO2 are given in Table 4. Table 4 Basicity of the Prepared Catalyst Measured by TPD-CO2 basic sites (mmol g–1) sample weak 320–500 K medium 500–750 K strong >750 K total basicity 13X 0.040 0.004 0.237 0.281 Cu13X-1 0.036 0.009 0.580 0.625 Cu13X-K1273-1 0.105 0.105 Weak, medium, and strong basic sites were identified in 13X and copper-modified 13X zeolites,19 which is explained by the application of the sodium form of the commercial zeolite for catalyst preparation as well as with the intrinsic (structural) basicity of oxygen atoms present in the zeolite.24 Copper-containing zeolite Cu13X-1 exhibited much higher quantities of desorbed CO2 related to strong basic sites (>750 K), indicating a more pronounced basicity of copper-exchanged zeolite. High temperature, however, can in general also lead to structural changes of the zeolite, thus preventing a straightforward assignment of high-temperature peaks to strong basic sites. This possibility was ruled out because only strong basic sites were seen for thermally stable Cu/13X-K1273-1. Acidity measurements were reported previously19 showing that copper-containing catalysts exhibited Lewis acidity, which can be explained by the presence of copper.2 Thermal treatment of Cu/13X-1 resulted in a decrease in acidity. Brønsted acid sites are degraded upon severe heat treatment above 773 K,24 whereas Lewis acidity from Cu2+ present in Cu/13X was diminished by the formation of copper oxide, showing a more basic character. As reported previously,19 higher acidity was measured for Cu/13X-K1273-1 compared to that for the copper-free counterpart. 3.2 Preliminary Catalytic Experiments and Analysis of Internal Mass Transfer Catalytic wet peroxide oxidation of OOMW was performed under mild reaction conditions. During preliminary studies, the extent of thermal decomposition of polyphenols present in the OOMW was investigated as well as the influence of catalyst addition on the reactant conversion rates. The possible catalytic activity of the parent Na-13X zeolite in the CWPO of phenol was excluded during our previous investigations of a model catalytic system.14 Preliminary results on catalytic oxidation were already reported,19 confirming the role of catalysts in reducing the amount of phenolics and decomposing hydrogen peroxide (Figure 6a,b). Thermal treatment of the catalyst at high temperature was effective in decreasing hydrogen peroxide decomposition, improving also the conversion of total phenols. Figure 6 Kinetic curves for hydrogen peroxide (a) and total phenols (b) (cHP,0 = 0.25 M, T = 353 K, N = 600 min–1, mCAT = 2.5 g, dB = 0.4–0.63 mm). These results indicate that the oxidant is probably inefficiently used in the reaction on Cu/13X-1 and that hydrogen peroxide is mainly consumed in the reactions where hydroxyl radicals are lost and are not used for degradation of the polyphenols. In CWPO, oxidation of organic compounds is attributed to the presence of hydroxyl radicals that are generated when hydrogen peroxide is decomposed. Reaction pathways can be presented with Reactions 1–6. In the initial stages of the reaction, hydroxyl and perhydroxyl radicals are produced by hydrogen peroxide decomposition on the catalyst25 1 2 Both radical species are capable of oxidizing the organic compounds; however, the reactivity of hydroxyl radicals is dominant.7 Catalytically produced hydroxyl radicals react with phenolic compounds, oxidizing them through a series of intermediates to carbon dioxide and water when complete mineralization is achieved 3 Hydroxyl radicals are very reactive, and they are involved in a number of competing side reactions such as scavenging hydrogen peroxide and termination between the hydroxyl and perhydroxyl radicals7 4 5 6 If the latter reactions of hydroxyl radicals are dominant, hydrogen peroxide will be consumed fast and majority of the generated hydroxyl radicals will be spent inefficiently in undesired side reactions. This could be considered a preferred reaction pathway if the intraparticle diffusion resistances for the phenolic molecules are present. In this case, only hydrogen peroxide would be adsorbed and decomposed on the catalytically active sites on the internal catalyst surface, whereas adsorption of polyphenols would be limited mostly to the outer surface of the catalyst. In the absence of organic compounds, the hydroxyl radicals formed inside the catalyst would for the most part react with one another and hydrogen peroxide. Taking into account the average pore sizes in the Cu/13X-1 catalyst (2 nm) and cross sections of hydrogen peroxide (0.15 nm) and polyphenols (1–2 nm), configurational diffusion limitations could be expected for the phenolic compounds found in the OOMW. In addition to configurational limitations, intraparticle resistances for hydrogen peroxide and polyphenols could be present. They were verified using the Weisz–Prater criterion26 7 where robs is the observed reaction rate, R is the particle radius, cs is the molar concentration of the solute at the catalyst surface, De is the effective diffusion coefficient of the solute, and n is the reaction order. For the porous media and the random pore model, the effective diffusion coefficient is defined as , where D is the diffusion coefficient, ε is the porosity, and τ is the tortuosity, which are connected to the structural characteristics of the catalyst and pore geometry. For the liquid–solid catalytic systems, only molecular diffusion was taken into account, D ≈ DABo. The molecular diffusion coefficient was calculated from the Wilke–Chang equation27 8 where Φ is the dimensionless association factor of the solvent (Φ = 2.6 for water), MB is the molar mass of the solvent, μB is the dynamic viscosity of the solvent in cP at temperature T (K), and Vb(A)0.6 is the liquid molar volume at the solute normal boiling point. For the purposes of this study, the liquid molar volumes at the solute’s normal boiling point were calculated from the Tyn and Calus equation 9 The calculations of the diffusion coefficients were performed for hydrogen peroxide and phenol diffusing in water using the data and expressions obtained from the thermodynamic properties databank.28 In the absence of thermodynamic data at the critical point for polyphenols such as hydroxytyrosol or tyrosol that are most commonly found in the OOMW, phenol was chosen as a model compound for the calculations. Because polyphenols are more complex and larger molecules than phenol, it is reasonable to expect that if the internal transfer limitations exist for phenol they would be even more pronounced for polyphenols. The obtained values of the diffusion coefficients at 353 K and normal pressure (typical reaction conditions) were 6.0 × 10–9 m2 s–1 for hydrogen peroxide and 3.5 × 10–9 m2 s–1 for phenol. Application of the Weisz–Prater criterion (eq 7) for the observed initial reaction rates of hydrogen peroxide decomposition (rHP,obs = 3.7 × 10–4 mol dm–3 s–1) and polyphenols oxidation (rTPh,obs = 5.3 × 10–5 mol dm–3 s–1) and their corresponding surface concentrations with the mean catalyst particle diameter of 0.515 mm and ratio of 0.1 (generally valid for zeolites) resulted in the values of the dimensionless Weisz modulus of 0.15 for hydrogen peroxide diffusion and 2.25 for phenol. With this result, the presence of intraparticle diffusion for hydrogen peroxide can be eliminated, whereas the same cannot be concluded for the phenolics, especially for the reaction orders higher than zero. For polyphenols in OOMW, larger pore diffusion limitations can be expected being the most probable cause of an inefficient use of hydrogen peroxide. Because there are no pore diffusion limitations for the oxidant, hydrogen peroxide mainly decomposes inefficiently inside the Cu/13X-1 catalyst pores where negligible amounts of polyphenols are present. On the other hand, because of the larger pore sizes of the calcined Cu/13X-K1273-1 catalyst (average pore size of 5 nm), the internal diffusion in this reaction should not be as significant as in the case of the Cu/13X-1 catalyst and faster decomposition of hydrogen peroxide and oxidation of polyphenols should be expected. However, this is not the case. The reason for a much slower radical generation rate lies in the fact that the postsynthesis thermal treatment induced migration of the catalytically active species (copper) toward the pellet surface, which was confirmed by SEM imaging in the backscattering mode of the cross sections of Cu/13X-1 and Cu/13X-K1273-1 pellets, as presented in Figure 5b,d. Hydrogen peroxide decomposition in the Cu/13X-K1273-1 catalyst takes place only in a narrow ring of few micrometers from the particle surface where the presence of copper is identified and where polyphenols are also present. In this case, it can be considered that the pore diffusion for polyphenols is not as significant as in the case of the Cu/13X-1 catalyst and that most of the generated hydroxyl radicals are reacting with the organic compounds and are not inefficiently spent in fast scavenging reactions inside the catalyst pellet (eqs 4–6). As a result, the rates of polyphenol oxidation are comparable for both catalysts with a higher extent of oxidation for Cu/13X-K1273-1 resulting in an almost complete removal of the phenolic content after 180 min of reaction. Comparison of Cu/13X and Cu/13X-K1273 during preliminary studies included also the investigation of their behavior in CWPO of OOMW, namely, measuring the extent of copper leaching during the reaction as well as by analyzing potential changes of the zeolite support after the reaction. XRD diffractograms of both catalysts prior and after catalytic experiments are close to each other (Figure 7a,b), indicating good stability of the support, while copper leaching was significantly different. Figure 7 XRD diffractograms of the fresh and spent Cu/13X-1 (a) and Cu/13X-K1273-1 (b) catalysts. By measuring the copper content in diluted reaction mixtures using atomic absorption spectroscopy, it was determined that after 180 min 38 wt % copper leached from the Cu/13X-1 catalyst in a striking contrast to only 2 wt % for its counterpart calcined at high temperature, indicating severe instability of Cu/13X-1. Contribution of the leached copper in the solution to the overall catalytic performance was discussed previously,29,30 concluding that it can be neglected due to inactivation of copper by carboxylic acids. However, in this case, when over 20% of copper leached from the catalyst before the reaction was initiated by addition of hydrogen peroxide, the homogeneous contribution should not be excluded because oxidation of organic compounds catalyzed by copper cations in the liquid phase is possible. The copper leaching results were confirmed by energy-dispersive X-ray spectroscopy analysis of the fresh and spent catalysts, showing 42 wt % loss of copper for the spent Cu/13X-1 catalyst and 4 wt % loss of copper for the Cu/13X-K1273-1 catalyst. Specific surface area measurements were also supporting the superior resistance against leaching of the thermally treated catalyst. For the Cu/13X-1 catalyst, the specific surface area and pore volume decreased from the initial SFRESH = 618 m2 g–1 and Vp,FRESH = 0.34 cm3 g–1 to SSPENT = 434 m2 g–1 and Vp,SPENT = 0.30 cm3 g–1, respectively, whereas the values for the Cu13X-K1273-1 catalyst did not significantly change before and after the reaction: SFRESH = 26 m2 g–1 and Vp,FRESH = 0.03 cm3 g–1 to SSPENT = 24 m2 g–1 and Vp,SPENT = 0.04 cm3 g–1, respectively. One of the possible explanations for large variations in stability between calcined and noncalcined catalysts could be the different copper speciation, namely, the presence of Cu+ in Cu/13X-1 as revealed by XPS. To our knowledge, no report on the differences in the stability of Cu+ and Cu2+ in the CWPO of phenolics has been published. However, different coordination of copper inside the zeolite lattice for Cu+ and Cu2+ cations was reported by Vanelderen et al.,31 which could have an impact on their catalytic properties as well. An alternative explanation was proposed by Taran et al. based on a study of Cu-ZSM-5.13 The authors have shown that copper catalysts with 1–2 wt % loading possessed the highest activity and reasonable stability, whereas an increase in copper resulted in a lower activity and stability. In the current work, for the noncalcined catalysts, the amount of Cu could have been too high to allow formation of a stable material. After calcination, the zeolitic structure has been destroyed, giving several new phases. It could be due to the fact that partial encapsulation of CuO particles makes the catalyst less prone to leaching. Whatever the explanation, elucidation of mass transfer influence and kinetic analysis was done for the Cu/13X-K1273 catalyst in which the more efficient use of the oxidant was proven to take place. 3.3 Mixing Efficiency and External Mass Transfer In the case of catalytic wet peroxide oxidation of polyphenols over a solid pelleted catalyst, following mass transfer processes should be considered: transport of the dissolved reactants from the liquid bulk to the catalyst outer surface and transport inside the pores of the pellet. These effects result in the concentration gradients of reactants and products across phase boundaries and within the catalyst particle, as present in Figure 8. Figure 8 Mass transfer in catalytic wet peroxide oxidation of polyphenols in OOMW. To evaluate all possible mass transfer limitations, a combined theoretical/experimental approach was adopted in this study. Mass transfer coefficients through the external boundary layer and inside the pores were calculated for hydrogen peroxide and model compound phenol, and the presence of diffusion limitations was evaluated by the application of external mass transport and internal pore diffusion criteria for the Cu/13X-K1273-1 catalyst. Additionally, the efficiency of mixing in the reactor was verified and evaluation of reaction conditions for achieving total suspension of the catalyst was performed. External mass transfer or the mass transfer in the thin boundary layer around the solid catalyst particle depends on hydrodynamic conditions in the reactor (stirring speed), physical properties of the liquids, and the size of the catalyst particles. In the catalytic reactions in which the suspended solid catalyst is used, external mass transfer resistances can be minimized by efficient mixing that establishes thorough dispersion of reactants and catalyst in the liquid and the use of the smaller catalyst particles. The first step in achieving this is ensuring that under the conditions of the catalytic experiments the solid catalyst is completely suspended in the liquid and no particles remain at the bottom of the reactor for longer than 1 s. The minimum stirrer speed necessary for total suspension can be calculated from the empirical Zweitering equation32 10 where g is the gravitational constant (cm s–2), dP is the particle diameter (cm), dM is the impeller diameter (cm), ρL and νL are the density and kinematic viscosity of water in g cm–3 and cm2 s–1, respectively, B is the percentage of the weight of the catalyst compared to the weight of the liquid, and Δρ = ρS – ρL. S is a dimensionless factor that depends on the reactor geometry and impeller type, , and is equal to 3.3 for the stirred baffled tank with a diameter of 6.5 cm and pitched four-blade turbine impeller 4.5 cm wide positioned at 2.5 cm from the bottom of the reactor.33 For achieving the total suspension of the Cu/13X-K1273-1 catalyst (ρS = 1.3 g cm–3, dP = 0.04 cm, B = 1%) in OOMW (approximated by water: ρL = 0.997 and νL = 0.009 cm2 s–1), the rotational speed of the stirrer should be minimum, 244 rpm. The absence of external mass transfer for the stirring speed of 600 rpm was verified by applying the external mass transport criterion that can be derived considering the mass transfer rate through the boundary layer of the catalyst particle equal to the observed reaction rate34 11 Assuming Cb – Cs < 0.05cb and the nth-order reaction, the criterion takes the form 12 where robs is the observed reaction rate, R is the particle radius, cb is the molar concentration of the solute in bulk, and kLS is the liquid/solid mass transfer coefficient. Liquid/solid mass transfer coefficients for hydrogen peroxide and phenol were calculated on the basis of the correlation between dimensionless Sherwood, Reynolds, and Schmidt numbers for slurry reactors34 13 where Re number is expressed as based on the Kolmogorov theory of turbulence. By rearranging eq 13, the following expression for estimating liquid/solid mass transfer coefficient kLS can be derived 14 In eq 14, ϵ denotes the energy of dissipation, D is the diffusion coefficient of the diffusing compound, dM is the impeller diameter, ρ and η are the density and dynamic viscosity of water, and dP is the particle diameter. The energy of dissipation or the maximum specific mixing power was calculated from 15 where P is the mixing power that depends on the impeller type and stirring speed, NP is the power number of the impeller, and ρL and VL are the density and volume of the liquid. For a 45° pitched four-blade turbine impeller 4.5 cm wide with NP = 1.3 in the turbulent region (Re > 103) at a stirring speed of 600 rpm mixing the volume of 250 cm3, the maximum specific mixing power was calculated to be 0.96 W kg–1. The mutual diffusion coefficients of solutes in water were calculated using the Wilke–Chang equation (eq 8), resulting in the values of 6.0 × 10–9 m2 s–1 for hydrogen peroxide and 3.5 × 10–9 m2 s–1 for phenol. Next, the external mass transfer coefficients for hydrogen peroxide and phenol were calculated from eq 14, resulting in kLS,HP = 5.6 × 10–4 m s–1 and kLS,Ph = 3.8 × 10–4 m s–1. The application of the external mass transfer criteria (eq 12) gave the values on the left side of the equation several orders of magnitude lower than those on the right side, showing that external mass transfer for both hydrogen peroxide and polyphenols is negligible even at the higher reaction orders and that the reaction mixture is effectively mixed at 600 rpm. The above theoretical approach results were experimentally verified by adopting the published procedures for the elimination of external mass transfer.35 To confirm the specific conditions under which the reaction was operating with negligible external mass transfer resistances, the influence of the stirring speed and particle size on the reaction rates of hydrogen peroxide decomposition and polyphenols oxidation was investigated. The results are presented in Figures 9, 10, and S1. From the results presented in Figure 9, it can be seen that already above 100 rpm there are no significant changes in the reaction rates of hydrogen peroxide decomposition and polyphenols oxidation and that the increase in the stirring speed above 600 rpm does not further increase them. This indicates that for the stirring speed above 600 rpm the external mass transfer resistances are minimized and that the mass transfer through the boundary layer proceeds faster that the surface reaction. Figure 9 Influence of the stirring speed on hydrogen peroxide (a) and total phenol (b) content with time (N = 50–800 min–1, cHP,0 = 0.25 M, T = 353 K, mCu/13X-K1273-1 = 2.5 g, dB = 0.4–0.63 mm). Figure 10 Influence of catalyst bead size on hydrogen peroxide (a) and total phenol (b) content with time (N = 600 min–1, cHP,0 = 0.5 M, T = 353 K, mCu/13X-K1273 = 2.5 g, dB = 0.315–2.00 mm). From the above-presented results, it can be concluded that the reaction mixture is most effectively mixed at the stirrer speed of 600 rpm and that decreasing the particle size below 0.8 mm resulted in only a slight improvement in the observed rate of phenol oxidation excluding the presence of external mass transfer limitations that could influence the reaction kinetics. 3.4 Influence of Catalyst Loading, Initial Concentration of Hydrogen Peroxide, and Temperature The subsequent experiments aimed at revealing the optimal initial concentration of hydrogen peroxide, catalyst loading, and reaction temperature were performed under the above-mentioned reaction conditions. The results of these studies presented in Figure 11 showed that the most significant influence on the extent of total phenols and TOC removal had the initial hydrogen peroxide concentration. Figure 11 Time dependence of hydrogen peroxide decomposition (a–c) and total phenol content (d–f) with points representing the experiment and lines representing the kinetic model. In (a) and (d), cHP,0 = 0–1.34 M, T = 353 K, mCu/13X-K1273-1 = 2.5; in (g), (b), and (e), cHP,0 = 0.5 M, T = 353 K, mCu/13X-K1273-1 = 0–5 g; and in (c) and (f), cHP,0 = 0.5 M, T = 323–353 K, mCAT = 2.5 g. In all cases, N = 600 min–1 and dB = 0.4–0.63 mm. By increasing the initial content of the oxidant, the rate and the extent of the total phenols, TOC, and COD removal increased. At higher initial concentrations of hydrogen peroxide (above 0.75 M), when all total phenols that constitute approximately 17 wt % in TOC loading are eliminated, no significant increase in the oxidation rate of polyphenols can be observed. This is considered to be the consequence of the intensification of side reactions of hydroxyl radicals and the scavenging effect of the oxidant as described earlier (eqs 4–6). However, oxidation of intermediates that are formed by polyphenol conversion becomes significant, further decreasing the organic content of the reaction mixture. The best results were obtained in the reaction conducted with the initial hydrogen peroxide concentration of 1.34 mol dm–3 at 353 K and with 2.5 g of catalyst when ∼97% of total phenols and 47% of TOC reduction were achieved with a rather small copper leaching (Figure 12). Figure 12 Influence of the initial hydrogen peroxide concentration on TOC and COD conversions and copper leaching in the reactions with the Cu/13X-K1273-1 catalyst (cHP,0 = 0–1.34 M, T = 353 K, N = 600 min–1, mCu/13X-K1273-1 = 2.5 g, dB = 0.4–0.63 mm). A higher catalyst bulk concentration gave more prominent hydrogen peroxide decomposition, and total phenol oxidation increased as expected (Figure 11b,e). In contrast to the reports published for similar catalytic systems,36 no limit of catalyst loading was observed and the reaction rates increased proportionally with the mass of the catalyst added to the reactor. The increase in the reaction temperature (Figure 11c,f) had a similar beneficial effect on the catalyst activity, yielding higher conversions of both reactants at elevated temperatures. 3.5 Kinetic Analysis In heterogeneous catalysis, intrinsic kinetics can be evaluated only if the external or internal mass transfer resistances are not affecting the surface reaction rate. The above-presented results and discussion of the diffusion influence in CWPO of polyphenols from OOMW over the Cu/13X-K1273-1 catalyst indicate that for a stirring speed of 600 rpm, catalyst size of 0.4–0.63 mm, and the catalyst loading of 2.5 g the external and internal mass transfer resistances for both the oxidant and the polyphenols are minimized and that the surface reaction can be presumed to be the slowest step in the overall reaction rate. As mentioned before, the CWPO reaction mechanism is very complex, consisting of numerous parallel and serial reactions involving different molecular and radical species. However, the following main reaction steps can be identified: initiation of catalytic decomposition of hydrogen peroxide, which results in the generation of hydroxyl radicals (eq 1), followed by the oxidation of polyphenols and intermediates (propagation, eq 3), and finally a loss of hydroxyl radicals in the nondesired side reactions (termination, eq 4–6). In a very simplistic form, the oxidation pathway can be represented as 16 Because of the complexity of the system, performing a detailed kinetic analysis is challenge even for the model wastewaters where most of the organic compounds present in the reaction mixture are known. The composition of the real wastewater effluent produced during olive oil extraction depends on the production process used, olive species, and climate region, but, in general, OOMW contains more than 30 different polyphenols as well as other prone to oxidation organic acids that can engage in the reaction with hydrogen peroxide and hydroxyl radicals. The presence of inorganic salts such as chlorides and phosphates complicates the matter further.3 The heterogeneity of the OOMW composition as well as the reaction scheme complexity make carrying out the detailed kinetic study next to impossible that would incorporate all individual reactions with all of the initial and intermediate compounds and radicals.37,38 Because of this, the kinetic modeling is often limited to some parameter that represents the group of targeted compounds or major constituents of the wastewater such as COD, TOC, or total phenol content. On the basis of the kinetic regularities and literature data for similar catalytic systems,39,40 the following kinetic model for polyphenol oxidation and decomposition of hydrogen peroxide was proposed 17 18 where cTPh is the molar concentration of total phenols expressed as gallic acid equivalent, cHP is the molar concentration of hydrogen peroxide, and γCAT is the catalyst loading in g dm–3. The total phenol content was divided into two fractions: more reactive (cTPh,1) and less reactive (cTPh,2) based on preliminary analysis of the obtained experimental data. In all performed experiments, two reaction phases could clearly be distinguished: a fast decrease of the total phenol content, which occurred within the first 30–60 min of the reaction, and slow oxidation of the remaining less-reactive fraction of total polyphenols present in the reaction mixture. The initial value of the concentration ratio of the two polyphenol fractions was set as 0.5 during parameter estimation analysis. Reaction constants (kTPh,1, kTPh,2, and kHP), reaction orders in reactants and the catalyst (n1, n2, n3, n4), and concentration ratio of the polyphenol fractions are the kinetic parameters that were estimated during modeling. These expressions take into account that the oxidation rate of polyphenols depends on the concentrations of both reactants and the catalyst loading and that the decomposition rate of hydrogen peroxide considers the contribution of not only the reaction with polyphenols but also the side reactions of hydrogen peroxide decomposition. The contribution of the noncatalytic hydrogen peroxide decomposition and polyphenol oxidation was also considered based on the data acquired during reactions without a catalyst. However, it was found that the noncatalytic contribution to the overall reaction rate was marginal. The estimation of the kinetic parameters was carried out by nonlinear regression analysis using simulation and parameter estimation software MODEST.41 Ordinary differential equations (eqs 17 and 18) were solved with the backward difference method. The sum of residual squares (Q) 19 was minimized with the hybrid Simplex–Levenberg–Marquardt method, where yexp represents experimental data and yest represents the estimated values, i.e., the concentrations. In the first iteration, the polyphenol oxidation reaction orders in polyphenols and hydrogen peroxide concentration were identified in a run with all of the kinetic parameters set as floating. In most cases, the polyphenol oxidation reaction orders with respect to phenol (n1), hydrogen peroxide (n2), and catalyst concentration (n3) were close to 1, whereas the order of hydrogen decomposition reaction with respect to hydrogen peroxide was close to 2 (n4). The second iteration of modeling was performed with fixed reaction orders, and the results are shown in Table 5 and Figures 11 and 13. Figure 13 Agreement of experimental and calculated data points for hydrogen peroxide (a) and total polyphenol (b) concentration. Table 5 Kinetic Modeling Results (n1 = n2 = n3 = l, n4 = 2) estimated parameter value standard error relative standard error (%) kTPh,1 (dm6 mol–1 min–1 gCAT–1) 14.9 2.9 19.5 kTPh,2 (dm6 mol–1 min–1 gCAT–1) 3.16 0.025 0.8 kHP (dm6 mol–1 min–1 gCAT–1) 0.186 0.00344 1.8 ETPH,1 (kJ mol–1) 62.2 19.6 31.5 ETPH,2 (kJ mol–1) 90.1 0.7 0.8 EHP (kJ mol–1) 30.1 1.8 6.1 cTPh,1/cTPh,2 0.345 0.00186 0.5 Taking into account the complexity of the reaction mixture and limitations of the analytical methods, the obtained results show good agreement of the experiment and the proposed kinetic model for both hydrogen peroxide decomposition and polyphenol oxidation. In general, the fit is better for hydrogen peroxide decomposition, whereas the worst agreement for the polyphenol oxidation was achieved for the reactions in which the initial concentration of hydrogen peroxide was the lowest, indicating that for these reactions one of the kinetic model assumptions does not hold. Activation energies for hydrogen peroxide decomposition and polyphenol oxidation over the pelleted Cu/13X-K1273-1 catalyst were determined from the temperature dependencies of the calculated rate constants described by a modified Arrhenius equation 20 where kav is the constant at the average temperature of the experiments Tav. The obtained values of activation energies for hydrogen peroxide decomposition and polyphenol oxidation of Ea,HP = 30.1 kJ mol–1, Ea,TPh,1 = 62.2 kJ mol–1 and ETPH,2 = 90.1 kJ mol–1 are in the range of values reported for similar model reaction systems using powdered catalysts,9,14,29,39 i.e., 45–140 kJ mol–1. 3.6 Catalyst Testing in Olive Oil Mill Wastewater Treatment The catalyst performance was finally tested in the prolonged reaction over 10 h to determine whether the catalyst gets deactivated with prolonged use. From the results presented in Figure 14, it can be seen that the oxidation of organic content continues after phenolics are eliminated, demonstrating the catalyst ability to enhancing peroxidation of not only phenols but also other organic compounds present in the olive oil mill wastewater, resulting in complete conversion of total phenols and 52% conversion of TOC. The Cu/13X-K1273-1 catalyst preserved its stability even after 10 h of reaction when only 3.3 wt % copper leached from the catalyst. By a comparison of the fresh and spent catalysts, it was observed that the catalyst maintained its initial surface area and pore volume (SFRESH = 26 m2 g–1 and Vp,FRESH = 0.03 cm3 g–1 to SSPENT-10h = 25 m2 g–1 and Vp,SPENT-10h = 0.03 cm3 g–1). Although the loss of 3.3 wt % of the initial copper content from the catalyst is not negligible, this result is very encouraging when compared to that of similar catalytic systems described in the literature. The extent of leaching of the Cu/13X-K1273 pelletized catalyst is generally lower when compared to that of other zeolite or zeolite-based catalysts9,13,42 and is comparable to leaching of copper-containing pillared clay catalysts according to the work of Inchaurrondo et al.29 However, most of these results are connected to CWPO studies of model or often highly diluted wastewaters, which should be taken into account when comparing them with the results of this study where a real industrial effluent was used because the extent of metal leaching from the catalyst depends not only on the catalyst type or support material but also very strongly on the reaction conditions such as temperature, pH, and particular organic compound species and their concentrations. Figure 14 Concentrations of hydrogen peroxide (a), total phenols, and TOC (b) in the reactor for a prolonged reaction time experiment with the Cu/13X-K1273-1 catalyst (cHP,0 = 0.5 M, T = 353 K, N = 600 min–1, dB = 0.40–0.63 mm, mCAT = 2.5 g, tR = 10 h). 4 Conclusions Postsynthesis thermal treatment was beneficial for catalytic behavior of the copper-containing 13X zeolite in catalytic wet peroxide oxidation of OOMW as it resulted in an increased stability against leaching, allowing better removal of total phenols TOC due to the presence of pore diffusion limitations for polyphenols in the noncalcined Cu/13X-1 catalyst. Results of mass transfer and diffusion investigation for the calcined Cu/13X-K1273-1 catalyst excluded the influence of both external and internal mass transfer limitations. It was found that the rate of phenol oxidation and hydrogen peroxide decomposition increased with the increase of stirrer speed, catalyst loading, initial hydrogen peroxide concentration, and reaction temperature and with the decrease of catalyst bead size. Kinetic analysis of the catalytic system was preformed, the reaction orders in reactants and in the catalyst were identified, and parameters of the proposed kinetic model and activation energies were determined. By treating the industrial olive oil mill wastewater in a catalytic wet peroxide oxidation process over thermally stabilized copper-containing zeolite-based catalyst under mild reaction conditions (353 K and atmospheric pressure), it is possible to achieve complete conversion of total phenols and over 50% conversion of TOC, substantially minimizing copper leaching. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00948.Observed reaction rate dependence on the stirring speed and catalyst bead size (PDF) Supplementary Material ao8b00948_si_001.pdf The authors declare no competing financial interest. Acknowledgments The authors acknowledge the support of the European Commission and the Croatian Ministry of Science, Education and Sports Co-Financing Agreement No. 291823 as well as Åbo Akademi Johan Gadolin Process Chemistry Centre. The contribution of Vilko Mandić to XRD analysis is highly appreciated. ==== Refs References Busca G. ; Berardinelli S. ; Resini C. ; Arrighi L. Technologies for the removal of phenol from fluid Streams: A short review of recent developments . J. Hazard. 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PMC006xxxxxx/PMC6644427.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145898410.1021/acsomega.8b01003ArticleComputational Study on Ring Saturation of 2-Hydroxybenzaldehyde Using Density Functional Theory Verma Anand Mohan Agrawal Kushagra Kishore Nanda Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India E-mail: nkishore@iitg.ac.in, mail2nkishore@gmail.com.01 08 2018 31 08 2018 3 8 8546 8552 15 05 2018 23 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Bio-oil produced from pyrolysis of lignocellulosic biomass consists of several hundreds of oxygenated compounds resulting in a very low quality with poor characteristics of low stability, low pH, low stability, low heating value, high viscosity, and so on. Therefore, to use bio-oil as fuel for vehicles, it needs to be upgraded using a promising channel. On the other hand, raw bio-oil can also be a good source of many specialty chemicals, e.g., 5-HMF, levulinic acid, cyclohexanone, phenol, etc. In this study, 2-hydroxybenzaldehyde, a bio-oil component that represents the phenolic fraction of bio-oil, is considered as a model compound and its ring saturation is carried out to produce cyclohexane and cyclohexanone along with various other intermediate products using density functional theory. The geometry optimization, vibrational frequency, and intrinsic reaction coordinate calculations are carried out at the B3LYP/6-311+g(d,p) level of theory. Furthermore, a single point energy calculation is performed at each structure at the M06-2X/6-311+g(3df,2p)//B3LYP/6-311+g(d,p) level of theory to accurately predict the energy requirements. According to bond dissociation energy calculations, the dehydrogenation of formyl group of 2-hydroxybenzaldehyde is the least energy demanding bond cleavage. The production of cyclohexane has a lower energy of activation than the production of cyclohexanone. document-id-old-9ao8b01003document-id-new-14ao-2018-01003cccc-price ==== Body 1 Introduction Due to declining fossil fuels and increasing pollution, there is a strong necessity to find an alternative and clean energy resource that can aid the present energy demands and reduce the pollution level. Currently, renewable energy resources, e.g., tidal energy, solar energy, biomass, wind energy, geothermal energy, and so on, are being largely utilized. They are providing a good relief for the present energy demand; however, their proper utilization still needs to be explored. Nevertheless, out of all renewable energy resources, only biomass has the ability to deliver sustainable carbon element.1−4 The sustainability of carbon element is necessary to achieve the production of transportation fuels or specialty chemicals.5 The lignocellulosic biomass is cheap, abundant, and easily available in most countries; therefore, the research based on lignocellulosic biomass conversion into biofuel has enormously increased in the last few decades.2 The lignocellulosic biomass basically contains three fractions, viz., lignin, hemicellulose, and cellulose.1,6 The research based on the cellulose and hemicellulose fractions of lignocellulosic biomass has received considerable attention in the past few years, but the lignin fraction has been ignored constantly because of its complex structure, although it has a high energy density compared to other two fractions.7 Nevertheless, there are various channels to convert the lignocellulosic biomass into bio-oil, e.g., pyrolysis, liquefaction, hydrolysis, and gasification, and the pyrolysis process has been reviewed to be the most economical and advantageous.3,8 However, the bio-oil produced from pyrolysis of lignocellulosic biomass comprises several hundreds of oxygenated compounds that lower its quality, endowing it with low heating value, low pH, low stability, low stability, high viscosity, and so on.4 Therefore, it needs to be upgraded to serve as a fuel for vehicles. Furthermore, unprocessed bio-oil is a great source of many specialty or platform chemicals, such as hydroxymethyl furfural, furfural, levulinic acid, dimethyl furan, and so on.1,5 As it has been pointed out that cellulose and hemicellulose fractions have received much attention compared to the lignin fraction, in this study, 2-hydroxybenzaldehyde, a component of phenolic fraction derived from lignin, has been selected as the bio-oil model compound. In the past, a few phenolic components, such as guaiacol,9−15 catechol,9,16 phenol,10,14,16,17 vanillin,18,19 anisole,14 and so on, have been studied both experimentally and numerically. This component has been recently considered by Verma and Kishore20 for its decomposition into benzene as the end product; however, there has been no further study regarding its ring saturation, which is more likely to occur in the presence of high hydrogen pressure-based hydrodeoxygenation conditions. Although a few authors21−24 have carried out pyrolysis of a few bio-oil model compounds and observed 2-hydroxybenzaldehyde as one of the products in the pyrolysis product mixtures, further experiments for upgrading 2-HB have not been carried out to date. For instance, Robichoud et al.21 carried out a pyrolysis study on dimethoxybenzene model compound and observed 2-hydroxybenzaldehyde as one of the products. Similarly, Zhang et al.24 also carried out pyrolysis study on the lignin dimer model compound and reported 2-hydroxybenzaldehyde as one of the products along with guaiacol, catechol, and others. Since 2-hydroxybenzaldehyde comprises two oxy-functionals, namely, hydroxyl and aldehyde groups, it still needs to be upgraded to achieve the nonoxygenated component. Therefore, 2-hydroxybenzaldehyde is selected as the bio-oil model compound and its conversion to lower fractions has been numerically attempted here. In this numerical study, 2-hydroxybenzaldehyde is allowed to undergo two reaction pathways producing cyclohexane and cyclohexanone, respectively. These reaction schemes are depicted in Figure 1. The notations in Figure 1 are labeled as X_Y, where X denotes the reaction pathway number and Y denotes the structure under that reaction pathway. Similarly, transition-state structures in the potential energy surfaces (PESs) are denoted as TSX_Y, where X is the reaction pathway number and Y denotes the transition-state number. Figure 1 Reaction schemes for the conversion of 2-hydroxybenzaldehyde. In Figure 1, it can be seen that the reaction pathway 1 produces cyclohexane. The preparatory part of reaction pathway 1 is the hydrogenation of carbon atom (para-positioned to OH group), followed by ring saturation of 2-hydroxybenzaldehyde using five further atomic hydrogenation reactions. The produced component 1_f after the ring saturation undergoes hydroxyl cleavage, followed by a single-step hydrogenation reaction to produce 1_h. Finally, structure 1_h follows two pathways. Under primary reaction pathway 1, the formyl group is cleaved, followed by a single-step hydrogenation reaction to produce cyclohexane, and under secondary reaction pathway 1, i.e., 1a, structure 1_h undergoes decarbonylation reaction to produce cyclohexane. On the other hand, reaction pathway 2 starts from the dihedral change of hydrogen atom of the hydroxyl group to initiate the keto–enol tautomerization reaction. The keto–enol tautomerization reaction of structure 2_a produces 2_b, which further undergoes ring saturation to produce structure 2_f. Finally, structure 2_f undergoes the decarbonylation reaction to produce cyclohexanone. All reaction steps of each reaction pathway are considered theoretically at the B3LYP/6-311+g(d,p) level of the theory under density functional theory (DFT) framework. The single point energy (SPE) calculation is performed for each structure at the M06-2X/6-311+g(3df,2p)//B3LYP/6-311+g(d,p) level of theory to predict energies accurately. 2 Results and Discussion 2.1 Bond Dissociation Energy (BDE) A thorough analysis of bond dissociation energy (BDE) of 2-hydroxybenzaldehyde with all possible bond cleavages was reported in our previous work.20 It has been reported that bond dissociation energies of 2-hydroxybenzaldehyde are quite high, i.e., in the range of 92–115 kcal/mol. The least energy demanding bond cleavage, i.e., D3 (dehydrogenation of the formyl group), requires 92.22 kcal/mol.20 Along with the most favorable bond dissociation site D3, dehydrogenation of the hydroxyl group (D2) was reported as the second most favorable bond cleavage site.20 It is also clear that dehydrogenation of phenyl ring of 2-HB is not favorable at all because the energy demand due to each hydrogen bond cleavage from phenyl ring of 2-HB is in the range of 112–115 kcal/mol.20 Therefore, cleavage of hydrogen atoms from phenyl ring is not advisable. In such scenario of high bond dissociation energies due to scissions of atoms/functionals of 2-HB, it is highly probable that the ring saturation of 2-HB may lead to a low energy demanding reaction pathway, which is the aim of this work. There are two ways of saturating the aromatic ring of 2-HB, i.e., first by direct ring hydrogenation and second by keto–enol tautomerization followed by ring hydrogenation. 2.2 Reaction Pathway 1 Reaction pathway 1 is about the direct ring hydrogenation reaction. The potential energy surfaces (PESs) of reaction pathways 1 and 1a are shown in Figure 2, and the corresponding optimized molecular structures are shown in Figure 3. Interatomic bond distances in the transition-state structures of Figure 3 are shown in angstrom (Å) units. The Cartesian coordinates of a few optimized molecular structures are given in the Supporting Information. Figure 2 Potential energy surfaces of reaction pathways 1 and 1a. Figure 3 Optimized molecular structures of reaction pathways 1 and 1a. The reaction pathway 1 starts with a single-step hydrogenation reaction at the fifth carbon position of the aromatic ring of the 2-hydroxybenzaldehyde structure (see structure 1_a in Figure 1), followed by another atomic hydrogenation reaction to saturate the generated radical by the first hydrogenation reaction. The first hydrogenation reaction requires 4.62 kcal/mol of activation barrier (see TS1_1 in Figures 3 and 4) according to single point energetics at the M06-2X/6-311+g(3df,2p)//B3LYP/6-311+g(d,p) level of theory. Figure 4 Potential energy surface of reaction pathway 2. As it is known that bond dissociation and radical recombination reactions do not possess any transition-state structure, the energy release during the recombination reaction of structure 1_a and H radicals is calculated by BDE approximation. According to BDE at the B3LYP/6-311+g(d,p) level of theory, the recombination of radicals 1_a and H to produce structure 1_b releases 73.58 kcal/mol of energy. Further, another transition-state structure is located for the hydrogenation reaction of structure 1_b to produce 1_c at potential energy surface (PES) as TS1_2 that requires only 2.89 kcal/mol of energy to surpass the barrier height. This follows through another radical recombination reaction of structure 1_c and H that releases 82.60 kcal/mol of energy. Similar to the reaction steps 2-HB → 1_a → 1_b, further hydrogenation reactions of structure 1_d are carried out as 1_d → 1_e → 1_f, where the activation barrier of 1_d → 1_e and energy release during 1_e → 1_f are calculated as 4.30 and 81.99 kcal/mol, respectively. The produced component after the aromatic ring saturation of 2-HB is identified as 2-hydroxycyclohexane-1-carbaldehyde. Further, the cleavage of hydroxyl group of structure 1_f is carried out with a calculated BDE of 86.57 kcal/mol, followed by a radical recombination reaction of structure 1_g and a hydrogen radical with an energy release of 94.49 kcal/mol. Afterward, the produced component, i.e., cyclohexanecarbaldehyde (structure 1_h), follows two pathways. The first pathway proceeds as the primary reaction pathway 1 (bold black arrows in Figure 1), which cleaves the formyl group from structure 1_h with a BDE of 71.43 kcal/mol, followed by saturation of generated radical with a hydrogen radical releasing 94.19 kcal/mol of energy to produce cyclohexane. On the other hand, the secondary reaction pathway 1 (nonbold black arrows in Figure 1) involves a decarbonylation reaction to directly produce cyclohexane. The barrier height of the decarbonylation is calculated to be 87.31 kcal/mol. In Figure 3, it can be seen that the highest uphill is due to the very first hydrogenation reaction of 2-HB, which is 4.62 kcal/mol at the potential energy surface, and other stationary states are in the negative side. Figure 3 clearly suggests an overall activation energy of only 4.62 kcal/mol of energy, which is a very favorable environment for this system even at lower temperature and pressure conditions. Also, it can be seen that the decarbonylation reaction of structure 1_h is slightly favorable for the direct cleavage of the formyl group followed by hydrogenation reaction. 2.3 Reaction Pathway 2 Reaction pathway 2 is about the production of cyclohexanone. The potential energy surface is shown in Figure 4 by a blue smooth line, and the corresponding molecular structures are shown in Figure 5. Figure 5 Optimized molecular structures of reaction pathway 2. Reaction pathway 2 starts with the dihedral change of hydrogen atom of the hydroxyl group. It can be seen from the potential energy surface of reaction pathway 2 (Figure 4) that 2-HB is the ground-state structure and 10.57 kcal/mol more stable than structure 2_a (energetically less stable configuration of 2-HB). The dihedral change of hydrogen atom occurs with a barrier height of 13.80 kcal/mol, and this reaction step is essential for further initiation of the keto–enol tautomerization reaction. The keto–enol tautomerization reaction step (2_a → 2_b), i.e., migration of hydrogen atom from hydroxyl group to the ortho-positioned carbon atom, produces 6-oxocyclohexa-1,3-diene-1-carbaldehyde with a barrier height of 73.90 kcal/mol. Further, a single-step hydrogenation reaction at structure 2_b is carried out with a barrier height of only 1.36 kcal/mol, followed by combination reaction of a hydrogen radical and structure 2_c releasing 77.53 kcal/mol of energy. Further, similar to the reaction steps 2_b → 2_c → 2_d, the reaction steps 2_d → 2_e → 2_f are performed with a barrier height of 1.75 kcal/mol for reaction step 2_d → 2_e and an energy release of 83.62 kcal/mol for reaction step 2_e → 2_f. The produced compound after the conversion of double bonds between carbon atoms into single bonds is recognized as 2-oxocyclohexane-1-carbaldehyde. Finally, to produce cyclohexanone from 2-oxocyclohexane-1-carbaldehyde, a decarbonylation reaction is carried out, which occurs with a barrier height of 71.04 kcal/mol. The production of cyclohexanone from 2-hydroxybenzaldehyde witnesses many reaction steps. It can be seen in Figure 4 that the energy state of TS2_2 is highest in the potential energy surface of reaction pathway 2; therefore, this particular energy state will be responsible for the overall activation energy. Therefore, the overall activation energy of reaction pathway 2 is 84.47 kcal/mol, which is very high to achieve at mild reaction conditions. Also, if compared, the production of cyclohexanone requires a considerably larger overall activation barrier (84.47 kcal/mol) than the overall activation barrier in the production of cyclohexane (4.62 kcal/mol). Therefore, it is clear that the possibility of production of cyclohexane from 2-HB will be considerably higher compared to the production of cyclohexanone. 3 Conclusions In this study, 2-hydroxybenzaldehyde (2-HB), a bio-oil oxygenated component that represents the phenolic fraction of bio-oil, is taken as the bio-oil model compound and its ring saturation is carried out to produce cyclohexane and cyclohexanone. The bond dissociation energy calculations of 2-hydroxybenzaldehyde do not suggest the cleavage of hydrogen atoms from either the phenyl ring or functional groups because of high energy requirements. Therefore, the conversion of 2-HB by ring saturation is carried out to produce cycloproducts. It is observed that the production of cyclohexane is highly favorable compared to the production of cyclohexanone because the overall activation energy of the former is only 4.62 kcal/mol, whereas the latter demanded 84.47 kcal/mol of overall activation energy. In other words, keto–enol tautomerization followed by ring hydrogenation process of 2-HB in gas phase is not preferred compared to direct ring hydrogenation process of 2-HB. 4 Computational Details The application of density functional theory (DFT) as a computational tool has enormously increased in the recent past due to its reliability and accuracy. Although there are various functionals under DFT, such as LSDA, B3LYP, BLYP, M05, M06-2X, etc., there has been much debate among researchers regarding their accuracy.25 Recently, a few research groups26,27 carried out an extensive survey of DFT functionals applied to various chemical systems. For instance, Goerigk et al.26 performed an extensive review to assess numerous DFT functionals and observed ωB97X-V functional as one of the best functionals among GGA, meta-GGA, and hybrid-GGA functionals. They reported an average performance of B3LYP functional compared to other DFT functionals. Furthermore, Mardirossian and Head-Gordon27 also performed an extensive assessment of numerous DFT functionals and reported root-mean-square deviation (RMSD) values for different data types. They reported ωB97M-V as the best functional because of low RMSD values for barrier heights (1.68) and thermochemistry (2.48) data types. However, an interesting observation in their study is that B3LYP functional performed excellently compared to many well-known DFT functionals, such as PBE, PBE-D3, TPSS-D3, M06-L, and so on. In addition, recently, Simón and Goodman28 have shown that the use of B3LYP functional is a better choice for organic covalent bond-forming reactions; therefore, all geometries and transition-state structures in this study are optimized using B3LYP29,30 functional under DFT31,32 framework. The basis set has been selected as 6-311+g(d,p).33 Normal-mode vibrational frequency calculations are carried out for all optimized structures to test the true natures of optimized structures. One and zero imaginary frequencies in the vibrational frequency result confirm the structure as first-order saddle point and minimum structure on potential energy surface (PES). Furthermore, an intrinsic reaction coordinate34 calculation is also carried out at each true transition-state structure to evaluate the minimum energy path. The single point energy (SPE) calculation is performed for each structure at the M06-2X/6-311+g(3df,2p)//B3LYP/6-311+g(d,p) level of theory to predict energies accurately. Bond dissociation energy calculations are performed for organic homolysis and radical recombination reactions because of the nonavailability of transition-state structures for such reactions. BDE provides a good approximation for the energy requirement to cleave a chemical bond. 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PMC006xxxxxx/PMC6644428.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145808610.1021/acsomega.8b01324ArticleElectronic Structure and Chemical Bonding of [AmO2(H2O)n]2+/1+ Hu Shu-Xian †Liu Hai-Tao ‡Liu Jing-Jing †Zhang Ping ‡Ao Bingyun §† Beijing Computational Science Research Center, Beijing 100193, China‡ Institute of Applied Physics and Computational Mathematics, Beijing 100088, China§ Science and Technology on Surface Physics and Chemistry Laboratory, Mianyang 621908, China* E-mail: hushuxian@csrc.ac.cn (S.-X.H.).* E-mail: zhang_ping@iapcm.ac.cn (P.Z.).23 10 2018 31 10 2018 3 10 13902 13912 13 06 2018 20 09 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Systematic americyl-hydration cations were investigated theoretically to understand the electronic structures and bonding in [(AmO2)(H2O)n]2+/1+ (n = 1–6). We obtained the binding energy using density functional theory methods with scalar relativistic and spin–orbit coupling effects. The geometric structures of these species have been investigated in aqueous solution via an implicit solvation model. Computational results reveal that the complexes of five equatorial water molecules coordinated to americyl ions are the most stable due to the enhanced ionic interactions between the AmO22+/1+ cation and multiple oxygen atoms as electron donors. As expected, Am–Owater bonds in such series are electrostatic in nature and contain a generally decreasing covalent character when hydration number increases. document-id-old-9ao8b01324document-id-new-14ao-2018-01324eccc-price ==== Body Introduction In the past several decades, a rapid increased resurgence of nuclear power in many countries for the goals of ensuring sustainable energy supplies has raised a possible hazard to the environment due to the problems of the radioactive waste and has increased the handling and reprocessing challenge of spent nuclear fuel and high-level nuclear waste.1−5 Especially, the long-term radioactivity of nuclear waste repositories is one of the main issues at stake and is in part determined by the presence of minor actinides, namely, americium (Am) and curium (Cm), with the half-life t1/2 = 7370 year for 243Am and t1/2 = 340 000 year for 248Cm.6,7 During the actinide waste storage, transportation, and separation, water is unavoidable. Therefore, the characterization and identification of the behavior of actinide ions in solution is of particular practical importance in addition to the fundamental actinide chemistry, such as the dynamics between the coordinated water molecules and the bulk solvent and the coordination flexibility of actinyl ions.8 So, the solution chemistry of actinide complexes has been considerably explored in experiment and theory; especially, the hydration of actinide ions has been widely investigated.9−12 However, such studies have mostly focused on early actinide ions due to their relatively substantial quantities and extensive applications. The uranyl UO22+ ion, for example, has received a large amount of interest because of its importance for environmental chemistry of radioactive elements and its role as a benchmark system for heavier actinides.13 Experimentally, direct structural information on the coordination of uranyl in aqueous solution has been mainly obtained by extended X-ray absorption fine structure or X-ray absorption near-edge structure measurements.9,14,15 Theoretically, the structural investigation has been performed via various ab initio studies of uranyl and related molecules, with a number of explicit water molecules or with a polarizable continuum model to mimic the environment over the past years, such as [UO2(CO3)3]4–, [Th(H2O)n]4+, [U(H2O)n]3+, UO2Fn(H2O)5–n2–n, and [UO2(H2O)n]n+.10,16−21 With the rapid development of quantum chemical methods, the less known transuranium elements have been increasingly studied, i.e., [NpO2(CO3)3]4–, [NpO2(CO3)m(H2O)n], [Cm(H3O)n]3+, [UO2(H2O)n]n+, [NpO2(H2O)n]n+, and [PuO2(H2O)n]n+.7,12,13,22−28 There appears to be no theoretical report about americyl (AmO22+/1+) in aqueous solution but one study on the water exchange mechanism in AmO2(H2O)52+ 15 years ago.29 In addition, the understanding of the coordination chemistry of americyl ions in solution can present the essentials to detect, characterize, and differentiate this metal ion from transition metals or lanthanide metals through the coordination characteristic. Hence, we will focus on speciation of the americyl ions (AmO22+ and AmO21+) in this paper, bridging the gap between the wide-established early actinides and the more complicate later actinides. However, an accurate description of solvent effects of the actinide complexes is inherently difficult due to their complicated electronic properties and the challengeable aqueous environment to theoretical chemists. There are two typical quantum chemistry strategies used to incorporate the solvent effects on the geometry and energy of the actinide complexes in aqueous solution. One is molecular dynamics simulations approach with a discrete model, where a number of explicit solvent molecules are included and treated at the same level of theory as that used for the solute.30 Although this supermolecule requires a substantial computational expense, the understanding improvement of the structural and chemical behavior of actinyl in solution is inconsequential because this method employs empirical potentials affecting the accuracy significantly, especially for those transplutoniums having few experimental data available. Another approach is the polarizable continuum model, where the solvent is described by a dielectric polarizable continuum.31 This model has shown a good agreement with experimental results on the geometric and electronic structures in actinyl–water complexes,15 where uranyl, neptunyl, and plutonyl coordinated to five water ligands in the equatorial plane have been computationally confirmed.13,32 In this contribution, we report a systematic investigation on the chemistry of hydrated americyl ions in the [AmO2(H2O)n]2+/1+ (n = 1–6) complexes using a suite of computational modeling and various bonding analysis methods. We address the equilibrium structures of [AmO2(H2O)n]2+/1+, as well as the hydration number of americyl ions and the bonding nature of americium ions and oxygen atoms. In addition to the knowledge for the periodicity in bonding properties, the influence of aqueous solvation of [AmO2(H2O)n]2+/1+ (n = 1–6) will provide a scientific comprehension to the actinide solution chemistry. Results and Discussion The static structures of hydrated complexes of AmO22+/1+ with one to eight water ligands have been regularly investigated via geometry optimizations in gas phase and in aqueous phase at the B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ level with or without the conductor-like screening solvation model (COSMO) method. First, concerning the water hydrogens lying either in the planes or perpendicular to the planes defined by the OylAmOyl, two possible gas-phase symmetric arrangements of water molecules for [AmO2(H2O)1]2+/1+ and [AmO2(H2O)2]2+/1+ have been optimized without symmetry constraints. As shown in Figure S1, the one with hydrogens perpendicular to the plane has one imaginary vibrational frequency (428i cm–1) showing the rotation of water ligand, indicating its instability compared with the one with hydrogens lying in the plane. Besides, the starting structure of [AmO2(H2O)7]2+/1+ has been assumed as a first-shell coordination number of seven when seven water molecules are bonded to AmO22+/1+, in which the seven equatorial water ligands lie rigorously in the equatorial plane. After optimization within density functional theory (DFT), seven water molecules have been arranged into two coordination shells; there are five water molecules in the first shell with a relatively shorter Am–Ow distance of <2.5 Å and two water molecules in the second shell at a longer distance of >4.4 Å. Therefore, the hydration number of AmO22+/1+ in the first shell is predicted to be less than seven. In addition, the optimized structural parameters and calculated binding energies of [AmO2(H2O)n]2+/1+ (n = 1–6) in the gas phase and aqueous solution on the basis of different levels of theory are listed in Tables S1–S4. As clearly shown in the Tables S3 and S4, the energy destabilization by roughly 32.0–128.2 kcal/mol for [AmO2(H2O)n]2+ (n = 1–6) complexes is due to the conductor-like polarized continuum model; a similar trend is also observed for [AmO2(H2O)n]1+ (n = 1–6) complexes. Upon inclusion of the surrounding effects, the binding energy decreases drastically to 62.4 kcal/mol for equatorial [AmO2(H2O)6]2+ and to −0.5 kcal/mol for equatorial [AmO2(H2O)6]1+ at scalar relativistic (SR)-B3LYP level of theory, indicating the instability of this structure. In terms of geometries of equatorial [AmO2(H2O)6]2+/1+, the six-water complexes with all six coordinated Ow atoms in the equivalent position are subject to the first-order Jahn–Teller instability and are only saddle points having imaginary frequencies. The Ci symmetry structure of [AmO2(H2O)6]2+ in which all six ligands are in the first coordination shell, shown in Figure S2, is stable with or without aqueous solution. The distance from the americium center to the water molecules located above and below the equatorial plane in a symmetric manner is longer (0.09 Å) than that for [AmO2(H2O)5]2+ and is significantly shorter (0.13 Å) than that reported for NpO2(H2O)62+ having a similar structure largely owing to the actinide contraction of ion radii. A somewhat different structure of [AmO2(H2O)6]1+ is obtained, whereas the sixth water is excluded to the second hydration shell owing to the weak electrostatic interaction between AmO21+ and water oxygen. Thus, the hydration number of AmO22+/1+ is five in resulting minimum-energy structures of both [AmO2(H2O)n]2+/1+ (n = 1–6) complexes. Since the solvation effect is significant on the geometry of [AmO2(H2O)n]2+/1+ and also plays a typical role in practical application, we only report the geometry optimization results under the effect of aqueous solvation in text. Table 1 presents the binding energies obtained at SR-B3LYP and CCSD(T) levels of theory for the following reaction of [AmO2]2+/1+ + nH2O → [AmO2(H2O)n]2+/1+ (n = 1–6) in solution. As shown in Table 1, each of these [AmO2(H2O)n]2+ (n = 1–6) complexes is significantly more stable than the corresponding [AmO2(H2O)n]1+ (n = 1–6). At the SR-B3LYP level of theory, the americyl–water binding energies steadily increase from 34.0 kcal/mol for [AmO2(H2O)1]2+ (19.1 kcal/mol for [AmO2(H2O)1]1+) to 119.0 kcal/mol for [AmO2(H2O)5]2+ (68.2 kcal/mol for [AmO2(H2O)5]1+), whereas at the spin–orbit (SO) level of theory, the binding energies show the same trend by increasing from 45.4 kcal/mol at n = 1 to 135.0 kcal/mol at n = 5 in the series of AmO2(H2O)n2+. Overall, the substantial stabilizations of [AmO2(H2O)5]2+/1+ relative to the isolated americyl and free water molecules favors the formation of a maximum of five water ligands in the equatorial plane in the first hydration shell. This five-coordination structure has been commonly observed in other actinyls in water environment in the available experimental studies and is in good accord with the published theoretical studies using quantum mechanical method or molecular dynamics approach. Table 1 Binding Energy (kcal/mol) for the Reaction of AmO22+/1+ + nH2O → [AmO2(H2O)n]2+/1+ (n = 1–6) in Solution at Different Levels of Theory with COSMO [AmO2(H2O)n]2+ [AmO2(H2O)n]1+ B3LYP CCSD(T) B3LYP CCSD(T) n ΔESR ΔESO ΔE ΔESR ΔESO ΔE 1 –34.0 –45.4 –64.7 –19.1 –31.0 –35.0 2 –64.6 –77.1 –120.9 –36.4 –49.7 –66.0 3 –88.5 –104.4 –173.5 –52.5 –68.4 –95.7 4 –110.2 –126.2 –213.1 –63.7 –93.6 –117.6 5 –119.0 –135.0 –68.2 –84.4 6 –113.7 –128.0 –65.1 –71.9 Geometric Structure of the [AmO2(H2O)n]2+/1+ (n = 1–5) Two different density functionals (Perdew–Burke–Ernzerhof (PBE) and B3LYP) were first employed for the geometrical optimization, which gave similar results, so only the results obtained at B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ level of theory are presented in the text and those obtained at the PBE/TZ2P level are detailed in the Supporting Information (Tables S1 and S2). As noted in the last section, the inclusion of solution therefore and the use of a solvation model are important in modeling the coordination structures of actinyl. So the calculated ground-state geometries for [AmO2(H2O)n]2+/1+ (n = 1–5) in aqueous solution using the COSMO solvation model are listed in Table 2 and shown in Figure 1. In terms of geometry, [AmO2(H2O)n]2+ and [AmO2(H2O)n]1+ have similar geometric structures; these are the water molecules coordinating to AmO22+/1+ on the equatorial plane of americyl with an increasing hydration number from n = 1–5, as expected, with the same hydration number as that of of uranyl, neputonyl, and plutonyl in aqueous solution. The calculated Am–Oyl distances in the [AmO2(H2O)n]2+ complexes elongate from 1.669 Å in pure americyl ion to 1.681 Å in [AmO2(H2O)1]2+ and increase all through to 1.711 Å in [AmO2(H2O)5]2+, whereas the Am–Oyl bonds in the [AmO2(H2O)n]1+ complexes appear to follow the same trend, consistent with the decrease in the bond order, attributed to the weakening of both ionic and covalent bonding interactions of Am–Oyl bond from n = 1–5. In all [AmO2(H2O)n]2+/1+ (n = 1–5) complexes, the americyl is coordinated by water O atoms (Ow) with bond Am–Ow distances longer than 2.2 Å, which implies weak bonding interactions. In addition, all Am–Ow bond lengths in the complexes are beyond the range of Am–O covalent single-bond lengths estimated by the sum of the self-consistent covalent radii derived by Pyykkö.33 Expectedly, the Am–Ow bond lengths in the [AmO2(H2O)n]2+ complexes are shorter than the corresponding bond lengths in the [AmO2(H2O)n]1+ complexes, owing to the stronger electrostatic interaction in dication complexes. The lengthening of the Am–Ow bond distances in the [AmO2(H2O)n]2+ complexes from 2.260 Å at n = 1 to 2.460 Å at n = 5 implies the reduction of bond strength and the weakening of bonding interaction of each Am–Ow bond along with increasing hydration numbers. Although each of the Am–Ow bonds is not particularly strong in comparison to Am–Oyl multiple bond, the overall bonding interaction is substantial, as indicated by significant binding energies (Table 1) and by the extreme redshift in the americyl asymmetric stretch frequency (Table 3). Figure 1 Optimized geometry and binding energy (kcal/mol, in parentheses) of AmO2(H2O)n2+/1+ (n = 1–5) at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ levels of theory. Table 2 Selected Optimized Geometrical Structures (Bond Length, Å, Angle, °) and Formation Energy (Ef, kcal/mol) for the Reaction of fϕ1fδ2 [AmO2(H2O)n]2+ and fϕ2fδ2 [AmO2(H2O)n]1+ (n = 0–5) at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ Levels of Theorya [AmO2(H2O)n]2+ [AmO2(H2O)n]1+ n state (symm.) Am–Oyl Am–Ow ∠OylAmOyl ∠OwAmOw state (symm.) Am–Oyl Am–Ow ∠OylAmOyl ∠OwAmOw 0 4Φ (D∞h) 1.669 5∑g(D∞h) 1.735 1 4B1 (C2v) 1.681 2.260 179.5 5A1 (C2v) 1.747 2.398 179.8 2 4B1 (C2v) 1.692 2.286 179.0 96.8 5A1 (C2v) 1.758 2.420 179.8 103.1 3 4A′ (Cs) 1.699 2.331, 2.326 179.8 127.8, 113. 2, 118. 7 5A′ (Cs) 1.768 2.448, 2.447, 2.445 179.9 122.2, 120.4, 117.4 4 4B2u (D2h) 1.707 2.371 180.0 92.7, 87.3 5Ag (C4h) 1.778 2.496 180.0 90.0 5 4A′ (Cs) 1.710 2.420, 2.462, 2.470 179.4 66.3, 74.5, 78.4 5A (C1) 1.781 2.562, 2.548, 2.586, 2.568, 2.589 179.4 72.0 a All optimizations are fully relaxed without constraints. Table 3 Calculated Vibrational Frequencies of [AmO2(H2O)n]2+/1+ (n = 1–5) at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ Levels of Theory with COSMO Am–Oyl Am–Ow Am–Oyl Am–Ow v3 v1 Δq(v3) v3 v1 v3 v1 Δq(v3) v3 v1 0 1053.7 925.1 925.6 828.0 1 1027.6 904.7 26.1 420.4 904.4 810.7 21.2 316.8 2 1009.1 891.6 18.6 385.7 412.4 882.9 792.7 21.4 309.8 307.6 3 994.8 880.7 14.2 360.8 367.5 866.0 777.7 16.9 260.1 290.0 4 980.9 868.4 13.9 312.0 351.7 849.5 764.7 16.4 246.9 275.0 5 970.4 855.9 10.5 273.9 314.2 839.1 754.1 10.4 239.6 252.6 For the monoaqua, the ground electronic states of [AmO2(H2O)]2+/1+ have been confirmed as 4B1 and 5A1 via a comparison among different multiplicities. Upon the coordination of Ow in [AmO2(H2O)]2+/1+, the linear OylAmOyl is broken into bent ∠OylAmOyl angles of 179.5 and 179.8°, respectively. The water molecule coordinates to americyl on the equatorial plane with the two hydrogen atoms lying symmetrically on both sides of the plane. The shortest Am–Ow bond distance of 2.26 Å among the [AmO2(H2O)n]2+ series is within the range of single covalent bond radii of Am–O (2.29 Å), mainly because of the strongest bonding. These distances are consistent with the bonding analysis detailed in the next section. The singly filled highest occupied molecular orbitals of [AmO2(H2O)]2+/1+ have roughly 98% Am 5fϕ and 90% Am 5fδ character, indicating that the Am 5f orbital is largely unperturbed by the coordination of the ligands. Thus, [AmO2]2+/1+ retain their electron configuration at Am ions in those [AmO2(H2O)]2+/1+ complexes, namely, fϕ1fδ2 and fϕ2fδ2, respectively. For the dual aqua, one additional water molecule locates in the equatorial plane with a bent ∠OwAmOw angle of 96.8° in [AmO2(H2O)2]2+ and 103.1° in [AmO2(H2O)2]1+, inducing a weakening of both axial and equatorial bond strength, as reflected by the increase of 0.10 and 0.02 Å in the Am–Oyl and Am–Ow bond distances compared to those in [AmO2(H2O)1]2+/1+. The optimized Am–Ow bond distance is 2.286 Å in [AmO2(H2O)2]2+ and 2.420 Å in [AmO2(H2O)2]1+; the slightly longer distance in the monocation complex is consistent with the small binding energy. Noticeably, the favorable bent versus linear geometries of the ∠OwAmOw reveals the covalent characteristic of Am–Ow bond, which pulls the two Ow atoms much closer, leading to an Ow–Ow distance of 3.420 Å in [AmO2(H2O)2]2+ and 3.790 Å in [AmO2(H2O)2]1+. The electrons singly occupy on Am 5f6d atomic orbital (AO)-based molecular orbitals (MOs) in [AmO2(H2O)]2+/1+, which provides extensive Am 5f → Ow 2p back-bonding, involving donation from an Am 5fϕ orbital into in-plane Ow 2p orbitals, thus leading to a three-center interaction. The effectiveness of the Ow 2p → Am 5f6d dative bonding and Am 5f → Ow 2p back donation is less obvious in [AmO2(H2O)2]1+ due to a longer Am–Ow distance, denoted by a relatively large ∠OwAmOw angle of 103.1°. In particular, the variety in the average effective charges of the Ow ligand, listed in Table 4, is an evidence to this statement. The average Hirshfeld charges on the Ow of [AmO2(H2O)2]2+ and [AmO2(H2O)2]+ are −0.16 \|e\| and −0.20 \|e\|, respectively, slightly greater relative to those of −0.15 \|e\| and −0.20 \|e\| in [AmO2(H2O)1]2+/1+, as the consequence of the Am-to-Ow back donation. Table 4 Average Hirshfeld, Voronoi Deformation Density (VDD) Charges, and Average Mulliken, Multipole Derived Charges (MDC_q) Spin Density on the B3LYP with COSMO-Optimized Structures of [AmO2(H2O)n]2+/1+ (n = 1–5) from the PBE/TZ2P Calculations [AmO2(H2O)n]2+ [AmO2(H2O)n]1+ charge spin density charge spin density n Hirshfeld VDD Mulliken MDC_q Hirshfeld VDD Mulliken MDC_q 0 Oyl –0.03 0.22 –0.22 –0.23 –0.26 –0.08 –0.23 –0.28 Am 2.06 1.55 3.44 3.46 1.52 1.17 4.46 4.57 1 Oyl –0.09 0.09 –0.21 –0.22 –0.30 –0.18 –0.23 –0.29 Ow –0.15 –0.15 –0.01 –0.09 –0.20 –0.15 –0.01 –0.09 Am 1.77 1.27 3.43 3.48 1.32 0.96 4.46 4.61 2 Oyl –0.13 –0.02 –0.20 –0.22 –0.33 –0.28 –0.22 –0.30 Ow –0.16 –0.16 –0.01 0.01 –0.20 –0.16 0.00 0.02 Am 1.53 1.04 3.42 3.46 1.14 0.79 4.46 4.61 3 Oyl –0.17 –0.13 –0.19 –0.15 –0.35 –0.35 –0.22 –0.06 Ow –0.18 –0.16 –0.01 0.01 –0.20 –0.15 0.00 0.10 Am 1.31 0.81 3.41 3.20 0.98 0.59 4.45 3.69 4 Oyl –0.19 –0.19 –0.19 –0.09 –0.37 –0.40 –0.21 0.00 Ow –0.19 –0.18 0.00 0.07 –0.21 –0.18 0.00 0.07 Am 1.18 0.75 3.39 2.94 0.89 0.54 4.41 3.52 5 Oyl –0.21 –0.20 –0.19 –0.01 –0.38 –0.41 –0.23 0.00 Ow –0.20 –0.20 0.00 0.04 –0.21 –0.19 0.00 0.06 Am 1.11 0.74 3.39 2.68 0.82 0.52 4.45 3.51 For the triaqua, one more water molecule added with the formation of [AmO2(H2O)3]2+/1+ further weakens the Am–O bonds, as identified by the longer Am–Oyl and Am–Ow bond distances than those in [AmO2(H2O)2]2+/1+. In gas phase, three coordinated water molecules are equivalent with the ∠OwAmOw angle of 120.0°, giving a C3v symmetry structure. However, when concerning the presence of aqua solvent, this symmetry has been broken into Cs symmetry with the ∠OwAmOw angles of 127.8, 113.2, and 118.7° in [AmO2(H2O)3]2+ and 117.4, 120.4, and 122.2° in [AmO2(H2O)3]1+ complexes, largely ascribed to the differences responding to the polarization of the hydration upon solvent inclusion. In essence, the solvent polarity is more effective in those complexes with larger polarity. As listed in Table 2, the deviation from the high-symmetry structure of the ∠OwAmOw angle in [AmO2(H2O)n]2+ is more obvious than that in [AmO2(H2O)n]1+, suggesting that the structure of dication ions could result in engaging in stronger hydrogen bonding with the bulk solvent than that of monocation ions. For example, the ∠OwAmOw angles are 92.7 and 87.3° in [AmO2(H2O)4]2+ and 66.3, 74.5, and 78.4° in [AmO2(H2O)5]2+, whereas 90.0° in [AmO2(H2O)4]1+ and 72.0° in [AmO2(H2O)5]1+ are retained, thus confirming that the solvent shows greater differential stabilization in [AmO2(H2O)n]2+ complexes. From our above discussion of [AmO2(H2O)n]2+/1+ structures, we would expect the additional water molecules to extend the Am–Oyl and Am–Ow bond lengths and to decrease the overall degree of dative bonding, consistent with the lower relative binding energy for the [AmO2(H2O)q]2+/1+ compared to that of the [AmO2(H2O)p]2+/1+ (5 ≥ q = p + 1). The maximum of five hydration number in the first coordination shell is in good agreement with the equilibrium structure having five water coordination of actinyl (UO22+, NpO22+, and PuO22+) in aqueous solution. In the equilibrium structure of [AmO2(H2O)5]2+/1+, four water molecules bonded to the center americyl are perpendicular to the equatorial plane and the fifth coordinated water molecule arranges within the equatorial plane where the dihedral angle between the equatorial water ligands and the equatorial plane is 0° (Figure 1). The bond length of the fifth Am–Ow bond is 2.420 Å in [AmO2(H2O)5]2+ and 2.548 Å in [AmO2(H2O)5]1+, the shortest one among five Am–Ow bond distances, thus leading to a slight bent of ∠OylAmOyl angles of 179.4°. Electronic Structure and Chemical Bonding of the [AmO2(H2O)n]2+/1+ (n = 1–5) To provide further insights into the interactions between americyl ion and the water ligands of [AmO2(H2O)n]2+/1+, several bonding analyses were performed, including the bond order analyses, population partitioning schemes, energy decomposition approach, Kohn–Sham orbital interaction, and electron localization function (ELF) method. The bond order analyses by three methods give a similar trend that is consistent with the previous detailed bond length trend. Taking the Mayer Am–O bond index as an example, the calculated Mayer bond orders (Table 5) of Am–Oyl and Am–Ow for the [AmO2(H2O)n]2+ complexes lie in two ranges, at averaged 1.9 and averaged 0.2, respectively, whereas those for [AmO2(H2O)n]1+ complexes lie in two ranges, at averaged 1.85 and averaged 0.2, respectively. This result indicates relatively weak Am–Ow bonding interactions compared with the Am–Oyl bonding in [AmO2(H2O)n]1+ complexes, which is resulted from not only the ionic bonding nature but also the somewhat longer bond distance induced by weaker orbital interaction. The calculated Am–Oyl bond order in [AmO2(H2O)n]2+ decreases gradually from 2.05 at n = 1 to 1.88 at n = 5, whereas the Am–Ow bond order decreases from 0.39 at n = 1 further to 0.19 at n = 5, suggesting there is a general decrease in Am–O bond strength with an increasing hydration number. In addition to the bond order, the effective atomic charges on Am and Ow ions can imply the variation of the ionicity of the Am–Ow bond. Several charge analyses were carried out on the B3LYP stationary structure in solution via COSMO. Although the absolute values are different on the basis of different methods used, the trend is the same. As listed in Table 4, the averaged Hirshfeld charges reveal that Am carries a considerable positive charge and the Ow ligands carry substantial negative charges in these [AmO2(H2O)n]2+/1+ complexes, which is in good agreement with the bond order analyses, indicating an ionic An–Ow bonding interaction. Generally, the Am ions become less charged and both Oyl and Ow atoms become more charged along with the increase of the hydration number, i.e., the calculated Hirshfeld charge of Am decreases from +2.06 \|e\| in pure AmO22+ to +1.77 \|e\| in monoaqua and further to +1.11 \|e\| in penta-aqua, accompanied by an increased charge of each Oyl atom from −0.03 \|e\| to −0.21 \|e\|; this is due to the charge rearrangement of americyl unit upon inclusion of explicit water in the first shell and implicit water by COSMO, which is in good agreement with the study of [UO2(H2O)m(OH)n]2–n.34 The averaged Hirshfeld charges of Ow atoms becoming greater from −0.15 \|e\| to −0.21 \|e\| suggests overall increasing of electron back donation into Ow ligand, thus leading to the Am–Oyl bond weakening with an increasing hydration number. In addition, the decreased electron density localized on americyl unit listed in Table 4 indicates that the charge transfer is from americyl unit to water ligands, owing to the Am-to-Ow back donation. Furthermore, the formal Am oxidation state of +VI and +V is not apparently affected by the hydration number in these [AmO2(H2O)n]2+/1+ complexes via population analyses. Table 5 Average Mayer Bond Orders of [AmO2(H2O)n]2+/1+ (n = 1–5) from the PBE/TZ2P Calculations [AmO2(H2O)n]2+ [AmO2(H2O)n]1+ Am–Oyl Am–Ow Am–Oyl Am–Ow n vacuum COSMO vacuum COSMO vacuum COSMO vacuum COSMO 0 2.12 2.12 1.99 2.00 1 2.06 2.05 0.37 0.39 1.94 1.94 0.26 0.27 2 2.00 2.00 0.34 0.34 1.90 1.90 0.21 0.21 3 1.96 1.95 0.31 0.32 1.86 1.86 0.18 0.19 4 1.91 1.90 0.22 0.22 1.83 1.83 0.13 0.14 5 1.89 1.88 0.17 0.19 1.80 1.81 0.11 0.12 To better understand the orbital interactions, Figure 2 shows the Kohn–Sham MO energy levels of [AmO2(H2O)n]2+ at SR-PBE/TZ2P level. In the linear AmO22+ moiety due to Am–Oyl bonding, the Am 5f and 6d orbitals transform as 5fu and 6dg species, respectively. Upon dative coordination with the water oxygen atoms in the equatorial plane, the Am–Ow orbital interactions cause to 6dg level further destabilization. While both dδg and fδu–fϕu orbitals lie around the equatorial plane, the Am–Ow orbital interactions mainly affect the 6d orbitals, indicating that the coordinated water molecules only slightly interact with the “nonbonding” contracted 5f orbitals. This bonding module is in good agreement with the well-known bonding principle proposed for actinides by Bursten, the FEUDAL (“f’s are essentially unaffected, d’s accommodate ligands”).35,36 Overall, the Am–Ow bonding in [AmO2(H2O)1]2+ is strongest among these [AmO2(H2O)n]2+ complexes and becomes progressively weaker on increasing the hydration number, consistent with the indirect evidence from geometries, vibrational frequencies, and bond order. These conclusions are consistent with the bonding compositions of the natural localized MOs (NLMOs, Table S5), with analysis detailed below. Figure 2 Kohn–Sham molecular orbital analyses of the AmO2(H2O)n2+ complexes based on SR-zero-order regular approximation (ZORA) B3LYP/TZ2P calculations. In addition to these chemical bonding analyses based on wave functions and electron densities, the energy decomposition analysis (EDA) based on canonical molecular orbitals and the extended transition state (ETS) method combined with the natural orbitals for chemical valence (ETS–NOCV) theory were further applied to reveal the intrinsic bonding mechanism in terms of the major contributions to the orbital interactions. The computed EDA results for AmO22+/1+ + nH2O → [AmO2(H2O)n]2+/1+ (Tables 6 and 7) show that electrostatic ionic interaction (ΔEelstat) accounts for almost 2/3 contribution to the total bonding energy (ΔEint), playing more significant roles than those of orbital interaction (ΔEorbital). The decreasing trend of both electrostatic interactions and covalent orbital interactions are the same as those of bonding energies, that is, generally increasing with the hydration number from monoaqua toward penta-aqua. The change of ΔEelstat or ΔEorbital is not significantly in tetra-aqua and penta-aqua, reflecting that the variation of both ionic and covalent interactions with the increasing number of the water molecules becomes smaller in aqueous solution. In contrast to the monotonous increase of ΔEelstat or ΔEorbital, the Pauli repulsion increases remarkably from 65.3 kcal/mol in monoaqua to 159.6 kcal/mol in tetra-aqua, then declines to 151.4 kcal/mol in penta-aqua, indicating the formation of the stable penta-aqua. The ETS–NOCV analyses additionally reveal the intrinsic bonding mechanism in terms of the contributions to the orbital interactions. The results of some representative contributions for [AmO2(H2O)n]2+/1+ are listed in Tables 6 and 7, and their deformation densities describing the density inflow are shown in Figures 3 and 4, respectively. The highest covalent character of each Am–Ow bond can be observed in monoaqua among these [AmO2(H2O)n]2+/1+ complexes in terms of the orbital interaction, which is decomposed into two types (π-type and σ-type) with several terms, taking orb1–orb3 as examples, providing energetic stabilizations of ΔEorb1 = −29.2 kcal/mol, ΔEorb2 = −14.4 kcal/mol, and ΔEorb3 = −6.4 kcal/mol, respectively. These natural orbitals denote the typical π- or σ-type orbital interactions achieved via two fragments of americyl and water molecules, relating to the Am 5f6d-hybridized atomic orbitals and π-MOs of water molecules contributed by Ow 2p atomic orbitals. Note that consistent with the reduced trend of bonding energy of each Am–Ow bond across the series, the trend of the donation character is also decreased accompanied by a decrease of major orbital contributions and a slight lengthening of Am–Ow bonds. The composition of the stabilization energy of each Am–Ow bond in [AmO2(H2O)n]2+/1+ complexes is small, but the summarization is substantial and increases across the series. Therefore, the orbital interactions between the An 5f6d hybrid orbitals and π-MOs of Ow result in a significant covalent bonding character not only stabilizing [AmO2(H2O)n]2+/1+ complexes beyond electrostatic ionic interaction but also effectively supporting the weak bonding characteristics of 5f orbital, which is consistent with the ELF result shown in Figure 5 and with the NLMO analyses (listed in Table S5). Figure 3 Contours of representative deformation densities (isovalue = 0.001 au) between the interacting fragments of AmO22+ and H2O unit from ETS–NOCV analysis, describing the density inflow (blue) and outflow (white). Figure 4 Contours of representative deformation densities (isovalue = 0.001 au) between the interacting fragments of AmO2+ and H2O unit from ETS–NOCV analysis, describing the density inflow (blue) and outflow (white). Figure 5 Two-dimensional ELF contours (isovalue = 0.03 au) for the Ow–Am–Ow planes containing the Am–Ow interactions. The results are based on the SR-ZORA calculated densities. Table 6 ETS–NOCV Energy Decomposition Analyses between the Interacting Fragments of AmO22+ and H2O Unit and Their Corresponding Energy ΔEiorb (kcal/mol) from Open-Shell PBE/TZ2P Calculations 1(4A) 2(4A) 3(4A) 4(4A) 5(4A) AmO22+ (4A) fϕ1fδ2 AmO22+ (4A) fϕ1fδ2 AmO22+ (4A) fϕ1fδ2 AmO22+ (4A) fϕ1fδ2 AmO22+ (4A) fϕ1fδ2 frag. H2O (1A)···8a2 H2O (1A)···16a2 H2O (1A)···24a2 H2O (1A)···32a2 H2O (1A)···40a2 ΔEint –68.5 –125.7 –177.8 –218.3 –241.9 ΔEPauli 65.3 114.6 143.9 159.6 151.4 ΔEelstat –76.5 –138.7 –190.0 –223.7 –229.6 ΔEorb –57.4 –101.6 –131.7 –154.2 –163.7 α β α β α β α β α β ΔE1 –14.5 –14.6 –17.5 –16.3 –14.2 –12.7 –19.3 –17.8 –16.6 –15.4 ΔE2 –7.8 –6.6 –10.5 –11.6 –13.8 –12.0 –10.5 –10.4 –14.2 –14.6 ΔE3 –3.1 –3.2 –11.4 –13.5 –8.5 –7.4 –11.5 –10.8 Table 7 ETS–NOCV Energy Decomposition Analyses between the Interacting Fragments of AmO21+ and H2O units and Their Corresponding Energy ΔEiorb (kcal/mol) from an Open-Shell PBE/TZ2P Calculation 1(5A) 2(5A) 3(5A) 4(5A) 5(5A) AmO21+ (5A) fϕ2fδ2 AmO21+ (5A) fϕ2fδ2 AmO21+ (5A) fϕ2fδ2 AmO21+ (5A) fϕ2fδ2 AmO21+ (5A) fϕ2fδ2 interacting fragments H2O (1A)···8a2 H2O (1A)···16a2 H2O (1A)···24a2 H2O (1A)···32a2 H2O (1A)···40a2 ΔEint –39.3 –69.7 –98.9 –121.7 –129.7 ΔEPauli 38.5 70.6 92.7 103.2 100.4 ΔEelstat –48.1 –89.7 –124.9 –147.9 –149.9 ΔEorb –29.8 –50.6 –66.7 –77.0 –80.2 α β α β α β α β α β ΔE1 –7.3 –6.6 –8.3 –7.1 –6.9 –6.7 –9.9 –8.7 –7.7 –6.9 ΔE2 –2.2 –2.0 –5.7 –5.2 –6.7 –6.7 –6.1 –5.7 –7.2 –6.5 ΔE3 –0.5 –0.5 Considering that the Am–Ow dative bond and back donation were observed from density deformation analyses, NLMO calculations were further explored to describe the extent of this bonding interaction. The localized σ- and π- MOs on Am–Ow are made up of Am 5f6d and O 2s2p hybrid orbitals, which are composed by roughly 95% O 2s2p lone-pair AO and ∼5% Am df hybrid orbitals in [AmO2(H2O)n]2+ and by ∼98% O 2s2p lone-pair AO and ∼2% Am df hybrid orbitals in [AmO2(H2O)n]1+ on the basis of the NLMO analyses. The enhanced stability of [AmO2(H2O)n]2+/1+ complexes across the series can only be explained as a result of the water coordinates inducing the increased electrostatic energy. Noticeably, the Am 6d participating in the dative oxygen bonding plays an important role over Am 5f due to both more extended radial distribution of the Am 6d orbitals and the better energy level matching between the An 6d and O 2p orbitals. The compositions of the NLMOs from Am valence atomic orbitals decline from around 3% of the σ orbitals and 7% of the π orbitals in [AmO2(H2O)1]2+ to 1% and 6% in [AmO2(H2O)5]2+. Conclusions Through relativistic quantum chemical calculations, the electronic and geometric structures of the [AmO2(H2O)n]2+/1+ (n = 1–6) complexes have been systematically studied. Full five-coordination binding of water ligands in the first shell is preferred in the aqua solution for [AmO2(H2O)n]2+/1+. Extensive chemical bonding analyses of the charges, energy decomposition analysis, and natural orbitals, all agree in that the Am–Ow interaction is predominantly ionic in nature, with a small extent of covalent bonding contribution resulted from the An 5f6d-hybridized orbitals and relevant Ow 2p orbitals. The covalent orbital interaction between the equatorial Ow and Am ions raises several interesting features in the bonding of Am–Ow: (1) a bonding interaction between the Am AOs and the Ow 2p orbitals arranges the water ligand deviation from linear in [AmO2(H2O)2]2+/1+ into bent; (2) the reduced dative bonding affects the Am–Ow bond length and bond order across the series; (3) the small back-bonding donations of Am 5f → Ow 2p decrease the population of americium ions. As noted, the implicit solvation model is unable to provide the dynamics information of actinide ions in solution or the exchange of ion-influenced solvent molecules with the bulk, which is important to some extent in practical application. Further quantum mechanics/molecular mechanics molecular dynamics simulation with explicit solvent is necessary to study statistically the equilibrium between americyl and water in different coordination shells. We hope that this investigation represents only the beginning of long-term research relevant to the mission in spent fuel reprocessing of the transplutonium, in addition to enriching our knowledge of actinide coordination chemistry. Methodology The calculations on the open-shell americyl complexes were carried out with unrestricted Kohn–Sham wave functions. As known, the molecular orbital (MO) diagram of pure americyl is similar to that of uranyl: the combinations of the 2p orbitals of the two oxygen with the 6d orbitals of the americium atom form occupied bonding orbitals, whereas the combinations of the O 2p orbitals with the 5f manifold orbitals of the actinide atom result in nonbonding δu and ϕu orbitals and antibonding σu* and πu* orbitals; the corresponding bonding σu and πu orbitals are found to have rather small 5f contributions. In AmO22+ and AmO21+, the americium has the configurations 5f3 and 5f4, respectively, and these electrons are spread in the nonbonding 5fδu and 5fϕu orbitals. Following Hund’s rule, the ground electronic states of hexavalent and pentavalent Am ions have been found to be of a high-spin character, that is, fδ2fϕ1 and fδ2fϕ2 configurations and 4Δ and 5Σ electronic states for AmO22+ and AmO21+, respectively. The spin–orbit coupling (SOC) effects should be considered during the calculations of the electronic structures of americyl complexes in solution.37 The scalar relativistic DFT calculations were performed with the Gaussian 09 and ADF 2017 softwares38−40 for the geometry optimizations and vibrational frequency analyses. First, for geometry optimizations by the Gaussian code, we used the scalar relativistic Stuttgart energy-consistent relativistic 32 valence electron pseudopotential and the associated ECP60MWB_SEG valence basis set41,42 for the americium atom. The split-valence triple-ζ basis sets with polarization functions (cc-pVTZ)43,44 were used for the oxygen and hydrogen atoms. The B3LYP hybrid density functional45,46 was used in these calculations. The solvent effects were approximated using the Gaussian 09 implementation of the conductor-like screening solvation model (COSMO) solvent model,47,48 where the solute is embedded in a shape-adapted cavity consisting of interlocking spheres centered on each solute atom or group. The combination of these pseudopotentials and basis sets with this functional (labeled as B3LYP/Am/ECP60MWB_SEG//O, H/cc-pVTZ level) has been shown to give accurate predictions of the properties and reaction energies of actinide complexes.19,20 At the same B3LYP theory level, vibrational frequency calculations were further performed to ensure that all force constants should be positive and all frequencies should be real. Ultrafine integration grids and very tight optimization were applied. The spin–orbital symmetry restrictions were relaxed, performing spin-unrestricted UDFT calculations. The coupled cluster with singles, doubles, and perturbative triples (CCSD(T)) method49−51 in MOLPRO 2012 program52 was used for single-point calculations on the optimized aqueous-phase B3LYP geometries to check whether the systems have multireference character and to acquire more accurate relative energies. The ECP60MWB pseudopotential with ECP60MWB_SEG basis set for Am and the cc-pVTZ basis for O and H were applied in the CCSD(T) calculations. However, limited by the computer resources, the nonsymmetry molecules of [AmO2(H2O)5]2+/1+ or [AmO2(H2O)6]2+/1+ cannot be handled by CCSD(T) due to the giant basis of those molecules. Further electronic structure and chemical bonding analyses at the DF level were performed with the ADF 2017. The scalar relativistic (SR) effects were taken into account by the zero-order regular approximation (ZORA).53,54 The SOC effects were taken into account by SO-ZORA. The frozen-core approximation was applied to the atomic [1s2–5d10] shell of Am and the [1s2] shells of the O atoms. The B3LYP combined with slater basis sets of triple-ζ plus two polarization function (TZ2P) quality were applied.55 Single-point calculations were carried out on the B3LYP/Am/ECP60MWB_SEG//O H/cc-pVTZ optimized aqueous-phase structures to determine the charge distribution and bond order. The bond order analyses based on the Mayer method,56 Gopinathan–Jug indices,57 and Nalewajski–Mrozek method58,59 were performed. The charge analyses based on the Mulliken method,60 Hirshfeld analysis,61 Voronoi deformation density (VDD),62 and multipole derived charges (MDC)63 were calculated as well. The energy decomposition analyses (EDA) based on canonical molecular orbitals and theoretical analyses via combined extended transition state (ETS) with the natural orbitals for chemical valence (NOCV) theory59,64−66 were carried out. The electron localization functions (ELF) were calculated to investigate the feature of the weak dative bonding. Weinhold’s natural bond orbital (NBO)67 and natural localized molecular orbital (NLMO)67−70 analyses were performed at the B3LYP/6-31G* level71 on optimized geometries from B3LYP calculations by using the NBO 5.0 program.72,73 In further geometry optimizations, the generalized gradient approximation with the PBE exchange–correlation functional74 was used in ADF 2017. Scalar (SR) and SO relativistic effects were accounted by applying the same stratagem as detailed in the above paragraph. The effects of water considered by COSMO were determined using single-point energy calculation on gas-phase structures. The atomic COSMO-default radii 210.0 pm was used.48 Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01324.Selected optimized geometrical structures (bond length Å and bond angle °) for the fϕ1fδ2 [AmO2(H2O)n]2+ (n = 0–6) at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ levels of theory with or without COSMO (in parentheses) (Table S1); selected optimized geometrical structures (bond length Å and bond angle °) for the reaction of fϕ2fδ2 [AmO2(H2O)n]1+ (n = 0–6) at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ levels of theory with or without COSMO (in parentheses) (Table S2); binding energy (kcal/mol) for the reaction of AmO22+ + nH2O → [AmO2(H2O)n]2+ (n = 0–6) at different levels of theory (Table S3); binding energy (kcal/mol) for the reaction of AmO21+ + nH2O → [AmO2(H2O)n]1+ (n = 0–6) at different levels of theory (Table S4); natural localized molecular orbital (NLMO) analyses of [AmO2(H2O)n]2+/1+ (n = 1–6) from open-shell B3LYP/cc-pVTZ/ECP60MWB_SEG calculations (Table S5); average Mayer, Gopinathan–Jug and Nalewajski–Mrozek bond orders for Am–Oyl and Am–Ow on the B3LYP-optimized geometry of AmO2(H2O)n2+/1+ (n = 0–6) at the SR–PBE/TZP level with COSMO (Table S6); average Mulliken and MDC_q charges and spin density on the B3LYP-optimized geometry of [AmO2(H2O)n]2+/1+ (n = 0–6) in solution from the PBE/TZP calculations (Table S7); calculated vibrational frequencies (cm–1) of [AmO2(H2O)n]2+/1+ (n = 0–6) in aqueous solution considered by COSMO at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ l level of theory (Table S8); ETS–NOCV energy decomposition analyses between the interacting fragments of AmO22+ and ligand units and their corresponding energy ΔEiorb (kcal/mol) on the B3LYP-optimized geometry of AmO2(H2O)n2+ (n = 1–5) in solution from an open-shell PBE/TZ2P calculation (Table S9); ETS–NOCV energy decomposition analyses between the interacting fragments of AmO21+ and ligand units and their corresponding energy ΔEiorb (kcal/mol) on the B3LYP-optimized geometry of AmO2(H2O)n1+ (n = 0–5) in solution from an open-shell PBE/TZ2P calculation (Table S10); two isomers (in-plane and perpendicular-to-plane) of water molecules for [AmO2(H2O)1]2+/1+ and [AmO2(H2O)2]2+/1+ at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ levels of theory (Figure S1); optimized geometry of D6h, Ci symmetry structures of [AmO2(H2O)6]2+ and [AmO2(H2O)7]2+ at SR-B3LYP/Am/ECP60MWB_SEG//O,H/cc-pVTZ levels of theory (Figure S2) (PDF) Supplementary Material ao8b01324_si_001.pdf The authors declare no competing financial interest. Acknowledgments We gratefully acknowledge the financial support for this research from the Science Challenge Project (No. TZ2018004) and the NSAF (U1530401), the Foundation of President of China Academy of Engineering Physics (No. YZJJSQ2017072), the Foundation for Development of Science and Technology of China Academy of Engineering Physics (No. 2015B0102020), and the National Natural Science Foundation of China (Nos. 21701006, 21771167, and 11874089). We are also grateful to the Beijing Computational Science Research Center for generous grants of computer time. ==== Refs References Gagliardi L. ; Roos B. O. Multiconfigurational quantum chemical methods for molecular systems containing actinides . Chem. Soc. Rev. 2007 , 36 , 893 –903 . 10.1039/b601115m .17534476 Li J. ; Bursten B. E. Relativistic density functional study of the geometry, electronic transitions, ionization energies, and vibrational frequencies of protactinocene, Pa(η8-C8H8)2 . J. Am. Chem. 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PMC006xxxxxx/PMC6644429.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145900010.1021/acsomega.8b01275ArticleDesign and Synthesis of Reactive Perylene Tetracarboxylic Diimide Derivatives for Rapid Cell Imaging Zhang Endong †‡Liu Libing †‡Lv Fengting †‡Wang Shu †‡† Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China‡ College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049, P. R. China* E-mail: liulibing@iccas.ac.cn (L.L.).* E-mail: wangshu@iccas.ac.cn (S.W.).06 08 2018 31 08 2018 3 8 8691 8696 07 06 2018 24 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A new water-soluble reactive perylene tetracarboxylic diimide derivative (PDI-pfp) is designed and synthesized that can realize fast imaging of the endoplasmic reticulum in living cells. The PDI-pfp comprises three functional moieties: perylene tetracarboxylic diimide as fluorescent backbone, poly(ethylene glycol) for providing good water disperse ability, and pentafluorophenol active ester as the reactive group under physiological condition. On the basis of covalent reaction between the active ester group of PDI-pfp and amine groups on cytomembrane, PDI-pfp can rapidly interact with cytomembrane, followed by uptake by living MCF-7 cells within 1 min and also exhibit low cell cytotoxicity. Furthermore, it is proved that PDI-pfp acts as a universal imaging agent for other types of cells. This fluorescent probe is of great potential for the application in the rapid imaging of organelles in cells. document-id-old-9ao8b01275document-id-new-14ao-2018-01275mccc-price ==== Body Introduction On account of the properties of intuition, multi-information, high sensitivity, and specificity,1−6 indispensable and emerging fluorescent probes and materials for cell imaging have been greatly developed.7−10 Compared to fluorescent proteins11 and fluorescent nanoparticles,12−16 organic dyes, which have smaller size, higher emission intensity, and broader available spectral range, play an important role in fluorescence imaging of cells.17−20 Furthermore, the design and synthesis of advantageous cell imaging materials with good water solubility, high fluorescence quantum yield, good biocompatibility, and low cytotoxicity are extremely imperative. Perylene tetracarboxylic diimide (PDI) and its derivatives with excellent redox and optical properties as well as superior photochemical and thermal stabilities21−24 have been extensively applied in organic pigments, electrophotography, and photovoltaic cells.25−28 Owing to the delocalized π-electronic rigid plane structure, PDI is endowed with high fluorescence quantum yield and, recently, has been widely utilized as a chromophore.29 However, the hydrophobic backbone of PDI results in its poor solubility in aqueous solution, which has greatly limited its application in biological systems. Two strategies have been employed so far to enhance the water solubility of PDI: 1) the attachment of hydrophilic chains at the imide nitrogen of PDI;30,31 and 2) the introduction of charge groups at the bay region of PDI.32 Pentafluorophenol active ester exhibits high reactivity with many functional groups,33−35 especially with amino groups under physiological conditions. Therefore, pentafluorophenol ester as an active group could be used to facilitate fluorescent probe to enter cells because of the widely distributed amino groups on the cell surfaces.36 In this work, we report the design and synthesis of a new water-soluble reactive perylene tetracarboxylic diimide derivative (PDI-pfp) with high brightness, good water solubility, and high chemical reactivity. By introducing oxylalkyl chains to PDI, the water solubility is enhanced without affecting its high fluorescence quantum yield because of the inappreciable influence of N-substituent for nodes of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals of PDI.21 Pentafluorophenol active ester offers PDI-pfp a high reactive group to interact with cells. It was demonstrated that the PDI-pfp exhibited low cytotoxicity and sufficient fluorescence ability for rapid imaging of the endoplasmic reticulum (ER) in living cells. Results and Discussion The synthetic procedure of PDI-pfp is shown in Scheme 1. The precursor molecule PDI–COOH was first synthesized by reacting perylene-3,4,9,10-tetracarboxylic dianhydride (PDI) and NH2-PEG10-COOH in imidazole at 130 °C for 5 h with a yield of 70%. PDI-pfp was then prepared by esterification of PDI–COOH with pentafluorophenol in the presence of N,N-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) in dichloromethane overnight with a yield of 53%. All of the chemical structures were confirmed by the 1H NMR and mass spectra. Scheme 1 Synthetic Procedure of PDI-pfp The photophysical properties of PDI–COOH and PDI-pfp were investigated in aqueous solution because the linkage of low toxic and uncharged alkoxy chains extremely enhanced their solubility. UV–vis absorption and fluorescence emission spectra of PDI and PDI-pfp were measured in aqueous solution. As shown in Figure 1, owing to the fact that both PDI–COOH and PDI-pfp consist of the same backbones, they exhibited quite similar absorption with a maximum peak at 498 nm. On the basis of Lambert Beer’s law, molar absorption coefficients of PDI–COOH and PDI-pfp were calculated to be 2.7 × 105 and 1.9 × 105 M–1 cm–1, respectively. Moreover, PDI-pfp has nodes in the orbital HOMO and LUMO at nitrogen atoms, which cause decoupling of single bonds between nitrogen atoms and substitutions.14 Thus, compared to the precursor molecule PDI–COOH, there is less influence on the fluorescence of PDI-pfp. PDI-pfp exhibited a bright green fluorescence with a maximum emission at 548 nm and an absolute fluorescence quantum yield of 0.35 in aqueous solution, which shows good potential for fluorescent imaging. Figure 1 UV–vis absorption and fluorescence emission spectra of PDI–COOH and PDI-pfp in aqueous solution. The excited wavelength of 507 nm was used for emission spectra. Low cytotoxicity is necessary for fluorescent probes to be utilized in cell imaging and biology applications.37 Cytotoxicities of PDI-pfp and PDI–COOH were investigated through a standard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, in which optical density values of formazan reduced from MTT by dehydrogenase in mitochondria of viable cells were measured. In this experiment, MCF-7 cells were incubated in 96-well culture plates treated with different concentrations of PDI and PDI-pfp for 30 min. Then, the supernatant was discarded, and MCF-7 cells were continuously cultured in fresh Dulbecco’s modified Eagle’s medium (DMEM) for the next 24 h, and cell viabilities were measured and calculated. As shown in Figure 2, PDI–COOH displayed nearly nontoxic behavior up to 10 μM concentration, whereas PDI-pfp also exhibited less cytotoxicity at concentrations lower than 10 μM, indicating their good biocompatibility for cell imaging. Figure 2 Viability of MCF-7 cells after incubating with PDI-pfp and PDI–COOH for 30 min treatment. [PDI-pfp] or [PDI–COOH] = 0–10 μM. The ability of imaging for living cells of PDI-pfp was then determined using MCF-7 cells. To investigate the effect of pentafluorophenol active esters of PDI-pfp on cell imaging, PDI–COOH without pentafluorophenol active esters was also studied as control. To accomplish this, considering the cell viability in Hank’s balanced salt solution (HBSS), the incubation time was set as 30 min. MCF-7 cells were treated with PDI–COOH and PDI-pfp for 30 min, and the treated cells were washed with Hank’s balanced salt solution (HBSS), followed by the addition of serum-free medium into the plates for confocal laser scanning microscopy (CLSM) characterization. As shown in Figure 3, PDI-pfp with pentafluorophenol active esters as terminal groups could uptake into MCF-7 cells and well stain cells. It is noted that MCF-7 cells internalized by PDI-pfp were observed in a good condition. Compared with PDI-pfp, PDI–COOH with carboxyl groups at the terminal position being negatively charged in aqueous solution is believed to be repelled by the negatively charged membrane of cells, leading to no interaction between PDI–COOH and MCF-7 cells. Furthermore, the imaging results indicated that PDI-pfp with higher concentration (10 μM) stains cells better than that with lower concentration (5 μM). Figure 3 CLSM images of MCF-7 cells incubated with PDI–COOH and PDI-pfp (5 and 10 μM) for 30 min. The excitation wavelength was 488 nm, and the fluorescence images were collected at the signals from 500 to 600 nm. To further confirm the localization of PDI-pfp inside cells, MCF-7 cells were incubated with PDI-pfp for 30 min, and then dyes using specific imaging of organelles in cells were added. It was observed that the location of PDI-pfp was well overlapped with that of ER Tracker Red, which is used for specific staining of the endoplasmic reticulum (ER) in cells (Figure 4a), while poor overlaps were seen with those of other organelle-specific dyes. A white line that was randomly selected in the fluorescent image of MCF-7 cells was chosen for obtaining the Pearson coefficient (Figure 4b). The value of the Pearson coefficient was calculated to be 0.79178, which showed that PDI-pfp was uptaken into cells and mainly located in the endoplasmic reticulum. Figure 4 (a) Colocalizing with endoplasmic reticulum-specific ER Tracker Red after treating MCF-7 cells with PDI-pfp for 30 min. (b) Line-series analysis of PDI-pfp with ER Tracker Red. The Pearson coefficients were calculated along the white line on the MCF-7 cells. PDI-pfp and ER Tracker Red were excited at the wavelengths of 488 and 559 nm, respectively. Rapid imaging of cells is an overwhelming superiority of biomaterials. The incubating times of cells with PDI-pfp at 1, 5, 10, 30, and 60 min were set as a time node to investigate the imaging ability of PDI-pfp toward cells. Figure 5a demonstrates that PDI-pfp could be internalized immediately and distributed in the cytoplasm of MCF-7 cells even within 1 min. With the extension of incubation time, the accumulation amount of PDI-pfp increased and maintained the same distribution in MCF-7 cells. It was noted that the accumulation amount of PDI-pfp did change up to 10 min. To prove the generality of PDI-pfp in rapid imaging toward cells, HeLa cells were chosen as the representative of cancer cells, and 293T cells were chosen as the representative of normal cells for imaging experiments. As shown in Figure 5b,c, similar imaging results for HeLa cells and 293T cells by PDI-pfp were observed as that of MCF-7 cells. Thus, PDI-pfp exhibits great potential for application in the rapid imaging of cells within 1 min. The uptake is the main driving force for the fast internalization of PDI-pfp, and the reaction between PDI-pfp containing pentafluorophenol active ester groups and amino groups on the surface of cells assists the process.31 Figure 5 CLSM images of cells treated with PDI-pfp (10 μM) with different incubation times: (a) MCF-7 cells; (b) HeLa cells; and (c) 293T cells. PDI-pfp were excited at the wavelength of 488 nm, and the fluorescence images were collected at the signals from 500 to 600 nm. Conclusions In summary, a new water-soluble reactive perylene tetracarboxylic diimide derivative, PDI-pfp, with pentafluorophenol active ester on the ends of the backbone was designed and synthesized. The alkoxy chains efficiently enhanced the solubility of PDI-pfp in aqueous solution. PDI-pfp exhibited low cell cytotoxicity and realized rapid imaging of the endoplasmic reticulum (ER) of living cells. The high reactivity between pentafluorophenol active ester groups and amino group on the cell surface led to the fast uptake of PDI-pfp into cells even within 1 min. PDI-pfp exhibited generality of rapid imaging toward different types of cells, including cancer cells (MCF-7 and HeLa cells) and normal cells (293T cells). This fluorescent probe is of great potential for application in the rapid imaging of organelles in cells. Experimental Section Materials and Measurements All chemicals were procured from Sigma-Aldrich Chemical Company, J&K Chemical Company or AMRESCO and used as received. All organic solvents were purchased from Beijing Chemical Works and used without further purification. NH2-PEG10-COOH were purchased from Yanyi Biotech Company Shanghai. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone/Thermo Fisher (Beijing, China). (3-(4,5′-Dimethylthiazol-2′yl)-2,5-dipehenyl-2H-tetrazolium hydrobromide) (MTT) was purchased from Xinjingke Biotech (Beijing, China) and dissolved in 1× phosphate-buffered saline (PBS) before use. The 1H NMR and 13C NMR spectra were recorded on Bruker ARX 300 and ARX 400 instruments with tetramethylsilane as the internal standard. High-resolution mass spectra (HRMS) were taken on a Bruker 9.4T Solarix FT-ICR-MS spectrometer. The UV–vis absorption spectrum was measured on a JASCO V-550 spectrophotometer. The fluorescence spectrum was taken on a Hitachi F-4500 fluorometer equipped with a Xenon lamp excitation source. Absolute fluorescence quantum yield was measured on Hamamatsu absolute photoluminescence quantum yield spectrometer C11347. The MTT assay was performed on a BIO-TEK Synergy HT microplate reader. Cell counting was performed on an automated cell counter (Countess, Invitrogen). Cell imaging was recorded by a confocal laser scanning microscope (FV 1000-IX81, Olympus, Japan). Synthesis of Compound PDI–COOH 3,4,9,10-Perylenetetracareboxylic dianhydride (254.89 mol, 100 mg), imidazole (36 mmol, 2.5 g), and NH2-PEG10-COOH (764.68 mol, 404.99 mg) were mixed together in a 10 mL flask. The solution was heated to 130 °C and refluxed for 5 h. The resulting mixture was cooled down to room temperature. After adding 5 mL of chloroform and 5 mL of 1 M HCl under sonication, the mixture was extracted with chloroform for three times. The combined organic layer was washed by 1 M HCl (5 mL × 3) and dried with anhydrous Na2SO4. The mixture was concentrated and filtrated to remove the solids. Then, the solvent was removed by vacuum, and the residue was a red oily liquid (yield: 254.2 mg, 70%). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.4 (s, 4H), 8.24 (s, 4H), 4.42 (s, 4H), 3.86 (t, J = 4 Hz, 8 Hz, 4H), 3.75 (t, J = 4 Hz, 8 Hz, 4H), 3.63–3.60 (m, 72H), 2.59 (t, J = 8 Hz, 8 Hz, 4H). HRMS (MALDI-TOF) m/z: [M + H]+ calcd 1437.619831, found: 1437.619519. Synthesis of Compound PDI-pfp PDI–COOH (254.2 mg, 179.58 mmol) and pentafluorophenol (99.16 mg, 538.73 mmol) were dissolved in dichloromethane. EDCI (89.51 mg, 466.9 mmol) was then added to the reaction mixture at low temperature using an ice bath, followed by the dropwise addition of DMAP (5.7 mg, 46.69 mmol). The reaction mixture was stirred overnight to ensure the completion of the reaction. Afterward, the reaction mixture was extracted by dichloromethane (20 mL × 3), and the combined organic layer was washed with distilled water (20 mL) and NaCl saturated solution (50 mL) separately. After drying with anhydrous Na2SO4, the solvent was removed, and the residue was purified by silica gel chromatography using dichloromethane/methanol (20:1) as the eluent to afford the red crystal. (Yield: 165.56 mg, 53%) 1H NMR (400 MHz, CDCl3) δ (ppm) 8.658 (d, J = 13.6 Hz, 4H), 8.93 (d, J = 10.8 Hz 4H), 4.47 (t, J = 0.8 Hz, 0.8 Hz 4H), 3.883 (t, J = 3.2 Hz, 5.2 Hz, 4H), 3.744 (t, J = 3.2 Hz, 5.6 Hz, 4H), 3.618–3.607 (m, 72H), 2.939 (t, J = 8 Hz, 8.4 Hz, 4H). HRMS (MALDI-TOF) m/z: [M + H]+ calcd 1769.588213, found: 1769.587103. Optical Experiment First, 10 μM PDI-pfp and 10 μM PDI–COOH in aqueous solution (with DMSO less than 1% to improve the water dispersibility) were separately used to investigate the photophysical properties, and the UV–vis absorption spectra and fluorescent emission spectra (excited at 507 nm) were obtained. The absolute fluorescence quantum yield of PDI-pfp in distilled water was measured at an excited wavelength of 507 nm. Cell Viability Assay Breast carcinoma cells (MCF-7 cells) were cultured in DMEM with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2 atmosphere. The cells were seeded in a 96-well culture plate at a concentration of 8 × 104 cells per mL and grown for 24 h. After washing with Hank’s balanced salt solution (HBSS), different concentrations of PDI–COOH and PDI-pfp dissolved in HBSS were added separately. Addition of HBSS was considered as the blank group. This was followed by washing with PBS and adding culture medium again. After incubating for 24 h and abandoning the culture medium, 100 μL of 1 mg mL–1 MTT in HBSS was added into each well and incubated at 37 °C under 5% CO2 atmosphere for 4 h. The supernatant was discarded, followed by the addition of 100 μL of DMSO per well to dissolve the formazan. A microplate reader measured the absorbance value of each well at a wavelength of 520 nm. The cell viability rates were calculated with the following equation where A is the absorbance value of the experimental group to which was added PDI or PDI-pfp. Ab is the absorbance value of the group without cells. A0 is the absorbance value of the control group with cells to which was added HBSS alone. Localization Analysis of PDI-pfp in MCF-7 Cells PDI-pfp was dissolved to prepare 10 μM solution in HBSS. First, 1 μM ER Tracker Red solution was prepared with HBSS. MCF-7 cells were incubated in 35 × 35 mm plates. After washing with HBSS twice, MCF-7 cells were incubated with 1 μM ER Tracker Red solution for 30 min. Then, the supernatant was discarded before washing with HBSS again. MCF-7 cells were incubated with 10 μM PDI-pfp solution for 30 min. Cells were washed with HBSS followed by adding serum-free culture medium, and then the fluorescence images of MCF-7 cells were recorded by CLSM; PDI-pfp and ER Tracker was, respectively, excited at the wavelengths of 488 and 559 nm. Cell Imaging with PDI-pfp and PDI–COOH Human cervical carcinoma cells (HeLa cells) and human epithelial cells (293T) were cultured in DMEM with 10% FBS at 37 °C under 5% CO2 atmosphere. Then, 5 μM and 10 μM of PDI–COOH and PDI-pfp solutions were prepared with HBSS, respectively. MCF-7 cells, HeLa cells, and 293T cells incubated in 35 × 35 mm plates were washed with HBSS twice before using. Washed MCF-7 cells were incubated with 5 and 10 μM of PDI–COOH and PDI-pfp solutions at 37 °C for 30 min. In addition, washed MCF-7 cells, HeLa cells, and 293T cells were incubated with the addition of 10 μM of PDI-pfp solutions at 37 °C for 1, 5, 10, 30, and 60 min. Discarding the supernatant and washing with HBSS twice was followed by adding serum-free culture medium, and then the fluorescence images of cells were recorded by CLSM using 488 nm as excitation wavelength. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. 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PMC006xxxxxx/PMC6644430.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145909910.1021/acsomega.8b00675ArticleNew Generation of N-Chloramine/QAC Composite Biocides: Efficient Antimicrobial Agents To Target Antibiotic-Resistant Bacteria in the Presence of Organic Load Ghanbar Sadegh †Kazemian Mohammad Reza ‡Liu Song †‡§†Department of Chemistry, Faculty of Science and ‡Department of Biosystems Engineering, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg R3T 2N2, Canada§ Department of Medical Microbiology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg R3E 0J9, Canada E-mail: Song.Liu@umanitoba.ca.22 08 2018 31 08 2018 3 8 9699 9709 10 04 2018 03 08 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. We previously reported that covalently joining an amide-based N-chloramine with a quaternary ammonium compound (QAC) can yield a new composite biocide with faster inactivation of various bacteria. Importantly, the composite biocide was found to reduce the risk for potential bacterial resistance associated with QAC. However, similar to other N-chloramines and QACs, this high-performance composite biocide becomes less potent against pathogenic bacteria in the presence of high protein fluids. In this study, we substituted the amide-based N-chloramine moiety in the previously reported composite biocide with a secondary amine-based N-chloramine to improve the biocidal efficacy in biological fluids. The N–Cl bond in the synthesized tetramethylpiperidine-based composite biocides is more stable in a high protein medium (HPM) than that in the hydantoin (amide)-based composite biocides. The composite biocide, 2-[4-(1-chloro-2,2,6,6-tetramethyl-piperidin-4-yloxymethyl)-[1,2,3]triazol-1-yl]-ethyl-dodecyl-dimethyl-ammonium chloride (6a), showed the best antibacterial activity in both phosphate-buffered saline and HPM among various composite biocides and benzyldodecyldimethylammonium chloride used in this study. document-id-old-9ao8b00675document-id-new-14ao-2018-00675hccc-price ==== Body Introduction Disinfection of hospital environment and food processing facilities has been an indispensable and valuable preventative measure for ensuring human health. In the context of rising antibiotic resistance and cross-resistance between antimicrobials and antibiotics,1−3 development of new potent non-resistance-inducing disinfectants becomes even more pivotal. Membrane active compounds, such as quaternary ammonium compounds (QACs), account for a big portion of disinfectants. They have been widely used in hospitals for infection control and by public for better hygiene. Reports on bacterial resistance to QACs indicate a concerning trend.4−7 In 2012, up to 83% of methicillin-resistant Staphylococcus aureus (MRSA) isolates carry qac resistance genes in comparison to 10.2% in 1992.4,5 A recent study suggests that besides QacA efflux pump, membrane mutation also greatly contributes to bacterial resistance against QACs. Such strategies as creating multicationic centers have met with certain success in combating bacterial resistance.8,9 Similarly, covalently joining QACs with other disinfectants with different modes of action might also serve as an effective strategy in thwarting bacterial resistance against QACs. Recently, we reported the synthesis of a series of composite biocides with both quaternary ammonium (QA) and N-chloramine moieties.10,11 The potential of one composite biocide, [3-(3-chloro-4,4-dimethyl-2,5-dioxo-imidazolidin-1-yl)-propyl]-dimethyl-tetradecyl-ammonium chloride (compound 2), and its corresponding precursor, [3-(4,4-dimethyl-2,5-dioxo-imidazolidin-1-yl)-propyl]-dimethyl-tetradecyl-ammonium chloride, (compound 1), in inducing bacterial resistance was studied.12 We were unsuccessful in isolating Pseudomonas aeruginosa mutants with decreased susceptibility to compound 2 despite arduous efforts of achieving the goal with two different strategies: recovering mutants from the zone of clearing and isolating mutants through exposure to gradually increasing concentrations of biocides. Interestingly, the presence of N-chloramine moiety in compound 2 seems potentially overcome the risk of selecting mutants of P. aeruginosa with reduced susceptibility against the quaternary ammonium part. The antimicrobial action of N-chloramines occurs via different pathways, such as formation of chlorine cover and attack of the bacterial cell wall proteins, penetration into the bacterial cell followed by attacking the intracellular vital component. N-chloramines attack various targets in living cells, particularly, sulfur-containing amino acids, such as cysteine and methionine, interfere with hydrogen bonding in proteins and affect protein functions governed by their structures.13,14 On the other hand, the attraction between the positively charged nitrogen on QACs and negatively charged head groups of bacterial membrane phospholipids kicks off the antibacterial action of QACs. As soon as this bond forms, the long alkyl chain penetrates and integrates into the membrane core.4 When the concentration of QAC is high, the bacterial membrane destruction (or hole generation) occurs due to micelles formation and aggregation.15 Also, QACs can disrupt and denature the structure of proteins and enzymes.4 Covalently joining a QAC with an N-chloramine can give a composite biocide that could inactive bacteria through all of the above-mentioned modes of action. This can not only minimize the chance of bacterial resistance but also enhance the overall biocidal activity. However, disinfectants often encounter interfering substances, such as proteins and carbohydrates, while fulfilling their tasks. Specifically, N-chloramines can be quenched by proteins through direct contact.16 This challenge presents a need to develop new antimicrobials, which are more resistant to interfering substances, such as proteins. Thiol-containing proteins can reduce N-chloramines and therefore quench their antimicrobial activity. Also, active chlorine (Cl+) in N-chloramines can transfer to amine, amide, and imide groups in proteins. In chlorine-transfer reaction (transchlorination), the oxidation capacity is a good measure for the ability of N-chloramines to exchange chlorine with another amine, amide, and imide groups. The reactivity of the active chlorine in N-chloramines is in the following order: amines < amides < imides, and therefore stability of N-chloramines follows the reverse order.17 The above-mentioned compound 2 contains an amide-based N-chloramine. Given the following stability sequence of N-chloramines: amines > amides > imides, an amine-based N-chloramine composite biocide was designed in this study to present improved antibacterial activity in a high protein medium (HPM) consisting of 5% fetal bovine serum (FBS). The proposed mode of action of a composite biocide with both N-chloramine and QA moieties is that the composite biocide punches holes in bacterial membrane due to the QA moiety (mode of action of QACs) and therefore facilitates the penetration of the whole composite molecule into the bacteria, allowing its N-chloramine component to exert oxidative stress inside cells causing fast inactivation of the bacterial cell. We hypothesized that an amine-based N-chloramine can better escape the quenching action of proteins and therefore be preserved to exert oxidative stress inside a bacterial cell to result a fast kill after the whole biocide gets into the cell through the holes in the bacterial membrane generated by the QA moiety (Scheme 1). Specifically, a secondary amine-based N-chloramine was linked to a QA moiety with an alkyl chain of 12 or 14 methylene units via a triazole ring linkage, and the antibacterial activity of these compounds was evaluated against Gram-negative and Gram-positive bacteria. This strategy led us to antimicrobial agents, which may be better suited for applications with high protein loadings, such as disinfection of wounds or food contact surfaces (Scheme 1). Scheme 1 (a) Proposed Mode of Action of the Secondary Amine-Based N-Chloramine and Its Resistance against Protein Quench, (b) Proposed Mode of Action of the Amide-Based N-Chloramine and Its Quench by Proteins Results Chemistry Synthesis We previously demonstrated the antibacterial enhancement effect of covalently bonding long alkyl chain QAC moieties to an amide-based N-chloramine (compound 2 in Scheme 2).10 In this study, we joined an amine-based N-chloramine to QACs with an alkyl chain of 12 or 14 carbon atoms (6a, 6b in Scheme 2) with an intention to improve the antibacterial efficacy of the composite biocide in the presence of organic matters, such as proteins. [3-(4,4-Dimethyl-2,5-dioxo-imidazolidin-1-yl)-propyl]-dimethyl-tetradecyl-ammonium chloride (compound 1), dodecyl-dimethyl-{2-[4-(2,2,6,6-tetramethyl-piperidin-4-yloxymethyl)-[1,2,3]triazol-1-yl]-ethyl}-ammonium chloride (5a), dimethyl-tetradecyl-{2-[4-(2,2,6,6-tetramethyl-piperidin-4-yloxymethyl)-[1,2,3]triazol-1-yl]-ethyl}-ammonium chloride (5b) are the corresponding N-chloramine precursors (i.e., containing only a QA moiety) of the composite biocide, and benzyldodecyldimethylammonium chloride (BC) was used as a QAC control in antibacterial tests. The chemical reactions pathway is represented in Scheme 3. An overnight reaction between long chain alkyl bromides and 2-azido-N,N-dimethyl-ethanamine led us to azido-functionalized QACs 3a and 3b. 2,2,6,6-Tetramethyl-4-(prop-2-ynyloxy)piperidine 4 was prepared using the method reported by Barnes et al.18 from 2,2,6,6-tetramethylpiperidinol. Piperidinol derivative 4 and two QACs 3a/3b were then linked together via a triazole bridge to get 5a and 5b with Br− counterion. 5a, 5b and compound 1 with Br− counterion were passed through an ion-exchange resin to replace the counterion Br– with Cl– followed by performing the chlorination reaction using t-BuOCl to obtain {2-[4-(1-chloro-2,2,6,6-tetramethyl-piperidin-4-yloxymethyl)-[1,2,3]triazol-1-yl]-ethyl}-dodecyl-dimethyl-ammonium chloride (6a), {2-[4-(1-chloro-2,2,6-trimethyl-piperidin-4-yloxymethyl)-[1,2,3]triazol-1-yl]-ethyl}-dimethyl-tetradecyl-ammonium chloride (6b), and [3-(3-chloro-4,4-dimethyl-2,5-dioxo-imidazolidin-1-yl)-propyl]-dimethyl-tetradecyl-ammonium chloride (compound 2), respectively. Scheme 2 Structures of Biocides Used in This Research Scheme 3 Chemical Reaction Pathways (a) NaN3, KOH, H2O, reflux, overnight, (b) R-Br, acetonitrile, reflux, overnight, (c) propargyl bromide, NaH, tetrahydrofuran (THF), N2, 60 °C, 5 h, (d) CuSO4, Cu, MeOH/H2O, room temperature, overnight, (e) anion-exchange resin (Amberlite R IRA-900, Cl–) tert-butyl hypochlorite, acetone/H2O, 0 °C, 1 h. Ra: Alkyl substitution on the compounds bearing the letter “a” in the name and Rb: alkyl substitution on the compounds bearing the letter “b” in the name. Copper Analysis Cu(II) was used as a catalyst in the click reaction to link 2,2,6,6-tetramethyl-piperidin with the long chain QA moiety. It is known that copper ion possesses antibacterial activity.19 To avoid unwanted interference in the antibacterial activity of the new composite biocides, copper ion was removed by using a chelating column made of Sorbtech CR20-01 beads. Cu327.395 in 5a and 5b was quantified to be 0.13 and 0.075 ppm, respectively. The amount of Cu324.754 also was measured and appeared to be 0.127 and 0.068 ppm for 5a and 5b. The amount of copper left in each compound found to be much less than the minimum concentration of copper required for antibacterial activity, reported to be 40 ppm by Gyawali et al.20 Redox Titration We proceeded to study the stability of N-chloramines in the HPM (5% FBS in phosphate-buffered saline (PBS)) using a redox titration assay, and the results are presented in Figure 1. The active chlorine content of compound 2 (amide-based N-chloramine) dropped to 80% within the first 5 min of contact, and the chlorine content reached a plateau at around 60% after 60 min. On the other hand, both 6a and 6b compounds (amine-based N-chloramines) were significantly more stable (p < 0.05), and their chlorine content was still maintained at >80% after 60 min. The loss rate of active chlorine in HPM was significantly lower (p < 0.05) for the two amine-based N-chloramines than the amide-based N-chloramine in the first 5 min. These results showed that the N–Cl bond of secondary amines (6a and 6b) was less reactive with proteins in FBS than that of amides. Figure 1 Stability of N-chloramines 6a, 6b, compound 2 as reflected by the remaining percent of active chlorine ([Cl+]%) in the high protein medium (HPM). Studies were performed at 23 °C and pH 7.4. Values represent the mean ± standard deviation (SD). n = 3 for all three compounds. Antibacterial Evaluation of the Synthesized Biocides Table 1 lists the time needed (total kill (Tk)) for the antimicrobial agents to inactivate all of the chosen bacteria (total kill) in different solutions (PBS and HPM). The total kill is claimed when the bacteria level is equal to or below the 1.82 log detection limit and equivalent to 4.18 log bacterial reduction with the starting bacterial concentration of 106 CFU/mL. Table 1 Time Required to Reach the Total Kill (Tk) (4.18 log Reduction) for Various Compounds in PBS and HPM against Four Strains of Bacteriaa time to total kill (Tk) (min) bacterial strain medium 5a 6a 5b 6b compound 1 compound 2 BC methicillin-resistant Staphylococcus aureus (MRSA) (70065) PBS >60 3 60 5 10 3 10 HPM >60 10 >60 30 5 10 20 Escherichia coli (25922) PBS >60 3 >60 3 30 3 3 HPM >60 3 >60 5 30 20 5 multidrug-resistant (MDR) P. aeruginosa (73104) PBS >60 <1 <1 <1 5 5 5 HPM >60 3 60 20 60 30 10 wild-type P. aeruginosa (PA01) PBS >60 3 5 3 5 5 5 HPM >60 20 60 >60 >60 >60 30 a Initial concentration of compounds in PBS and HPM are 141.0 and 423.1 μM, respectively. n = 3 for all compounds. As can be found in Table 1, when the medium was changed from PBS to HPM, increase in Tk for 6a and 6b was 9 min on average, whereas Tk increase in the case of compound 2 was 16 min (excluding data of PA01). This means that the deterioration of antimicrobial activity in the case of piperidine (a secondary amine)-based N-chloramine compounds (6a, 6b) was less than compound 2. On the other hand, the performance of compound 2 in PBS was better than compound 1, but their activity was almost the same in HPM, which indicates that [Cl]+ on compound 2 is reduced in HPM, and compound 2 is partially converted back to compound 1, as revealed in Figure 1. Figures 2–5 present the quantitative antibacterial results over the contact time against four bacteria in various media. The composite biocides appeared to be more effective against the Gram-negative strains (E. coli and P. aeruginosa) than Gram-positive one (MRSA). Figure 2 Bacterial viability (log) as a function of contact time between biocides and methicillin-resistant S. aureus (MRSA) in the high protein medium (HPM, 5% FBS in PBS) and PBS. Studies were performed at 23 °C and pH 7.4. Values represent the mean ± SD. n = 3 for all of the compounds (the starting inoculum size is indicated in the bracket after each compound in the legend). Against MRSA (Figure 2), 6a and compound 2 showed the best performance in PBS. As expected, their activity deteriorated in HPM, but the extent of deterioration varied with antimicrobial agents. Among all biocides used in this study, compound 1 showed the best antibacterial activity in HPM. Biocides demonstrated different antibacterial behavior against Gram-negative bacteria E. coli and MDR P. aeruginosa. The activity of compound 2 against E. coli significantly dropped (p < 0.05) in HPM in comparison to PBS (Figure 3). However, 6a and 6b showed insignificant change in their activity (p > 0.05) in two media and were significantly faster in inactivating E. coli in HPM than compound 2 (Tk = 3, 5 versus 20 min). In the case of MDR P. aeruginosa (Figure 4), 6a and 6b illustrated significantly superior killing than others in the first 5 min (p < 0.05) in PBS. In HPM, 6a displayed the least decline in their activity among the chlorinated biocides. compound 1 presented a slow killing kinetics and the most severe deterioration of antimicrobial activity in HPM against MDR P. aeruginosa. Figure 3 Bacterial viability (log) as a function of contact time between biocides and E. coli in the high protein medium (HPM, 5% FBS in PBS) and PBS. Studies were performed at 23 °C and pH 7.4. Values represent the mean ± SD. n = 3 for all of the compounds (the starting inoculum size is indicated in the bracket after each compound in the legend). Figure 4 Bacterial viability (log) as a function of contact time between biocides and MDR P. aeruginosa in the high protein medium (HPM, 5% FBS in PBS) and PBS. Studies were performed at 23 °C and pH 7.4. Values represent the mean ± SD. n = 3 for all of the compounds (the starting inoculum size is indicated in the bracket after each compound in the legend). Wild-type P. aeruginosa was the least susceptible strain among the tested Gram-negative bacteria. All biocides, except 5a, were effective against this strain in PBS and reached total kill within 5 min of contact (Figure 5). However, only 6a (in 20 min), BC (30 min) and 5b (60 minutes) reached total kill of PA01 in HPM, and all other biocides failed to reach total kill in HPM in the tested time frame (60 min). 6a clearly stands out as the most potent biocide among all of the tested ones in both PBS and HPM when being challenged with the least susceptible bacterium. Figure 5 Bacterial viability (log) as a function of contact time between biocides and wild-type P. aeruginosa PA01 in the high protein medium (HPM, 5% FBS in PBS) and PBS. Studies were performed at 23 °C and pH 7.4. Values represent the mean ± SD. n = 3 for all of the compounds and results are significantly different (p < 0.05) (the starting inoculum size is indicated in the bracket after each compound in the legend). MRSA, P. aeruginosa, E. coli, and wild-type P. aeruginosa were also challenged by sodium hypochlorite in PBS at 141.0 and 282.1 μM (equivalent to 5 and 10 ppm [Cl+]) and in HPM at 423.1 and 864.3 μM (equivalent to 15 and 30 ppm [Cl+]) for 10, 20, 30, and 60 min. The initial bacterial concentrations for the above bacteria were 4.2 × 106, 4.8 × 106, 5.4 × 106, and 3.6 × 106 CFU/mL, respectively. No significant bacterial reduction was observed at the tested concentrations within 1 h of contact of all of the bacteria (p > 0.05) except for one case: 23% reduction of MRSA achieved by 282.1 μM sodium hypochlorite (equivalent to 10 ppm [Cl+]) in PBS within 60 min of contact (p = 0.027 < 0.05). Discussion Antimicrobial efficacy of multiple QACs compounds was tested against different bacteria in PBS and HPM. It was observed from Table 1 that in PBS, chlorinated compounds (6a, 6b, and compound 2) performed better than nonchlorinated ones (5a, 5b, and compound 1). This indicates that the introduction of either amide or amine-based N-chloramine boosts the antibacterial activity of the whole composite biocides. However, in HPM, the antimicrobial efficacy of all biocides (chlorinated and nonchlorinated) (except compound 1 against MRSA) deteriorated, and Tks of all of the tested biocides (except compound 1 against MRSA) increased in comparison to those obtained in PBS. In the case of QACs, this deterioration in activity might be the result of ionic and hydrophobic interactions between QAC and negatively charged peptides and hydrophobic domains on proteins, which resulted in binding of QAC molecules to proteins and deactivation of QAC.21 On the other hand, N-chloramines could transfer their active chlorine to proteins through direct contact or be reduced by sulfur-containing amino acids, such as cysteine and methionine in HPM.22 The reaction kinetics of N-chloramine with proteins varies from one compound to another. Active chlorine loading on the composite biocides was monitored as a function of time in PBS and HPM (Figure 1). It is confirmed that secondary amine-based composite biocides (6a and 6b) are more resistant to protein quench than their amide-based counterpart (compound 2). This correlates well with their superior activity against E. coli in HPM as compared with compound 2 (Table 1). But the result that Tks of 6a against the other three bacteria in HPM were significantly shorter (p < 0.05) than those of both 6b and compound 2 reveals the existence of other important factors affecting their antibacterial activity in HPM. The proper balance of hydrophobicity and hydrophilicity in the composite biocide 6a with a C12 alkyl chain might contribute to more effective membrane damage (mode of action of QACs) and be one of the other factors. One more possible factor could be associated with less protein binding of a QAC with a C12 alkyl chain (6a) via hydrophobic interaction, as reported by Jono and co-workers.21 They found that C14-benzalkonium chloride was deactivated more than C12-benzalkonium chloride in the presence of proteins. This led to less quench of the biocide by proteins in HPM through the deactivation mechanism of QAC: electrostatic and hydrophobic interactions. Gilbert and Moore15 have shown that antibacterial activity of QACs is a function of their structure, alkyl chain length, and strain of bacteria. For example, to be effective against Gram-positive bacteria, the alkyl chain of QACs should consist of 12–14 carbon atoms, however, in the case of Gram-negative bacteria, the number of carbon atoms in the alkyl chain should be between 14 and 16. N-chloramines are more hydrophobic than their amine/amide precursors, and the added hydrophobicity in N-chloramine/QAC composite biocides might move the optimum alkyl chain length against Gram-negative bacteria from 14–16 to 12. In antibacterial test against Gram-negative bacteria E. coli and P. aeruginosa, the antibacterial efficacy of 6a and 6b appeared to be less compromised by HPM than compound 2, whereas compound 2 and 6a showed similar changes in antibacterial kinetics in two media (PBS and HPM) against MRSA. These results still correlate with the stability of N-chloramine in HPM and can be explained using our hypothesized mechanism. Proteins in HPM neutralize the amine-based N-chloramine at a slower pace than the amide-based N-chloramine so that the amine-based N-chloramine is preserved to exert oxidative stress inside a bacterial cell after the whole molecule diffuses into the cell through the holes in the bacterial membrane due to the mode of action of the QA moiety. However, to what degree this advantage of better preserved amine-based N-chloramine can be manifested is also dependent on how effectively bacterial cells can adsorb the biocides, which is the first step of QACs’ action of disrupting bacterial membranes. E. coli has a much higher number density of negative charges (0.145 m–3 at pH 7) than S. aureus (0.025 m–3 at pH 7),23 which might be the reason why this advantage of better preserved amine-based N-chloramine is not clearly shown in the challenge test against MRSA in HPM (Figure 2). The thick cell wall of MRSA might also play a role by delaying the interaction of the positively charged biocides with bacterial cell membrane. As can be seen from Figures 2 and 3, BC showed a faster killing kinetics against E. coli than S. aureus. 5a and 5b were very ineffective against E. coli even in PBS (Figure 3). One possible reason is that the piperidine functional group is very basic (pKa of its conjugate acid is around 11)14 and become protonated in pH 7.4 PBS, greatly increasing the hydrophilicity of the whole molecules of 5a and 5b. QACs act on bacterial membranes by inserting their hydrophobic tails into the hydrophobic membrane core. The enhanced hydrophilicity due to the protonated piperidine functional group might impede this interaction, hence compromising the antibacterial potencies of both 5a and 5b. Chlorination of the synthesized QACs boosts their antibacterial activity via two different mechanisms. First, chlorine changes the hydrophilicity of QAC compounds by transforming the positively charged piperidine functional group in biological pH (7.4) to a neutral moiety (decreased basicity due to the presence of chlorine). Therefore, the hydrophilicity of the compounds drops, allowing QACs to better interdigitate into the bacterial membrane and negatively impact on its osmoregulatory and physiological functions.24 Secondly, active chlorine also attacks targets in bacterial cells. As reflected from the antibacterial results of sodium hypochlorite, active chlorine [Cl+] alone at the tested concentrations within the tested time frame (1 h) in either PBS or HPM is not effective in killing those bacteria. This indicates again that the N-chloramine moiety together with the QA moiety attacks bacterial cells in a potentially synergistic manner. Conclusions Nowadays, prevention of bacterial contaminations in food industries has become difficult due to long food production line, large production volume of food, and antimicrobial resistance.25 Also, the number of effective antimicrobial agents with a good performance in high protein containing environment is limited.26 In this study, two amine-based N-chloramine/QAC composite biocides were synthesized. These synthesized biocides were challenged with various bacterial strains, including Gram-positive and Gram-negative bacteria MRSA, E. coli, MDR P. aeruginosa, and wild-type P. aeruginosa (PA01) in both PBS and HPM. The results indicate that when a secondary amine as opposed of an amide-based N-chloramine is used, the loss of active chlorine in high protein-loaded fluids decreases, which results in less deterioration of antibacterial activity. The composite biocide 6a demonstrated the best antibacterial efficacy in both PBS and HPM among all biocides studied in this study. The relatively better antimicrobial activity of the amine-based N-chloramine (6a) in HPM is due to the relatively high stability of the active chlorine as compared to the amide-based N-chloramine. Transchlorination between the amine-based N-chloramines (6a, 6b) and proteins in the medium occurs at a slow rate as compared to the amide-based N-chloramine (compound 2). The amine-based N-chloramine then has a better chance to exert oxidative stress inside a bacterial cell after the whole molecule finds its way into the cell due to the membrane disruption action of the QA moiety. Interestingly, the amine-based composite biocide with a QAC unit of C12 alkyl chain (6a) is more potent in the protein medium than its counterpart with a QAC unit of C14 alkyl chain (6b). This is opposite to the general order of antibacterial activity of QACs with different alkyl chain lengths: C14 > C12 previously reported18,27 due to the presence of the piperidine-based N-chloramine moiety in the composite biocide. These interesting findings bring us one step closer to the design and synthesis of more potentially non-resistance-inducing biocides potent in real world where organic matters might be inevitable. Experimental Section Materials All of the reagents and solvents, which were analytical grades and no further purification been used, were purchased from suppliers namely, Sigma, Fisher, and VWR. NMR spectra were performed using Bruker Avance 300 MHz NMR spectrometer at room temperature and in 5 mm NMR tubes. High-resolution mass measurements were recorded by AB SCIEX Triple TOF 5600+ (ESI-MS), Concord, ON with the direct infusion method. Multidrug-resistant (MDR) P. aeruginosa #73104 and community-associated-MRSA #70065 and wild-type P. aeruginosa (PA01), which were obtained from the CANWARD (Canadian Ward Surveillance) study assessing antimicrobial resistance in Canadian hospitals, http://www.canr.ca. Escherichia coli ATCC 25922 was sourced from ATCC. Synthesis and Analysis (2-Azido-ethyl)-dimethyl-amine (2) (Scheme 3) 2-Chloro-N,N-dimethylethylamine hydrochloride (1) (20 g, 139 mmol) was dissolved in 50 mL of water, and then 45.13 (0.69 mol, 5 equiv) of sodium azide was added to the solution and reaction continued overnight at reflux. Then, 7g of potassium hydroxide added to the mixture followed by extraction with dichloromethane (DCM) (3 × 100 mL). After solvent evaporation, 10.3 g (95 mmol, 65% yield) of a yellow liquid was achieved. 1H NMR (CDCl3, 300 Hz) δ [ppm]: 3.35 (t, J = 6.1 Hz, 2H: N–N=NCH2−), 2.5 (t, J = 6.2 Hz, 2H: −CH2N), 2.27 (s, 6H: −N(CH3)2). 13C NMR (CDCl3, 75 Hz) δ [ppm]: 46.6, 46.6, 48.7, 65.8; HRMS (ESI-TOF) m/z: [M + H]+ calculated for C4H10N4+, 114.0905; found: 114.0911. 3a, 3b (Scheme 3) 2-Azido-N,N-dimethylethanamine (2) (5.0 g, 44 mmol) was dissolved in 50 mL of acetonitrile, and then 48.4 mmol of 1-bromoalkane, 1-bromotetradecane (3a), or 1-bromotetradecane (3b) was added to the solutions and the reactions continued overnight at reflux. After solvent evaporation, compounds were dissolved in 5 mL of methanol followed by participation in 150 mL of 50:50 ethyl acetate/hexane 3a (11.20 g, 35.11 mmol, 79% yield) and 3b (11.6 g, 33.4 mmol, 76% yield) of white solids were achieved. 3a: 1H NMR (D2O, 300 Hz) δ [ppm]: 4.04 (t, J = 6.2 Hz, 2H: N–N=NCH2−), 3.62 (t, J = 6.2 Hz, 2H: −CH2N+), 3.43 (t, J = 7.8 Hz, 2H: N+CH2−), 3.22 (s, 6H: (CH3)2N+), 1.73–1.9 (m, 2H: −CH2CH2), 1.23–1.48 (m, 18H: −(CH2)9CH3), 0.91 (t, J = 6.3 Hz, 3H: −CH3). 13C NMR (D2O, 75 Hz) δ [ppm]: 13.8, 22.6, 26.2, 29.4, 29.5, 29.7, 29.8, 31.9, 44.9, 51.9, 61.6, 64.5; HRMS (ESI-TOF) m/z: [M – Br]+ calculated for C16H35N4+, 283.2856; found: 283.2864. 3b: 1H NMR (D2O, 300 Hz) δ [ppm]: 4.04 (t, J = 6.2 Hz, 2H: N–N=NCH2−), 3.62 (t, J = 6.2 Hz, 2H: −CH2N+), 3.43 (t, J = 7.5 Hz, 2H: N+CH2−), 3.22 (s, 6H: (CH3)2N+), 1.73–1.9 (m, 2H: −CH2CH2), 1.23–1.48 (m, 22H: −(CH2)11CH3), 0.91 (t, 3H: −CH3). 13C NMR (D2O, 75 Hz) δ [ppm]: 13.9, 22.7, 26.2, 29.2, 29.7, 29.9, 30.1, 32.0, 45.0, 52.1, 61.5, 64.3; HRMS (ESI-TOF) m/z: [M – Br]+ calculated for C18H39N4+, 311.3169; found: 311.3174. 2,2,6,6-tetramethyl-4-(prop-2-ynyloxy)piperidine (4) (Scheme 3) 2,2,6,6-tetramethylpiperidin-4-ol (9.0 g, 57 mmol) was added to 100 mL of anhydrous THF and kept under a nitrogen atmosphere for 30 min, stirring at room temperature, followed by addition of 2.26 g (57.0 mmol) of NaH (60%). After 30 min of mixing, the flask sealed with a rubber stopper and transferred into a 60 °C oil bath where 6.356 mL (57.00 mmol) of propargyl bromide (80%) was added to the mixture and the reaction continued overnight at 60 °C. Afterward, solid settlements were removed by filtration and THF evaporated using rotary evaporator. After solvent removal, the compound dissolved in 50 mL of 1N HCl solution and was washed with 3 × 50 mL of DCM. Then, sodium hydroxide was used to increase the pH to 10 (in an ice bath). Then, the aqueous solution was washed with DCM (3 × 50 mL). The organic layer was dried on sodium sulfate, and the solvent was evaporated. 5.10 g (26.2 mmol, 46% yield) of yellow solid was achieved. 1H NMR (CDCl3, 300 Hz) δ [ppm]: 4.17 (s, 2H: OCH2−), 3.83–3.96 (m, 1H: −CHO), 2.39 (s, 1H: −CH), 1.97–1.96 (d, 1H), 1.93–1.92 (d, J = 4 Hz, 2H: (CHCH)2), 1.17 (s, 6H: NH(C(CH3CH3))2), 1.12 (s, 6H: −(CH3CH3)2), 0.99 (t, J = 11.8 Hz, 2H: (CHCH)2−). 13C NMR (CDCl3, 75 Hz) δ [ppm]: 29.0, 35.0, 44.5, 51.6, 54.9, 71.9, 73.9; HRMS (ESI-TOF) m/z: [M + H]+ calculated for C12H22NO+, 196.1696; found: 196.1716. Click Reaction, 5a, 5b (Scheme 3) 4: (2.91 g, 15.0 mmol) was dissolved in 15 mL of MeOH. Then, 12.5 mmol of azide 3a (4.62 g) or 3b (4.92 g) was added to the solution. Afterward, 0.312 g (1.95 mmol, 10%) of CuSO4 was dissolved in 2 mL of water and added to the solutions. Then, 2.38 g (37.4 mmol) of Cu was added to the solutions and reactions continued overnight at room temperature. After filtration to remove the copper particles, the solution passed through Sorbtech CR20-01 to remove Cu(I) and the anion-exchange resin to replace Br− with Cl−. 5a: 1H NMR (D2O, 300 Hz) δ [ppm]: 8.22 (s, 1H: −CHN–N=N), 5.057 (s, 2H: OCH2−), 4.72 (t, J = 6.1 Hz, 2H: −CH2), 4.01 (t, J = 6 Hz, 2H: −CH2), 4.09–4.24 (m, 1H: −CHO), 3.31 (t, J = 7.4 Hz, 2H: N+CH2−), 3.18 (s, 6H: N+(CH3)2), 2.30 (d, J = 3.5 Hz, 1H: −CHCH), 2.25 (d, J = 3.5 Hz, 1H: −CHCH), 1.61 (m, 2H: −CH2CH2), 1.50 (s, 6H: C(CH3CH3)2), 1.48 (s, 6H: C(CH3CH3)2), 1.23–1.38 (m, 18H: −(CH2)9CH3), 1.18 (t, J = 11.1 Hz, 2H, (CHCH)2−), 0.88 (t, J = 5.5, 3H: −CH3). 13C NMR (D2O, 75 Hz) δ [ppm]: 13.8, 22.5, 26.3, 28.7, 29.4, 29.5, 30.3, 31.8, 41.9, 44.1, 51.6, 54.5, 61.4, 64.4, 71.7, 125.4, 145.1; HRMS (ESI-TOF) m/z: [M – Cl]+ calculated for C28H56N5O+, 478.4479; found: 478.4482. Purity based on QNMR result (internal standard: maleic acid) > 99%. 5b: 1H NMR (D2O, 300 Hz) δ [ppm]: 8.22 (s, 1H: −CHN–N=N), 5.057 (s, 2H: OCH2−), 4.72 (t, J = 6.1 Hz, 2H: −CH2), 4.01 (t, J = 6 Hz, 2H: −CH2), 4.09–4.24 (m, 1H: −CHO), 3.31 (t, J = 7.4 Hz, 2H: N+CH2−), 3.18 (s, 6H: N+(CH3)2), 2.30 (d, J = 3.5 Hz, 1H: −CHCH), 2.25 (d, J = 3.5 Hz, 1H: −CHCH), 1.61 (m, 2H: −CH2CH2), 1.50 (s, 6H: C(CH3CH3)2), 1.48 (s, 6H: C(CH3CH3)2), 1.23–1.38 (m, 22H, −(CH2)11CH3), 1.18 (t, 2H, J = 11.1 Hz, 2H, (CHCH)2−), 0.88 (t, J = 5.5, 3H: −CH3). 13C NMR (D2O, 75 Hz) δ [ppm]: 14.0, 22.7, 27.2, 29.1, 29.5, 29.7, 30.2, 32.0, 42.9, 43.1, 51.9, 52.8, 60.8, 64.13, 72.6, 125.4, 145.4; HRMS (ESI-TOF) m/z: [M – Cl]+ calculated for C30H60N5O+, 506.4792; found: 506.4788. Purity based on QNMR result (internal standard: maleic acid) > 97%. Chlorination of Compounds 5a and 5b (Scheme 3) 5a or 5b (500 mg) was dissolved in 10 mL of water/acetone (2:8 by volume), then 3 equiv of tert-butyl hypochlorite (450 μL) was added to vials, which completely wrapped with aluminum foil at 0 °C. Reactions continued for 60 min. Afterward, air flow was employed to remove acetone and unreacted tert-butyl hypochlorite, and water was removed using vacuum. 6a: 1H NMR (CDCl3, 300 Hz) δ [ppm]: 8.53 (s, 1H: −CHN–N=N), 5.28 (s, 2H: OCH2−), 4.55 (t, J = 6.3 Hz, 2H: −CH2), 4.36 (t, J = 6.4 Hz, 2H: −CH2), 3.68–3.81 (m, 1H: −CHO), 3.4 (t J = 7.1 Hz, 2H: N+CH2−), 3.34 (s, 6H: N+(CH3)2), 2.1 (d, J = 3.1 Hz, 1H: −CHCH), 2.05 (d, J = 3.1 Hz, 1H: CHCH), 1.52 (m, 2H: −CH2CH2), 1.23–1.38 (m, 32H: −(CH2)9CH3, C(CH3)2 and −CHCH), 0.88 (t, J = 6.5, 3H: −CH3). 13C NMR (CDCl3, 75 Hz) δ [ppm]: 14.1, 22.5, 26.3, 29.1, 29.3, 29.5, 30.9, 31.9, 33.2, 44.2, 45.6, 51.5, 62.6, 70.4, 77.1, 125.1, 145.8; HRMS (ESI-TOF) m/z: [M – Cl]+ calculated for C28H55ClN5O+, 512.4090; found: 512.4080. Purity based on QNMR result (internal standard: maleic acid) > 98%. 6b: 1H NMR (CDCl3, 300 Hz) δ [ppm]: 8.5 (s, 1H: −CHN–N=N), 5.24 (s, 2H: OCH2−), 4.58 (t, J = 6.4 Hz, 2H: −CH2), 4.32 (t, J = 6.4 Hz, 2H: −CH2), 3.7–3.83 (m, 1H: −CHO), 3.4 (t, J = 7.6 Hz, 2H: N+CH2−), 3.33 (s, 6H: N+(CH3)2), 2.10 (d, J = 3.3 Hz, 1H: −CHCH), 2.05 (d, J = 3.3 Hz, 1H: CHCH), 1.54 (m, 2H: −CH2CH2), 1.11–1.38 (m, 36H: −(CH2)11CH3, C(CH3)2 and −CHCH), 0.88 (t, J = 6.5, 3H: −CH3). 13C NMR (CDCl3, 75 Hz) δ [ppm]: 14.1, 22.5, 26.2, 29.2, 29.4, 29.6, 29.7, 31.9, 33.3, 44.4, 45.6, 51.6, 62.7, 70.4, 77.1, 125.1, 145.8; HRMS (ESI-TOF) m/z: [M – Cl]+ calculated for C30H59ClN5O+, 540.4403; found: 540.4386. Purity based on QNMR result (internal standard: maleic acid) > 95%. Copper Analysis Antibacterial effect of copper has been reported in many studies.28 Copper has been used as the catalyst for click reaction, and this copper can interfere the antibacterial activity of compounds. Therefore, copper removal to the noneffective concentration was crucial. To this end, Sorbtech CR20-01 beads have been used. Copper concentration in compounds was measured using ICP optical emission spectrometer, Varian 725-ES. Redox Titration Redox titration is a titration technique in which a redox reaction takes place between an analyte and a titrant.19 A redox indicator and conductometer were used during the titration. In this study, redox titration was carried on, to track the chlorine lost in high protein-loaded fluid (5% FBS) for 6a, 6b, and compound 2. To this end, 0.25 mmol of each compound dissolved in 10 mL of Milli-Q water to have an aqueous solution of each compound. Afterward, 50 μL of fetal bovine serum (FBS) was added to 950 μL of the prepared solution and mix them on an orbital shaker for different time frames (1, 5, 20, 60, 90, and 120 min). Mixture (100 μL) was added to a mixture of 30 mL of Milli-Q water, 2 mL of 5% acetic acid buffer solution, 1 g (6 mmol) potassium iodide, and five drops of freshly prepared 1% starch solution to get a solution with a dark purple color. Sodium thiosulfate standard solution (0.001 N) was used to titrate (dropwise addition) the mixture using buret. The titration stopped when the solution color turned clear, and the chlorine content was calculated as below where Mw, m, and Vt stand for molecular weight (g/mol), mass of the compound (g), and buret reading (mL), respectively. Quantitative Antimicrobial Assay (in PBS and High Protein Medium) A bacterial suspension was prepared at a concentration of 108 CFU/mL in phosphate-buffered saline (PBS, 0.1 M, pH 7.4) using 0.5 McFarland standard. After 100 times dilution, 20 μL of diluted suspension was added to 60 mL of different broths based on the bacterial strains, Tryptone Soy Broth in the case of MRSA and Mueller Hinton Broth for P. aeruginosa and E. coli, followed by 18 h incubation at 37 °C. After incubation, 50 μL of bacterial suspension was added to a solution of 5 [Cl]+ ppm (141.0 μM) to 20 mL of PBS (0.1 M, pH 7.4) and 15 [Cl]+ ppm (423.1 μM) to a solution of 5% FBS in PBS and incubated on the orbital mixer at 37 °C. At each time point (1, 3, 5, 10, 20, 30, 60 min), 150 μL of mixture was added to 150 μL of neutralizer solution (PBS buffer consisting of 1.4% (w/v) l-α-phosphoatidylcholine, 10% (w/v) Tween 80, 1% peptone, and 0.316% sodium thiosulfate) to deactivate antimicrobial agent. Bacterial suspension (30 μL) and its serial dilutions were plated on Tryptone Soya Agar plates and incubated for 24 h, followed by colony counting. To eliminate the effect of bacterial growth in HPM and/or bacterial death due to malnutrition in PBS at each time point, there was a negative control. The bacterial log reduction was calculated as following Lecithin, tween, and peptone have been proven to be effective in quenching QACs.11,29,30 We have also run adequate inactivation tests to confirm thorough quenching of our biocides at the tested concentrations (141.0 and 423.1 μM). Specially, we plated bacterial suspensions of one series of antibacterial experiments 30 and 90 min after mixing with the neutralizer, and no further decrease of bacterial concentration was observed from the enumeration results, indicating thorough quench of all biocides. To compare the antibacterial efficacy of the synthesized biocides with free chlorine, an antibacterial test was also conducted against methicillin-resistant S. aureus (MRSA), P. aeruginosa, E. coli, and wild-type P. aeruginosa using sodium hypochlorite at two different concentrations (5 and 10 ppm [Cl+] (141.0 and 282.1 μM)) in PBS and two concentrations (15 and 30 ppm [Cl+] (423.1 and 864.3 μM)) in HPM. The authors declare no competing financial interest. Acknowledgments The authors are grateful for the financial support from the Collaborative Health Research Project (CHRP) operating grant (Grant no.: CHRP 413713-2012) and the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (Grant no.: RGPIN/04922-2014). ==== Refs References Neill J. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations . In Review on Antimicrobial Resistance ; O’Neill J. , Ed.; Wellcome Trust : London , 2014 . Davin-Regli A. ; Pagès J. 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PMC006xxxxxx/PMC6644431.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145782710.1021/acsomega.7b00429ArticleBinding and Selectivity of Essential Amino Acid Guests to the Inverted Cucurbit[7]uril Host Gao Zhong-Zheng †Kan Jing-Lan ‡Chen Li-Xia †Bai Dong †Wang Hai-Yan †Tao Zhu †Xiao Xin †† Key Laboratory of Macrocyclic and Supramolecular Chemistry of Guizhou Province, Guizhou University, Guiyang 550025, P. R. China‡ College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, P. R. China E-mail: gyhxxiaoxin@163.com (X.X.).08 09 2017 30 09 2017 2 9 5633 5640 16 06 2017 24 08 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Interactions between inverted cucurbit[7]uril (iQ[7]) and essential amino acids have been studied at pH = 7.0 by 1H NMR spectroscopy, electronic absorption spectroscopy, isothermal titration calorimetry, and mass spectrometry. The interactions can be divided into three binding types at pH = 7.0. Experimental results from the present study showed that the host displays a strong binding to the aromatic amino acids, Trp and Phe, and the guests of Lys, Arg, and His lie outside the cavity portal of the host. Meanwhile, the alkyl moieties of the guests Met, Leu, and Ile were accommodated within the cavity of iQ[7], but there was no significant interaction between iQ[7] and Thr or Val. The complexation behavior of iQ[7] with essential amino acids was explored at pH = 3, and the binding of Lys, Arg, and His revealed an unexpected behavior, with their side chains located in the cavity of iQ[7], whereas those of the aromatic Trp and Phe were deeper within the iQ[7] cavity. The alkyl side chains of the guests Met, Leu, Ile, Thr, and Val were also located inside the iQ[7] cavity and formed the host–guest complexes. document-id-old-9ao7b00429document-id-new-14ao-2017-00429sccc-price ==== Body Introduction In recent years, molecular recognition in biomolecules has attracted considerable attention,1−4 especially regarding amino acids and aromatic peptides. As the building blocks of proteins, amino acids are essential components of life processes and play critical roles in metabolism growth and development. A lack of any essential amino acids can lead to an abnormal physiological function and eventually to diseases such as nutritional imbalances, Alzheimer’s, and pancreatitis. Research on amino acid and aromatic peptide complexation and recognition by cyclodextrins,5 calixarenes,6 pillararenes,7 and other macrocyclic receptors has been reported. The novel family of macrocycles, cucurbit[n]urils (Q[n]s, where n = 5–8, 10, and 13–15),8−12 can selectively accommodate and interact with various organic molecules. Several examples of interactions of amino acids with different members of the cucurbit[n]urils (Q[n]s)13−18 have been described. Thuéry built chiral assemblies l-Cys-lanthanide–Q[6] complexes using l-cysteine as a chiral linker;19a Gamal-Eldin’s work on the selective molecular recognition of methylated lysines and arginines by Q[7] had been reported;19b and supramolecular structures of tryptophan with Q[6] showed a very interesting and peculiar structure.20 Kim et al. explored the specific high-affinity binding of Q[7] to amino acids (Lys, Arg, and His and Phe, Tyr, and Trp) in water.21 Urbach and co-workers observed the 1:1 binding of phenylalanine derivatives to Q[7] and the binding of aromatic amino acids to Q[8].22 Nau and co-workers did some work of monitoring of amino acids based on self-assembly.23 Scherman’s group reported the first example of the recognition of a selected amino acid epitope within a protein by Q[8] complexation.24 Isaacs’s group obtained ‘‘turn-on’’ fluorescent sensors for amino acids using fluorescent cucurbituril derivatives.25 In 2006 and 2016, our group reported supramolecular receptors for the detection and recognition of amino acids by TMeQ[6],26 Q[7, 8],27 and twisted cucurbit[14]uril.28 This area has seen a large expansion in the number of publications on the binding of amino acids. In 2005, Isaacs and Kim reported the isolation, characterization, and recognition properties of inverted cucurbit[n]urils (iQ[n]s, where n = 6 and 7),29 and some related properties of iQ[n]s were also described.30−32 However, to date, few reports have focused on the newest member of the Q[n]s, iQ[7]. Our group studied the coordination chemistry of an iQ[7] with a series of metal ions.33−35 To investigate the host–guest chemistry of the iQ[7], we investigated the binding interaction of an inverted cucurbit[7]uril with α,ω-alkyldiammonium guests36 and 4,4′-bipyridine derivatives.37,38 In a continuation of this research, herein, we explored the binding of essential amino acids to the iQ[7] host to expand our knowledge of the supermolecular chemistry of iQ[n]s. We studied the binding interactions of iQ[7] with 10 essential amino acids (Scheme 1) in buffered solution by 1H NMR spectroscopy, isothermal titration calorimetry (ITC), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Meanwhile, we also characterized the interaction between iQ[7] and basic amino acids at pD = 3 by 1H NMR spectroscopy. The experimental results provide new insights into the interactions of amino acids and iQ[7]. Scheme 1 Structures of iQ[7] and Essential Amino Acids Investigated in This Work Results and Discussion NMR Spectroscopy The complexation of iQ[7] with essential l-α-amino acids was first examined by 1H NMR spectroscopy of host–guest mixtures. Figure 1 shows the 1H NMR spectra of Lys recorded in the absence and presence of approximately 1.0 equiv of the host in D2O, and D2O was adjusted to pD = 7.0 with sodium phosphate. In the presence of iQ[7], the peaks for all methylene protons of Lys display a substantial downfield shift compared with those of the free guest, indicating that all methylene protons interact with the carbonyl groups of iQ[7]. It has been reported that the 1H NMR peaks of the guest protons inside the low-polarizability cavity of Q[n] shift upfield, and interactions with the carbonyl oxygen molecules of Q[n] result in downfield shifts owing to the deshielding of the protons.21,39,40 Additionally, the signal corresponding to the α-proton of the amino acid (Hα) is shifted downfield (Δδ = 0.37 ppm). This indicates that the guest is located just outside the portal of the host. Figure 1 1H NMR spectra (400 MHz, pD = 7.0) of iQ[7] in the absence (A) and presence of 0.15 (B), 0.40 (C), 0.70 (D), 0.82 (E), and 1.02 (F) equiv of Lys and free guest Lys (G) at 20 °C. Similar iQ[7] complexation-induced 1H NMR changes (downfield shifts and peak splitting) were observed for another two essential amino acids, Arg and His, indicating similar binding modes. The results of titration 1H NMR spectroscopy obtained using a fixed amount of iQ[7] and various equivalents of Arg are shown in Figure S1. The side-chain proton (Hβ and Hγ) signals for Arg showed a downfield shift of 0.04 and 0.18 ppm, respectively, and the signal for the proton Hα showed a downfield shift of 0.29 ppm when the iQ[7]–Arg ratio reached 1:1.02. As shown in Figure S2, the imidazole proton signal of His is shifted downfield compared to that of the free guest, as are the peaks for methylene protons Hβ and Hα. This may reflect the fact that Arg and His lie outside the portal of the host, unlike the interaction of Q[7] with Lys and Arg.21 The possible reason leading to the difference lies in the smaller cavity of iQ[7] which contains a single inverted glycoluril unit. The binding behavior of iQ[7] with the aromatic amino acids Trp and Phe clearly departs from our observations with Lys, Arg, and His. As shown in Figure 2, all aromatic protons (Hγ–Hη) of Trp move upfield considerably and are broadened compared with those of the free guest as a consequence of inclusion-induced shielding effects. This indicated that they were averaged signals of the free and bound guest molecules due to a rapid exchange rate of binding and release on the NMR time scale. Meanwhile, one of the CH2 protons of Trp is moved upfield, which indicates that it is located inside the cavity. By contrast, the proton Hα of Trp moved downfield by 0.07 ppm when the iQ[7]–Trp ratio reached 1:1.05, which indicates that it is located outside the cavity. It is noted that the two methine protons (H1 on Scheme 1) of the inverted glycoluril unit in the cavity of iQ[7] were shifted upfield with increasing amounts of Trp, suggesting that Trp also interacts with these methine protons (H1) in the cavity of iQ[7]. These observations suggest that the CH2 group and indole moiety of the Trp guest are encapsulated in the cavity of the iQ[7] host. This is also the case for the binding interactions of iQ[7] with Phe; as shown in Figure S3, the aromatic ring protons of Phe are clearly subject to upfield shifts upon binding to iQ[7]. Figure 2 1H NMR spectra (400 MHz, pD = 7.0) of iQ[7] in the absence (A) and presence of 0.18 (B), 0.40 (C), 0.64 (D), 0.85 (E), and 1.05 (F) equiv of Trp and free guest Trp (G) at 20 °C. Meanwhile, the signal corresponding to the α-proton of the bound amino acid (Hα) is shifted obviously downfield (Δδ = 0.13 ppm), similar to the binding interactions of iQ[7] with Trp. This result is consistent with the binding behavior of Q[7] with aromatic amino acids.21 The interaction of iQ[7] with Ile could be conveniently monitored by 1H NMR. A slight upfield shift of the signals of the protons of the alkyl chain (Hβ–Hδ) and a slight downfield shift of the signal of the Hα were observed upon the addition of iQ[7] (Figure 3), suggesting that there is a weak interaction between iQ[7] and Ile. Similar 1H NMR spectra for the interaction of iQ[7] and the guests Leu and Met were also recorded (Figures S4 and S5). This indicates that the alkyl moiety of the guests was accommodated within the cavity of iQ[7] but the interaction is weak. Meanwhile, no obvious shift was observed when mixing the host with the essential amino acids, Val or Thr (Figures S6 and S7). Figure 3 1H NMR spectra (400 MHz, pD = 7.0) of iQ[7] in the absence (A) and presence of 0.45 (B), 0.68 (C), 0.85 (D), and 1.05 (E) equiv of Ile and free guest Ile (F) at 20 °C. To compare the binding patterns of the essential amino acids with iQ[7] under different conditions, we also investigated the interactions between the protonated forms of the amino acids and iQ[7] by 1H NMR titration at pD = 3. We first studied the binding behavior of three basic amino acids with iQ[7]. A trace amount of the acid was added together with iQ[7] to ensure the formation of the complexes. With 1.0 equiv of Lys, Arg, or His, the proton of the side chains of these amino acids showed a significant upfield displacement (Figures 4, S8, and S9), indicating the formation of complexes. Lys, Arg, and His showed unexpected changes at pD = 3 compared with D2O because 1H NMR experiments confirmed that these amino acids formed inclusion complexes with iQ[7]. This change in behavior is likely due to a change in the protonation state.21 We concluded that the side chains of Lys, Arg, and His were predominantly located in a shielded environment.21,39,40 This is because the binding to iQ[7] causes His, Lys, and Arg to favor the fully protonated state. As Kim reported before, paying the thermodynamic penalty for the protonation of the carboxylate group is favored over the binding to iQ[7] as the deprotonated form. Figure 4 1H NMR spectra (400 MHz, pD = 3) of iQ[7] in the absence (A) and presence of 0.35 (B), 0.75 (C), and 1.05 (D) equiv of Lys and free guest Lys (E) at 20 °C. For Trp and Phe, the amino groups of the guests remained protonated at pD = 3. Next, we investigated the binding interactions of the aromatic amino acids Trp and Phe with iQ[7]. The interaction of Trp with iQ[7] was studied first (Figure 5), and the results showed that the proton of the indole moiety was shifted upfield, suggesting that the indole moiety of Trp was located inside the cavity, as concluded previously. Upon comparing the differences, it became apparent that the protons Hα and Hβ of Trp were shifted upfield, indicating that the methylene and methine groups of the guest were also encapsulated in the iQ[7] cavity. This conclusion was also reached for Phe. As shown in Figure S10, all protons of the benzyl moiety underwent a considerable upfield shift; meanwhile, the Hα protons also experienced a small upfield shift. These iQ[7]-induced shift patterns suggested that the benzene ring and alkyl chain moiety of Phe were situated inside the iQ[7] cavity. Overall, the results suggest that Trp and Phe guests were buried deeper within the iQ[7] cavity. This indicates that the aromatic amino acids can maintain their binding affinities reasonably well even if their carboxyl groups are deprotonated, which is consistent with the results of the binding of Q[7] with aromatic amino acids. Figure 5 1H NMR spectra (400 MHz, pD = 3) of iQ[7] in the absence (A) and presence of 0.35 (B), 0.70 (C), 0.85 (D), and 1.05 (E) equiv of Trp and free guest Trp (F) at 20 °C. The host–guest interactions of iQ[7] with charged amino acids (Ile, Leu, Met, Val, and Thr) at pD = 3 were also investigated by 1H NMR spectroscopy. The 1H NMR spectra of Ile and Ile bound to iQ[7] are shown in Figure 6. All protons of the alkyl side chain of Ile were clearly shifted upfield by between 0.23 and 0.41 ppm, indicating burial within the iQ[7] cavity. Similarly, when the amino acids were added to iQ[7] at pD = 3, the alkyl side-chain protons of Leu, Met, Val, and Thr experienced a significant upfield shift, suggesting a deep inclusion in the cavity of iQ[7] due to the formation of inclusion complexes (Figures S11–S14). Upon comparing with nuclear magnetic titration experiments in D2O, it became apparent that the alkyl side-chain protons of the guests were shifted upfield, confirming that these four amino acids were more likely to form inclusion complexes at pD = 3. Major binding differences were observed (using NMR spectroscopy) between pD = 7 and 3, which can be explained as a consequence of the different protonation state for the carboxylate group. Upon the protonation of the carboxylate group, the amino acid inclusion into the cucurbituril cavity is favored. Figure 6 1H NMR spectra (400 MHz, pD = 3) of iQ[7] in the absence (A) and presence of 0.30 (B), 0.65 (C), 0.85 (D), and 1.05 (E) equiv of Ile and free guest Ile (F) at 20 °C. Ultraviolet–Visible Absorption and Fluorescence Emission Spectra The interaction of iQ[7] with Trp was also examined by UV absorbance spectrophotometry and fluorescence spectroscopy. According to the UV absorption spectroscopic results (Figure 7A), the gradual addition of iQ[7] to Trp in buffered solution (pH = 7) was accompanied by a significant decrease in the intensity at 218 nm and a slight bathochromic shift because of the strong interaction between iQ[7] and Trp. As can be seen in Figure 7B, Trp displayed an emission peak at 366 nm at an excitation wavelength of 269 nm. Successive addition of iQ[7] caused a decrease and a hypsochromic shift from 359 to 350 nm in the fluorescence intensity at 359 nm. These substantial changes in the emission profile further confirm the strong host–guest interaction between iQ[7] and Trp. From the ultraviolet–visible (UV–vis) absorption and fluorescence intensity, the binding constant (Ka) for iQ[7]–Trp could be determined to be 2.32 × 104 M–1 and 2.68 × 104 M–1. Furthermore, Job’s plots (Figure 7C,D) based on the continuous variation method clearly showed that the UV spectra and fluorescence spectra of Trp fitted well with 1:1 stoichiometry of the host–guest inclusion complexes. Figure 7 Electronic absorption (A) and fluorescence emission spectra (B) of Trp (2 × 10–5 mol·L–1) upon the addition of increasing amounts (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 equiv) of iQ[7]. ΔA (C) and ΔF (D) vs NiQ[7]/(NiQ[7] + NTrp) plots. Isothermal Titration Calorimetry To better understand the host–guest interactions between iQ[7] and the 10 essential l-α-amino acids, we carried out at least two ITC experiments at 25 °C in 10 mM sodium phosphate (pH 7.0). Table 1 and Figures S15 and S16 show the equilibrium association constants (Ka) and thermodynamic parameters for iQ[7]–amino acid interaction systems for Lys, Arg, His, Trp, and Phe. The experimental results revealed Ka values ranging from ∼103 to ∼105 M–1 and negative ΔG° values ranging from −25.4 to −28.2 kJ/mol for iQ[7]–amino acid interactions. Thus, these l-α-amino acids could effectively bind to the iQ[7] host. However, the amino acids Met, Ile, Leu, Thr, and Val showed no effective interaction with iQ[7] (Figure S17). The revealed Ka values indicated a strong binding with the aromatic amino acids Trp and Phe, among which iQ[7] binds with Phe with the highest binding affinity, which is consistent with the binding behavior of Q[7] with aromatic amino acids (Table 2).21,41 The observations can be explained in terms of a combination of ion-dipole and electrostatic interactions between the positively charged side chains of the amino acids and the polar carbonyl groups of iQ[7] and hydrophobic interactions between the aromatic moieties of the amino acids and the macrocyclic cavity. From the ΔH° and TΔS° values shown in Table 1, all intermolecular complexation interactions between the iQ[7] host and l-α-amino acids guests appear to be driven by favorable enthalpy changes, accompanied by small negative (unfavorable) entropy changes. According to the NMR data, the interactions between iQ[7] and Met, Ile, and Leu are relatively weak; meanwhile, iQ[7] with Thr and Val shows no interaction. These facts might be a reasonable explanation why the ITC experiments showed no effective interaction of iQ[7] with these amino acids Met, Ile, Leu, Thr, and Val. Table 1 Complex Stability Constant (Ka), Enthalpy (ΔH°), Entropy Changes (TΔS°), and Gibbs Free Energy (ΔG°) for iQ[7]–Guest Interactions in Buffered Solution at pH = 7 guest Ka (×104 M–1) ΔH° (kJ/mol) TΔS°(kJ/mol) ΔG° (kJ/mol) Lys 0.87 ± 0.15 –31.2 ± 14.0 –4.17 –27.1 Arg 1.60 ± 0.97 –32.4 ± 9.55 –4.23 –28.2 His 0.66 ± 0.13 –33.8 ± 11.0 –7.93 –25.9 Trp 2.83 ± 0.66 –37.4 ± 5.71 –12.0 –25.4 Phe 10.7 ± 0.24 –37.6 ± 2.25 –9.62 –28.0 Table 2 Complex Stability Constant (Ka), Enthalpy (ΔH°), and Entropy Changes (TΔS°) for Q[7]–Guests guest Ka (M–1) ΔH° (kJ/mol) TΔS° (kJ/mol) Lys a2.1(±0.7) × 102 –4.4 ± 0.3 8.8 ± 0.3 Arg anot available b327 ± 16 –5.0 ± 0.1 9.2 ± 0.1 His anot available Trp a1.2(±0.1) × 103 –28.9 ± 0.6 –11.3 ± 0.6 Phe a1.8(±0.5) × 105 –30.5 ± 2.8 –0.6 ± 2.8 a Measured in sodium phosphate buffer at pH = 7.0, ref (21). b Measured in sodium phosphate buffer at pH = 6.0, ref (41). Mass Spectrometry We further studied the formation of the inclusion complexes of iQ[7] and guests for 10 of the essential l-α-amino acids by MALDI-TOF MS. In the resultant MALDI-TOF MS spectra (Figure S18), major signals at m/z = 1309.012, 1336.526, 1317.864, 1367.793, 1328.397, 1294.573, 1294.498, and 1312.410 were observed, corresponding to Lys–iQ[7] (calculated 1309.151), Arg–iQ[7] (calculated 1337.164), His–iQ[7] (calculated 1318.118), Trp–iQ[7] (calculated 1367.189), Phe–iQ[7] (calculated 1328.152), Ile–iQ[7] (calculated 1294.136), Leu–iQ[7] (calculated 1294.136), and Met–iQ[7] (calculated 1312.175), respectively. These intense signals provide direct support for the formation of 1:1 stoichiometric host–guest inclusion complexes for these eight amino acids. It is noted that no significant host–guest interaction signals were observed between iQ[7] and Thr or Val in the MS spectra. The results of the mass spectra are consistent with the results from the NMR experiments. Conclusions We explored the binding interactions between 10 essential l-α-amino acid guests and the iQ[7] host using a variety of characterization methods in buffered solution (pH = 7). The experimental results indicated a strong binding with the aromatic amino acids Trp and Phe, and iQ[7] binds with Phe with the highest binding affinity, which is consistent with the binding behavior of Q[7] with aromatic amino acids. Lys, Arg, and His guests lie outside the portal of the host, whereas the alkyl moieties of Met, Leu, and Ile guests were accommodated within the iQ[7] cavity, and there was no significant interaction between iQ[7] and Thr or Val. Additionally, interactions between the protonated form of Lys, Arg, and His with iQ[7] were also investigated at pH = 3, and unexpectedly, the side chains were located in the cavity of iQ[7] under acidic conditions. Furthermore, the aromatic amino acids Trp and Phe were more deeply buried in the iQ[7] cavity at the lower pH. An upfield chemical shift for the protons of the alkyl side chains of Met, Leu, Ile, Thr, and Val guests indicated that they were located inside the iQ[7] cavity and hence formed host–guest complexes. These results not only enhance our knowledge of the molecular recognition of amino acids but may also be of significance for the design and synthesis of new macrocyclic compounds for biological identification and simulation. Experimental Section Materials and Reagents Ten essential l-α-amino acids were purchased from Aldrich. iQ[7] was prepared and purified according to our previously published procedure.33 All other reagents were of analytical grade and were used as received. Double-distilled water was used for all experiments. Nuclear Magnetic Resonance Measurements All 1H NMR spectra, including those for titration experiments, were measured on a Varian INOVA-400 NMR spectrometer with SiMe4 as an internal reference at 20 °C. D2O was used as a field-frequency lock, and the observed chemical shifts are reported in parts per million (ppm) relative to that for the internal standard (TMS at 0.0 ppm). The ratio of amino acids versus iQ[7] was calculated by the ratio of their integral areas for special peaks. The concentrations of the amino acids were 1.0 × 10–4 mol/L in the NMR experiments. D2O was adjusted to pD = 7.0 with sodium phosphate. The value was verified on a pH meter calibrated with two standard buffer solutions. D2O was adjusted to pD = 3.0 with 1 M DCl. The pD = 3 of the solution was also verified on a calibrated pH meter. UV–Vis Absorption and Fluorescence Emission Spectra UV–vis absorption spectra of the host–guest complexes were recorded with an Agilent 8453 spectrophotometer at room temperature. Fluorescence spectra measurements were performed on a Varian Cary Eclipse fluorescence spectrophotometer equipped with a xenon discharge lamp at room temperature. The absorption and fluorescence titration experiments were performed as follows: 200.0 μL of 1.0 × 10–3 mol/L stock solution of Trp and various amounts of 1.0 × 10–4 mol/L iQ[7] aqueous solution were transferred into a 10 mL volumetric flask, and then the volumetric flask was filled to the final volume with distilled water. The pH was adjusted to pH = 7 with sodium phosphate. ITC Measurements Microcalorimetric experiments were performed using an isothermal titration calorimeter Nano ITC (TA, USA). The heat evolved was recorded at 298.15 K. The heat of the reaction was corrected for the heat of the dilution of the guest solution determined in separate experiments. All solutions were degassed prior to the titration experiment by sonication. A stock solution (1.0 × 10–3 mol/L) of amino acids and 1.0 × 10–4 mol/L stock solution of iQ[7] were prepared with 10 mM sodium phosphate (pH 7.0). A typical ITC titration was carried out by titrating the l-α-amino acid solution (pH = 7, 1.0 × 10–3 mol/L, 6 μL of aliquots, at 250 s intervals) into an iQ[7] solution. The concentration of iQ[7] in the sample cell (1.3 mL) was 1.0 × 10–4 mol/L at pH = 7. Computer simulations (curve fitting) were performed using the Nano ITC analyze software. First points in the ITC data were excluded when fitting the model to acquire the binding constant, enthalpy change, and entropy change. MALDI-TOF MS MALDI-TOF MS spectra were recorded on a Bruker BIFLEX III ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometer with α-cyano-4-hydroxycinnamic acid as the matrix. The MALDI-TOF experiments were carried out by adding the l-α-amino acid solution (1.0 × 10–3 mol/L, 100 μL) into an iQ[7] solution (1.0 × 10–4 mol/L, 1.0 mL). The solution concentration was about 1.0 × 10–4 mol/L (l-α-amino acids–iQ[7] = 1:1). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00429.NMR spectra, MALDI-TOF MS spectra of inclusion complexes, and ITC profiles of iQ[7] with guests (PDF) Supplementary Material ao7b00429_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (no. 21561007), the Innovation Program for High-level Talents of Guizhou Province (no. 2016-5657), the Science and Technology Fund of Guizhou Province (no. 2016-1030), and the Scientific Research Foundation of Guizhou University (no. 2015-62). ==== Refs References Joseph R. ; Rao C. P. Ion and molecular recognition by lower rim 1,3-di-conjugates of calix[4]arene as receptors . Chem. Rev. 2011 , 111 , 4658 –4702 . 10.1021/cr1004524 .21513269 Zheng B. ; Wang F. ; Dong S. ; Huang F. 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PMC006xxxxxx/PMC6644432.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145889310.1021/acsomega.8b00411ArticleSynthesis of an Alleged Byproduct Precursor in Iodixanol Preparation Haarr Marianne Bore †Lindbäck Emil †Håland Torfinn ‡Sydnes Magne O. †† Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, NO-4036 Stavanger, Norway‡ GE Healthcare AS, NO-4521 Lindesnes, Norway* E-mail: torfinn.haaland@ge.com (T.H.).* E-mail: magne.o.sydnes@uis.no (M.O.S.).05 07 2018 31 07 2018 3 7 7344 7349 05 03 2018 20 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. N1,N3-Bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-(N-(oxiran-2-ylmethyl)acetamido)isophthalamide (1), the alleged precursor of several minor byproducts formed when the X-ray contrast agent iodixanol is synthesized from 5-acetamido-N1,N3-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide (2), has been successfully prepared with an overall yield of 25%. Epoxide 1 enabled the confirmation of its presence in the reaction mixture during the preparation of iodixanol when amide 2 was used as the starting material. document-id-old-9ao8b00411document-id-new-14ao-2018-00411pccc-price ==== Body 1 Introduction Fully understanding an industrial process is of great importance when process improvements are to be made.1 Small quantities of byproducts formed in a reaction, which on a laboratory scale synthesis are neglectable, become important to minimize when the reaction takes place on a ton-scale on a daily basis. From an industrial perspective, it is therefore important to fully understand how byproducts are formed in a given process. With such knowledge in hand, it could be possible to alter the reaction conditions to diminish the byproduct formation, or even better, remove them totally. X-ray-contrast agent iodixanol (Figure 1) can be prepared by several methods.2−4 If amide 2 is used as the starting material under basic conditions, its anion would react with epichlorohydrin, a reagent in the reaction, to give epoxide 1 as an intermediate in a two-step process. Because of the reactive nature of epoxides,5 and the presence of several nucleophilic sites in amide 2, epoxide 1 could potentially be a precursor for the formation of several minor byproducts in addition to the major and desired product iodixanol. Figure 1 Structure of iodixanol, epoxide 1, and amide 2. Previous results have shown that amide 2 is present in two anionic forms, compounds 2a and 2b (Figure 2a), upon treatment with base where anion 2a is the dominating species.6 Anion 2b can react with epoxide 1, which results in the formation of the imidate byproduct depicted in Figure 2b. The formation of the imidate has previously been proved experimentally.6 Another plausible pathway to this product could be via the intermediate, as shown in Figure 2c. This byproduct and other byproducts1 are then removed from crude iodixanol during work-up and purification processes, resulting in pure iodixanol. Figure 2 (a) Two anionic forms of amide 2; (b) byproduct formed if epoxide 1 reacts with the anionic form of amide 2; and (c) potential intermediate from the reaction between anionic amide 2 and epichlorohydrin. Epoxide 1 is expected to have a short lifetime under the reaction conditions, and its isolation from the reaction mixture is therefore not a viable option when substantial amount of material is needed for reactivity studies and for studies directed toward elucidating its role in byproduct formation. Herein, we communicate our synthetic approach for the preparation of epoxide 1, which also allowed us to confirm its presence in small amounts during iodixanol synthesis when amide 2 is used as starting material. 2 Results and Discussion 2.1 Synthesis Initially, we attempted a protection group-free synthesis of epoxide 1 by performing a regioselective N-alkylation7 of the acetamide moiety within substrate 2, followed by a direct epoxidation of the resulting alkene by a Prileschajew reaction.5 However, to our disappointment, we quickly ended up with solubility problems of the material under solvent conditions required to conduct the necessary synthetic transformations. To get around the solubility issue, protection of the free hydroxyl groups was inevitable. Substrate 2 was therefore converted to the tert-butyl(dimethyl)silyl (TBS)-protected product 3 in 96% yield, utilizing standard reaction conditions (Scheme 1).8 The TBS protection group was thought to be suitable for the chemistry required for the formation of epoxide 1 and, at the same time, to be relatively easy to remove in the final step, utilizing a global deprotection strategy. Silyl ether 3 was then subjected to a regioselective N-alkylation of the acetamide, resulting in the formation of the desired alkene 4 in 90% isolated yield. Scheme 1 Synthetic Pathway toward Epoxide 1 Attempts to convert substrate 4 directly to epoxide 7 upon oxidation by m-CPBA in a range of solvents9 and with hydrogen peroxide in glycine buffer10 were all unsuccessful. A more labor-intensive route was therefore required to prepare the target epoxide. Thus, alkene 4 was subjected to an Upjohn dihydroxylation in acetone/water (6:1) resulting in a clean conversion to the corresponding diol 5 (96% yield).11−13 Treating glycol 5 with methanesulfonyl chloride (MsCl) under conditions described by O’Donnel and Burke14 gave predominant mesylation of the primary hydroxyl group, resulting in the isolation of mesylate 6 in 73% yield. In addition, 10% of the product resulting from mesylation of both hydroxyl groups (compound 8 in Scheme 1) was isolated from the reaction. The 1H and 13C NMR chemical-shifts for the mesyl groups in compounds 6 and 8 were in good agreement with literature reports for similar compounds.14−16 Treating compound 6 with 2 M NaOH (aq) in ether at room temperature, according to the Izuhara and Katoh procedure,17 resulted in the formation of epoxide 7 (70% isolated yield) over the course of 1 h. Finally, subjecting substrate 7 to tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) at room temperature gave target compound 1 in 86% isolated yield after a tedious purification process,18 which involved three rounds of flash chromatography to fully remove all salt from the product. Attempts to remove salts and excess TBAF from the product by using a mix of mildly acidic Ca2+ and basic ion-exchange resins were only partly successful,19 and unfortunately also resulted in significant loss of product. Epoxide 1 was found to be stable under neutral conditions and when stored neat. However, when exposed to acidic conditions, the epoxide underwent ring opening readily. Comparing the high-performance liquid chromatography (HPLC) chromatograms of compound 1 prepared herein and the reaction mixture during industrial production of iodixanol enabled us to confirm that epoxide 1 was indeed present in small quantities during the preparation of iodixanol (see Figures S1 and S2 in the Supporting Information), thus opening up for potentially being the precursor of byproducts formed during production. Interestingly, we also found that epoxide 1 partly ring-opens to iohexol (Figure 3) on the analytical HPLC column. Figure 3 Structure of iohexol. 2.2 Rotational Conformation The predominant rotational conformation of the acetamide bond CO–N in compounds 2 and 3 was in the endo-form, where the carbonyl moiety is directed toward the ring, as shown in Figure 4.20 However, the most favored rotational isomer of the alkylated acetamide in compounds 4–7 and 1 was the exo-form. The exo-conformation is also the dominating species in iohexol, where the acetamide is alkylated.21 Isomerism of all compounds was determined by the split acetyl signal in 1H NMR into one major and one minor peak, with the endo-peak positioned downfield of the exo-peak, because of ring-current effects.22 For compound 3, the endo-/exo-peaks were positioned at chemical shifts 2.01 and 1.58 ppm in a 27:1 ratio, respectively, and the equivalent peaks for compound 4 were located at 2.18 and 1.73 ppm in a 1:20 ratio (see Figure S3 in the Supporting Information). Figure 4 Endo-/exo-isomerism of the acetamide attached to the triiodinated benzene ring. 1H NMR analysis conducted at 400 K (127 °C) showed that rotation of the amide bond was more restricted in compound 4 (containing a tertiary acetamide moiety) compared with compound 3. At 400 K, the endo- and exo-peaks in compound 3 overlapped forming one broad singlet. However, for alkene 4, the endo- and exo-peaks remained separated, with an endo-/exo-ratio of 1:7 at 127 °C, compared to a ratio of 1:20 obtained at room temperature. Further evidence of rotational isomerism was observed, but not investigated, in this work. For more information on the subject, we refer to relevant literature on the topic.20−23 3 Conclusions Epoxide 1 was prepared over six synthetic steps with an overall yield of 25% using a strategy, where the hydroxyl groups were protected with TBS groups. Our synthesis of compound 1 enabled us to confirm the presence of epoxide 1 in the reaction mixture in small quantities during the synthesis of iodixanol making compound 1 a potential starting point for byproduct formation. Work is now going on in these laboratories to study whether compound 1 is involved in the byproduct formation when iodixanol is synthesized from amide 2. 4 Experimental Section 4.1 General All commodity chemicals and reagents were purchased from commercial suppliers and used without further purification. Petroleum ether (pet. ether, 40–65 °C) was used for column chromatography. Silica gel NORMASIL 60 40–63 μm was used for flash chromatography. Proton (1H) and carbon (13C) NMR spectra were acquired at 20 °C. Proton and carbon NMR spectra were recorded using an Ascend 400 NMR spectrometer from Bruker (Ascend 400), operating at 400 and 100 MHz. NMR spectra are referenced to residual dimethyl sulfoxide (DMSO) (δ 2.50 ppm, 1H; δ 39.52 ppm, 13C). 1H NMR data are recorded as follows: chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), and relative integral] where multiplicity is reported as: s = singlet; d = doublet; t = triplet; q = quartet; quint = quintet; dt = doublet of triplet; m = multiplet; and bs = broad singlet. For 13C NMR spectra, the data are given as chemical shift (δ), (protonicity), where the number of protons is defined as: C = quaternary (where ArC = quaternary aromatic carbon); CH = methyne; CH2 = methylene; and CH3 = methyl. The assignment of signals in various NMR spectra was often assisted by correlation spectroscopy (COSY), nuclear Overhauser effect and exchange spectroscopy, heteronuclear single quantum COSY, and/or heteronuclear multiple bond COSY. Analytical HPLC was performed on an Agilent 1100 or a TSP (Thermo Separation products) instrument with UV detection at 254 nm using an RP-18 column (YMC 150 × 4.6 mm, 5 μm). The following linear gradient program with mixtures of water (A) and acetonitrile (B) as eluent was applied. Program: 3.0% B (0–2.7 min), 3.0–7.2% B (2.7–5.5 min), 7.2% B (5.5–16.5 min), 7.2–13.0% B (16.5–19.5 min), 13.0–45.0% B (19.5–26.5 min), 45.0% B (26.5–31.5 min). The flow rate was 1.25 mL/min. No acid was mixed with the eluents unless otherwise stated. The amount of each component is given in percentage, based on its relative area. 4.1.1 5-Acetamido-N1,N3-bis(2,3-bis((tert-butyldimethylsilyl)oxyl)propyl)-2,4,6-triiodoisophthalamide (3) Imidazole (6.58 g, 96.7 mmol), 4-dimethylaminopyridine (DMAP) (3.27 g, 26.8 mmol), and tert-butyldimethylsilyl chloride (13.38 g, 88.8 mmol, 6.6 equiv) were added to a solution of amide 2 (10.0 g, 13.5 mmol) in dimethylformamide (100 mL). The reaction was left stirred at 60 °C under a nitrogen atmosphere for 5 h. The reaction was then quenched with NaHCO3 (sat. aq solution, 100 mL) and extracted with EtOAc (150 mL × 3), and the organic layers were washed with brine (100 mL × 2) and water (50 mL). The crude product was purified by flash column chromatography using petroleum ether/EtOAc, 7:3 → 1:4 (gradient elution). Isolation of the appropriate fractions (Rf = 0.32 in petroleum ether/EtOAc 7:3) gave 15.6 g (96%) of the O-silylated product 3 as a white solid, mp 241.0–241.5 °C. IR (KBr) νmax: 3280, 2929, 2857, 2602, 2489, 2359, 1648, 1540, 1472, 1256, 1139, 1101, 984, 938, 835, 777, 668 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 9.97 and 9.52 (2br s in ratio 93:4, 1H), 8.70–8.71, 8.56–8.58, 8.32–8.37, and 8.19–8.24 (2m + 2m in ratio 78:80:18:12, 2H), 3.89–3.95 (m, 2H), 3.78–3.80, and 3.51–3.55 (2m in ratio 1:1, 4H) 3.38–3.36, 3.25–3.23, 3.15–3.13, and 3.01–2.99 (4m in ratio 1:1:1:1, 4H), 2.01 and 1.58 (2br s in ratio 27:1, 3H), 0.88, 0.87, 0.86, and 0.84 (4br s in ratio 8:6:3:1, 36H), 0.12, 0.08, 0.03, −0.05 (3br s and 1s in ratio 6:6:12:1, 24H); 13C NMR (400 MHz, DMSO-d6): δ 169.9 (C), 168.2 (C), 150.3 (ArC), 78.6 (CH), 66.6 (CH2), 42.8 (CH2), 26.4 (CH3), 23.5 (CH3), 18.6 (C), 18.3 (C), −2.5 (CH3), −4.0 (CH3), −4.2 (CH3), −4.8 (CH3) (specific aromatic carbons were not visible because of slow relaxation and rotamerization). HRMS found: [M + H]+, 1204.1972. C40H77I3N3O7Si4 requires [M + H]+, 1204.1967. 4.1.2 5-(N-Allylacetamido)-N1,N3-bis(2,3-bis((tert-butyldimethylsilyl)oxy)propyl)-2,4,6-triiodoisophthalamide (4) To a solution of compound 3 (15 g, 12.5 mmol) in EtOH (100 mL) and isopropyl alcohol (50 mL), a solution of H3BO3 (1.55 g, 25 mmol) in water (15 mL) was added with pH adjusted to approx. 12.5 by dropwise addition of 10 M KOH. Allyl bromide (1.08 mL, 12.5 mmol, 1 equiv) was added, and the reaction was left stirred for 3 h at 40 °C, whereas the pH was monitored and constantly adjusted to approx. pH 12.5 by dropwise addition of 10 M KOH. A second addition of allyl bromide (0.54 mL, 6.25 mmol, 0.5 equiv) was conducted, and the reaction was left stirred for an additional 20 h. EtOH was then removed in vacuo, the product was extracted with EtOAc (50 mL × 2), and the combined organic fractions were washed with water (40 mL). The crude product was then purified using a prepacked silica gel column (HP silica 50 μm) from PuriFlash (Interchim 215, petroleum ether/EtOAc, 95 → 50% petroleum ether (gradient elution), 35 mL/min) and yielded 13.9 g (90%) of the N-alkylated product 4 as white crystals, mp 115–116 °C. IR (KBr) νmax: 3420, 3287, 3082, 2953, 2929, 2885, 2857, 2360, 2339, 2244, 1654, 1546, 1528, 1502, 1471, 1314, 1139, 1101, 982, 937, 921, 835, 778, 734, 668 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.58–8.57 (m, 2H), 5.91–5.86 (m, 1H), 5.17–5.16, 5.12, 5.05, and 5.03 (4m in ratio 1:1:1:1, 2H), 4.14–4.12 and 4.06–4.04 (2m in ratio 1:1, 2H), 3.94–3.93 and 3.88–3.86 (2m in ratio 1:1, 2H), 3.78–3.75 and 3.56–3.52 (2m in ratio 1:1, 4H), 3.37–3.33, 3.26–3.24, 3.20–3.18, and 3.07–3.05 (4m in ratio 1:1:1:1, 4H), 2.1,9 2.18, 1.74 and 1.73 (4br s in ratio 1:1:20:20, 3H), 0.88, 0.87 (2br s in ratio 1:1, 36H), 0.12, 0.11, 0.08, and 0.03 (4br s in ratio 1:1:2:4, 24H); 13C NMR (400 MHz, DMSO-d6): δ 169.3 (C), 168.5 (C), 150.8 (ArC), 146.8 (ArC), 132.6 (CH), 119.0 (CH2), 101.1 (ArC), 100.6 (ArC), 90.8 (ArC), 71.7 (CH), 66.0 (CH2), 50.7 (CH2), 42.0 (CH2), 25.9 (CH3), 22.8 (CH3), 18.1 (C), 17.9 (C), −4.7 (CH3), −5.2 (CH3); HRMS found: [M + H]+, 1244.2287. C43H81I3N3O7Si4 requires [M + H]+, 1244.2280. 4.1.3 N1,N3-Bis(2,3-bis((tert-butyldimethylsilyl)oxy)propyl)-5-(N-(2,3-dihydroxypropyl)acetamido)-2,4,6-triiodoisophthalamide (5) To a solution of substrate 4 (12 g, 9.65 mmol) in acetone (150 mL) and water (25 mL) at room temperature were added NMO (1.8 g, 15.4 mmol, 1.6 equiv) and OsO4 in tBuOH (1.95 mL, 2 mol %). The reaction was left stirred for 20 h and then quenched by the addition of Na2S2O3 (20 mL, 1 M aq). The product was then extracted with EtOAc (50 mL × 3), and the combined organic fractions were concentrated to dryness under reduced pressure. The crude product was purified on a prepacked silica gel column (HP silica 50 μm) from PuriFlash (Interchim 215, petroleum ether/EtOAc, 95 → 40% petroleum ether (gradient elution), flow rate 35 mL/min). Isolation of the fractions with Rf = 0.19 (pet. ether/EtOAc, 11:9) gave 11.8 g (96%) of compound 5 as white crystals, mp 122–123 °C. IR (KBr) νmax: 3274, 2953, 2929, 2857, 2885, 2357, 2337, 1652, 1471, 1392, 1255, 1139, 1100, 835 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.62–8.57 (m, 2H), 4.60–4.56 (m, 2H), 3.97–3.94, 3.89–3.86 (2m in ratio 1:1, 2H), 3.91–3.88, 3.82–3.79 (2m in ratio 1:1, 1H), 3.79–3.77, 3.57–3.55 (2m in ratio 1:1, 4H), 3.38–3.42 (m, 2H), 3.83–3.81, 3.69–3.67, 3.34–3.32, 3.15–3.13 (4m in ratio 1:1:1:1, 4H), 2.24, 2.22, 1.78, and 1.77 (4br s in ratio 1:1:15:15, 3H), 0.89–0.88, 0.87–0.86 (2m in 1:1 ratio, 36H), 0.12, 0.11, 0.09, 0.04 (4br s in ratio 1:1:2:4, 24H); 13C NMR (400 MHz, DMSO-d6): δ 170.6 (C), 169.9 (C), 151.5 (ArC), 148.6 (ArC), 100.7 (ArC), 90.9 (ArC), 72.2 (CH), 71.9 (CH), 70.4 (CH), 66.4 (CH2), 65.0 (CH2), 53.9 (CH2), 42.5 (CH2), 26.4 (CH3), 23.3 (CH3), 18.6 (C), 18.3 (C), −4.01 (CH3), −4.72 (CH3); HRMS found: [M + H]+, 1278.2340. C43H82I3N3O9Si4 requires [M + H]+, 1278.2335. 4.1.4 3-(N-(3,5-Bis((2,3-bis((tert-butyldimethylsilyl)oxy)propyl)carbamoyl)-2,4,6-triiodophenyl)acetamido)-2-hydroxypropyl Methanesulfonate (6) To a solution of diol 5 (9.5 g, 7.44 mmol) in dichloromethane (DCM) (100 mL) at 0 °C was added pyridine (5.5 mL, 68.0 mmol, 9.1 equiv), DMAP (5 mol %), and MsCl (0.92 mL, 11.9 mmol, 1.6 equiv). The reaction was left stirred for 22 h at 5 °C and was then quenched with water (50 mL), extracted with DCM (80 mL × 3), washed with HCl (75 mL, 2 M), K2CO3 (75 mL, sat. aq solution), and water (75 mL), dried over MgSO4, and concentrated under reduced pressure. Purification was conducted on a prepacked silica gel column (HP silica 50 μm) from PuriFlash (Interchim 215, petroleum ether/EtOAc, 95–30% petroleum ether (gradient elution), flow rate 35 mL/min). Excess pink/orange salt was further removed from product by filtration through silica gel (petroleum ether/EtOAc, 9:1). Isolation of the fractions with Rf = 0.19 (petroleum ether/EtOAc 1:1) gave 7.3 g (73%) of the primary mesylate 6 as white crystals, mp 146–149 °C. IR (KBr) νmax: 3368, 3293, 2953, 2929, 2886, 2857, 2360, 2341, 1653, 1540, 1472, 1394, 1361, 1254, 1174, 1139, 1100, 982, 938, 835, 777 cm–1; 1H NMR (400 mHz, DMSO-d6): δ 8.63–8.59 (m, 2H), 5.43–5.38 and 5.36–5.30 (2m in ratio 1:1, H), 4.36–4.34 and 4.12–4.10 (2m in ratio 1:1, 2H), 4.10–4.08 and 4.02–3.99 (2m, 2H), 4.02–4.00, 3.97–3.95, and 3.02–3.00 (3m, 2H), 3.96–3.94 and 3.89–3.86 (2m, H), 3.79–3.77 and 3.57–3.54 (2m in ratio 1:1, 4H), 3.42–3.41, 3.25–3.24, and 3.04–3.03 (3m, 4H), 3.17 (br s, 3H), 2.25, 2.24, 1.80, and 1.79 (4br s in ratio 2:2:13:13, 3H), 0.88 and 0.87 (2br s in ratio 1:1, 36H), 0.12, 0.09, and 0.04 (3br s in ratio 1:1:2, 24H); 13C NMR (400 MHz, DMSO-d6): δ 170.6 (C), 169.9 (C), 73.7 (CH2), 72.3 (CH), 71.9 (CH), 67.4 (CH), 66.4 (CH2), 53.0 (CH2), 42.6 (CH2), 37.0 (CH3), 26.4 (CH3), 23.3 (CH3), 22.7 (CH3), 18.4 (C), −4.0 (CH3), −4.1 (CH3), −4.7 (CH3) (aromatic carbons were not visible because of slow relaxation and rotamerization); HRMS found: [M + Na]+, 1378.1935 C44H84I3N3O11SSi4 requires [M + Na]+, 1378.1930. Isolating of the fractions with Rf = 0.41 (petroleum ether/EtOAc 1:1) gave the dimesylated product 8 as white crystals (1.1 g, 10%), mp 152–154 °C. IR (KBr) νmax: 3368, 2953, 2929, 2886, 2857, 2362, 2341, 1654, 1541, 1472, 1396, 1361, 1254, 1174, 1143, 1102, 982, 938, 834, 777 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.60–8.58, 8.52–8.50, and 8.48–8.45 (3m in 10:3:3 ratio, 2H), 4.65–4.60 and 4.45–4.43 (2m, 2H), 4.45–4.41 and 4.41–4.37 (2m, HCOMs), 4.37–4.29, 4.32–4.24, 3.44–3.36, and 3.27–3.20 (4m, 2H), 3.95–3.88 and 3.87–3.80 (2m, 2H), 3.97–3.92 and 3.91–3.86 (2m in 1:1 ratio, 4H), 3.78–3.73 and 3.30–3.19 and 3.11–2.99 (3m, 4H), 3.25 (2br s, 3H), 3.17 (br s, 3H), 2.25, 2.24, 1.80, and 1.79 (4br s in ratio 2:2:13:13, 3H), 0.88 and 0.87 (2br s in ratio 1:1, 36H), 0.12, 0.09, and 0.04 (3br s in ratio 1:1:2, 24H); 13C NMR (400 MHz, DMSO-d6): δ 170.3 (C), 169.4 (C), 152.3 (ArC), 151.6 (ArC), 147.7 (ArC), 101.1 (ArC), 99.4 (ArC), 91.5 (ArC), 71.7 (CH), 71.4 (CH), 65.9 (CH2), 56.7 (CH2), 52.6 (CH2), 42.1 (CH2), 36.9 (CH3), 25.9 (CH3), 25.8 (CH3), 22.5 (CH3), 18.1 (C), 17.9 (C), −4.5 (CH3), −4.7 (CH3), −5.3 (CH3). 4.1.5 N1,N3-Bis(2,3-bis((tert-butyldimethylsilyl)oxy)propyl)-2,4,6-triiodo-5-(N-(oxiran-2-ylmethyl)acetamido)isophthalamide (7) To a solution of mesylate 6 (6.5 g, 4.8 mmol) in Et2O (150 mL) was added NaOH (10 mL, 2 M aq). After stirring for 23 h at room temperature, the mixture was extracted with Et2O (100 mL × 2). The extract was washed with water (75 mL) and dried over MgSO4, filtered, and concentrated. The crude product was purified by flash column chromatography (petroleum ether/EtOAc, 3:1). Concentration of the fractions with Rf = 0.22 (petroleum ether/EtOAc 7:3) yielded 4.2 g (70% yield) of epoxide 7 as white crystals, mp 119–120 °C. IR (KBr) νmax: 3288, 3059, 2929, 2885, 2857, 2246, 1655, 1542, 1472, 1389, 1316, 1256, 1139, 1101, 981, 938, 910, 836, 778, 734, 668 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 8.62–8.58 (m, 2H), 3.96–3.94 and 3.90–3.88 (2m in ratio 1:1, 2H), 3.78–3.76 and 3.57–3.55 (2m in ratio 1:1, 4H), 3.70–3.69, 3.52–3.51, and 3.28–3.27 (3m, 2H), 3.41–3.39, 3.25–3.24, and 3.06–3.05 (3m, 4H), 3.28–3.26 and 3.22–3.20 (2m, 1H), 2.73–2.72, 2.51–2.49, and 2.48–2.46 (3m, 2H), 2.24, 2.23, 1.80, and 1.79 (4br s in ratio 1:1:24:24, 3H), 0.89 and 0.88 (2br s in ratio 1:1, 36H), 0.12, 0.10, and 0.05 (3br s in ratio 1:1:2, 24H); 13C NMR (400 MHz, DMSO-d6): δ 169.3 (C), 169.2 (C), 151.0 (ArC), 147.3 (ArC), 100.4 (ArC), 91.0 (ArC), 71.7 (CH), 71.4 (CH), 65.9 (CH2), 52.0 (CH2), 51.3 (CH2), 49.2 (CH), 48.7 (CH), 46.4 (CH2), 42.0 (CH2), 25.9 (CH3), 24.1 (CH3), 22.4 (CH3), 18.1 (C), 17.9 (C), −4.5 (CH3), −4.7 (CH3), −5.2 (CH3); HRMS found: [M + H]+,1260.2235. C43H81I3N3O8Si4 requires [M + H]+, 1260.2229. 4.1.6 N1,N3-Bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-(N-(oxiran-2-ylmethyl)acetamido) Isophthalamide (1) To a solution of epoxide 7 (1.64 g, 1.3 mmol) in THF (35 mL) was added 1 M TBAF in THF (7.2 mL, 7.2 mmol, 5.5 equiv). After 2 h, the solvent was removed by vacuum. Three subsequent purifications using flash column chromatography EtOAc/MeOH (3:1) (Rf = 0.26) yielded 895 mg (86% yield) of epoxide 1 as a white powder, mp 285 °C (dec). IR (KBr) νmax: 3343, 2926, 2350, 1730, 1650, 1429, 1393, 1347, 1316, 1270, 1173, 1110, 1044, 981, 921, 727 cm–1; 1H NMR (400 mHz, DMSO-d6): δ 8.57 (br s, 2H), 4.77–4.75 and 4.55–4.53 (2m, 2H), 3.71–3.68 (m, 2H), 3.45–3.42 (m, 4H), 3.69–3.68, 3.57–3.56, 3.49–3.48, and 3.32–3.31 (4m, 2H), 3.49–3.48, 3.30–3.29, and 3.07–3.06 (3m, 4H), 3.26–3.24 and 3.22–3.19 (2m, 1H), 2.72–2.71, 2.52–2.51, and 2.47–2.46 (3m, 2H), 2.24, 2.22, 1.80, and 1.79 (4br s in ratio 1:1:11:11, 3H); 13C NMR (400 MHz, DMSO-d6): δ 169.6 (C), 169.5 (C), 151.1 (ArC), 146.6 (ArC), 100.5 (ArC), 92.1 (ArC), 70.2 (CH), 64.0 (CH2), 51.9 (CH2), 51.3 (CH2), 49.3 (CH), 48.7 (CH), 46.5 (CH2), 42.6 (CH2), 22.5 (CH3), 22.1 (CH3); HRMS found: [M + H]+, 803.8779. C19H25I3N3O8 requires [M]+, 803.8776. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00411.Components of the reaction mixture during preparation; HPLC chromatogram of synthesized epoxide 1 and ring-opening product iohexol; endo-/exo-configuration of compounds 3 and 4 at 298 K and 400 K; and 1H, 13C NMR, and/or 2D spectra for all new compounds (PDF) Supplementary Material ao8b00411_si_001.pdf The authors declare no competing financial interest. Acknowledgments The University of Stavanger, the research program Bioactive, and GE Healthcare are thanked for the financial support of the project. Dr. Holmelid, University of Bergen, is also thanked for recording mass spectra. Professor Tanaka, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan, is thanked for excellent working conditions during a sabbatical stay. ==== Refs References Håland T. ; Sydnes L. K. 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PMC006xxxxxx/PMC6644433.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145899210.1021/acsomega.8b01375ArticleHierarchical Nickel–Cobalt Dichalcogenide Nanostructure as an Efficient Electrocatalyst for Oxygen Evolution Reaction and a Zn–Air Battery Hyun Suyeon Shanmugam Sangaraju Department of Energy Science Engineering, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, The Republic of Korea E-mail: sangarajus@dgist.ac.kr. Phone: +82-53-785-6413 (S.S.).02 08 2018 31 08 2018 3 8 8621 8630 19 06 2018 20 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. A unique three-dimensional (3D) structure consisting of a hierarchical nickel–cobalt dichalcogenide spinel nanostructure is investigated for its electrocatalytic properties at benign neutral and alkaline pH and applied as an air cathode for practical zinc–air batteries. The results show a high oxygen evolution reaction catalytic activity of nickel–cobalt sulfide nanosheet arrays grown on carbon cloth (NiCo2S4 NS/CC) over the commercial benchmarking catalyst under both pH conditions. In particular, the NiCo2S4 NS/CC air cathode shows high discharge capacity, a narrow potential gap between discharge and charge, and superior cycle durability with reversibility, which exceeds that of commercial precious metal-based electrodes. The excellent performance of NiCo2S4 NS/CC in water electrolyzers and zinc–air batteries is mainly due to highly exposed electroactive sites with a rough surface, morphology-based advantages of nanosheet arrays, good adhesion between NiCo2S4 and the conducting carbon cloth, and the active layer formed of nickel–cobalt (oxy)hydroxides during water splitting. These results suggest that NiCo2S4 NS/CC could be a promising candidate as an efficient electrode for high-performance water electrolyzers and rechargeable zinc–air batteries. document-id-old-9ao8b01375document-id-new-14ao-2018-01375jccc-price ==== Body Introduction Splitting water into pure hydrogen and oxygen to generate sustainable green hydrogen energy has been intensively studied in recent years, which can replace fossil fuel use.1,2 However, the efficiency of water splitting has so far been limited by the lack of sustainable catalysts toward the oxygen evolution reaction (OER) that can accelerate the kinetics.3−5 So far, IrOx and RuO2 are the best-known OER catalysts, although their high cost and scarcity limit their widespread use.4 Meanwhile, some promising attempts have been devoted to developing an efficient nonprecious metal OER catalyst under alkaline conditions. However, an almost harsh alkaline medium presents severe corrosion and related environmental issues.6,7 In this regard, someday, the splitting of water at neutral pH from ocean or river would be the target goal to satisfy renewable future hydrogen energy.4 It is thus highly required to develop efficient OER electrocatalysts that can operate in both alkaline and neutral media for overall water splitting even though it is relatively tough searching for those catalysts. Nowadays, various nonprecious transition metal-based catalysts are being explored, for example, transition metals,8 transition-metal oxides,9,10 chalcogenides,11−14 phosphides,15−17 hydroxides/oxyhydroxides,18,19 carbides,20 borides,21 and so on. Although lots of established catalysts have been reported concerning their excellent OER activity under alkaline conditions, only a few of them could still maintain their catalytic activity in neutral media. Cai et al. reported that the amorphous cobalt sulfide porous nanocubes showed a low OER onset potential of 1.5 V, comparable to that of RuO2 (1.49 V).22 However, a still substantial overpotential of 570 mV is needed to generate 4.59 mA cm–2 in phosphate-buffered solutions (PBSs; pH 7.0), whereas it could generate 10 mA cm–2 current density at 290 mV in 1 M KOH (pH 14.0). Similarly, sulfur-incorporated NiFe2O4 nanosheets (NSs) on nickel foam (S–NiFe2O4/NF) developed by Liu et al. exhibited a remarkably enhanced water-splitting performance for both OER and hydrogen evolution reaction (HER) as a bifunctional electrode under both alkaline and neutral conditions.23 The S–NiFe2O4/NF still requires 1.921 V to deliver 10 mA cm–2 in 1 M PBS (pH 7.4) for overall water splitting in three electrode systems, mostly occurring during OER with an overpotential of 494 mV. As air cathode catalysts for Zn–air batteries, Prabu et al., demonstrated a highly active one-dimensional structure of a spinel NiCo2O4 catalyst in rechargeable Zn–air batteries and Li–O2 batteries.24,100 Recently, Meng et al. constructed Co0.85Se nanocrystals in situ coupled with N-doped carbon with a metal–nitrogen–carbon (M–N–C) structure and short diffusion pathways for the transport of electron/ion to improve the Zn–air battery performance.25,26 For instance, Wu et al. reported zinc cobalt sulfide, the nanoneedle (NN) arrays grown on the carbon fiber paper electrode catalyst, which enables the Zn–air battery operation with an overpotential of 0.85 V and a long cycle life time of up to 200 cycles at 10 mA cm–2 as well as comparable water-splitting performance.27 Wang et al. proposed Co3FeS1.5(OH)6 hydroxysulfides serving as a superb air electrode catalyst with a low overpotential of 0.84 V and prolonged cyclability over 36 h test for 108 cycles at 2 mA cm–2.28 In pursuit of high electrochemical performance in water electrolyzers and for a Zn–air battery application, the spinel bimetallic sulfide NiCo2S4 with abundant redox chemistry has been considered to be the most promising electrochemically active material, which exhibits 2 orders of magnitude larger than that of NiCo2O4 and ∼104 times better electric conductivity than conventional single-metal compounds.12,29 Moreover, the stable spinel structures of bimetallic sulfide with a formula of AB2S4 possess plentiful exposed edge sites, leading to a higher electrochemical activity. Therefore, it has been widely applied for supercapacitors, Li-ion batteries, and a counter electrode for dye-sensitized solar cells as well as in water electrolyzers.30 For example, in our group, NiCo2S4 nanowire arrays were directly grown on 3D Ni foam (NiCo2S4 NW/NF) as a water-splitting catalyst and applied in an alkaline water electrolyzer. Because of its intrinsic properties, large surface area, and well-separated NW structures, NiCo2S4 NW/NF afforded continuous water-splitting reaction of generating hydrogen and oxygen gas at a cell voltage of only 1.63 V to generate 10 mA cm–2 current density.31 Ma et al. developed 3D networked porous NiCo2S4 nanoflakes on NF, which can offer more exposed active sites and easy transport of electrons and ions, thereby leading to significantly improved HER activity and stability.32 The outstanding OER performance of the spinel bimetallic sulfide NiCo2S4 has also become a promising application for rechargeable Zn–air batteries involving reversible OER and ORR. Also, hybridizing NiCo2S4 with the 3D structure of NS or NN with a carbon cloth (CC) (NiCo2S4 NS/CC or NN/CC) substrate by an in situ growth hydrothermal approach can be an efficient way to enhance the robustness of the electrode. It could also help to minimize the agglomeration of NiCo2S4 nanostructures and the detachment during long-term operation, and make faster ion/electron kinetics. Also, the advantages of CC, such as flexibility, high conductivity, and corrosion/dissolution resistivity might lead to enhanced catalytic activity and stability in a wide pH range.33 Meanwhile, tuning the nanostructure and the morphology, as well as porosity, can be another promising strategy to produce numerous exposed catalytic active sites on the catalyst surface.17 On the basis of our knowledge, we describe the physical and electrochemical properties of NiCo2S4 NS/CC as a highly active OER catalyst in both neutral and alkaline media, which have not been thoroughly investigated so far. Mainly, this is the first time a bimetallic sulfide, the NiCo2S4-based material, is reported to catalyze OER under a neutral condition. The catalytic performance of NiCo2S4 NS/CC is remarkably enhanced compared to the recent reports, particularly under a neutral condition. The NiCo2S4 NS/CC electrocatalyst exhibits the lowest OER overpotentials of 260 and 402 mV to generate 10 mA cm–2 in alkaline and neutral media, respectively. Specifically, it exhibits a low Tafel slope of 123 mV dec–1 and a high turnover frequency (TOF) of 8.17 × 10–3 s–1 at 1.63 V applied potential to drive 10 mA cm–2 current density under neutral conditions, confirming superior intrinsic activity with a substantial electrochemical active surface area (ECSA) of NiCo2S4 NS/CC compared with commercial RuO2/CC and other previously reported OER electrocatalysts. In addition, the constructed NiCo2S4 NS/CC air cathode for primary and rechargeable Zn–air batteries exhibits high discharge capacity, a narrow overall overpotential, and a long cycling life time exceeding the benchmark for precious metal-based electrodes. Experimental Methods Material Synthesis Cobalt nitrate hexahydrate [Alfa Aesar, Co(NO3)2·6H2O], nickel nitrate hexahydrate [Sigma-Aldrich, Ni(NO3)2·6H2O], urea (Sigma-Aldrich, CH4N2O), and sodium sulfide hydrate (Sigma-Aldrich, Na2S·xH2O) were used to synthesize the electrodes. A piece of CC (NARA CELL-TECH, 0.7 cm × 0.7 cm) was utilized with further treatment with ethanol. The NiCo2S4 NSs grown on CC (NiCo2S4 NS/CC) were prepared through a two-step hydrothermal process. A nickel nitrate of 0.004 M and cobalt nitrate of 0.008 M was dissolved in 120 mL of deionized water; further, 0.012 M urea was added. The obtained solution was transferred into a Teflon-lined stainless steel autoclave of 200 mL capacity, and a piece of CC was immersed in the solution. The autoclave was heated at 120 °C for 8 h in an electric oven. After the first step, the electrode was washed with deionized water several times to eliminate unreacted residues. Consequently, sodium sulfide flakes were dissolved in deionized water to prepare a 0.2 M sulfide solution for a sulfurization process. This sulfur-containing solution was again transferred to the autoclave and heated at 160 °C for 8 h in an electric oven. After cooling down to room temperature naturally, we washed the synthesized electrode several times with ethanol and deionized water, followed by the drying step in the vacuum oven at 60 °C overnight. For comparison, NiCo2S4 NN arrays on CC (NiCo2S4 NN/CC) were prepared by changing the temperature of the first hydrothermal step from 120 to 130 °C and maintaining the heating time of 8 h. To synthesize NiCo2O4 with NSs morphology, which is grown on CC (NiCo2O4 NS/CC), the electrode after the first step of the hydrothermal growth process was annealed at 450 °C for 2 h in an air atmosphere. Microstructural Characterizations The morphology and element compositions were studied on a field-emission scanning electron microscopy (FE-SEM, Hitachi-S4800, 3 kV) system equipped with a Horiba Scientific energy dispersive spectrometer and using transmission electron microscopy (TEM, Hitachi HF-3300, 300 kV). The crystal structures of all catalysts were examined by powder X-ray diffraction (XRD, Rigaku MiniFlex600). The composition of the catalyst was studied using X-ray photoelectron spectroscopy (XPS, Thermo-Scientific ESCALAB 250Xi). Electrochemical Measurements For electrochemical measurements, the OER catalytic performance was evaluated by linear sweep voltammetry (LSV) with a low scan rate of 1 mV s–1 in an electrolyte of 1 M KOH and a PBS without purging oxygen. The OER performance was evaluated in a three-electrode configuration directly using synthesized electrodes such as NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC as a working electrode (1.0 mg cm–2), a saturated calomel electrode (SCE) as a reference electrode, and a Pt wire as the counter electrode. Similarly, commercial RuO2 cast onto CC was used as a working electrode (1.4 mg cm–2), Pt wire as a counter electrode, and SCE as a reference electrode. The potentials reported were converted to the reversible hydrogen electrode (RHE). All electrochemical results were iR-corrected, considering the ohmic resistance from the electrolyte. The current densities presented in this paper are normalized concerning the geometric surface area of the electrode. The cyclic voltammetry (CV) was performed in N2-saturated 1 M KOH at room temperature with a scan rate of 10 mV s–2. Electrochemical impedance spectroscopy (EIS) was performed within a frequency range of 0.01 Hz to 0.1 MHz. The ECSA is calculated by following an established methodology reported in the literature.31 In detail, through the cyclic voltammogram obtained in a non-faradaic region at various scan rates (1, 2.5, 5, 10, 20, and 50 mV s–1), double-layer capacitance (Cdl) can be estimated. By plotting the anodic and cathodic current densities against the scan rate, the obtained linear slope value is Cdl. Finally, the ECSA can be obtained from the following equation: Cs denotes the specific capacitance of a flat, smooth surface of the electrode material, which is assumed to be 26 μF cm–2 for Ni- and Co-containing materials. Zinc–Air Battery Fabrication and Testing For full-cell zinc–air battery evaluation, the as-synthesized NiCo2S4 NS/CC or commercial catalyst of RuO2 + Pt/C/CC was used as an air-breathing cathode (1.54 cm2). Typically, the NiCo2S4 NS/CC air cathode was prepared with a loading amount of 1.0 mg cm–2. The commercial catalyst-based cathode was fabricated by coating with 2 mg of RuO2 and 40 wt % Pt/C on CC to achieve a loading of 1.30 mg cm–2. A polished zinc plate with a thickness of 0.1 mm, 6 M KOH solution with 0.2 M zinc acetate, and a Whatman glass microfiber filter membrane were prepared as an anode, an electrolyte, and a separator, respectively, to assemble the zinc–air battery with a coin cell (MTI Korea) configuration. The specific capacity and the charge–discharge curves were reported with a battery analyzer (BST8-3), which consumed ambient air. The discharge capacity of the primary zinc–air cell was normalized to the consumed mass of Zn metal, whereas the current density was normalized to the area of the electrode. Results and Discussion The hierarchical bimetallic sulfide NS arrays/CC hybrids were developed using an in situ two-step hydrothermal method, as graphically represented in Scheme 1. In the first step of the hydrothermal process, nickel nitrate hexahydrate and cobalt nitrate hexahydrate in a stoichiometric ratio were dissolved in deionized water and then urea was added. The solution was transferred into an autoclave and heated at 120 °C for 8 h, forming cobalt–nickel carbonate hydroxide hydrate NS arrays on a CC substrate. Subsequently, after oxidation reaction, a solution of sodium sulfide flakes dissolved in deionized water was prepared for the next sulfurization process. The anion exchange reaction from Co32–/OH– anions to S2– anions occurred at 160 °C for 6 h, thereby leading the complete phase transformation from cobalt–nickel carbonate hydroxide hydrate to nickel–cobalt sulfide on the CC. The morphology and composition of NiCo2S4 NS/CC were studied by FE-SEM and TEM. The bare CC consists of interconnected fibers with a smooth surface, as shown in Figure 1a. After the first hydrothermal reaction, numerous NS arrays are stacked onto the surface of CC with a rough surface shown in Figures 1b and S1. Consequently, the second hydrothermal process for sulfurization treatment is conducted, and its NS-like morphology perpendicular to a substrate with a rough surface still preserves its architecture (Figure 1e). From the top view of NiCo2S4 NS/CC, as shown in Figure 1c,d, the NiCo2S4 NSs that are interconnected with each other and form a porous architecture with submicron-size pores, providing ample space for the fast diffusion of redox ions during the reaction, uniformly cover the surface of CC. For comparison, the needle-like shape of NiCo2S4 vertically grown on the surface of CC with the average length of 3 μm is also synthesized to understand the effect of morphology on catalytic activities in both alkaline and neutral medium (Figure S2a–c). Also, the NiCo2O4 NS/CC was fabricated to have the same morphological characteristics as NiCo2S4 NS/CC (Figure S2d–f). Figure 1 FE-SEM images of (a) bare CC, (b) NiCo-precursor/CC after the first step in the hydrothermal process, (c–f) NiCo2S4 NS arrays grown on the surface of CC at different magnifications. Scheme 1 Schematic Preparation Process of Self-Supported NiCo2S4 NS/CC Nanostructures The XRD patterns of NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC are presented in Figure 2a. After oxidation reaction in the first step of the hydrothermal process, nickel–cobalt carbonate hydroxide hydrate NS arrays are uniformly formed on the CC, as shown in Figure S1 (ICDD 00-040-0216). During the sulfurization in the second hydrothermal step, the nickel–cobalt carbonate hydroxide hydrate phase is completely transformed into spinel nickel–cobalt sulfide without destroying the original nanostructures. The diffraction peaks of NiCo2S4 NS/CC and NiCo2S4 NN/CC at 16.2°, 26.7°, 31.4°, 38.1°, 50.3°, and 55.1° are assigned to the (111), (220), (311), (400), (511), and (440) planes of cubic-phase NiCo2S4, respectively (ICDD 00-043-1477). The high-resolution TEM image in Figure 2b reveals the interplanar distance of 0.28, 0.23, 0.16, 0.33, and 0.18 nm, corresponding to the (311), (400), (440), (220), and (511) planes of NiCo2S4, respectively, confirming the successful formation of NiCo2S4. The formation of NiCo2S4 NS/CC is confirmed based on the energy-dispersive X-ray (EDX) using TEM, which shows that the atomic percentages of nickel, cobalt, and sulfur are 15.2, 30.7, and 54.1 at. %, respectively, as shown in Figure S3. Note that the two characteristic peaks at 26° and 43° for all prepared electrodes are attributed to the CC (ICDD 01-074-2329). The diffraction peaks of the NiCo2O4 NS/CC catalyst obtained after the first step in the hydrothermal process and heat treatment are consistent with the standard pattern of NiCo2O4 (ICDD 01-073-1702). Meanwhile, the NN-like morphology of NiCo2S4 NN/CC can be confirmed from the TEM elemental mapping images shown in Figure S4. Figure 2 (a) XRD patterns of NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC catalysts. (b) High-resolution TEM images of NiCo2S4 NS/CC. High-resolution XPS deconvolution spectra of the NiCo2S4 NS/CC catalyst for (c) Ni 2p, (d) Co 2p, (e) S 2p, and (f) C 1s. To further characterize the chemical composition of electrodes, the XPS analysis is carried out, and results are given in Figure 2c–f. As shown in Figure 2c, the Ni 2p spectrum consists of two spin–orbit doublets of Ni2+ and Ni3+, including Ni2+ at 853.8 eV for Ni 2p3/2 and 873.8 eV for Ni 2p1/2, and Ni3+ at 857.7 eV for Ni 2p3/2 and 875.6 eV for Ni 2p1/2.24,30,34,35 The XPS spectrum of Co 2p (Figure 2d) contains well-resolved peaks of Co2+ 2p3/2, Co3+ 2p3/2, Co2+ 2p1/2, and Co3+ 2p1/2 at 798.8, 793.9, 783.0, and 778.9 eV, respectively, implying the co-presence of Co2+ and Co3+ species in NiCo2S4 NS/CC.23,34 The S 2p XPS spectrum (Figure 2e) shows two peaks at 163.0 and 161.8 eV, which are assigned to metal–sulfur bonds and the low coordination state sulfur ion that exists at the surface of NiCo2S4 NS/CC, respectively, with the satellite peak appearing at 170.1 eV in Figure 2e.34,35 The C 1s spectrum of NiCo2S4 NS/CC is deconvoluted into four peaks located at 284.8, 285.5, 287.0, and 291.3 eV, which correspond to the C 1s orbital of C–C (sp2), C–C (sp), C–O, and π–π interactions, respectively (Figure 2f). The additional π–π interaction indicates the strong interactions between NiCo2S4 NSs arrays and the CC, which can minimize contact resistance to generate a direct electron pathway.29 The binding energy values of Ni 2p, Co 2p, and S 2p are matched well with the previous reports on NiCo2S4-based materials. The OER catalytic activity of NiCo2S4 NS/CC was first evaluated with a three-electrode setup using a low scan rate of 1 mV s–1 to eliminate the capacitive current effects in alkaline solution (1 M KOH, pH = 14). For comparison, NiCo2S4 NN/CC, NiCo2O4 NS/CC, bare CC, and RuO2/CC benchmarking OER catalysts were also tested under the same condition. All the synthesized electrodes in this work are directly used as free-standing oxygen-evolving electrodes, including conventional RuO2/CC, to avoid possible influencing factors. The LSV polarization curves and Tafel plots for all samples are revealed, as shown in Figure 3a,b. The NiCo2S4 NS/CC exhibits a superior catalytic activity toward OER with a low onset potential of only 180 mV. Moreover, the overpotential of 260 mV is required to generate 10 mA cm–2, which is smaller than that of NiCo2S4 NN/CC (316 mV), NiCo2O4 NS/CC (368 mV), RuO2/CC (322 mV), and bare CC (484 mV). Also, it requires only a 280 mV overpotential to afford 10 mA cm–2 when the scan rate is 5 mV s–1 (Figure S5). It is lower than those of many other reported nonprecious metal-based OER electrocatalysts tested under 1 M KOH conditions, such as NiCo2S4 NWs/graphdiyne foam (300 mV), NiCo2S4/NF (306 mV)—, NiCo2S4 NAs/CC (310 mV), and so on.22−24,29,31,36−39 A detailed comparison is further summarized in Table S1. The cyclic voltammograms for NiCo2S4 NS/CC and NiCo2O4 NS/CC in the potential region from 1.0 to 1.8 V show a broad peak at 1.36 and 1.22 V, indicating the redox behavior of Ni2+ and Ni3+, respectively (Figure S7). The catalytic kinetics of OER is evaluated by Tafel plots in alkaline medium. The Tafel slope of NiCo2S4 NS/CC is 72 mV dec–1, which is lower than that of all other electrodes, such as RuO2/CC (87 mV dec–1), NiCo2S4 NN/CC (84 mV dec–1), NiCo2O4 NS/CC (114 mV dec–1), and bare CC (178 mV dec–1), indicating a more favorable rate of OER at the NiCo2S4 NS/CC electrode. The favorable kinetics of OER on NiCo2S4 NS/CC is also supported by EIS analysis to measure the charge transfer resistance during OER (Figure S6). The charge transfer resistance of NiCo2S4 NS/CC, which forms NSs morphology is 8.94 Ω at 1.5 V versus RHE, smaller than that of NiCo2S4 NN/CC with NN-like architectures (16.3 Ω). In contrast, NiCo2O4 NS/CC shows at least five times higher charge transfer resistance than that of NiCo2S4 NS/CC and NiCo2S4 NN/CC under the same applied potential because of the lower electrical conductivity of NiCo2O4 NS/CC. Figure 3 OER in alkaline media (1 M KOH, pH = 14): (a) LSV polarization curves for OER and (b) Tafel plots of NiCo2S4 NS/CC, along with NiCo2S4 NN/CC, NiCo2O4 NS/CC, RuO2/CC, and CC electrodes for comparison. (c) Cyclic voltammogram measured in a non-faradaic region at various scan rates for the NiCo2S4 NS/CC electrode. The inset shows the plot of anodic and cathodic charging current density vs different scan rates. (d) Current density based on intrinsic catalytic activity vs voltage curves. (e) TOF and the specific (intrinsic) activities of NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC electrodes at η = 350 mV. (f) Time dependence of the current density for NiCo2S4 NS/CC at a fixed potential of 1.55 V for 162 h. To better understand the different OER catalytic activities of NiCo2S4 NS/CC, including NiCo2S4 NN/CC and NiCo2O4 NS/CC catalysts, the ECSA and roughness factor (RF) of all electrodes are determined to estimate the real catalytic activities in the same pH condition. It can be easily calculated based on the double-layer capacitance (Cdl) through CV in a non-faradaic region at different scan rates of 1, 2.5, 5, 10, 20, and 50 mV s–1 (Figure 3c). The NiCo2S4 NS/CC electrode shows over twofold higher ECSA value of 5.5 mF cm–2 than that of NiCo2S4 NN/CC (2.0 mF cm–2) and NiCo2O4 NS/CC (2.3 mF cm–2), respectively (Figure S8). This indicates that plenty of catalytically active sites for OER might form on NiCo2S4 NS/CC. The surface roughness for all electrodes was also calculated by dividing the estimated ECSA to the geometric area of the electrode, and the values of 27.5, 10, and 11.5 were achieved for each electrode, NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC, respectively. On the basis of these results, 2D NS architecture arrays can offer larger space and have a rougher surface; hence, they lead to more electrochemical active sites on the catalyst surface. As a result, the excellent electrocatalytic performances of the NiCo2S4 NS/CC electrode can be partially ascribed to the high ECSA and consequently highly exposed active sites. We further calculate the TOF, which could provide the intrinsic OER catalytic activities of NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC electrodes. The TOF of various electrocatalysts was derived using the following equation: J is the geometric current density at a specific overpotential. A denotes the geometric area of the electrode. The number of electrons consumed for generating 1 mol of O2 from water is 4. F is the Faraday constant value of 96 485 C mol–1. m denotes the mole numbers of active materials.40 On the basis of this calculation, at an overpotential of 350 mV, the TOF of NiCo2S4 NS/CC and NiCo2S4 NN/CC is calculated as 7.46 × 10–2 and 4.12 × 10–2 mol O2 s–1, respectively (Figure 3e). In sharp contrast, NiCo2O4 NS/CC has the lowest TOF value of 1.19 × 10–2 s–1. Moreover, it is almost fourfold higher than that of the IrOx catalyst (0.89 × 10–2 s–1), indicating that the NiCo2S4 NS/CC is highly efficient toward OER.41 The specific activity of catalysts with different surface areas or loading is calculated with current normalization by the catalyst RF. NiCo2S4 NS/CC can deliver a high specific current density of 2.67 mA cm–2, whereas NiCo2O4 NS/CC can produce only 0.96 mA cm–2 at the same overpotential of 350 mV. Even though their morphology appears to be similar, that is, the density and the distribution of NiCo2S4 NSs on the CC are virtually the same as those of NiCo2O4 NSs on CC, NiCo2S4 NS/CC outperforms NiCo2O4 NS/CC. This result shows that NiCo2S4 NS arrays on CC have a higher intrinsic OER catalytic activity than that of NiCo2O4 NS arrays on CC (Figure 3d), which can be explained by the difference in the crystal structure of NiCo2S4 and NiCo2O4. NiCo2S4 that formed closely packed arrays of large S2– anions with nickel and cobalt metal cations in different oxidation states occupying the tetrahedral and octahedral sites, respectively, possesses more octahedral active sites of Co(III) concerning NiCo2O4, which has smaller anions of O2– in the spinel structure.31,42 On the basis of the previous literature, σ* orbital (eg) occupation–related metal cations at octahedral sites are mostly coordinated with electrocatalytic activities.43 In view of this point, NiCo2S4 NS/CC might afford better OER intrinsic activity compared to NiCo2O4 NS/CC. The electrocatalytic activity toward the OER of NiCo2S4 NS/CC is also evaluated in PBS (pH = 7) as well as control samples as shown in Figure 4a. Similar to the OER activity trend in alkaline media, the NiCo2S4 NS/CC catalyst exhibits the highest OER performance compared with other electrodes. Surprisingly, NiCo2S4 NS/CC requires only 321 and 402 mV to afford 5 and 10 mA cm2, respectively. However, RuO2/CC as a state-of-the-art OER catalyst needs an extremely large overpotential of 700 mV to deliver 5 mA cm–2 current density. At the same time, NiCo2S4 NN/CC and NiCo2O4 NS/CC require at least 368 and 460 mV to produce 5 mA cm–2, respectively. Bare CC shows negligible OER performance. The catalytic activity of NiCo2S4 NS/CC for OER in neutral media is exceptional compared to that of many electrodes reported recently, such as CoS4.6O0.6 (η = 570 mV for 5 mA cm–2),19 ultrathin Co3S4 NS (η = 650 mV for 3.27 mA cm–2),40 Co3O4 nanorod (η = 385 mV for 1 mA cm–2),44 Co-Pi NA/Ti foam (η = 450 mV for 10 mA cm–2),15 Co–Bi NSs/graphene (η = 570 mV for 14.4 mA cm–2),18 and Fe–Ni–P (η = 429 mV for 10 mA cm–2).14 The detailed comparison is summarized in Table S2. Figure 4b shows the Tafel plots of all electrodes for a better understanding of the obtained catalytic behavior. The Tafel slope of 123 mV dec–1 in a neutral electrolyte for NiCo2S4 NS/CC is achieved. It is the smallest value among NiCo2S4 NN/CC (125 mV dec–1) and NiCo2O4 NS/CC (203 mV dec–1) and comparable to that of RuO2/CC (115 mV dec–1), which in turn favors the kinetics of OER. Notably, in comparison with NiCo2O4 NS/CC, the NiCo2S4 NS/CC catalyst presents a lower Tafel slope value, originating from the increase in electrical conductivity as well as more plentiful electrocatalytic active sites correlated with its intrinsic activities. Figure 4 OER in neutral media (phosphate buffer, pH = 7): (a) LSV polarization curves for OER. (b) Corresponding Tafel plots of NiCo2S4 NS/CC, along with NiCo2S4 NN/CC, NiCo2O4 NS/CC, RuO2/CC, and CC electrodes for comparison. (c) Cyclic voltammogram measured in a non-faradaic region at various scan rates for the NiCo2S4 NS/CC electrode. (d) Anodic and cathodic current density vs scan rate plot of NiCo2S4 NS/CC. (e) Mass activities and TOF of NiCo2S4 NS/CC, NiCo2S4 NN/CC, and NiCo2O4 NS/CC electrodes in PBS. (f) Time dependence of the current density for NiCo2S4 NS/CC at a fixed potential of 1.6 V for 11 h. We further measure the double-layer charging of electrodes via scan-rate-dependent CVs to estimate the effective surface areas for catalytic activity. The potential range in which the non-faradaic region was chosen with the potential window of 0.04 V centered at an open-circuit voltage (OCV) of each system.34 The electrochemical double-layer capacitance for NiCo2S4 NS/CC is 0.044 mF cm–2, whereas those for NiCo2S4 NN/CC and NiCo2O4 NS/CC are 0.025 and 0.023 mF cm–2, respectively, indicating the rougher surface of the NiCo2S4 NS/CC electrode. It is noticeable that NiCo2S4 NS/CC still possesses almost twofold higher electrochemical double-layer capacitance than NiCo2O4 NS/CC in neutral media, which is probably because of the well-aligned hierarchical NSs architecture and the formation of numerous electrochemically active sites. The TOF at the overpotential of 400 mV in neutral media is evaluated to compare the intrinsic activities of NiCo2S4 NS/CC with those of other comparison electrodes. The calculated TOF for NiCo2S4 NS/CC is 9.89 × 10–3 s–1, which is much larger than those for previously reported cobalt-based catalysts, including Co3S4 (1.32 × 10–3 s–1 at η = 500 mV),40 Co–Pi (∼2 × 10–3 s–1 at η = 410 mV),45 Co–Bi (1.5 × 10–3 s–1 at η = 400 mV),46 and Co3O4 (≥0.8 × 10–3 s–1 at η = 414 mV),40 further suggesting the remarkable OER catalytic activity of NiCo2S4 NS/CC under neutral conditions. The NiCo2S4 NN/CC for which NiCo2S4 NN arrays are grown on CC indicates a TOF of 4.83 × 10–3 s–1, implying its lower intrinsic activities compared with that of the NiCo2S4 NS/CC electrode, whereas the TOF of NiCo2O4 NN/CC is calculated as 2.47 × 10–3 s–1, which shows the lowest value among the three electrodes. Therefore, the remarkable electrocatalytic activity of NiCo2S4 NS/CC can partially originate from the higher electrochemical surface area and the direct contact between NiCo2S4 NS arrays and the CC, which facilitate fast electron transfer as well as enhanced mass transportation. In addition, (i) the intrinsic electrocatalytic activity of the NiCo2S4 with larger anions compared with NiCo2O4 so as to expose more cation active sites; (ii) the enough void space among interconnected NiCo2S4 NSs, which allows facile redox ion diffusion; (iii) the 2D morphology of NiCo2S4 NSs that yields a large contact area between the catalyst and the electrolyte; and (iv) the formation of the nickel–cobalt (oxy)hydroxide active layer on its surface, which will be discussed later, all contributed to the superb performance of NiCo2S4 NS/CC in the OER. The long-term operation of the OER catalyst is a critical issue for practical application. Figure 3f exhibits the chronoamperometric (CA) curve of the NiCo2S4 NS/CC measured at 1.55 V potential in alkaline media (1 M KOH). After the 29 h CA test, the electrode entirely stabilizes and retains a current density of 10 mA cm–2 (without iR corrected) and then is reduced to 85% of its original activity over 160 h long-term operation. The inset of Figure 3f shows the linear polarization curves of NiCo2S4 NS/CC before and after 1000 CV cycles with 50 mV s–1 scan rate and a potential range from 1.2 to 1.7 V. Accordingly, there is no difference in the LSV curve recorded after 1000 CV cycles, indicating its high stability. Meanwhile, we detected losses of larger than 24, 29, and 47% in their current densities within 50 h for NiCo2S4 NN/CC, NiCo2O4 NS/CC, and RuO2/CC, respectively, at the same potential of 1.55 V in 1 M KOH solution. We also performed the durability test for same electrodes under the neutral condition. NiCo2S4 NS/CC presents excellent durability for 11 h, achieving 6 mA cm–2 at 1.6 V versus RHE with only 10% loss. During CA, O2 gas bubbles were visibly observed from the NiCo2S4 NS/CC electrode and dissipated quickly into the electrolyte. The NiCo2S4 NN/CC and NiCo2O4 NS/CC electrodes show a dramatic catalytic activity loss of 45 and 54%, respectively. Moreover, RuO2/CC almost lost its catalytic activity after 4 h durability test. The morphological robustness of NiCo2S4 NS/CC was examined by post-OER FE-SEM analysis under alkaline and neutral conditions (Figure S12). Maintaining morphology with negligible damage is another convincing evidence of the structural robustness of NiCo2S4 NS/CC observed in the FE-SEM micrographs. Recently, Li et al.46 reported that the nonoxide transition metal-based chalcogenides, especially cobalt selenide catalysts, usually oxidize during the OER under the basic condition and progressively transform to the corresponding TM (oxy)hydroxides, which is proposed to be the true active species to catalyze the OER.47 In the case of the Co3Se4/CF electrode, the XPS peak intensity of Se virtually disappears after a 3 h chronopotentiometric electrolysis duration, and after 12 h, Co3Se4 is converted to CoOOH. Similarly, in our study, we investigated the composition of the electrode after 160 h CA operation by XPS (Figure S11) to confirm the real surface species of NiCo2S4 NS/CC. The XPS Ni 2p shows that Ni2+ at 853.8 eV for Ni 2p3/2 and 873.8 eV for Ni 2p1/2 visibly disappears and the peaks located at 855.7 and 873.2 eV are assigned to Ni3+ species of the nickel (oxy)hydroxide.31,48 Moreover, the binding energy shift of Ni 2p for 1.4 eV reveals the occurrence of electron transfer during extended CA electrolysis. Similarly, the XPS Co 2p3/2 peak is deconvoluted into two peaks of 780.7 and 782.3 eV, which represent the formation of Co(OH)2 and CoOOH, implying the formation of a higher valence state of cobalt (Co3+).49,50 Meanwhile, the peak intensity of S 2p was weakened, whereas the two strong peaks of O 1s spectra were observed at 531.3 and 532.7 eV, indicating the O–H bond in NiCoOOH and the adsorption of H2O on the surface of NiCo2S4 NS/CC, respectively.48−50 The XPS results demonstrate that in situ electrochemical tuning of nickel–cobalt sulfide to nickel–cobalt mixed (oxy)hydroxide phase occurred, which is highly active for the OER catalytic activity attributed to the enhanced surface area and electrochemically active sites. This transformation might change the electronic states and the interactions with intermediate products during OER. Hence, it leads to the catalyst becoming more catalytic active for OER, which is also shown in other chalcogenide materials.46,50 The post-OER durability measurement for over 11 h in the neutral medium was also carried out using XPS analysis to confirm the chemical composition (Figure S12). Similarly, in an alkaline environment, the Ni2+ peak disappeared from the surface of catalyzed NiCo2S4 NS/CC and transformed to Ni3+ of TM (oxy)hydroxides with binding energy values at 855.7 and 873.2 eV as well as satellite peaks at 865.1 and 879.9 eV. Also, the new peaks formed at 779.9 and 781.1 eV are also assigned to the (oxy)hydroxides phase.47−50 It is noticeable that similar phenomena of in situ electrochemical tuning for NiCo2S4 NS/CC have occurred under a neutral condition, achieving an increase in the surface area as well as electrochemically active sites for primarily improved catalytic activity for OER. To validate the practical application of the NiCo2S4 NS/CC catalyst, a primary zinc–air battery was demonstrated and fully discharged to 0.6 V at a current density of 5 mA cm–2 (Figure 5a). The NiCo2S4 NS/CC cathode shows an OCV of 1.18 V with a specific capacity of 722 mA h g–1, which is almost 88.1% utilization of theoretical capacity (∼820 mA h g–1), whereas the commercial catalyst-based cathode shows an OCV of 1.31 V with a discharge capacity of 590 mA h g–1. Moreover, the galvanostatic discharge–charge cycling performance was evaluated at a current density of 5 mA cm–2 with a 5 min discharge followed by 5 min charge for each cycle (Figure 5b). For the initial cycle of NiCo2S4 NS/CC, the rechargeable battery discharged at 1.11 V versus Zn, with the corresponding charging potential of 1.90 V giving an overall overpotential of 0.79 V, which increased only 0.04 V (1.95 V for charge and 1.12 V for discharge potential) after 30 h battery operation (173 cycles). However, in the case of RuO2 + Pt/C/CC, the potential gap between charge and discharge increased continuously from 0.61 to 1.00 V even after 1350 min (135 cycles) cycling. The superior cycling durability over 173 cycles with a high discharge capacity of 722 mA h g–1 indicates the excellent electrocatalytic activity and stability of NiCo2S4 NS/CC for zinc–air batteries. Figure 5 Zn–air battery performance: (a) specific discharge capacities of primary zinc–air batteries with NiCo2S4 NS/CC and commercial RuO2 + Pt/C/CC air cathodes. (b) Comparative galvanostatic charge–discharge profiles of rechargeable zinc–air batteries based on NiCo2S4 NS/CC and RuO2 + Pt/C/CC air cathodes at 5 mA cm–2 in 10 min interval cycles. A two-electrode alkaline water electrolyzer was developed for full water splitting with NiCo2S4 NS/CC and Pt/C/CC as the anode and cathode, respectively, in 1 M KOH solution (Figure 6). To achieve a current density of 10 mA cm–2, a voltage of 1.53 V was needed for water splitting with gas evolution on both electrode surfaces, showing more advantages to split water than precious metal-based electrodes, which requires higher cell voltages of 1.64 V. Figure 6 Overall water electrolysis: the polarization curves based on NiCo2S4/CC//PtC/CC and commercial RuO2/CC//Pt/C/CC electrodes with a scan rate of 5 mV s–1 in 1 M KOH solution. The inset is the photograph of the two-electrode configuration. Conclusions In summary, the hierarchical spinel bimetallic sulfide nanostructures in situ grown on the CC were investigated for their electrochemical properties in different pH media and evaluated for their capability in practical primary and rechargeable zinc–air batteries. The most active NiCo2S4 NS/CC electrode can catalyze the OER at an overpotential of 260 mV at 10 mA cm–2 with good durability of over 160 h operations under an alkaline condition. Moreover, the NiCo2S4 NS/CC electrode still maintained its superior OER catalytic activity under the neutral condition. The enhanced intrinsic catalytic properties, morphology-based advantages of nanostructures, and the generation of the Ni–Co oxyhydroxide active layer were considered responsible for the excellent OER performance in water splitting. Especially, the in situ fabricated NiCo2S4 NS/CC-integrated air cathode exhibits excellent durability and electrocatalytic activity in zinc–air batteries compared with the precious metal-based catalyst. This work supports a snapshot of the rational design and construction of nonprecious electrode materials with excellent catalytic activity and durability for the future practical system toward OER in water electrolyzers and zinc–air batteries. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01375.Experimental section; XRD patterns; FE-SEM images; TEM EDX pattern and mapping images; LSV curves; EIS spectra; CV curves measured in the OER region; CV curves and their corresponding charging current density versus different scan rates; postmortem FE-SEM and XPS analysis; and comparison of OER performance with the recently reported literature (PDF) Supplementary Material ao8b01375_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was supported by the DGIST R&D Program of the Ministry of Science, ICT and Future Planning of Korea (18-IT-02), the Korea Institute of Energy Technology Evaluation and Planning (KETEP), and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (no. 20174030201590). 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PMC006xxxxxx/PMC6644434.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145783210.1021/acsomega.7b01051ArticleFree-Standing Sandwich-Structured Flexible Film Electrode Composed of Na2Ti3O7 Nanowires@CNT and Reduced Graphene Oxide for Advanced Sodium-Ion Batteries Li Zhihong †Ye Shaocheng †Wang Wei †Xu Qunjie †Liu Haimei †Wang Yonggang ‡Xia Yongyao ‡† Shanghai Key Laboratory of Materials Protection and Advanced Materials in Electric Power, College of Environmental and Chemical Engineering, Shanghai University of Electric Power, Shanghai 200090, China‡ Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Institute of New Energy, Fudan University, Shanghai 200433, China* E-mail: liuhm@shiep.edu.cn. Tel: +86 021 35303476 (H.L.).* E-mail: ygwang@fudan.edu.cn. Tel: +86 021 51630319 (Y.W.).12 09 2017 30 09 2017 2 9 5726 5736 26 07 2017 17 08 2017 Copyright © 2017 American Chemical Society2017American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A free-standing flexible anode material for sodium storage with sandwich-structured characteristics was fabricated by modified vacuum filtration, consisting of stacked layers of Na2Ti3O7 nanowires@carbon nanotubes (NTO NW@CNT) and graphene oxide. The NTO NWs have a larger specific surface area for Na+ insertion/extraction with shortened ion diffusion pathways, accelerating the charge transfer/collection kinetics. The added CNTs both facilitate the uniform dispersion of the nanowires and nanotubes and also contribute to the connectivity of the nanowires, improving their conductivity. More importantly, the unique sandwichlike layered-structured film not only provides large numbers of electron-transfer channels and promotes the reaction kinetics during the charging and discharging process but also ensures the structural stability of the NTO NWs and the electrode. Electrochemical measurements suggest that this rationally designed structure endows the electrode with a high specific capacity and excellent cycling performance. A satisfactory reversible capacity as high as 92.5 mA h g–1 was achieved after 100 cycles at 2C; subsequently, the electrode also delivered 59.9 mA h g–1 after a further 100 cycles at 5C. Furthermore, after the rate performance test, the electrode could be continuously cycled for 100 cycles at a current density of 0.2C, which demonstrated that durable cyclic capacity with a high reversible capacity of 114.1 mA h g–1 was retained. This novel and low-cost fabrication procedure is readily scalable and provides a promising avenue for potential industrial applications. document-id-old-9ao7b01051document-id-new-14ao-2017-01051fccc-price ==== Body Introduction In modern society, portable and wearable electronics, such as flexible sensors and hand-held displays, are highly desired, which has motivated research into the necessary lightweight and flexible high-performance power sources.1−3 At present, the study of flexible power sources is mainly focused on lithium-ion batteries (LIBs)4−6 and supercapacitors (SCs). In the energy-storage field, great attention has been focused on flexible SCs because of their convenient processability, fast charging and discharging ability, excellent rate capability, and their superior cycle lifetime.7−9 However, the energy density of SCs is not very high considering the need for long-term resilience, and thus flexible LIBs perhaps hold greater promise for smart electronics, rollup displays, wearable devices, and other applications.10−12 Flexible LIBs are of particular interest due to their higher energy storage densities. However, at present, with the commercialization of electric vehicles powered by LIBs, the limited availability of lithium storage will progressively increase its cost. As a result, low-cost sodium ion batteries (SIBs) have become a very popular topic in the research field of energy storage and conversion devices. Sodium ion batteries have attracted interesting attention because they have similar electrochemical properties to LIBs in addition to their low cost and environmental friendliness.13,14 However, flexible SIBs are still rarely reported at present. It is well-known that characteristics of batteries, such as flexibility, specific capacity, and rate capability, are mainly determined by their electrode materials.15 Therefore, it is highly desirable to search for suitable electrode materials with appropriate flexibility for use in SIBs. Actually, some endeavors have been made to study flexible electrode materials for flexible SIBs, such as MoS2,16,17 Sb,18 and Li4Ti5O12.19 Among the various anode materials, Na2Ti3O7 (NTO) has been most promising on account of its high specific capacity (177 mA h g–1) and its low charging/discharging voltage plateau of 0.3 V versus Na/Na+.20 Nevertheless, the structural instability and the low electronic conductivity of Na2Ti3O7 limit its further practical application. To solve these problems, lots of efforts have been made to design and fabricate Na2Ti3O7 nanostructures, including nanorods, nanoparticles, microspheres, nanotubes, and nanocomposites with carbon.21−26 However, the design and fabrication of reliable flexible electrodes with high storage capacity, high rate capability, and excellent stability remain challenging. For the fabrication of SIB electrodes, conventional methods usually involve mixing, casting, and pressing the mixed compounds. These compounds comprise a cathode or an anode material for sodium storage, the metal current collectors, a conductive agent to maintain the electrode conductivity onto the metal current collectors, and a binder to inhibit the shedding of the active materials from metal current collectors.27−29 Such electrodes usually have little flexibility due to their rigid structure. Moreover, they contain a significant amount of inert components (e.g., binders) and cumbersome current collectors are used in the electrodes, inevitably compromising the energy density of the devices. Lightweight flexible substrates, such as textiles30 or plastics,31 have been proposed as replacements for metal substrates. To address sluggish Na reaction kinetics, Li et al. first designed the surface-engineered Na2Ti3O7 nanoarrays on flexible Ti substrates by using the atomic layer deposition and hydrogenation process, through which the Na-ion batteries demonstrated the high-rate and long-cycled operation.32,33 Zhang et al. reported that NTO nanosheet array/carbon textiles exhibited excellent performance when used as the anode materials of sodium-ion pseudocapacitors.34 Our previous work also showed that ultralong NTO nanowires@carbon cloth as a binder-free flexible electrode demonstrated a large capacity and long lifetime for sodium-ion batteries.35 However, the preparation of these flexible electrodes usually requires a substrate, such as carbon cloth, carbon paper, etc., and they are difficult to develop for large-scale production. In addition, the loading mass of the active material in these flexible electrodes is limited and difficult to control. Herein, by introducing an architectural design strategy, a hybrid free-standing and sandwich-structured electrode was developed, which is a combination of one-dimensional nanowires together with nanotubes and two-dimensional graphene sheets in hierarchically structured composites. In this work, we demonstrate a simple method of hydrothermal-assisted vacuum filtration to prepare Na2Ti3O7 nanowires@CNT@reduced graphene oxide (NTO NW@CNT@rGO) sandwich-structured flexible film electrodes for flexible SIBs for the first time. The unique sandwich-structured NTO NW@CNT@rGO flexible electrode materials were prepared by layer-by-layer vacuum filtration. This not only affords stability and robustness in the electrodes but also facilitates the diffusion of the electrolyte and improves their electrical conductivity, thus enhancing the electrochemical properties of Na2Ti3O7. In addition, this method is suitable for large-scale production due to the low cost of the raw materials and the fact that the amount of active material can be modulated at will. Results and Discussion The free-standing and sandwich-structured flexible NTO NW@CNT@rGO film electrode was prepared using a two-step fabrication method. First, NTO NW@CNT was synthesized by a hydrothermal method. In the hydrothermal process, the carbon nanotubes are combined with Na2Ti3O7 nanowires and can be used as bridges between nanowires to improve the conductivity of the nanowires. NTO NW@CNT@rGO flexible electrode materials with a sandwich structure were then prepared via layer-by-layer vacuum filtration (Figure 1). The graphene therein has a large specific surface area and good electrical conductivity. The NTO NW@CNT layer is sandwiched between graphene layers during the process of vacuum filtration. On the one hand, this method is beneficial as it increases the contact area between nanowires and graphene and improves the electrical conductivity of the material. On the other hand, it can improve the infiltration of the electrolyte and increase the ion conductivity of the electrode material, thereby improving the electrochemical properties of Na2Ti3O7. Figure 1 (a–c) Schematic illustration and ideal electron-transfer pathway of a free-standing and sandwich-structured flexible film electrode. Meanwhile, the great flexibility of the film can be clearly observed in Figure 2a. The as-prepared electrodes can remain stable while being folded, which show great potential for use in highly flexible devices. To investigate the morphological and structural characteristics of the film, scanning electron microscopy (SEM) was employed. SEM images showing cross-sectional and top views of the S–NTO NW@CNT@rGO film are presented in Figure 2b–e. Figure 2b,c shows cross-sectional SEM images of the as-formed S–NTO NW@CNT@rGO film and the corresponding EDX mapping. As can be seen from Figure 2b, the thickness of the film is about 20 μm and the structure of the cross section is clearly considered as a layer-by-layer structure. Energy-dispersive X-ray (EDX) spectrometry mapping analysis was further employed for one layer of the film, and the results are shown in Figure 2c. The main elements of this layer are C (red), O (green), Na (cyan), and Ti (pink) and are shown in Figure 2c. C refers to the graphene on the upper surface and is evenly distributed. The shallow red color indicates that the graphene layer is relatively thin. Na, Ti, and O are the main elements of NTO and are evenly distributed. The color of the elements is deep, indicating that NTO is the main constituent of this layer. The structure of each layer is similar to the sandwich structure, which is consistent with the effect we expect to achieve by layered titration and filtration. Subsequently, the upper surface of the film was observed and is shown in Figure 2d,e. The top-view SEM image reveals that the thin graphene layer is uniformly distributed on the upper surface of the film on a large scale and through the graphene layer, interdigitated NTO nanowires can be clearly seen. At a greater magnification shown in Figure 2e, the graphene layer is indeed observed, and NTO NW@CNT composites exist under the graphene layer. Thus, it can be seen that in the as-formed S–NTO NW@CNT@rGO film, the CNT of the NTO NW@CNT layer serves mainly to increase the connections between nanowires, thus extending the pathway of electron transfer of the NTO nanowires. The upper and lower graphene layers serve mainly to increase the contact area with the NTO nanowires and improve the electrical conductivity of the crossed nanowires. Figure 2 (a) Digital optical image of the bending state of the NTO NW@CNT@rGO film; (b, c) SEM images of the cross section and corresponding EDX mapping of C (red), O (green), Na (cyan), and Ti (pink); and (d, e) SEM images of the upper surface. In addition, in Figure 2b, we also see that there is a certain gap between the layers in the as-formed S–NTO NW@CNT@rGO film, which can facilitate the penetration of the electrolyte. But how did these gaps form? To illustrate the problem, the graphene film and the NTO NW@CNT film were prepared by the same process and then observed and analyzed by SEM. Figure S1 shows the SEM images of NTO NW@CNT. It can be seen in Figure S1a that NTO nanowires with the large aspect ratio are very uniform on the upper surface of the film. The distributions of NTO nanowires and CNT are also uniform. It is regrettable that after vacuum filtration the NTO NW@CNT film does not form a multilayer structure that could be seen in the cross section. However, multilayer graphene is clearly observed in the cross section of the film after vacuum filtration, as shown in Figure S2b, which means that the formation of the multilayered S–NTO NW@CNT@rGO film may be related to the graphene itself. After filtration, graphene oxides are formed in the film and then undergo a high-temperature process where some of the oxygen-containing functional groups between the layers become vaporized oxygen, leading to the emergence of a gap between the layers, as shown in Figure S2. Therefore, the gaps in the as-prepared sandwich-structured film containing the graphene oxide occur due to the volatilization of oxygen after the high-temperature treatment. It is precisely because the film prepared by our method exhibits a certain gap that the film both improves the electrical conduction of the electrode and facilitates the penetration of the electrolyte. To further understand the structure of the S–NTO NW@CNT@rGO film electrode, transmission electron microcopy (TEM) was applied to observe the Na2Ti3O7 nanowires, CNT, and rGO of the film. As shown in Figure 3a,b, the interdigitated long NTO nanowires, including several hundred nanometers or even up to several micrometers in length, form a three-dimensional (3D) network structure, resulting in lots of porous structures. As we all know, these porous structures of the electrode materials likely facilitate the penetration of the electrolyte and also promote sufficient contact between the NTO nanowires and the electrolyte, providing shortened ionic diffusion pathways that may contribute to the improvement of the electrochemical performance in sodium storage. At the same time, it is clear that the distribution of the homogeneous NTO nanowires and the CNT is very uniform. CNT wrapped around the NTO nanowires increases the connectivity between the nanowires and improves their electrical conductivity. In Figure 3b, the graphene above the NTO nanowires and CNT can be seen to completely cover the nanowires having a large contact area with nanowires that greatly improves the conductivity of the nanowires. Figure 3c shows an HRTEM image of the NTO nanowires. The lattice fringes show interplanar spacings of 0.84 and 0.35 nm, corresponding to the distances of the (001) and (102) lattice planes in the NTO structure. The figure also shows that the NTO nanowires have good crystallinity, which is beneficial to the electrochemical performance of NTO. The colored diagrams in Figure 3d show the EDX mapping of NTO nanowires. It can be seen that the distribution of Na (cyan), O (green), and Ti (red) comprising Na2Ti3O7 is consistent with the orientation of the nanowires, indicating that the nanowires are made of sodium trititanate. Figure 3 (a, b) Low-magnification TEM images of the NTO NW@CNT@rGO film; (c) HRTEM image; and (d) TEM and corresponding EDX mapping of Na (cyan), O (green), and Ti (red). To quantify the amount of NTO and carbon in the flexible film, TGA was carried out in air. The sample was heated from 25 to 800 °C at a rate of 10 °C min–1. Figure 4a shows the TGA curve of the S–NTO@CNT@rGO film. The weight loss of the sample is about 16.3%, indicating that the carbon content of the film is about 16.3%, in line with the requirements for the carbon content of a flexible film material. In contrast, the NTO nanowires comprise approximately 83.6% of the mass and are the main component of the film. This shows that the as-prepared film has a certain flexibility, the key being that the nanowires themselves are also easily prepared as part of a flexible film. To gain further knowledge of the crystallographic phase and structure of the different samples, X-ray diffraction (XRD) tests were performed. As can be seen in Figure 4b, all of the diffraction peaks of NTO NW@CNT powder correspond to those of layered Na2Ti3O7 (JCPDS card no. 31-1329). In addition, for the as-prepared NTO NW@CNT@GO film and the NTO NW@CNT@rGO film, the sharp and well-defined peaks correspond to those of Na2Ti3O7 according to the standard PDF card data. However, compared to that of the NTO NW@CNT sample, the intensity of the diffraction peak of NTO NW@CNT@GO becomes stronger at 10.5° and weaker near 25 and 30°. The reason for this phenomenon is that graphene oxide has a strong diffraction peak at 10.5°, as shown in Figure 4c.36 The diffraction peaks of Na2Ti3O7 and graphene oxide are superimposed at 10.5° so that the intensity of the diffraction peak is enhanced, whereas those at 25 and 30° are weakened. After high-temperature treatment, graphene oxide is reduced to graphene (Figure 4c) and the intensity of the diffraction peak of the S–NTO NW@CNT@rGO film becomes weaker at 10.5°, consistent with that of NTO NW@CNT powder. Figure 4d displays the Raman scattering spectra of the rGO film and the S–NTO NW@CNT@rGO film. Two distinctive characteristic carbon bands between 1200 and 1700 cm–1 can be observed. The asymmetrical shape of the carbon characteristic bands implies the existence of two types of bonding states of carbon.37 The Raman scattering spectra near 1300 and 1600 cm–1 are the characteristic bands of the C-atom crystals corresponding to the disorder-induced phonon mode (D band) and the graphite band (G band) of the sp2 carbon, which represents the lattice defect of the carbon atom and the in-plane stretching vibration of the sp2 hybrid of the carbon atom, respectively.38,39 Figure 4 (a) TGA of the NTO NW@CNT@rGO film; (b) XRD patterns of various samples; (c) XRD of GO and rGO; (d) Raman spectra of rGO and NTO NW@CNT@rGO film. To quantify the excellent electrochemical performance of the free-standing sandwich-structured film prepared by vacuum filtration, the electrochemical sodium storage characteristics of the flexible S–NTO NW@CNT@rGO film were evaluated by assembling an appropriate mass of the material in a Na-haft cell. The electrical energy storage capability of the film electrode was first examined using cyclic voltammograms (CVs). Figure 5a shows the CV curve at a scanning rate of 0.02 mV s–1 in the potential range between 2.5 and 0.01 V versus Na/Na+. A couple of redox peaks located at 0.25 V (cathodic peak) and 0.63 V (anodic peak) versus Na/Na+ can be observed, representing typical Na+ insertion/extraction in the Na2Ti3O7 crystal lattice. The distinct and well-defined redox peaks of Na2Ti3O7 indicate its excellent kinetics performance in the S–NTO NW@CNT@rGO film electrode. Moreover, the CV of the S–NTO NW@CNT@rGO electrode in a voltage window of 0.01–2.5 V at various sweep rates ranging from 0.01 to 0.1 mV s–1 is presented in Figure S3. The redox peaks in the potential range of about 0.2–0.6 V in the CV data could be attributed to the redox of the Ti4+/Ti3+ couple. With increasing cycles and increasing scanning rates, the peak current and the voltage of the electrodes increased continuously, which is consistent with the characteristics of the electrode material itself, indicating the great stability of the as-prepared electrode materials and the good reversibility of the de/intercalation of sodium ions. The charge/discharge curves of S–NTO NW@CNT@rGO were calculated at 1C in the voltage range 0.01–2.5 V for 1st, 10th, 20th, 50th, 70th, and 100th cycles, as shown in Figure 5b. The initial discharge and charge capacities of the S–NTO NW@CNT@rGO film were 372.2 and 184.4 mA h g–1, respectively, which are higher than the theoretical capacity of 177 mA h g–1. Similar phenomena have been observed from self-assembled TiO2–graphene hybrid nanostructures40 and graphene-wrapped porous Li4Ti5O12 nanofiber composites.41 The higher capacity suggests additional interfacial Na storage at the NTO/electrolyte (solid–liquid) and NTO/graphene (solid–solid) interfaces. The interfacial reaction between the electrode and the electrolyte led to the formation of a solid electrolyte interphase (SEI) film, causing a large irreversible capacity loss. In addition, the electrode displays sloped reaction plateaus located at 0.6 V (charge) and 0.2 V (discharge), in keeping with the CV curves (Figure 5a) and showing the typical charge/discharge profiles of the Na2Ti3O7 electrode in SIBs.26,34 Figure 5 Electrochemical measurements of the as-formed NTO NW@CNT@rGO electrode in a range of 0.01–2.5 V. (a) Cyclic voltammograms at a scan rate of 0.02 mV s–1; (b) charge–discharge curves at different cycles; and (c–f) cycling performance at current densities of 0.5C, 1C, 2C, and 5C. To investigate the cycling stability of the film electrodes, a cycling performance test was performed at different current densities. Figure 5c–f shows the cycling performance of the S–NTO NW@CNT@rGO film when the current densities were 0.5C, 1C, 2C, and 5C, respectively. As can be seen from Figure 5c–e, the film electrodes show good discharge specific capacity and Coulombic efficiency. At lower current densities 0.5C, 1C, and 2C, S–NTO NW@CNT@rGO delivers a high initial discharge capacity, and capacities of 419, 372.2, and 380.5 mA h g–1 can be respectively achieved. Remarkably, after 100 cycles, the electrode retains high capacities of 107.2, 101, and 97.9 mA h g–1, respectively, which confirms the excellent properties of Na storage capacity and cycling stability. For the NTO@CNT film electrode, its initial discharge capacity is only 141.7 mA h g–1, and after 70 cycles, a capacity of 34.2 mA h g–1 is retained, as shown in Figure S4. Similarly, the cycling performance of the NTO@CNT+rGO film electrode is also very poor. As shown in Figure S5, the NTO@CNT+rGO electrode delivers only 65.3 and 26.5 mA h g–1 at 0.5C and 1C, respectively, after 100 cycles. On the other hand, the most impressive observation is that the S–NTO NW@CNT@rGO electrode still retained a discharge capacity of 80.6 mA h g–1 after 150 cycles at the highest current density of 5C, which is a relatively high discharge capacity when Na2Ti3O7 is presently used as the anode material of SIBs under the same conditions.23,24,42 To further explore the effectiveness of the sandwich-structured film in improving the electrochemical performance of Na2Ti3O7 electrodes, the long-term cycling performance and the rate capability were investigated, as shown in Figure 6. In Figure 6a, the S–NTO NW@CNT@rGO film electrode starts at 363.8 mA h g–1 and maintains at 105.7 mA h g–1 after initial precycling. From 11 cycles, the discharge capacity has remained in a stable range at 2C, and it is satisfactory that a reversible capacity as high as 92.5 mA h g–1 is still achieved after 100 cycles. Subsequently, the S–NTO NW@CNT@rGO electrode also delivers 59.9 mA h g–1 after a further 100 cycles at 5C. This experiment fully demonstrates that the as-prepared S–NTO NW@CNT@rGO electrode possesses better capacity retention and longer lifetime. Figure 6b shows the rate capabilities of S–NTO NW@CNT@rGO at various current densities. For each stage, the process was taken from 0.2C to 10C over five cycles. Compared with the S–NTO NW@CNT+rGO electrode (Figure S6), the S–NTO NW@CNT@rGO electrode delivers discharge capacities of 204.8, 162, 115.5, 86.5, 56, and 31.9 mA h g–1 at current densities of 0.2C, 0.5C, 1C, 2C, 5C, and 10C, respectively. When cycled back to 5C, 2C, 1C, 0.5C, and 0.2C from 10C, the discharge capacity of the electrode can still reach up to 59.7, 91.7, 133.6, 166, and 196.8 mA h g–1, indicating the excellent reversibility of the S–NTO NW@CNT@rGO electrode. To further confirm the excellent electrochemical performance of the film, the electrode was even continuously cycled for 100 cycles at a current density of 0.2C, which showed that durable cyclic capacity with a high reversible capacity of 114.1 mA h g–1 was retained. The above results indicate that the S–NTO NW@CNT@rGO electrode has a significant advantage in terms of lifetime and capacity retention. Figure 6 Electrochemical measurements of the as-formed S–NTO NW@CNT@rGO electrode in a range of 0.01–2.5 V. (a) Long cycling performance at different current densities from 2C to 5C; and (b) rate performance at various current densities from 0.2C to 10C and then cycling performance at 0.2C. From the above results, it can be seen that the as-prepared S–NTO NW@CNT@rGO flexible electrode materials have excellent electrochemical performance, which is mainly attributed to the design strategy and fabrication method of the electrodes. First, the absence of a binder is beneficial because of the increased electrical conductivity of the electrodes. The conventional electrode preparation process involves mixing the active materials, the electronic conductor, and the binder with the n-methyl-2-pyrroludone solvent and coating the resultant slurry on copper foil as a current collector.43,44 However, our film electrode without the binder or current collector can not only improve the electrical conductivity but also effectively decrease the weight of the SIB system and enhance its energy density. Second, the Na2Ti3O7 nanowires grown in situ using the hydrothermal method have many advantages, including a larger specific surface area for Na+ insertion/extraction and shortened ion diffusion pathways that promote the charge transfer/collection kinetics. In addition, many porous structures are formed by intertwined nanowires, which are more conducive to the infiltration of the electrolyte, increasing the ion diffusion rate. Third, in the hydrothermal process, the added nanotubes not only facilitate the uniform dispersion of nanowires and nanotubes but also contribute to the formation of Na2Ti3O7 nanowires, in which the nanotubes wrap around the nanowires or connect them, improving the conductivity of the nanowires themselves. Fourth, to ensure the largest contact area between the graphene and Na2Ti3O7 nanowires, the process of the as-prepared film is repeated first with the filtration of graphene and then with that of the NTO NW@CNT. If this step is not performed, the contact area between graphene and nanowires is very small because most of the nanowires are inside the graphene layer rather than lying on the graphene surface, as seen in Figure S7b,c, leading to the poor conductivity of the material even with the addition of graphene. Fifth, the sandwichlike layered-structured film is formed via vacuum filtration. As can be seen in Figures 2b and S2, there is a certain gap between two layers because of the volatilization of oxygen in the reduction process of graphene oxide, which is beneficial to the infiltration of the electrolyte. Of course, the gap does not exist for the NTO NW@CNT+rGO sample in Figure S7a. Finally, the biggest advantage of sandwich-structured NTO NW@CNT@rGO is its ability to improve the electrical conductivity itself. Figure 1 shows the schematic representation and ideal electrotransfer pathway for the as-prepared S–NTO NW@CNT@rGO film. It can be seen that the electrons can transfer at will in the graphene layer and between the graphene layer and NTO NW@CNT layers. In addition, in the NTO NW@CNT layer, CNT connecting the nanowires can allow the electrons to move freely, implying that many electron-transfer channels are added. All of these benefits endow the electrode with the proposed flexible, current collector-free, binder-free, and sandwich-structured anode configuration, which significantly improve the cycling performance and the rate capability of the electrode. To further confirm the structural integrity of the electrodes, the battery was disassembled after the charge and discharge cycles and the electrode material was analyzed by both XRD and SEM (Figure 7). In Figure 7a, compared with the diffraction peaks before cycling, the diffraction peak of the electrode material after charging and discharging is very obvious at 10.5°, which is the typical peak of the Na2Ti3O7 pattern corresponding to JCPDS no. 31-1379. This proves that the electrode material itself does not change after undergoing a rigorous process of intercalation and deintercalation of sodium ions. SEM images of a cross section of the S–NTO NW@CNT@rGO film electrode after cycling are shown in Figure 7b,c. The hierarchical structure is still obviously maintained, and the graphene and NTO nanowires of the interlayer are still observed, indicating that the sandwich-structured film is very stable. Meanwhile, as seen in the top view in Figure 7d,e, the morphology of the NTO nanowires remains intact, and there is still a graphene layer covering the surface of the NTO nanowires. This means that the graphene layer not only protects the multilayer structure of the electrode from damage and improves the electrode conductivity but also ensures that the NTO nanowires are not destroyed during the charging and discharging process. Figure 7 (a) XRD patterns of the NTO NW@CNT@rGO film electrode before and after the cycling test and morphology and structure of the NTO NW@CNT@rGO film electrode after the cycling test: (b, c) SEM images of the cross section and (d, e) SEM images of the upper surface. Conclusions In summary, we have presented a facile approach to produce free-standing and sandwich-structured NTO NW@CNT@rGO flexible film anode electrodes for high-performance sodium ion batteries by hydrothermal-assisted modified vacuum filtration. The as-obtained S–NTO NW@CNT@rGO flexible electrodes are not only binder-free and current-collector-free but also exhibit excellent electrochemical performances with a long cycle lifetime and large capacity for NIBs. At current densities of 0.5C, 1C, and 2C, the discharge capacities of the electrodes can be maintained at 107.2, 101, and 97.9 mA h g–1 after 100 cycles, respectively. The S–NTO NW@CNT@rGO electrode still retain a discharge capacity of 80.6 mA h g–1 after 150 cycles at a higher current density of 5C. During a long-term cycling performance test, after 100 cycles at a current density of 2C, a reversible capacity of 92.5 mA h g–1 was achieved; the discharge capacity could reach 59.9 mA h g–1 at 5C after 100 cycles. More importantly, after the rate performance test, a high reversible capacity of 114.1 mA h g–1 was retained after a further 100 cycles at 0.2C. The superior electrochemical performance of the electrodes is believed to be in connection with the unique sandwich architecture formed by the ultralong Na2Ti3O7 nanowires@CNT layer and highly conductive graphene layer. Furthermore, this general strategy explores exciting new methods for the design and fabrication of other flexible films applying to catalysis, energy storage, and other energy transformation processes. Experimental Section Synthesis of Graphene Oxide (GO) GO was prepared from natural graphite by a modified Hummers method.45,46 Briefly, 3 g of graphite powder was slowly added into a mixture of 50 mL 98% H2SO4, 3 g of K2S2O8, and 3 g of P2O5 and then the solution was stirred continuously at 80 °C for 5 h. The prepared preoxidized product was cleaned using deionized water and dried in a vacuum oven at 80 °C for 12 h. Afterward, it was mixed with 150 mL of 98% H2SO4, forming a black uniform dispersion. KMnO4 (15 g) was slowly added at a temperature below 20 °C, then the ice bath was removed, and the mixture was stirred at room temperature for 1 h. After slowly heating to 35 °C for 2 h, additional ice–water mixture and 20 mL of 30% H2O2 were slowly added into the mixed solution to completely react with the excess KMnO4. After 10 min, a bright yellow solution appeared. The resulting mixture was washed with diluted aqueous HCl (1/10 v/v) solution and H2O. Graphite oxide was prepared after freeze-drying in a vacuum oven. Synthesis of Na2Ti3O7 Nanowires@CNT (NTO NW@CNT) Na2Ti3O7 nanowires@CNT was synthesized via a simple hydrothermal process. In brief, 20 mL of 0.2 M tetrabutyl titanate ([CH3(CH2)3O]4Ti) solution in ethanol was mixed with 20 mL of 10 M aqueous NaOH solution. After adding 0.0403 g of CNT into the solution, the mixed solution was magnetically stirred and then transferred into a Teflon-lined stainless steel autoclave. It was heated in an oven at 190 °C for 12 h. After being air-cooled to room temperature (25 °C), the precipitate was collected by centrifugation, washed with deionized water and ethanol several times, and dried in air at 80 °C for 24 h, finally obtaining Na2Ti3O7 nanowires@CNT. Fabrication of Free-Standing and Sandwich-Structured Na2Ti3O7 Nanowires@ CNT@Reduced Graphene Oxide (S–NTO NW@CNT@rGO) Flexible Film Electrode The flexible S–NTO NW@CNT@rGO film was fabricated by the vacuum filtration method. Typically, 20 mg of GO and 10 mg of NTO NW@CNT were respectively dispersed in 50 mL ethanol and then sonicated for 8 h to form solutions A and B. Subsequently, a sandwich-structured film was prepared by adding the solution dropwise to a vacuum filter with a 0.2 μm porous PTFE membrane according to the order “ABAB···”. Finally, the obtained filter film was peeled off from the microporous membrane and vacuum-dried at 100 °C for 12 h to obtain a free-standing film. The film was cut into pieces with a diameter of 10 mm and vacuum-dried at 200 °C for 4 h to convert graphene oxide to reduced graphene oxide. For comparison, a Na2Ti3O7 nanowires@CNT (NTO NW@CNT) film and a Na2Ti3O7 nanowires@CNT+rGO (NTO NW@CNT+rGO) film were also prepared by vacuum filtration. A dispersion with Na2Ti3O7 nanowires@CNT powder dispersed in ethanol by ultrasonication was filtered to produce a NTO NW@CNT film. The NTO NW@CNT+rGO film was obtained by filtering a mixture of the ethanol solution with Na2Ti3O7 nanowires@CNT powder and graphene oxide. Material Characterization The X-ray diffraction patterns of the prepared samples were obtained using a Bruker D8 advance instrument at 40 kV and 40 mA with Cu Kα radiation (λ = 0.154 nm) (the scanning rate: 3 min–1; the 2θ range: 5–70°). The morphology and particle size of the samples were investigated using field-emission scanning electron microscopy (FESEM, JSM-7800F) and energy-dispersive X-ray spectroscopy (EDX). TEM and HRTEM images were obtained using a JEM-2100F instrument under a different resolution transmission electron microscope. Raman spectra were recorded on a PerkinElmer system at a laser wavelength, λ, of 532 nm. Thermogravimetric analysis (TGA) was carried out using a STA409 PC thermogravimetric analyzer to determine the carbon content in the flexible samples. Electrochemical Measurements The electrochemical performance of the as-prepared samples was investigated by CR2016-type coin half-cells using Na metal foil as the counter electrode. The free-standing and flexible S–NTO NW@CNT@rGO films were directly used as the working electrodes without the involvement of any metal current collector, conductive additive, or polymeric binder. The half-cells were assembled in an argon-filled glove box. The mass loading of the active material in each coin cell was typically 1.0–1.5 mg cm–2. NaClO4 (1 M) in a mixture (1:1, volume) of propylene carbonate (PC) and ethylene carbonate (EC) was used as the electrolyte, and a glass microfiber filter (Whatman GF/D) was used to separate the contacts of the cathode and anode electrode materials. Galvanostatic charge/discharge measurements were conducted using a LAND CT2001A test system (Wuhan, China) under various current densities in the potential range 0.01–2.5 V (vs Na+/Na) at room temperature. Cyclic voltammetry (CV) measurements were performed using a CHI650D (Chenhua, Shanghai) electrochemical workstation. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01051.SEM images of the as-formed NTO NW@CNT film electrode and as-formed rGO film, CV curves of the S–NTO NW@CNT@rGO electrodes at various sweep rates, cycling performance of the as-formed NTO NW@CNT film electrode at the current density of 0.2C, cycling performance of the as-formed NTO NW@CNT+rGO film electrode at current densities of 0.5C and 1C, rate performance of the as-formed NTO NW@CNT+rGO film electrode, SEM images of the as-formed NTO NW@CNT+rGO film electrode (PDF) Supplementary Material ao7b01051_si_001.pdf The authors declare no competing financial interest. Acknowledgments This work was financially supported by the National Nature Science Foundation of China (Nos. 21543015, 21336003, and 21371021), the National Key Research Program of China (No. 2016YFB0901501), and Science and Technology Commission of Shanghai Municipality (No. 14DZ2261000). ==== Refs References Miller J. R. ; Simon P. Electrochemical capacitors for energy management . Science 2008 , 321 , 651 –652 . 10.1126/science.1158736 .18669852 Xiao X. ; Yuan L. ; Zhong J. ; Ding T. ; Liu Y. ; Cai Z. ; Rong Y. ; Han H. ; Zhou J. ; Wang Z. L. High-strain sensors based on ZnO nanowire/polystyrene hybridized flexible films . Adv. Mater. 2011 , 23 , 5440 –5444 . 10.1002/adma.201103406 .22002439 El-Kady M. F. ; Strong V. ; Dubin S. ; Kaner R. B. Laser scribing of high-performance and flexible graphene-based electrochemical capacitors . Science 2012 , 335 , 1326 –1330 . 10.1126/science.1216744 .22422977 Ye J. ; Li Y. X. ; Zhang L. ; Zhang X. P. ; Han M. ; He P. ; Zhou H. S. 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PMC006xxxxxx/PMC6644435.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145911310.1021/acsomega.8b01508ArticleHybrid Noble-Metals/Metal-Oxide Bifunctional Nano-Heterostructure Displaying Outperforming Gas-Sensing and Photochromic Performances Tobaldi David Maria †Leonardi Salvatore Gianluca ‡Movlaee Kaveh ‡§Lajaunie Luc ∥#¶Seabra Maria Paula †Arenal Raul ∥⊥Neri Giovanni ‡Labrincha João António †† Department of Materials and Ceramics Engineering/CICECO−Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal‡ Department of Engineering, University of Messina, C.da Di Dio, 98166 Messina, Italy§ Center of Excellence in Electrochemistry, School of Chemistry, College of Science, University of Tehran, 14155-6455 Tehran, Iran∥ Laboratorio de Microscopías Avanzadas, Instituto de Nanociencia de Aragón, Universidad de Zaragoza, 50018 Zaragoza, Spain⊥ ARAID Foundation, 50018 Zaragoza, Spain E-mail: david.tobaldi@ua.pt and david@davidtobaldi.org. Phone: +351 234 370 041.24 08 2018 31 08 2018 3 8 9846 9859 01 07 2018 10 08 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. As nanomaterials are dominating 21st century’s scene, multiple functionality in a single (nano)structure is becoming very appealing. Inspired by the Land of the Rising Sun, we designed a bifunctional (gas-sensor/photochromic) nanomaterial, made with TiO2 whose surface was simultaneously decorated with copper and silver (the Cu/Ag molar ratio being 3:1). This nanomaterial outperformed previous state-of-the-art TiO2-based sensors for the detection of acetone, as well as the Cu–TiO2-based photochromic material. It indeed possessed splendid sensitivity toward acetone (detection limit of 100 ppb, 5 times lower than previous state-of-the-art TiO2-based acetone sensors), as well as reduced response/recovery times at very low working temperature, 150 °C, for acetone sensing. Still, the same material showed itself to be able to (reversibly) change in color when stimulated by both UV-A and, most remarkably, visible light. Indeed, the visible-light photochromic performance was almost 3 times faster compared to the standard Cu–TiO2 photochromic material—that is, 4.0 min versus 10.8 min, respectively. It was eventually proposed that the photochromic behavior was triggered by different mechanisms, depending on the light source used. document-id-old-9ao8b01508document-id-new-14ao-2018-01508accc-price ==== Body 1 Introduction As nanostructured materials are dominating the scene in the 21st Century, their potential influence on future products and services is enormous. This has led to a huge advance in research and development efforts related to nanoscale materials and devices. As such, researchers are willing to engineer complex multifunctional and, to a certain degree, smart devices and systems. There is consequently a request for a higher level of complexity in materials design, that is, in other words, combining multiple functionalities in the same nanostructure.1 Metal oxides (MOs) are a very important family of nanomaterials, having unique properties and advantageous functionalities that clearly are attractive for a number of applications ranging from electronic devices to energy conversion.2 Titanium dioxide (titania, TiO2) nanoparticles (NPs), because of their versatility, are a well-investigated class of MOs. Indeed, they are widely used in everyday applications, becoming by far the most popular material in light-to-energy conversion and storage.3 The “sun” of TiO2 as photocatalyst rose indeed in Japan more than 4 decades ago, when a paper by Honda and Fujishima about semiconductor photo-electrochemistry was published in 1972.4 This initiated research on liquid junction solar cells and semiconductor-assisted photocatalysis.5 The photocatalytic (PC) mechanism is now well known: when the surface of a semiconductor material is irradiated with photons having energy higher than or equal to its band gap energy (Eg), photo-electrons e– and photo-holes h+ are generated.6 Taking advantage of this phenomenon, multifunctional nanomaterials might be engineered—that is, simultaneous PC and antibacterial activities have been shown to exist in silver-modified titania.7 Furthermore, Japan was also the cradle of that phenomenon that was named “multicolour photochromism” by their discoverers, Tatsuma’s group and, once again, Fujishima’s group.8 This was found exploiting the PC phenomenon in anatase TiO2 films loaded with silver NPs when triggered by UV-light. This outcome led Parkin et al. to demonstrate a relationship between the photochromic state of a Ag–TiO2 thin-film and its UV-light PC activity,9 thus showing multifunctionality within the Ag–TiO2 system. Following on from these findings, Tobaldi and co-authors, first reported the photochromism and PC activity of multifunctional silver-modified TiO2 NPs, triggered by a purely visible white-light.10 It has also been recently shown that a nanomaterial exhibiting reversible color properties can be engineered by coupling copper and TiO2, and the redox process from Cu2+ to Cu+ linked to a change in color, can be simply controlled by regulating the light source (UV or visible light) and exposure time.11 Hence, decorating titania with a noble metal can yield to a purely inorganic photochromic noble-metal/MO hybrid nanomaterial. Conversely, most of the existing photochromic systems are based on polymers or organogels containing organic moieties, thus displaying limitations for being implemented on commercial devices.12 Besides, TiO2 nanomaterials have also been extensively employed for sensing applications,3 because of their unique chemical and physical properties as well as the nontoxic and environmentally friendly nature, excellent biocompatibility, and stability. In particular, it has been widely demonstrated that resistive gas sensors based on TiO2 nanostructures are highly effective in detecting both reducing [i.e., H2, CO, volatile organic compounds (VOCs)],13 and oxidizing (e.g., NO2, O3) gases.14 TiO2 nanostructures—because of the extremely high surface to volume ratio and their ability to absorb reactive oxygen species at their surface15—can provide a large number of active sites for the interaction with the gas, thus resulting in high sensitivity. Despite this, some drawback related to high working temperatures and long recovery times are the main disadvantages of that material.3 Modifications of TiO2 by doping and/or the formation of metal/TiO2 or MO/TiO2 heterostructures are amongst the simplest ways to enhance the sensitivity and selectivity yet shortening the response/recovery times.16 Our previous works were inspired by light and its ability to trigger redox reactions on the surface of a semiconductor nanomaterial when this is decorated with noble-metals NPs (i.e., silver and copper, as in refs (10) and (11), respectively). Those redox reactions are indeed associated to a reversible change in color of the (photochromic) material. Color is definitely an essential characteristic of Japanese traditional metal art and craft.17 As a result, this research was greatly inspired by the Land of the Rising Sun—photocatalysis,4 photochromism,8 and Japanese traditional alloys.17Shibuichi—a copper–silver alloy used mainly for sword ornaments, that is, tsuba—is one of the most representative Japanese traditional alloys. Shibuichi means literally “one-fourth”, to indicate its standard formulation of a copper alloy containing 25% of silver.17 Hence, we designed a multifunctional photochromic/gas-sensing nanomaterial, made with TiO2 decorated expressly with copper and silver having the Cu/Ag molar ratio constrained to be 3:1—that is, one part of Ag, and three parts of Cu. As far as we know, this is the first research of its kind involving multifunctionality (i.e., UV and most remarkably visible-light photochromism as well as gas-sensing properties) on TiO2 modified with Ag NPs and CuxO nanoclusters, certainly without neglecting the particular ratio of the noble metals used in here. As a matter of fact, previous research works involved TiO2 modified with Cu and Ag, but solely with a view limited to PC or antibacterial applications.18,19 2 Results and Discussion 2.1 QPA and Microstructural Analyses: XRD and HR-STEM X-ray diffraction (XRD) patterns of the specimens are shown in Figure 1. Results of the XRD-quantitative phase analysis (QPA) analysis are listed in Table 1, and a graphical output of a Rietveld QPA refinement is shown in Figure S1. Unmodified titania is composed of all the three main natural occurring TiO2 polymorphs/anatase (56.5 wt %), rutile (19.8 wt %), and brookite (23.6 wt %). The presence of brookite has to be linked to the strongly acidic synthesis conditions.20 The modification with both copper and silver led to a huge decrease in the weight fraction of rutile and brookite, meaning a delay in the anatase-to-rutile phase transition (ART). Specimen SBC1, for instance, is composed of 94.5 wt % anatase, 4.4 wt % rutile, and 1.2 wt % brookite. By contrast, SBC10 contains 93.7 wt % anatase, 3.7 wt % rutile, and 2.6 wt % brookite, see Table 1. Actually, in Shibuichi specimens, the trend with the increase in the Ag + Cu mol % content is a slight decrease in the anatase and rutile fractions (from 94.5 to 93.7 wt %, and from 4.4 to 3.7 wt %, respectively) and a small increase in brookite amount—from 1.2 to 2.6 wt %. The delay in the ART, when including Ag + Cu in the system, can be explained as being caused by a grain-boundary pinning (Zener drag),21 which culminates in delaying the ART—that is a nucleation-growth mechanism.22 Microstructural information, as extracted by means of whole powder pattern modeling (WPPM), is listed in Tables 2 and 3; an example of WPPM graphical output is shown in Figure S2. From the negligible differences in unit cell volumes, it can be inferred that neither Cu nor Ag entered the TiO2 lattice. Indeed, the ionic radii of [VI]Cu1+/2+, [VI]Ag1+/2+, and [VI]Ti4+ are 0.77/0.73, 1.15/0.94, and 0.61 Å, respectively.23 Modification of TiO2 with Ag + Cu also led to a decrease in the mean average diameter of the crystalline domains, cf Table 3 and Figure 2a–c. Anatase domains in unmodified TiO2 have an average diameter of 10.4 nm, rutile of 14.4 nm, and brookite averages of 7.0 nm. Shibuichi modification of TiO2 led not only to smaller crystalline domain diameters, but also to narrower size distribution of those crystalline domains, refer to Table 3 and Figure 2a–c. The average crystalline domain diameter in anatase and rutile that are in SBC1 is 6.7 and 11.2 nm, respectively; on the other hand, that of anatase and rutile in SBC10 is 4.6 nm for both the polymorphs. This outcome means that copper and silver NPs likely nucleate on the grain boundaries, pinning them, thus limiting their further growth. This also explains the observed delay of the ART and crystal growth. The decrease in the size of anatase nanocrystals is also confirmed by Raman analysis: the Raman Eg mode of anatase is shifted toward higher energies in the Ag + Cu specimens, thus confirming their reduced dimensions (cf Table S1).24 Figure 1 XRD patterns of synthesized specimens. The vertical bars represent the XRD reflections of anatase (black, JCPDS-PDF card no. 21-1272), rutile (blue, JCPDS-PDF card no. 21-1276), and brookite (red, JCPDS-PDF card no. 29-1360—only the three most intense reflections were reported here). Figure 2 Size distribution, as obtained from the WPPM modeling of (a) anatase, (b) rutile, and (c) brookite, contained in the synthesized specimens. Table 1 Rietveld Agreement Factors and Crystalline Phase Composition of the Unmodified and Cu/Ag-Modified TiO2a agreement factors phase composition (wt %) sample no. of variables RF2 (%) Rwp (%) χ2 anatase rutile brookite Ti450 20 3.54 4.03 1.74 56.5 ± 0.1 19.8 ± 0.2 23.6 ± 0.6 SBC1 19 2.27 3.01 1.59 94.5 ± 0.1 4.4 ± 0.2 1.2 ± 0.2 SBC2 18 2.04 3.48 1.56 93.4 ± 0.1 4.8 ± 0.2 1.8 ± 0.2 SBC5 16 2.72 3.42 1.66 93.6 ± 0.1 4.2 ± 0.2 2.2 ± 0.3 SBC10 17 4.22 3.16 1.64 93.7 ± 0.1 3.7 ± 0.2 2.6 ± 0.4 a There were 2285 observations for every refinement; the number of anatase, rutile, and brookite reflections was 32, 31, and 154, respectively. Table 2 WPPM Agreement Factors and Unit Cell Parameters for the Three Phases in the Synthesized Samples unit cell parameters agreement factors anatase rutile brookite sample Rwp (%) Rexp (%) χ2 a = b (nm) c (nm) V (nm3) a = b (nm) c (nm) V (nm3) a (nm) b (nm) c (nm) V (nm3) Ti450 6.17 1.96 3.15 0.3791(1) 0.9515(1) 0.137(1) 0.4598(1) 0.2959(1) 0.063(1) 0.5440(2) 0.9206(4) 0.5157(1) 0.258(1) SBC1 2.83 2.04 1.39 0.3790(1) 0.9516(2) 0.137(1) 0.4608(1) 0.2955(1) 0.063(1) SBC2 3.56 1.88 1.89 0.3790(2) 0.9514(4) 0.137(1) 0.4596(3) 0.2959(4) 0.062(1) 0.5440(42) 0.9057(26) 0.5153(8) 0.254(3) SBC5 2.26 1.76 1.28 0.3793(2) 0.9508(11) 0.137(1) 0.4608(4) 0.2959(5) 0.063(1) 0.5519(62) 0.9276(117) 0.5107(34) 0.261(8) SBC10 2.13 1.62 1.32 0.3797(2) 0.9504(13) 0.137(1) 0.4613(5) 0.2959(8) 0.063(1) 0.5525(43) 0.9138(62) 0.5141(17) 0.260(5) Table 3 Mean Crystalline Domain Size of Anatase (Ant), Rutile (Rt) and Brookite (Brk)—Defined as the Mean of the Lognormal Size Distributions; Maximum Values and Skewness of the Lognormal Size Distributions mean crystalline domain diameter mode of the size distribution skewness of the size distribution sample ⟨Dant⟩ (nm) ⟨Drt⟩ (nm) ⟨Dbrk⟩ (nm) Ant (nm) Rt (nm) Brk (nm) Ant (nm) Rt (nm) Brk (nm) Ti450 10.4 ± 0.7 14.4 ± 0.6 7.0 ± 0.1 9.4 ± 0.6 9.9 ± 0.4 5.3 ± 0.1 0.8 ± 0.1 1.8 ± 0.1 1.4 ± 0.1 SBC1 6.7 ± 0.3 11.2 ± 0.1 5.3 ± 0.2 9.4 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 SBC2 5.2 ± 0.8 9.4 ± 0.3 5.3 ± 1.4 4.0 ± 0.1 8.2 ± 0.3 4.6 ± 1.2 1.4 ± 0.1 0.9 ± 0.1 1.0 ± 0.1 SBC5 5.1 ± 0.1 5.8 ± 0.8 5.7 ± 0.9 3.9 ± 0.1 3.4 ± 0.5 5.0 ± 0.8 1.4 ± 0.1 2.0 ± 0.1 1.0 ± 0.1 SBC10 4.6 ± 0.1 4.6 ± 0.8 5.8 ± 1.2 3.5 ± 0.1 3.2 ± 0.6 5.5 ± 0.1 1.4 ± 0.1 1.8 ± 0.1 0.6 ± 0.1 High-resolution scanning transmission electron microscopy (HR-STEM) experiments were performed on the Shibuichi samples with the highest Ag + Cu concentrations (SBC5 and SBC10) in order to investigate the possible formation of a metallic@TiO2 heterostructure and to determine their morphology and microstructure. Figure 3a shows a low-magnification STEM–high-angle annular dark-field imaging (HAADF) micrograph of the sample SBC5, in which two distinct kinds of NPs, displaying two distinct contrasts, can be clearly seen. A single NP displaying a brighter contrast (highlighted by the green arrow) can be observed in contact with a matrix of agglomerated NPs showing a darker contrast. NPs displaying the brighter contrast are scarce and hard to find. To get more information on the nature of the NPs, they were investigated by a combination of energy dispersive X-ray spectroscopy (EDS) and high-resolution TEM (HR-TEM) techniques, that is, a powerful tool to extract structural and chemical information at the nanoscale.25 A darker contrast in STEM–HAADF images highlights materials having lower atomic number and/or lower mass density and/or lower thickness. Thus, these NPs showing a darker contrast correspond to TiO2 NPs. This is confirmed by the EDS spectrum acquired on these NPs (displayed in red in Figure 3b). The EDS spectrum acquired at the center of the brighter particle (displayed in blue in Figure 3b) highlights the presence of Ag. It has to be highlighted that Cu signal can be seen in both the NPs. However, Cu signal can emerge from the specimen, but it might also come from the TEM sample-holder or even from the TEM column, thus making a univocal conclusion difficult. Figure 3c displays the HR-STEM ADF micrograph of a facetted NP standing in the vacuum and showing a brighter contrast. These NPs have a diameter typically between 7 and 10 nm and, as it can be clearly seen from Figure 3c, they are crystalline. Automatic indexation of the fast Fourier transform (FFT) pattern (displayed in the inset) performed on one of the facet revealed that the NP corresponds to the Ag face-centered cubic structure in the [011] zone axis. Same results have been obtained on all the investigated NPs, showing this kind of bright contrast. This is also confirmed by the EDS spectrum acquired at the center of the NP (displayed in Figure 3d), which highlights the presence of the Ag lines. A HR-STEM–HAADF micrograph of the TiO2 NPs is shown in Figure S3. All of the TiO2 NPs are crystalline with a diameter between 1.8 and 8 nm. A similar analysis is shown in Figure S4 for SBC10 sample. The surface of this sample is flecked due to the presence of smaller but more abundant Ag NPs (diameter between 1.5 and 8 nm). These results are in good agreement with the WPPM modeling and clearly show the formation of (at least) an Ag@TiO2 heterojunction for the Shibuichi samples. Figure 3 (a) Low-magnification STEM–HAADF micrograph of sample SBC5. The green arrow highlights the presence of a NP having brighter contrast in contact with a matrix of NPs with a darker contrast. (b) EDS spectra acquired on the NPs with the darker and brighter contrasts highlighted by a red and blue circle in the STEM–ADF micrograph displayed in the inset, respectively. (c) HR-STEM ADF micrograph of a NP with a brighter contrast standing in the vacuum. The inset displays the FFT pattern obtained on the area highlighted by the red square. (d) EDS spectrum acquired at the center of the NP displayed in (c). 2.2 Optical Properties: Photochromism Diffuse reflectance spectroscopy (DRS) spectra of fresh un-irradiated samples are depicted in Figure 4. As per Ti450, a single absorption band is observable at <360 nm, due to the band-to-band transition in titania.26Shibuichi-modified specimens, besides these bands, also display other absorption features. The absorption band located at >600 nm is assigned to the d–d electronic transitions in Cu2+. The absorption at around 450 nm belongs to the electron transferring from the valence band (VB) of TiO2 to CuxO clusters that are around titania (interfacial charge transfer, IFCT);27,28 consistent with what was reported in an earlier work, this IFCT band increases itself as the amount of Cu in the specimens increases.11 Furthermore, when the Cu amount is higher than 1.50 mol % (i.e., specimens SBC5 and SBC10), an absorption tail extending itself up to ∼575 nm, assigned to interband absorptions in Cu2O, is also noticeable.29,30 Optical signatures of metallic Ag0 (i.e., the surface plasmon resonance band, SPR) are not seen at this un-irradiated stage. This is likely because silver is present as Ag2O, thus displaying just its IFCT feature mingling itself with that of CuxO clusters around TiO2.31 Figure 4 Absorption data, derived by Kubelka–Munk analysis, of the optical spectra (un-irradiated specimens). When the Ag + Cu-modified specimens are exposed to UV-A light, progressive changes are observable to their optical spectra, as shown in Figure S5a–d. As per SBC1, as the UV-A irradiation time increases, there is also a gradual decrease in the Cu2+ d–d band, thus being accompanied by a continuing increase in the band due to the IFCT. Interestingly, with only 0.25 s of UV-A irradiation time, also the band belonging to the interband absorptions in Cu2O starts to appear (and its magnitude increases with the irradiation time as well). These phenomena imply that upon UV-A irradiation, the Cu2+ is swiftly and gradually reduced to its cuprous oxidation state.11 There is also no signature of the appearance of the Ag0 SPR band, likely because of the much reduced amount of silver. With higher mol % amounts of Ag + Cu (specimens SBC2, SBC5, and SBC10), qualitatively the same change in the optical features as in SBC1 was observed. It is worth to highlight that the SPR band in Ag0 is not detected even with higher amounts of silver, probably because the optical bands of copper cover that of Ag0 SPR. However, an indirect evidence of the reduction of silver into Ag0 after light exposure is given by Raman spectroscopy. As shown in Figure S6, the Raman signal is enhanced in the specimen that has been subjected to UV-A irradiation, thus indeed proving the presence, in this latter, of metallic Ag, giving rise to the surface-enhanced Raman scattering effect.32 These changes in the optical properties (i.e., photochromism) are caused, mechanistically, by the PC process in titania: when TiO2 specimens are irradiated with a light having supra Eg energy, the electron that migrated to the conduction band (CB) is able to reduce the CuO oxides (and allegedly also the Ag2O) that are clustered around the semiconductor surface. Looking at the evolution of the increase in the IFCT band area in the Shibuichi samples after UV-A irradiation, Figure 5a–d, it is seen that these all follow a third-order exponential function, for all the Ag + Cu modified specimens. This being partly consistent with our previous investigations: indeed, when dealing only with copper-modified TiO2 and Ag–TiO2 NPs, the best fit was always obtained adopting a double exponential function.11,31 This suggesting that, in the case of Ag + Cu modified TiO2, the simultaneous adoption of two noble metals increases the degree of complexity of the photochromic phenomenon—there is indeed an electron “pumping” from TiO2 to CuO and Ag2O NPs decorating it. Figure 5 Evolution of the increase in the IFCT band area in the Shibuichi samples after UV-A irradiation. (a) SBC1; (b) SBC2; (c) SBC5; and (d) SBC10. The coefficients of determination R2 of the third-order exponential function adopted for the fittings were ≥0.9984. When specimens are irradiated with the white lamp, the same changes in the optical properties happen, though in a lesser extent, Figure S7a–d. This is better shown in Figure 6a–d, and in Table 4, where is listed the time needed to halve the IFCT integrated area—data obtained using both UV-A and visible light are compared. As listed in Table 4, for instance, the time to reach half of the IFCT integrated area in SBC1 using the UV-A radiation is equal to 0.23 min, while, with the visible light, that time is 4.04 min (∼18 times faster using the UV-A lamp). This latter is indeed a striking result, being the time to halve the IFCT-integrated area in a specimen made only of 1 mol % Cu and TiO2 and using the very same visible-light source, equal to 10.8 min11—that is, almost 3 times faster by adding only 0.25 mol % of silver and decreasing from 1 to 0.75 mol % the Cu content. Figure 6 Evolution of the increase in the IFCT band area in the Shibuichi samples after visible-light exposure. (a) SBC1; (b) SBC2; (c) SBC5; and (d) SBC10. The coefficients of determination R2 of the third-order exponential function adopted for the fittings were ≥0.9923. Table 4 Time to Reach One-Half of the Final Extent of the Integrated IFCT Area, After UV-A and Vis-Light Irradiation t1/2 IFCT band (min) sample UV-A Vis-light SBC1 0.23 4.04 SBC2 0.44 11.96 SBC5 4.65 15.89 SBC10 9.34 33.29 On the other hand, specimen SBC10 and UV-A lamp, the time needed to reach half of the final extent of the IFCT integrated area is 9.34 min, versus 33.29 min when using the Vis-light (approximately 4 times faster). This means that a lamp irradiating with an energy that is ≥TiO2Eg triggers in a faster way the photochromic effect, through the PC mechanism. However, this also means that the specimens show themselves to be photochromic, even under visible-light exposure. This is explained invoking a different phenomenon, that is, photosensitization of the Cu(II)/TiO2 system. Indeed, as suggested by Irie et al., visible light triggers IFCT from the VB of titania to the Cu(II) that are grafted onto the surface of TiO2 NPs.27 That is to say that the electrons in the VB of TiO2 are directly transferred to the CuO decorating the TiO2 NPs, reducing Cu2+ to Cu+, as spectroscopically shown by the decrease in the Cu2+ d–d transitions and the resultant increase in the extent of the IFCT band—and visually by the change in color of the specimens, that is, photochromism. A proposed mechanism of these two distinct phenomena is given in Figure 7a–c, where the energy band diagrams of TiO2, whose surface is modified with CuO and Ag2O, are given—the electrochemical potentials of the band edges of TiO2, CuO, and Ag2O, with respect to the absolute vacuum scale, were taken from the literature.33 [It has to be stressed that, although the data in Figure 7a–c might not reflect a rigorous picture of the absolute values of CB and VB potentials of the materials studied here, they provide a reasonable estimate of the relative band edge positions.] In both cases (UV-A and visible-light sources), TiO2 acts itself as an electron shuttle.34 Figure 7 Proposed mechanism for the UV-A and visible-light photochromism. (a) After UV-A exposure, an electron is promoted from the VB to the CB of TiO2, a CB transferring from TiO2 to CuO is then favored, thus reducing Cu2+ to Cu+. (b) Visible light triggers IFCT from the VB of titania directly to the CB of Cu2+ that are grafted on the TiO2 surface, reducing it and promoting the photochromic effect. The same proviso, considering their band edge positions, can be made with TiO2 and Ag2O. (c) After UV-A and visible-light irradiation, (most of) the Ag(I) and Cu(II) oxides have been reduced into Ag0 and Cu2O. This situation promotes charge separation, as shown by the yellow arrow in the energy band diagram: electrons move themselves from Ag0 to the CB of TiO2 and from the CB of Cu2O to that of TiO2. Photochromism is, per se, a reversible change in color (and/or optical absorption spectra) under electromagnetic radiation.35 Reversible photoswitches of specimens SBC1 and SBC2 were thus investigated. Specimens were irradiated for 10 or 30 s with visible light (irradiance = 50 W m–2), then the reversibility of the process was triggered by annealing the irradiated specimens in a dark oven at 100 °C for 15 min. As displayed in Figure 8a–d, SBC1 and SBC2 exhibit similar changes, both specimens show an increase in the ΔRt value from the un-irradiated state after the first exposure to visible light. This is followed by a partial recovery of the initial virgin state after annealing the samples in the dark at 100 °C for 15 min. However, after that initial loss in the initial R % value, very close reflectance values are obtained from the 4th switching cycle on, as a clear and repeatable gap is noticeable for both specimens, exhibiting a high degree of stability and repeatability in the switching values for repeated cycles (up to a total of 14), as reported in Figure 8a,b. This initial instability seems to be originated from the swift decrease from the virgin state: the time granted for the annealing did not seem enough to fully reverse it. Figure 8 Photochromic recovery switches with repeated visible light (red spheres)/dark@100 °C for 15 min (green spheres) cycles; the blue sphere represent the un-exposed specimen. (a) SBC1 upon 10 s Vis and 15 min@100 °C; (b) SBC1 upon 30 s Vis and 15 min@100 °C; (c) SBC2 upon 10 s Vis and 15 min@100 °C; and (d) SBC2 upon 30 s Vis and 15 min@100 °C. 2.3 Electrical and Gas-Sensing Properties The electrical behavior of the prepared sensors was first investigated by recording the resistance of the sensitive films directly printed on the alumina substrates, in the temperature range of 100–400 °C in the presence of dry air. In order to favor the complete desorption of humidity and other species likely adsorbed during environmental exposure, each sensor was first allowed to stabilize at 400 °C, and then, the resistance was recorded during a 50 °C step decrease of the temperature. The plots of the resistance versus temperature of pure TiO2 and Ag + Cu (Shibuichi)-modified samples are reported in Figure 9. All samples show a decrease in the resistance with increase in the temperature, according to typical semiconductor behavior in the whole range of temperatures. Ti450 shows two linear regions with different slopes, in the range 150–250 and 250–400 °C, respectively. This can be attributed to a strong adsorption of oxygen species which, in turn, act increasing the electrical resistance. As known, MOs are able to adsorb oxygen species as ions (•O2–, O–, O2–) on their surface as a function of the temperature.36 This implies that electrons are subtracted from the bulk, increasing the electrical resistance. Furthermore, a different equilibrium between adsorption and desorption of these species was established as a function of the temperature. Therefore, if adsorption prevails over desorption, a large quantity of oxygen is adsorbed on the MO surface, which leads to a stronger removal of electrons from the bulk. For an n-type semiconductor as TiO2, this results in an increase of the resistance.37 According to this, it is plausible that Ti450 is able to strongly adsorb oxygen in the temperature range of 150–250 °C showing two different slopes of the log resistance versus temperature in the investigated range. Figure 9 Electrical resistance as a function of the temperature of the as-fabricated gas sensors recorded in dry air. On the contrary, as per Shibuichi samples (SBC1, SBC2, SBC5, and SBC10), the trend of resistance fluctuation with the temperature is linear in the entire investigated range of temperatures, suggesting a lower ability in adsorbing oxygen species. In addition, the effect of Ag + Cu modification is to decrease the electrical resistance in comparison to Ti450 when the load is higher than 2 mol % as for SBC2, SBC5, and SBC10 samples. In this regard, it was possible to record the resistance of these samples up to 100 °C, while for Ti450 and SBC1 the resistance was much higher than the detection limit of the apparatus employed. The effect of Ag + Cu modification of TiO2 on the sensing performance toward acetone was first investigated at different working temperatures. Figure 10 shows the responses of the prepared sensors toward 20 ppm of acetone as a function of the temperature, in the range 100–400 °C. Owing to the high resistance of both Ti450 and SBC1, it was not possible to record the response at a temperature lower than 150 °C. As observed, all sensors show a Gaussian-like behavior, where a first rise of the responses up to 150–200 °C is followed by a decrease with further increase of the temperature. These typical response behaviors can be simply explained, bearing in mind the effect of different activation energies for the gas adsorption/desorption and reaction processes occurring at the surface. At a low operating temperature, the activation energy is not enough to promote the reaction of acetone molecules with the active species present on the material surface. This leads to a very weak or no alteration of the electrical properties of the sensitive material and, consequently, to a weak response.38 On the other hand, the adsorption ability of acetone molecules onto the surface is reduced at higher operating temperatures, thus leading again to a lower response.39 Figure 10 Gas responses toward 20 ppm acetone in dry air of Ti450- and Shibuichi-based sensors at different operating temperatures. Among Shibuichi-based sensors, SBC2 is the one that shows the best performances and have the maximum response S = 9 at the lowest operating temperature, 150 °C. A further increase of Ag + Cu modification of TiO2 over 5 mol % as for the SBC10 sample resulted in a strong decrease of the response to acetone. However, all Shibuichi-based sensors showed lower response in comparison to Ti450, in the whole range of investigated temperatures. However, response is not the sole parameter that is investigated to define the performances of a gas sensor. In this regard, Figure 11 shows a comparison of the transient responses recorded for each sensor at the operating temperature of 200 °C to a pulse of 20 ppm acetone. Still in a relatively low temperature, all Shibuichi-based sensors show fast signal variations after acetone exposure, reaching the equilibrium state in less than 30 s, which is faster than that observed for Ti450-based sensor (Figure S8a). When the sensors are again exposed to pure air, a fast recovery of the 90% of response is obtained in less than 30–40 s on the Shibuichi-based sensors. As per the Ti450-based sensor, it is worth noting that the time at 90% of response recovery is close to that of the SBC2-based sensor (Fig S8b); nevertheless, the time to the full recovery of the baseline signal is much higher, thus leading to a serious drawback in real applications. Therefore, a positive effect of the incorporation of Ag + Cu into TiO2 is to ease the desorption of subproducts of acetone reaction from the surface of the material. In this regard, Dutta et al.,40 investigating the CO-sensing performance of anatase TiO2, suggested that CuO incorporation, favoring the decomposition of carbonate intermediate species, aids the desorption of subproduced CO2 and then the recovery time of the sensor. It is well known that the typical sensing mechanism of reducing gases with MOs involves the interaction of gas molecules with chemisorbed oxygen species at the surface.41 At temperatures lower than 200 °C, the predominant chemisorbed oxygen species on MOs are O2– and O–. Therefore, because of the lower activation energy of these species than the more reactive species, a proposed sensing mechanism of acetone might involve the quasi–chemical reactions, as given by eqs 1 and 2(42) 1 2 Figure 11 Transient responses toward 20 ppm acetone in dry air of Ti450- and Shibuichi-based sensors at the operating temperature of 200 °C. Accordingly, because the reaction of acetone with surface-adsorbed oxygen species may involve the formation of carbonates before the evolution to CO2 and H2O, a similar catalytic effect coming from CuO and/or AgO nanoclusters could be the reason of faster recovery times of Shibuichi-TiO2 based sensors in comparison to Ti450. All investigated sensors show a reduction in the electrical resistance when exposed to acetone, according to n-type semiconductor MOs when they interact with reducing gases. This suggests that the addition of such load of p-type species, like copper, is not enough to alter the typical n-type behavior of anatase TiO2.43 Teleki et al.,44 showed a p-type behavior in a heat-treated 100 wt % rutile TiO2 sensors toward CO at 500 °C, while the same authors observed a n-type response for a 5 at. % Cu/TiO2-based sensor, having a rutile content of 50 wt %.45 On the other hand, Savage et al.46 investigated the sensing behavior of mixed anatase–rutile TiO2 toward CH4 and CO gases. They reported stable n-type response for rutile phase less than 75 wt % and p-type response for pure rutile. As all Shibuichi samples show contents of anatase higher than 90 wt % and about 56 wt % for Ti450 (cf Table 1), this might explain the stable n-type behavior of all the sensors reported here. Among Ag–Cu samples, because of the higher response, lower operating temperature, and reduced electrical resistance, the sensor based on SBC2 was chosen to be investigated for further sensing performances. Figure 12a shows the transient response recorded during subsequent exposure of the SBC2-based sensor to different concentrations of acetone ranging from 1 to 40 ppm in dry air at the working temperature of 150 °C. The sensor shows a fast reduction of the electrical resistance after acetone exposure. In addition, this sensor is able to quickly and fully recover the baseline resistance after each pulse, displaying a perfect reversible behavior. Figure 12b shows the calibration curve extrapolated by the above transient responses. In the range of investigated concentration, the response follows a typical log–log behavior, which can be fitted by eq 3 3 Figure 12 (a) Transient response of the SBC2-based sensor during consecutive exposures ranging from 1 to 40 ppm of acetone in dry air at the operating temperature of 150 °C; (b) calibration curve. In eq 3, S is the response and c is the concentration of acetone. The determination coefficient (R2 = 0.994) suggests a good linear trend between the logarithm of response and the logarithm of concentration, in the studied range. Using this data, a limit of detection lower than 100 ppb of acetone can be estimated, that is 5 times lower compared to the actual state-of-the-art TiO2-based sensors reported in the literature (i.e., 500 ppb).47 Aiming at exploring the reliability and stability of the sensor, the reproducibility of response was investigated by recording consecutive pulses of acetone in the same and different days, respectively. Figure S9a displays the perfect repeatability and reversibility of response when the sensor was exposed to five consecutive pulses of 5 ppm of acetone in air. In addition, as observed in Figure S9b, the response to 20 ppm acetone, recorded during 3 different days after various cycles of heating–cooling and exposure to different gases, is almost similar, also suggesting excellent long-time reproducibility and stability of the sensing material. The selectivity of the sensor was also investigated at the optimal operating temperature for acetone, by analyzing the response toward common organic and inorganic gases. The sensor shows low response to typical concentration of the investigated gases (Figure 13a). Only the response to ethanol at the same concentration of the acetone was very similar, suggesting this sensor as a possible candidate for VOCs detection. Finally, in practical applications, acetone coexists with humidity, whose typical aftermath is to decrease the sensitivity of the sensor toward VOCs. The effect of humid air was thus evaluated:48Figure 13b shows a comparison of calibration curves recorded in dry and humid air (50% RH at 25 °C) operating at the temperature of 150 °C. The presence of humidity causes a decrease in response to acetone from 2- to 5-folds compared to that obtained in dry condition. Regardless of that, the sensor was still able to show appreciable response to a concentration of acetone as low as 500 ppb. Figure 13 (a) Gas responses of the SBC2-based sensor toward common reducing and oxidizing gases in dry air, operating at 150 °C; (b) comparison of SBC2-based sensor responses toward different concentrations of acetone in dry and humid air (50 RH % at 25 °C) at the operating temperature of 150 °C. 3 Conclusions Inspired by the Land of the Rising Sun, TiO2 NPs have been decorated with one part of silver to three parts of copper—Shibuichi. This made a multifunctional nanomaterial, which shows simultaneously photochromism and gas-sensing abilities. It has been shown that photochromism might be triggered by UV-A and, most strikingly, also visible light. However, two distinct phenomena are responsible of the same effect. When using a UV-A lamp (i.e., an energy ≥ Eg), photocatalysis sensu strictu happened to activate photochromism. On the other hand, when employing a visible light, a photosensitization of Cu(II)/TiO2 system happens, triggering photochromism. Gas-sensing tests showed that the modification of TiO2 NPs with Ag + Cu leads to an improvement of the transient response to acetone, shortening the response an recovery times in comparison to the pure TiO2-based sensor even working at a relatively low temperature of 150 °C. Furthermore, it also exhibited a lower detection limit for acetone (5 times lower) compared to state-of-the-art TiO2-based sensors reported in the literature, for example, 100 ppb in this work versus 500 ppb in previous work. Still, among the Shibuichi samples, a gas sensor based on TiO2 decorated with 2 mol % Ag + Cu showed excellent sensing performance toward VOCs, exhibiting good sensitivity, stability, and selectivity in comparison with other inorganic gases, as well as capability of being implemented at lower operating temperature. All these characteristics together make our material attractive for real commercial application. 4 Experimental Section 4.1 Sample Preparation An adapted aqueous sol–gel method, developed by the authors, was used for the synthesis of TiO2-based nanomaterials; precise detail of it can be found elsewhere.49 To do the Ag/Cu-modified TiO2, stoichiometric amounts of silver nitrate (1 M aqueous solution, Sigma-Aldrich) and copper(II) nitrate trihydrate (Aldrich, ≥98.5%) were added to the sol, which had a concentration of 1 M Ti4+. The Ag/Cu molar ratio was strictly constrained to be 1:3, to follow the Shibuichi formulation. Four Ag/Cu-modified sols were prepared, with (Ag + Cu) molar amounts equal to 1, 2, 5 and 10 mol % [i.e., in the case of (Ag + Cu) = 1 mol %, Ag molar amount was 0.25 mol %, and Cu was 0.75 mol %, with Ag/Cu = 0.25:0.75 = 1:3]. Afterward, dried gels were thermally treated at 450 °C under a static air flow, using an electric muffle furnace. The heating/cooling rate was 5 °C min–1, with a 2 h dwell time at the selected temperature. Samples were referred to as Ti450 (unmodified TiO2), SBC1 (Ag + Cu = 1 mol %), SBC2 (Ag + Cu = 2 mol %), SBC5 (Ag + Cu = 5 mol %), and SBC10 (Ag + Cu = 10 mol %). 4.2 Sample Characterization Information about the microstructure of the specimens was attained via advanced X-ray methods. The Rietveld method was used to obtain semiquantitative information about the phase composition of the samples (i.e., not accounting for the presence of amorphous fraction). XRD patterns for QPAs were recorded on a θ/θ diffractometer (PANalytical X’Pert Pro, NL), equipped with a fast RTMS detector (PIXcel 1D, PANalytical), with Cu Kα radiation (45 kV and 40 mA, 20–80° 2θ range, with a virtual step scan of 0.02° 2θ and a virtual time per step of 200 s). Rietveld refinements were accomplished by means of GSAS-EXPGUI software suite,50,51 following the refining strategy that we reported previously;31 structure models of anatase, rutile, and brookite were taken from the literature.52−54 WPPM formalism,55 through the PM2K software package,56 was used to extract microstructural information from the XRD data. In this latter case, aiming to manipulate data with a high signal-to-noise ratio, XRD patterns were recorded in the same instrument and setup as described above, but in the 20–145° 2θ range, with a virtual step of 0.1° 2θ, and virtual time per step of 500 s. The instrumental contribution was obtained by parameterizing the profile of 14 hkl reflections from the NIST SRM 660b standard (LaB6), according to the Caglioti et al. relationship.57 The following parameters were refined: background (modeled with a sixth-order Chebyshev polynomial), peak intensities, specimen displacement, lattice parameters, and mean and variance of the size distributions. Crystalline domains were approximated to be spherical with their diameter distributed according to a lognormal curve. HR-STEM experiments were performed using an FEI Titan low-base microscope operated at 300 kV and equipped with an EDS detector, a CESCOR Cs probe corrector, an ultrabright X-FEG electron source, and a monochromator. HR-STEM imaging was performed using HAADF and ADF detectors. The inner and outer angles for most of the micrographs recorded with the HAADF and ADF detector were 48 and 200 mrad and 22 and 120 mrad, respectively. Automatic indexation of the FFT patterns was performed by using the JEMS software.58 For TEM studies, samples were dispersed in ethanol in an ultrasound bath for a few minutes and a drop of the suspension was placed onto a molybdenum/nickel grids coated with carbon membrane. Optical properties of the specimens were investigated by means of DRS on a Shimadzu UV-3100 spectrometer (JP), equipped with an integrating sphere, and a white reference material made of Spectralon; the UV–Vis spectral range (250–850 nm) was investigated, with 0.2 nm in resolution. To explore the photochromic properties of the specimens, they were exposed (0.1 g), for different irradiation times, to either UV-A or visible light. A protocol described in more detail in our previous works10,11,31 was severely followed to get comparable data. The UV-A and visible-light lamps were placed above (vertically) the specimen, adjusting the distance between the lamp and the specimen to get the desired irradiance values. The intensity of the radiation reaching the samples, measured with a radiometer (Delta OHM, HD2302.0, IT), was estimated to be ∼22 W m–2 in the UVA range (315 nm < λ < 400 nm) and ∼50 W m–2 in the visible region (λ > 400 nm, being nil in the UVA); irradiation times were considered to be consecutive and absolute. Reflectance data were transformed into pseudo-absorption spectra F(R) by means of the Kubelka–Munk transformation:59F(R) = α = (1 – R)2/2R, where R is the reflectance. To explore the reversibility of the photochromic process, fresh un-irradiated samples were exposed to visible light (as above) for periods ranging from 10 to 30 s, at room temperature, and their DRS spectra were recorded immediately. They were then placed in a dark oven at 100 °C for 15 min to reverse the process, and the optical spectra were measured again. The degree of photoswitching has been described as the difference between the maximum value in reflectance at time zero (R0) and the reflectance after each irradiation/annealing cycle (Rt): ΔRt (%) = (R0 – Rt)/R0 × 100. Raman spectra were measured using a RFS 100/S (Bruker, DE) equipped with a 1064 nm Nd:YAG laser as the excitation source, in the 50–1000 cm–1 wavenumber range, with 2 cm–1 resolution. 4.3 Gas Sensing Gas-sensing performance of the synthesized materials was investigated by fabricating resistive gas sensor devices. The screen printing method has been used to make reproducible thick films (3 × 3 mm2 area, and ∼10 μm in thickness). The synthesized nanopowders were mixed with an appropriate volume of double distilled water to obtain a paste. Appropriate water amounts have been added to have an ink paste with suitable rheological properties. Then, the ink has been deposited by means of a hand screen printer over the interdigitated area of the sensor. This latter was made of an alumina substrates (6 × 3 mm2 × 0.5 mm), supplied by a pair of Pt interdigitated electrodes on one side and a Pt heater on the other side. The film has been printed with the appropriate geometric characteristic by using a proper plastic mask. Sensing measurements were carried out in the range of temperature 100–400 °C under a controlled atmosphere, by placing the sensors in an optimized stainless steel test chamber where a constant gas steam of 100 scc/min was allowed to flow. All gases and acetone vapor (coming from certified bottles) can be progressively diluted in air using mass flow controllers, in order to obtain the desired working concentrations. Electrical resistance of the sensitive films was recorded by a Keithley 6487 picoammeter/voltage source, while an Agilent 3632A was employed to power the heater of the sensor substrate in order to control the operating temperature. The gas sensor response, S, is defined as the ratio Rair/Rg, where Rair is the electrical resistance of the sensor in reference air (baseline resistance) and Rg is the electrical resistance at the fixed target gas concentration. The response and recovery times, unless otherwise indicated, were calculated at 90% of response variation. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01508.Graphic output of the Rietveld refinement of the sample SBC1; graphic output of the WPPM modeling of SBC1; HR-STEM–HAADF micrographs (samples Ti450 and SBC10); DRS spectra of irradiated samples (UV-A and visible light); Raman spectra before and after UV-A irradiation; response time at 90% signal variation as a function of temperature (Ti450- and SBC2-based sensors); recovery times of Ti450- and SBC2-based sensors; reproducibility of the SBC2-based sensor; and position and full width half-maximum of Raman Eg mode of anatase (PDF) Supplementary Material ao8b01508_si_001.pdf Author Present Address # Departamento de Ciencia de los Materiales e Ingeniería Metalúrgica y Química Inorgánica, Facultad de Ciencias, Universidad de Cádiz, Campus Río San Pedro S/N, Puerto Real 11510, Cádiz, Spain. Author Present Address ¶ Instituto Universitario de Investigación de Microscopía Electrónica y Materiales (IMEYMAT), Facultad de Ciencias, Universidad de Cádiz, Campus Río San Pedro S/N, Puerto Real 11510, Cádiz, Spain. The authors declare no competing financial interest. Acknowledgments This work was developed within the scope of the project CICECO–Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. The STEM measurements were performed in the Laboratorio de Microscopias Avanzadas (LMA) at the Instituto de Nanociencia de Aragon (INA)—Universidad de Zaragoza (Spain). R.A. gratefully acknowledges the support from the Spanish Ministerio de Economia y Competitividad (MAT2016-79776-P), from the Government of Aragon, and the European Social Fund under the project “Construyendo Europa desde Aragon” 2014–2020 (grant number E/26). ==== Refs References Key attributes of nano-scale materials and functionalities emerging from them . In Nanoscale Multifunctional Materials: Science and Applications ; Mukhopadhyay S. M. , Ed.; John Wiley & Sons, Inc. : Hoboken, NJ, USA , 2011 ; pp 3 –33 . Zhang J. Z. Understanding the Growth of Metal Oxide Nanostructures . J. Phys. Chem. Lett. 2012 , 3 , 2920 –2921 . 10.1021/jz301355a . Bai Y. ; Mora-Seró I. ; De Angelis F. ; Bisquert J. ; Wang P. Titanium Dioxide Nanomaterials for Photovoltaic Applications . Chem. Rev. 2014 , 114 , 10095 –10130 . 10.1021/cr400606n .24661129 Fujishima A. ; Honda K. Electrochemical Photolysis of Water at a Semiconductor Electrode . Nature 1972 , 238 , 37 –38 . 10.1038/238037a0 .12635268 Kamat P. 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PMC006xxxxxx/PMC6644436.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145880010.1021/acsomega.8b01065ArticleSynthesis of (Arylmido)niobium(V) Complexes Containing Ketimide, Phenoxide Ligands, and Some Reactions with Phenols and Alcohols Srisupap Natta Wised Kritdikul Tsutsumi Ken Nomura Kotohiro Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, 1-1 Minami Osawa, Hachioji, Tokyo 192-0397, Japan E-mail: ktnomura@tmu.ac.jp.07 06 2018 30 06 2018 3 6 6166 6181 19 05 2018 28 05 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. Reactions of Nb(NAr)(N=CtBu2)3 (3a, Ar = 2,6-Me2C6H3) with 1.0, 2.0, or 3.0 equiv of Ar′OH (Ar′ = 2,6-iPr2C6H3) afforded Nb(NAr)(N=CtBu2)2(OAr′), Nb(NAr)(N=CtBu2)(OAr′)2, or Nb(NAr)(OAr′)3, respectively (at 25 °C), whereas the reaction with 2.0 equiv of 2,6-tBu2C6H3OH afforded Nb(NAr)(N=CtBu2)2(O-2,6-tBu2C6H3) upon heating (70 °C) without the formation of bis(phenoxide) and the reaction of 3a with 2.0 equiv of 2,4,6-Me3C6H2OH afforded Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H2)2(HN=CtBu2). Similar reactions of 3a with 1.0 equiv of (CF3)3COH or 2.0 equiv of (CF3)2CHOH afforded Nb(NAr)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) or Nb(NAr)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2), respectively. On the basis of their structural analyses and the reaction chemistry, it was suggested that these reactions proceeded via coordination of phenol (alcohol) to Nb and the subsequent proton (hydrogen) transfer to the ketimide (N=CtBu2) ligand. The reaction of Nb(NAr)(N=CtBu2)2(OAr′) with 1.0 equiv of 2,4,6-Me3C6H2OH gave the disproportionation products Nb(NAr)(N=CtBu2)(OAr′)2 and Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H2)2(HN=CtBu2) with 1:1 ratio, clearly indicating the presence of the above mechanism and the fast equilibrium (between the ketimide and the phenoxide). The reaction of 3a with 1.0 or 2.0 equiv of C6F5OH afforded Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) as the sole isolated product, which was formed from once generated Nb(NAr)(N=CtBu2)2(OC6F5)(HN=CtBu2) by treating with C6F5OH. document-id-old-9ao8b01065document-id-new-14ao-2018-010659ccc-price ==== Body Introduction Arylimido ligands have been widely used to stabilize the high-oxidation-state early transition-metal complexes,1−10 which are especially used as catalysts or catalyst precursors for carbon–carbon bond formation exemplified in olefin metathesis,6−10 and olefin polymerization.11,12 Introduction of a steric bulk in the arylimido ligand should be effective not only to stabilize the oxidation state, but also to avoid the formation of dimeric species through the bridged imido ligand or certain dimeric decomposition pathways in metal-alkylidenes;4,6−9 the ligand modification is also effective to control the electronic nature through the metal–nitrogen double bond.6−8,10,13 Monodentate anionic ancillary donor ligands (such as phenoxy,10,14−16 ketimide,15−19 imidazolin-2-iminato,20 and others15,16,21,22) have also been known to play a role to stabilize the oxidation state as well as control of electronic/steric natures by the ligand modification.10,15,16 Studies on the synthesis and some reaction chemistry of half-sandwich niobium complexes containing arylimido ligands,23−31 which would exhibit metallocene-like reactivity through the “isolobal” relationship,25 were known. Moreover, synthesis of some (called nonmetallocene type) (arylimido)niobium(V) complexes,32−40 especially those containing monoanionic chelate guanidinato ligand (Scheme 1),37−39 was also known. However, in contrast to a number of reports of (tert-butyl-imido)niobium complexes especially containing β-diketiminate ligand (Scheme 1),41−44 reports concerning the synthesis and reaction chemistry of (arylimido)niobium(V) complexes containing “monodentate” anionic ancillary donor ligands still have been limited so far (Scheme 1).32−34,40 Since certain (arylimido)vanadium(V) complexes containing anionic donor ligands (such as phenoxy, ketimide, imidazoline-iminato) exhibit promising characteristics as catalysts or catalyst precursors for olefin metathesis9,10,13,45 and coordination/insertion polymerization12,46−49 and also (arylimido)niobium(V)-alkylidene complexes containing fluorinated alkoxo ligand catalyze metathesis polymerization of cyclic olefins and disubstituted acetylene (Scheme 1),40 synthesis and some reactions of (arylimido)niobium(V) complexes containing ketimide and aryloxo ligands have been a promising subject.50−52 Scheme 1 Reported Examples for (Arylimido)niobium(V) Complexes,32−34,38−40 and Selected Four-, Five-, and Six-Coordinate (t-Butylimido)niobium Complexes41,42 In this study, we thus focus on the synthesis of (arylmido)niobium(V) complexes containing ketimide (N=CtBu2) ligand, not only because use of this ligand plays a role to stabilize the oxidation state,50 as demonstrated by the synthesis of a series of (arylimido)vanadium(V) complexes,9,53−55 but also because, as described below, the solvent-free tris(ketimide) complex, Nb(NAr)(N=CtBu2)3 (3a), could be isolated from Nb(NAr)Cl3(dme) (Ar = 2,6-Me2C6H3, dme = 1,2-dimethoxyethane) and subsequent substitution with phenol was expected.54,55 Moreover, on the basis of the reaction chemistry of V(NAr)Me(N=CtBu2)2 with phenols, it was demonstrated that the reactions of ketimide with phenols proceed by coordination with phenol and not by protonation (protonolysis, H+) and also proceed by coordination of phenol to the electron-deficient metal center trans to the methyl group to give a pentacoordinated trigonal bipyramidal species and subsequent proton (hydrogen) transfer to the aryloxide/ketimide affording ketimine/phenol dissociation.55−58 Therefore, we herein present our explored reaction chemistry of the (arylimido)niobium(V)-ketimide complexes with phenols and the unique contrast in the reactivity between the vanadium and niobium complexes. Results and Discussion Synthesis of (Arylimido)niobium(V) Complexes Containing Phenoxide, Ketimide Ligands, and Some Reactions with Phenols and Fluorinated Alcohols Reactions of (imido)niobium(V) trichloride, Nb(NR′)Cl3(dme) [R′ = 2,6-Me2C6H3 (Ar),40 2,6-iPr2C6H3 (Ar′),32,33 1-adamantyl (Ad),32,33 dme = 1,2-dimethoxyethane] with LiOAr′ in Et2O afforded corresponding Nb(NR′)Cl2(OAr′)(dme) (Scheme 2, R′ = Ar (1a), Ar′ (1b), Ad (1c)) in high yields (76–84%), and the reaction of Nb(NAr)Cl3(dme) with Li(O-2,6-Ph2C6H3) also afforded Nb(NAr)Cl2(O-2,6-Ph2C6H3)(dme) (1d).a These complexes were identified by NMR spectra and elemental analysis (except 1b),a and the structures of 1a and 1d were determined by X-ray crystallography as a distorted octahedral geometry around niobium.b It turned out that both 1a and 1d showed low catalytic activities for ethylene polymerization in the presence of methylaluminoxane (MAO) (activities for 1a and 1d = 23 and 12 kg PE/(mol Nb h), respectively, ethylene 8 atm at 25 °C for 1 h).c The reason for the low catalytic activities would be coordination of dme, which seems difficult to cleave from Nb, as described below. Scheme 2 Synthesis of (Imido)niobium(V) Complexes Containing Aryloxide, Ketimide, and Triflate (OTf) Ligands (Detailed Synthetic Procedures Are Shown in the Supporting Information (SI))a,b It turned out that dme was strongly coordinated to Nb in CDCl3 even at 100 °C (variable-temperature spectra are shown in Figure S2-1, Supporting Information (SI)),a and attempted treatments of 1a with NiBr2 or ZnCl2 (3 equiv) in toluene at 50 °C recovered 1a. Treatment of 1a with 2 equiv of AgOTf (OTf = CF3SO3) in CH2Cl2 afforded corresponding Nb(NAr)(OAr′)(OTf)2(dme) (2a) without liberation of dme (Scheme 2); 2a was identified by NMR spectra and elemental analysis.a Similarly, Nb(NAr)Cl3(dme) was treated with 3 equiv of AgOTf in CH2Cl2 to afford Nb(NAr)(OTf)3(dme) (2b), which was also isolated as the sole product confirmed by NMR spectra.a Structure of 2a was suggested on the basis of that of 1a (confirmed by X-ray crystallography) and the NMR spectra, but two resonances were observed in the 19F NMR spectrum when the CDCl3 solution was kept at room temperature (25 °C) for 21 h, probably due to isomerization of the OTf ligand (trans to cis, the spectra are shown in Figure S2-3, SI).a In contrast, reactions of Nb(NAr)Cl3(dme) with 3.0 equiv of Li(N=CtBu2) in toluene afforded the corresponding four-coordinate tris(ketimide) analogue, Nb(NAr)(N=CtBu2)3 (3a), and treatment of NbCl5 with 3.0 equiv of Li(N=CtBu2) in toluene also afforded trans-NbCl2(N=CtBu2)3 (3b) as the sole isolated product (Scheme 2).a These complexes (3a and 3b) were identified by NMR spectra and elemental analysis.a As described in Introduction, reports concerning synthesis and reaction chemistry of (arylimido)niobium(V) complexes containing monodentate anionic donor ligands (without solvent coordination) still have been limited (Scheme 1).32−34,40,50 Since the ketimide (N=CtBu2) ligand in the (arylimido)vanadium(V) complexes were known to be replaced with phenoxide (alkoxide) upon addition of phenol (alcohol), reactions of Nb(NAr)(N=CtBu2)3 (3a) with phenols were thus conducted.a It turned out that reaction of 3a with 1.0 equiv of Ar′OH in n-hexane afforded the mono phenoxide, Nb(NAr)(N=CtBu2)2(OAr′) (4a), as the sole isolated product (Scheme 3).a The similar reactions with 2.0 and 3.0 equiv of Ar′OH afforded the corresponding bis- and tris(phenoxide) complexes, Nb(NAr)(N=CtBu2)(OAr′)2 (5) and Nb(NAr)(OAr′)3 (6), respectively, in high yields (Scheme 3);a the reaction of Nb(NAr)Cl3(dme) with 3.0 equiv of LiOAr′ in Et2O also afforded 6. Complexes 4a, 5, and 6 were identified by NMR spectra and elemental analysis, and the structure of 4a was determined by X-ray crystallography (Figure 1).a,d Figure 1 ORTEP drawings for Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a, left) and Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-tBu2C6H3OH) (4b, right). Thermal ellipsoids are drawn at the 50% probability level, and H atoms are omitted for clarity. Detailed analysis data are shown in the Supporting Information.d Scheme 3 Reactions of Nb(N-2,6-Me2C6H3)(N=CtBu2)3 (3a) with Phenols (Detailed Synthetic Procedures Are Shown in the Supporting Information)a In contrast, the similar reaction of 3a with 1.0 equiv 2,6-tBu2C6H3OH (in n-hexane at 25 °C) afforded a mixture of 3a and the corresponding mono phenoxide, Nb(NAr)(N=CtBu2)2(O-2,6-tBu2C6H3) (4b), even after stirring overnight (Figure S2-4, SI).a The reaction did not reach full conversion of 3a upon heating at 70 °C overnight, and 4b could be isolated from the mixture after purification, including separation of 2,6-tBu2C6H3OH by extraction of 4b with n-hexane.a It turned out that monitoring the reaction of 3a with 2.0 equiv of 2,6-tBu2C6H3OH at 70 °C (by 1H NMR spectra in C6D6) showed resonances ascribed to 4b (and those in the phenol) solely with complete conversion of 3a, and further reaction did not take place even after 1 week (Figures S2-5 and S2-6, SI).a Moreover, 1H NMR spectrum of the reaction of 3a with 2.0 equiv of 2,6-tBu2C6H3OH in toluene-d8 at 100 °C for 1 day also showed resonances ascribed to 4b with complete conversion of 3a (Figure S2-8).a Complex 4b was identified by NMR spectra and elemental analysis, and the structure was determined by X-ray crystallography (Figure 1).a,d As shown in Scheme 3, the resultant complexes (4a, 4b, 5, and 6) are four-coordinate (arylimido)niobium complexes without coordination of HN=CtBu2 (product by reaction with phenol). In contrast, it should be noted that the similar reaction of 3a with 1.0 equiv of C6F5OH in n-hexane afforded Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7, isolated from the mixture), and the same reaction with 2.0 equiv of C6F5OH also afforded 7 as the sole isolated product from the mixture (Scheme 4, 33% isolated yield). The results clearly suggest that the reaction of arylimido ligand in 3a with C6F5OH took place, whereas the reaction of the ketimide ligand in 3a cleanly took place with 2,6-iPr2C6H3OH, 2,6-tBu2C6H3OH (and 2,4,6-Me3C6H2OH shown below). The complex 7 was identified by NMR spectra and elemental analysis, and the structure was determined by X-ray crystallographic analysis.a,d Scheme 4 Reactions of Nb(N-2,6-Me2C6H3)(N=CtBu2)3 (3a) with C6F5OH, (CF3)3COH, and (CF3)2CHOH (Detailed Synthetic Procedures Are Shown in the Supporting Information)a It turned out that reactions of 3a with 1.0 equiv of (CF3)3COH afforded the mono alkoxide, Nb(NAr)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) (8), which coordinates HN=CtBu2 to Nb trans to the arylimido ligand (Scheme 4). The fact could strongly suggest that (CF3)3COH coordinates to Nb through the N(arylimido)–N(ketimide)–N(ketimide) face and the ligand exchange took place with proton transfer (not protonation) from (CF3)3COH to the ketimide ligand. However, it was difficult to reach complete conversion of 3a upon heating monitored by 1H NMR spectra, and the reaction reached 50% conversion of 3a when the mixture was heated at 40 °C overnight. In contrast, the reaction of 3a with 2.0 equiv of (CF3)2CHOH in n-hexane (at 25 °C) afforded the bis(alkoxide), Nb(NAr)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2) (9, Scheme 4). The complexes 8 and 9 were identified by NMR spectra and elemental analysis, and their structures were determined by X-ray crystallography (Figure 2).a,d It is thus clear that the reaction with the arylimido ligand did not take place, whereas the reaction with the arylimido ligand took place in the reaction of 3a with C6F5OH (described above). It also turned out that coordinated HN=CtBu2 in 9 could be removed by treatment with NiBr2 (ca. 3 equiv) in toluene; the 1H NMR spectrum of the reaction mixture (after filtration and removal of toluene) suggested the formation of Nb(NAr)(N=CtBu2)[OCH(CF3)2]2 (10) due to disappearance of resonances ascribed to tBu protons (and shift of resonances in 19F NMR spectra, Figures S1-28 and S1-29, SI).a Figure 2 ORTEP drawings for Nb(N-2,6-Me2C6H3)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) (8, left), Nb(N-2,6-Me2C6H3)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2) (9, middle), and Nb(N-2,6-Me2C6H3)(N=CtBu2)2(OC6F5)(HN=CtBu2) (11, right). Thermal ellipsoids are drawn at the 50% probability level, and H atoms are omitted for clarity. Detailed analysis data are shown in the Supporting Information.d To explore why reaction of 3a with C6F5OH afforded Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7) as the isolated product, we conducted reaction of 3a with 0.50 equiv of C6F5OH under diluted conditions (details are shown in the SI).a Interestingly, upon careful dropwise addition of n-hexane solution containing C6F5OH under diluted conditions at −30 °C, the reaction mixtures consisted of Nb(N-2,6-Me2C6H3)(N=CtBu2)2(OC6F5) (11), which could be isolated from the mixture and was identified by NMR spectra and X-ray crystallographic analysis, and 3a was monitored by NMR spectra (Scheme 5, data shown in Figures S2-9–12, SI).a,d The results strongly suggest that the reaction to afford 7 proceeded via formation of 11 as the initial product, which would be formed by coordination of C6F5OH to Nb through N(arylimido)-N(ketimide)-N(ketimide) face in 3a (and the subsequent proton transfer from C6F5OH to the ketimide ligand). In fact, further careful experiments of dropwise (and two- or three-step) addition of C6F5OH afforded 11 even in the reaction with 0.8 equiv of C6F5OH (monitored by NMR spectra, Figures S2-13 and S2-14, SI).a Importantly, after the above experimental procedure (careful three-step addition of C6F5OH under highly diluted conditions), further addition of 1.0 equiv of C6F5OH afforded Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7) as the major observed product on NMR spectra (Scheme 5, Figures S2-15 and S2-16, SI).a Although the equivalence of C6F5OH was uncertain, the results could suggest that 7 was formed from the mono phenoxide (11) by reaction with the arylimido ligand. Scheme 5 Reactions of Nb(N-2,6-Me2C6H3)(N=CtBu2)3 (3a) with C6F5OH under Diluted Conditions (Detailed Synthetic (Reaction) Procedures Are Shown in the Supporting Information)a Structural Analysis of (Arylimido)niobium(V) Complexes Containing Phenoxide and Ketimide Ligands Figure 1 shows ORTEP drawings for Nb(NAr)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a) and Nb(NAr)(N=CtBu2)2(2,6-tBu2C6H3OH) (4b) determined by X-ray crystallography,d and the selected bond distances and angles are summarized in Table 1. These complexes fold a distorted tetrahedral geometry around Nb, and the Nb–N(1) distances in the imido ligands for 4a and 4b [1.783(3) and 1.790(9) Å, respectively] are rather longer than those in Nb(NAr)Cl2(O-2,6-iPr2C6H3)(dme) (1a) [1.7643(15) Å] and Nb(NAr)Cl2(O-2,6-Ph2C6H3)(dme) (1d) [1.7569(16) Å] (shown in the SI)b and Nb(N-2,6-iPr2C6H3)(NMe2)2[η3-tBuNC(NMe2)NEt] (shown in Scheme 1, 1.768(3) Å),39 but are close to that in Nb(N-2,6-iPr2C6H3)(NMe2)2[η2-(NMe2)C=NtBu] (shown in Scheme 1, 1.783(2) Å).38 The Nb–N(1)–C(arylimido) bond angles in 4a and 4b [174.5(2) and 170.1(7)°, respectively] are rather smaller than those in 1a and 1d [176.53(12) and 175.74(14)°, respectively]b and Nb(N-2,6-iPr2C6H3)(NMe2)2[η2-(NMe2)C=NtBu] [177.3(2)°],38 but larger than that in Nb(N-2,6-iPr2C6H3)(NMe2)2[η3-tBuNC(NMe2)NEt] [167.3(2)°].39 The Nb–N bond distances in the ketimide ligands in 4a and 4b [Nb–N(2) and Nb–N(3): 1.946(8)–1.957(3) Å] are close to or slightly longer than those reported [1.937(2) and 1.939(2)] in Nb(N=CtBu2)4,50 and the Nb–N–C bond angles [173.4(3)–178.9(3)°] are close to those reported in Nb(N=CtBu2)4 [176.5(2)°].50 Table 1 Selected Bond Distances and Angles for Complexes 4a, 4b, 8, 9, and 11d 4a 4b 8 9 11 Bond Distances in Angstrom Nb–O(1) 1.939(3) 1.965(7) 2.0642(13) 1.9986(12) 2.066(3) Nb–O(2) 1.9879(14) Nb–N(1)imido 1.783(3) 1.790(9) 1.7877(15) 1.790(2) 1.796(3) Nb–N(2)ket 1.957(3) 1.946(8) 1.9647(18) 1.9399(16) 1.963(4) Nb–N(3)ket 1.947(3) 1.949(8) 1.9670(17) 1.965(4) Nb–N(H)CtBu2 2.5448(17), Nb(1)–N(4) 2.440(2), Nb(1)–N(3) 2.443(3), Nb(1)–N(4) Bond Angles in deg Nb–O(1)–C(1) 168.6(2) 148.2(6) 145.75(11), C(39) 134.76(12), C(27) 123.9(2), C(36) Nb–O(2)–C 129.15(15), C(30) Nb–N(1)–C(imido) 174.5(2), C(13) 170.1(7), C(15) 177.56(13), C(1) 177.89(14), C(1) 170.1(3), C(1) Nb–N(2)–C(ket) 173.4(3), C(21) 173.4(7), C(23) 178.00(17), C(9) 174.01(12), C(9) 175.6(3), C(9) Nb–N(3)–C(ket) 178.9(3), C(30) 177.0(8), C(32) 177.13(14), C(23) 178.8(3), C(18) Nb–N(4)H–C 159.78(14), Nb(1)–N(4)–C(18) 157.30(12), Nb(1)–N(3)–C(18) 160.7(3), Nb(1)–N(4)–C(27) N(1)–Nb–O(1) 114.86(12) 112.3(3) 101.32(6) 98.64(7) 93.68(13) N(1)–Nb–O(2) 98.38(7) N(1)–Nb–N(2) 106.01(12) 102.9(4) 99.88(7) 99.42(8) 103.15(13) N(1)–Nb–N(3) 104.88(12) 102.9(3) 99.14(7) 99.00(15) N(1)–Nb–N(4) 174.27(6) 170.26(6), N(1)–Nb–N(3) 165.90(13) N(2)–Nb–N(3) 118.09(12) 120.4(3) 125.68(7) 125.63(14) O(1)–Nb–N(2) 107.28(11) 108.2(3) 111.69(6) 116.07(6) 111.40(13) O(1)–Nb–N(3) 106.09(11) 109.9(3) 113.58(6) 115.97(12) O(1)–Nb–N(4) 73.34(5) 72.16(6), O(1)–Nb(1)–N(3) 72.68(11) 83.62(6), O(2), –Nb(1)–N(3) The Nb–O(1) bond distance in 4b [1.965(7) Å] is longer than that in 4a [1.939(3) Å], but these distances are longer than those in 1a [1.8878(12) Å] but within the range of those in 1d [1.9231(9) Å],b NbCl2[(O-2,4-Me2C6H2-6-CH2)3N] [1.8838(19)–1.925(2) Å],59 NbCl[2,2′-CH3CH(O-4,6-tBu2C6H2)2]2(CH3CN)2 [1.8768(18)–1.9355(18) Å],60 and CpNbCl[(O-C6H4-6-CH2)3N] [1.970(5)–2.009(6) Å].61 The Nb–O–C(phenyl) bond angles in 4a and 4b [114.86(12) and 112.3(3)°, respectively] are smaller than those in 1a and 1d [158.00(10) and 159.159(6)°, respectively];b these values are influenced by the steric bulk around the metal center.15,16 Figure 2 shows ORTEP drawings for Nb(NAr)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) (8), Nb(NAr)(N=CtBu2)-[OCH(CF3)2]2(HN=CtBu2)(9), and Nb(NAr)(N=CtBu2)2(OC6F5)(HN=CtBu2) (11) determined by X-ray crystallographic analysis,d and the selected bond distances and angles are summarized in Table 1. These complexes fold a distorted trigonal bipyramidal structure around niobium consisting of N(arylimido)–Nb–N(in HN=CtBu2) axis [N(1)–Nb–N(4) (for 8 and 11) or N(1)–Nb–N(3) (for 9) bond angles: 174.27(6), 165.90(13), and 170.26(6)°, respectively] and a N–N(O)–O plane from alkoxide (or OC6F5) and N=CtBu2 ligand [sum of N(2)–Nb–N(3) or N(2)–Nb–O(2), O(1)–Nb–N(2), and O(1)–Nb–N(3) or O(1)–Nb–O(2): 350.95° (8), 353.09° (9), 353.0° (11)]. The Nb–N(arylimido) bond distances in complexes 8, 9, and 11 [1.7877(15)–1.796(3) Å] are longer than those in 1a and 1d [1.7643(15) and 1.7730(8) Å, respectively],b but close to those in 4a and 4b [1.783(3) and 1.790(9) Å, respectively]. The Nb–N(1)–C(phenyl) bond angles in 8 and 9 [177.56(13) and 177.89(14)°, respectively] are larger than those in 11 [170.1(3)°] and 4a and 4b [174.5(2) and 170.1(7)°, respectively] and are relatively close to those in 1a and 1d [176.53(12) and 175.806(3)°, respectively].b The Nb–N bond distances in the ketimide (N=CtBu2) ligand [1.9399(16)–1.9670(17) Å] in 8, 9, and 11 are within the range of 4a and 4b [1.946(8)–1.957(3) Å], but apparently shorter than the Nb–N(H)=CtBu2 bond distances [2.440(2)–2.5448(17) Å];d the Nb–N(4)–C or Nb–N(3)–C bond angles in Nb–(H)N=CtBu2 [157.30(12)–160.7(3)°] are apparently small compared to the Nb–N(2)–C bond angle in the ketimide ligand [174.01(12)–178.8(3)°]. The Nb–O bond distances in 8, 9, and 11 [1.9879(14)–2.066(3) Å] are longer than those in 1a and 1d [1.8878(12) and 1.9231(9) Å, respectively],b4a and 4b [1.939(3) and 1.965(7) Å, respectively], and NbCl2[(O-2,4-Me2C6H2-6-CH2)3N] [1.8838(19)–1.925(2) Å],59 but close to those in CpNbCl[(O-C6H4-6-CH2)3N] [1.970(5)–2.009(6) Å].61 As shown in Figure 3, ORTEP drawing for Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7), the complex folds a distorted octahedral geometry around niobium, and both OC6F5 ligands and two of ketimide (N=CtBu2) ligands are positioned trans [172.10(14), 168.32(12)°]. These four ligands fold a plane [sum of O(1)–Nb–N(2), N(2)–Nb–O(3), O(3)–Nb–N(3), and O(1)–Nb–N(3): total 360.97°] against the O(in OC6F5)–Nb–N(in HN=CtBu2) axis [O(2)–Nb–N(1): 178.35(14)°]. The Nb–O bond distances [1.950(3)–1.976(3) Å] are within the range of those introduced above, and the Nb–N bond distances in the ketimide ligands [1.965(3) and 2.098(4) Å] are shorter than the Nb–(H)N=CtBu2 bond distance [2.234(4) Å], clearly suggesting a difference of nature of anionic donor or neutral donor ligand. Figure 3 ORTEP drawings for Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7). Thermal ellipsoids are drawn at the 50% probability level, and H atoms are omitted for clarity. Detailed analysis data are shown in the Supporting Information. Selected bond distances in 7 (Å): Nb(1)–N(1) 1.965(3), Nb(1)–N(2) 2.098(4), Nb(1)–N(3) 2.234(4), Nb(1)–O(1) 1.950(3), Nb(1)–O(2) 1.996(3), Nb(1)–O(3) 1.976(3). Selected bond angles in 7 (deg): Nb(1)–N(1)–C(1) 178.7(3), Nb(1)–N(2)–C(10) 167.1(3), Nb(1)–N(3)–C(19) 163.0(3), Nb(1)–O(1)–C(28) 168.3(3), Nb(1)–O(2)–C(34) 177.3(3), Nb(1)–O(3)–C(40) 173.3(3), N(1)–Nb(1)–N(2) 96.51(15), N(1)–Nb(1)–N(3) 95.04(14), N(2)–Nb(1)–N(3) 168.32(12), O(1)–Nb(1)–N(1) 87.80(13), O(1)–Nb(1)–N(2) 91.66(14), O(1)–Nb(1)–N(3) 90.48(13), O(1)–Nb(1)–O(2) 93.10(12), O(1)–Nb(1)–O(3) 172.10(14), O(2)–Nb(1)–O(3) 91.66(12), O(2)–Nb(1)–N(1) 178.35(14), O(2)–Nb(1)–N(2) 84.84(13), O(2)–Nb(1)–N(3) 83.58(13), O(3)–Nb(1)–N(1) 87.29(13), O(3)–Nb(1)–N(2) 95.04(14), O(3)–Nb(1)–N(3) 83.79(13). Exploration for Reaction Mechanisms of Nb(NAr)(N=CtBu2)3 (3a) and Nb(NAr)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a) with Phenols As described above, the reaction of Nb(NAr)(N=CtBu2)3 (3a) with 1.0 or 2.0 equiv of 2,6-iPr2C6H3OH in n-hexane afforded Nb(NAr)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a) or Nb(NAr)(N=CtBu2)(O-2,6-iPr2C6H3)2 (5), respectively, as the sole isolated product, whereas similar reactions with 2,6-tBu2C6H3OH (2.0 equiv) afforded Nb(NAr)(N=CtBu2)2(O-2,6-tBu2C6H3) (4b, Scheme 6) upon heating. On the basis of reactions of 3a with fluorinated alcohols (and C6F5OH under dilute conditions), these reactions would proceed via coordination of phenol (or alcohol) to Nb through the N(arylimido)–N(ketimide)–N(ketimide) face in 3a and the subsequent proton transfer from the phenol (or alcohol) to the ketimide (N=CtBu2) ligand, without accompanying protonolysis of the N=CtBu2 group with the alcohols (phenols). Although, as described in Introduction, this is the similar hypothesis confirmed in the reactions of V(NAr)Me(N=CtBu2)2 with phenols affording another methyl complex54 and the reaction of V(CHSiMe3)(NAd)(CH2SiMe3)(PMe3)2 with phenol;57 however, the reaction chemistry in the niobium complexes have never been reported. We thus conducted reaction of 4a with a different phenol (2,4,6-Me3C6H2OH) to get some information concerning the mechanism. Scheme 6 Reactions of Nb(N-2,6-Me2C6H3)(N=CtBu2)3 (3a) with 2.0 equiv of Phenols (Detailed Synthetic Procedures Are Shown in the Supporting Information)a It turned out that the reaction of 3a with 2.0 equiv of 2,4,6-Me3C6H2OH in n-hexane afforded the corresponding bis(phenoxide) with coordination of HN=CtBu2, Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H2)2(HN=CtBu2) (12, Scheme 6), identified by NMR spectra and elemental analysis.a As observed in 9, treatment of the complex 12 with NiBr2 (ca. 3 equiv) in toluene (at 25 °C for 3 days) would afford Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H2)2 (13) due to disappearance of resonances ascribed to methyl protons in the 1H NMR spectrum of the reaction mixture (after filtration and removal of toluene, Figure S1-32, SI).a We note that the reaction mixture of 4a with 1.0 equiv of 2,4,6-Me3C6H2OH in n-hexane (after 2 h) afforded a 1:1 mixture of 5 and 12 (and residual 4a) without the formation of Nb(NAr)(N=CtBu2)(O-2,6-iPr2C6H3)(O-2,4,6-Me3C6H2) (Scheme 7 and Figure 4); the reaction seemed complete and further stirring did not improve the conversion of 4a even after 1 day (1H NMR spectra are shown in Figures S2-17–19, SI). The fact clearly indicates not only that certain disproportionation (phenoxy exchange reactions) proceeded in situ, but also that the reaction should proceed via fast coordination (shown in bracket in Scheme 7), proton transfer, and dissociation of phenols. We do not have a clear explanation of why the reaction only afforded the disproportionation products (5 and 12) at this moment. Figure 4 1H NMR spectra (in C6D6 at 25 °C) for (a) Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H3)2(HN=CtBu2) (12), (b) Nb(NAr)(N=CtBu2)(O-2,6-iPr2C6H3)2 (5), (c) Nb(NAr)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a), and (d) the reaction mixture of 4a with 1.0 equiv of 2,4,6-Me3C6H2OH. Detailed conditions are shown in the Supporting Information. (More spectra are shown in Figures S2-17–19.)a Scheme 7 Reactions of Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a) with 1.0 equiv of 2,4,6-Me3C6H2OH (Detailed Synthetic Procedures Are Shown in the Supporting Information)a Concluding Remarks We have prepared a series of four-, five-, and six-coordinate (arylimido)niobium(V) complexes containing ketimide (N=CtBu2), phenoxide, or fluorinated alkoxide ligands. Unique reactivities of the tris(ketimide) analogue, Nb(NAr)(N=CtBu2)3 (3a), with various phenols (2,6-iPr2C6H3OH, 2,6-tBu2C6H3OH, 2,4,6-Me3C6H2OH, and C6F5OH), (CF3)2CHOH, and (CF3)3COH have been demonstrated as summarized below. Reactions of Nb(NAr)(N=CtBu2)3 (3a) with 1.0, 2.0, or 3.0 equiv of 2,6-iPr2C6H3OH afforded Nb(NAr)(N=CtBu2)2(O-2,6-iPr2C6H3), Nb(NAr)(N=CtBu2)(O-2,6-iPr2C6H3)2, or Nb(NAr)(O-2,6-iPr2C6H3)3, respectively (in n-hexane at 25 °C), whereas a similar reaction with 1.0 equiv of 2,6-tBu2C6H3OH afforded a mixture of 3a and Nb(NAr)(N=CtBu2)2(O-2,6-tBu2C6H3) (4b) (even after overnight) and the reaction with 2.0 equiv of 2,6-tBu2C6H3OH at 70 °C in C6D6 (or 100 °C in toluene-d8) afforded 4b without the formation of the bis(phenoxide); the reaction with 2.0 equiv of 2,4,6-Me3C6H2OH afforded Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H3)2(HN=CtBu2) with coordination of HN=CtBu2. Similar reactions of 3a with 1.0 equiv of (CF3)3COH or 2.0 equiv of (CF3)2CHOH afforded Nb(NAr)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) or Nb(NAr)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2), respectively. In addition to the structural analyses data, it was assumed that these reactions proceeded via coordination of phenol (alcohol) to Nb and the subsequent proton transfer to the ketimide ligand. In contrast, the reaction of 3a with 1.0 or 2.0 equiv of C6F5OH afforded Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7) as the sole isolated product (with reaction of the arylimido ligand). It turned out that the careful dropwise addition of C6F5OH to 3a under diluted conditions afforded Nb(NAr)(N=CtBu2)2(OC6F5)(HN=CtBu2) (by reaction of 3a with the ketimide) and subsequent treatment with C6F5OH afforded 7. The reaction of Nb(NAr)(N=CtBu2)2(O-2,6-iPr2C6H3) with 1.0 equiv of 2,4,6-Me3C6H2OH afforded the disproportionation products Nb(NAr)(N=CtBu2)(O-2,6-iPr2C6H3)2 and Nb(NAr)(N=CtBu2)(O-2,4,6-Me3C6H2)2(HN=CtBu2) with 1:1 ratio, clearly indicating that the reaction proceeded via coordination of phenol to Nb and the subsequent fast proton transfer (between ketimide and phenoxide), not by protonation. As described in Introduction, the synthesis and reaction chemistry of (arylimido)niobium(V) complexes containing monodentate anionic donor ligands were less compared to those in vanadium complexes and (tert-butylimido)niobium complexes containing β-diketiminate ligand. Therefore, the results introduced here should provide important information in the reaction chemistry of niobium for better understanding. We are now exploring another route for the synthesis of the four-coordinate (arylimido)niobium(V) dichloride or dialkyl complexes as promising catalyst precursors for olefin polymerization and promising precursors for the alkylidene complexes for olefin metathesis. Experimental Section General Procedures All experiments were carried out under nitrogen atmosphere in a vacuum atmosphere dry box unless otherwise specified. All chemicals used were of reagent grade and were purified by the standard purification procedures. Anhydrous-grade n-hexane, dichloromethane, diethyl ether, and toluene (Kanto Chemical Co., Inc.) were transferred into bottles containing molecular sieves (mixture of 3 Å 1/16 and 4 Å 1/8, and 13× 1/16) under a nitrogen stream in the dry box and used without further purification. Nb(N-2,6-Me2C6H3)Cl3(dme),40 Nb(N-2,6-iPr2C6H3)Cl3(dme),33 and Nb(NAd)Cl3(dme)33 (Ad = 1-adamantyl) were prepared according to a published procedure.33,40 Ethylene for polymerization was of polymerization grade (purity >99.9%, Sumitomo Seika Co., Ltd.) and was used as received. Elemental analyses were performed using EAI CE-440 CHN/O/S Elemental Analyzer (Exeter Analytical, Inc.). All 1H and 13C NMR spectra were recorded on a Bruker AV500 spectrometer (500.13 MHz for 1H NMR, 125.77 MHz for 13C NMR). All spectra were obtained in the solvent indicated at 25 °C unless otherwise noted. Chemical shifts are given in parts per million and are referenced to SiMe4 (δ 0.00 ppm, 1H NMR, 13C NMR) and CFCl3 (δ 0.00, 19F NMR). Coupling constants are given in hertz. Synthesis of (Imido)niobium(V) Dichloride Complexes Containing Phenoxy Ligands A series of (imido)niobium(V) dichloride complexes, Nb(NR)Cl2(OAr′)(dme) [Ar′ = 2,6-iPr2C6H3, R = 2,6-Me2C6H3 (1a); 2,6-iPr2C6H3 (1b); 1-adamantyl (1c); R = 2,6-Me2C6H3, Ar′ = 2,6-Ph2C6H3 (1d); dme = 1,2-dimethoxyethane], were prepared from Nb(NR)Cl3(dme) by treating with 1.0 equiv of LiOAr′ in Et2O. These complexes were identified by NMR spectra and elemental analysis, and some structures were determined by X-ray crystallography. Synthesis of Nb(N-2,6-Me2C6H3)Cl2(O-2,6-iPr2C6H3)(dme) (1a) To an Et2O solution (70 mL) containing Nb(N-2,6-Me2C6H3)Cl3(dme) (1501 mg, 3.674 mmol), 2,6-iPr2C6H3OLi (677 mg, 3.674 mmol) was added at −30 °C. The reaction mixture was warmed slowly to room temperature and then the mixture was stirred for 17 h. Volatiles were removed under reduced pressure to give an orange solid. The residue was extracted with toluene (ca. 40 mL) to afford an orange solution. The solution was filtered through a Celite pad, and the filter cake was washed by toluene. The combined filtrate and the wash were removed in vacuo. Recrystallization from CH2Cl2/pentane at 30 °C gave yellow microcrystals. Yield 1546 mg (76%). 1H NMR (CDCl3): δ 7.05 (d, J = 7.60 Hz, 2H, Ar-H), 6.93 (t, J = 7.60 Hz, 1H, Ar-H), 6.82 (d, J = 7.46 Hz, 2H, Ar-H), 6.70 (t, J = 7.46 Hz, 1H, Ar-H), 4.13, 4.11 (br, 4H, −OCH2), 4.02 (br, 3H, −OCH3), 3.92 (m, 2H, −CH(CH3)2), 3.82 (br, 3H, −OCH3), 2.49 (s, 6H, −CH3), 1.08 (d, J = 6.90 Hz, 12H, −CH(CH3)2). 13C NMR (CDCl3): δ 159.9, 152.8, 138.0, 136.6, 127.3, 125.0, 123.5, 122.8, 74.0, 70.9, 67.6, 61.8, 25.8, 24.2, 19.1. Anal. Calcd for C24H36Cl2NNbO3: C, 52.38; H, 6.59; N, 2.55. Found: C, 52.60; H, 6.61; N, 2.55. Synthesis of Nb(N-2,6-iPr2C6H3)Cl2(O-2,6-iPr2C6H3)(dme) (1b) In a dry box, to an Et2O solution (10 mL) in a round-bottom flask containing Nb(N-2,6-iPr2C6H3)Cl3(dme) (250 mg, 0.54 mmol), Et2O solution (5 mL) containing Li(O-2,6-iPr2C6H3) (83 mg, 0.57 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred overnight. The volatiles were then evaporated in vacuo, the resultant mixture was extracted with toluene, and the toluene solution was filtered through a Celite pad. The filtrate was placed in a rotary evaporator under vacuum. The resultant mixture was then dissolved in minimum amount of CH2Cl2, and the solution was placed in a freezer (−30 °C). The chilled solution afforded an orange-yellow solid. Yield: 209 mg (80.0%). 1H NMR (CDCl3): δ 7.02 (d, 2H, J = 7.6 Hz, Ar), 6.96 (d, 2H, J = 8.0 Hz, Ar), 6.90 (t, 1H, J = 8.0 Hz, Ar), 6.89 (t, 1H, J = 8.2 Hz, Ar), 4.13 (b, 2H, CH2OCH3), 4.11 (b, 2H, CH2OCH3), 4.01 (m, 2H, CH(CH3)2), 3.96 (s, 3H, CH2OCH3), 3.89 (m, 2H, CH(CH3)2), 3.81 (s, 3H, CH2OCH3), 1.07 (t, 24H, J = 5.4 Hz, CH(CH3)2). 13C NMR (CDCl3): δ 159.3, 150.0, 146.8, 137.8, 125.6, 123.5, 122.8, 122.7, 74.0, 70.9, 67.1, 61.7, 28.0, 25.9, 24.6, 24.3. Synthesis of Nb(NAd)Cl2(O-2,6-iPr2C6H3)(dme) (1c) In a dry box, to an Et2O solution (6 mL) in a round-bottom flask containing Nb(NAd)Cl3(dme) (30 mg, 0.07 mmol), Et2O solution (4 mL) containing Li(O-2,6-iPr2C6H3) (13 mg, 0.07 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred overnight. The volatiles were then evaporated in vacuo, the resultant mixture was extracted with toluene, and the toluene solution was filtered through a Celite pad. The filtrate was placed in a rotary evaporator under vacuum. The resultant mixture was dissolved in minimum amount of CH2Cl2 and layered by n-hexane, and the solution was placed in a freezer (−30 °C). The chilled solution afforded a fresh yellow solid. Yield: 33 mg (84.0%). 1H NMR (CDCl3): δ 7.08 (d, 2H, J = 7.3 Hz, Ar), 6.93 (d, 2H, J = 7.3 Hz, Ar), 4.04 (b, 6H, CH2OCH3), 3.88 (m, 2H, CH(CH3)2), 3.70 (b, 3H, CH2OCH3), 1.90 (s, 3H, Ad), 1.69 (s, 6H, Ad), 146 (m, 6H, Ad), 1.20 (d, 12H, J = 6.7 Hz, CH(CH3)2). 13C NMR (CDCl3): δ 160.7, 137.4, 123.3, 122.0, 73.7, 71.8, 70.8, 67.8, 61.2, 43.4, 36.1, 29.3, 26.1, 24.1. Anal. Calcd. for C26H42Cl2NNbO3: C, 53.80; H, 7.29; N, 2.41. Found(1): 53.39; H, 7.60; N, 2.38. Found(2): 53.99; H, 7.65; N, 2.38. Synthesis of Nb(N-2,6-Me2C6H3)Cl2(O-2,6-Ph2C6H3)(dme) (1d) To an Et2O solution (40 mL) containing Nb(N-2,6-Me2C6H3)Cl3(dme) (333 mg, 0.815 mmol), Li(O-2,6-Ph2C6H3) (206 mg, 0.815 mmol) was added at −30 °C. The reaction mixture was warmed slowly to room temperature and then the mixture was stirred for 17 h. Volatiles were removed under reduced pressure to give an orange solid. The residue was extracted with toluene (ca. 40 mL) to afford an orange solution. The solution was filtered through a Celite pad, and the filter cake was washed by toluene. The combined filtrate and the wash were removed in vacuo. Recrystallization from CH2Cl2/pentane at 30 °C gave orange solids. Microcrystals suitable for X-ray crystallographic analysis were obtained by recrystallization in benzene at room temperature. Yield 340 mg (67%). 1H NMR (CDCl3): δ 7.41 (d, J = 7.30 Hz, 4H, Ar-H), 7.17 (m, 8H, Ar-H), 7.03 (t, J = 7.45 Hz, 1H, Ar-H), 6.77 (d, J = 7.34 Hz, 2H, Ar-H), 6.71 (t, J = 7.34 Hz, 1H, Ar-H), 3.79 (br, 4H, −OCH2), 3.66 (br, 3H, −OCH3), 3.18 (br, 3H, −OCH3), 2.23 (s, 6H, −CH3). 13C NMR (CDCl3): δ 160.3, 152.5, 139.2, 137.2, 133.1, 131.1, 130.6, 127.7 127.0, 126.5, 124.9, 122.3, 73.7, 70.5, 67.0, 61.3, 19.0. Anal. Calcd for C30H32Cl2NNbO3: C, 58.27; H, 5.22; N, 2.27. Found: C, 58.51; H, 5.52; N, 2.35. Synthesis of Nb(N-2,6-Me2C6H3)(CF3SO3)2(O-2,6-iPr2C6H3)(dme) (2a) To a chilled CH2Cl2 (12 mL, −30 °C) solution containing Nb(N-2,6-Me2C6H3)Cl3(dme) (100 mg, 0.182 mmol), AgOTf (94 mg, 0.363 mmol) was added. The reaction was warmed slowly to room temperature (25 °C) and stirred for 3 h. After the reaction, the volatiles were evaporated in vacuo and the resultant mixture was extracted with toluene. The solution was filtered through a Celite pad. The filtrate was placed in a rotary evaporator under vacuum. The resultant mixture was dissolved in minimum amount of Et2O layered with hexane, and the solution was placed in a freezer (−30 °C). The resultant orange solids were isolated as an analytically pure orange precipitate. Yield: 86 mg (61%). 1H NMR (C6D6): δ 7.07 (d, J = 7.64 Hz, 2H, Ar-H), 6.95 (t, J = 7.64 Hz, 1H, Ar-H), 6.61 (d, J = 7.53 Hz, 2H, Ar-H), 6.52 (t, J = 7.53 Hz, 1H, Ar-H), 3.71 (s, 3H, −OCH3), 3.69 (m, 2H, −CH(CH3)2), 3.51 (t, 2H, −OCH2), 3.22 (s, 3H, −OCH3), 3.21 (t, 2H, −OCH2), 2.41 (s, 6H, −CH3), 1.21 (d, J = 6.80 Hz, 12H, −CH(CH3)2). 19F NMR (C6D6): δ −76.89. 13C NMR (C6D6): δ 160.7, 153.4, 137.5, 136.4, 127.0, 124.7, 124.3, 120.1 (q, 1JCF = 317.9 Hz), 73.8, 70.5, 70.3, 62.6, 26.4, 24.2, 18.4. Anal. Calcd for C26H36F6NNbO9S2: C, 40.16; H, 4.67; N, 1.80. Found: C, 40.29; H, 4.60; N, 1.75. Synthesis of Nb(N-2,6-Me2C6H3)(CF3SO3)3(dme) (2b) In a dry box, to a dichloromethane solution (18 mL) in a round-bottom flask containing Nb(N-2,6-Me2C6H3)Cl3(dme) (490 mg, 1.20 mmol), a dichloromethane solution (8 mL) containing AgOTf (OTf = CF3SO3, 925 mg, 3.60 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred overnight. The volatiles were then evaporated in vacuo, and the resultant mixture was extracted with toluene. The toluene solution was filtered through a Celite pad. The filtrate was placed in a rotary evaporator under vacuum. The resultant mixture was dissolved in minimum amount of CH2Cl2 and layered by n-hexane and the solution was placed in a freezer (−30 °C). The chilled solution afforded a purple solid. Yield: 550 mg (61.2%). 1H NMR (CDCl3): δ 6.99 (d, 2H, J = 7.6 Hz, Ar), 6.91 (t, 1H, J = 7.1 Hz, Ar), 4.71 (t, 2H, J = 5.1 Hz, CH2OCH3), 4.58 (s, 3H, CH2OCH3), 4.26 (t, 2H, J = 4.9 Hz, CH2OCH3), 3.80 (s, 3H, CH2OCH3), 2.64 (s, 6H, ArCH3). 19F NMR (CDCl3): δ −75.82(s), −76.29(s). 13C NMR (CDCl3): δ 152.6, 139.9, 130.03, 127.94, 122.84, 120.40, 120.31, 117.79, 115.26, 78.26, 73.56, 70.38, 63.89, 53.56, 18.71. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)3 (3a) To a toluene solution (14 mL) containing Nb(N-2,6-Me2C6H3)Cl3(dme) (100 mg, 0.25 mmol), a toluene solution (6 mL) containing Li(N=CtBu2) (110 mg, 0.75 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred overnight. The volatiles were then evaporated in vacuo, and the resultant mixture was extracted with toluene. The toluene solution was filtered through a Celite pad. The filtrate was placed in a rotary evaporator under vacuum. The resultant mixture was dissolved in minimum amount of n-hexane, and the solution was placed in a freezer (−30 °C). The chilled solution afforded an orange solid. Yield: 61 mg (39.0%). 1H NMR (C6D6): δ 7.08 (d, 2H, J = 7.9 Hz, Ar), 6.80 (t, 1H, J = 7.6 Hz, Ar), 2.63 (s, 6H, ArCH3), 1.30 (s, 54H, CCH3). 13C NMR (CDCl3): δ 195.3, 131.5, 128.4, 126.8, 119.5, 44.6, 30.8, 19.9. Anal. Calcd. for C35H65N4Nb: C, 66.43; H, 10.04; N, 8.85. Found: C, 64.22; H, 10.13; N, 8.57. Rather low C value would be due to incomplete combustion during analysis run. Synthesis of trans-NbCl2(N=CtBu2)3 (3b) To a toluene solution (40 mL) containing NbCl5 (600 mg, 2.22 mmol), a toluene solution (18 mL) containing Li(N=CtBu2) (982 mg, 6.67 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred overnight. The volatiles were then evaporated in vacuo, the resultant mixture was extracted with toluene, and the toluene solution was filtered through a Celite pad. The filtrate was placed in a rotary evaporator under vacuum. The resultant mixture was dissolved in minimum amount of n-hexane, and the solution was placed in a freezer (−30 °C). The chilled solution afforded an orange solid. Yield: 213 mg (16.4%).1H NMR (C6D6): δ 1.36 (s, 54, C(CH3)3). 13C NMR (C6D6): δ 188.9, 46.0, 31.1. Anal. Calcd. for C27H54Cl2N3Nb: C, 55.48; H, 9.31; N, 7.19. Found: C, 55.70; H, 9.12; N, 6.97. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a) To an n-hexane solution (34 mL) containing Nb(NAr)(N=CtBu2)3 (3a) (560 mg, 0.89 mmol), an n-hexane solution (8 mL) containing 2,6-iPr2C6H3OH (158 mg, 0.89 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-pentane. The solution was then placed in a freezer (−30 °C), and the chilled solution afforded a yellow solid. Yield: 408 mg (69.0%). 1H NMR (CDCl3): δ 6.99 (d, 2H, J = 7.7 Hz, Ar), 6.92 (d, 2H, J = 7.4 Hz, Ar), 6.80 (t, 1H, J = 7.6 Hz, Ar), 6.70 (t, 1H, J = 8.8 Hz, Ar), 3.45 (m, 2H, CH(CH3)2), 2.40 (s, 6H, ArCH3), 1.28 (s, 36H, CCH3), 1.05 (d, 12H, J = 6.9 Hz, CH(CH3)2). 13C NMR (CDCl3): δ 158.1, 137.0, 131.8, 127.0, 122.8, 121.6, 119.6, 53.6, 45.1, 30.7, 28.6, 26.7, 23.4, 19.5, 14.3. Microcrystals suitable for X-ray crystallographic analysis were prepared by recrystallization. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-tBu2C6H3OH) (4b) To a sealed Schlenk glass tube containing Nb(NAr)(N=CtBu2)3 (3a) (1095 mg, 1.73 mmol) and toluene (25 mL), a toluene solution (18 mL) containing 2,6-tBu2C6H3OH (375 mg, 1.82 mmol) was added slowly at −30 °C. The reaction mixture was stirred at 70 °C overnight under N2. The volatiles were then evaporated in vacuo, and the resultant mixture was extracted by minimum amount of n-hexane. The solution was then filtered through a filter paper for removal of remaining 2,6-tBu2C6H3OH. The resultant solution was placed in a freezer (−30 °C), and the chilled solution afforded a yellow solid. Yield: 531 mg (44.0%). 1H NMR (C6D6): δ 7.28 (d, 2H, J = 8.3 Hz, Ar), 6.99 (d, 2H, J = 7.8 Hz, Ar), 6.84 (t, 1H, J = 7.8 Hz, Ar), 6.79 (t, 1H, J = 7.5 Hz, Ar), 2.60 (s, 6H, ArCH3), 1.53 (s, 18H, ArC(CH3)3), 1.27 (s, 36H, C(CH3)3). 13C NMR (C6D6): δ 197.9, 196.8, 138.7, 128.4, 127.6, 125.9, 122.6, 119.8, 44.7, 35.4, 31.8, 30.7, 20.0. Anal. Calcd. for C40H66N3NbO: C, 68.84; H, 9.53; N, 6.02. Found: C, 68.11; H, 9.53; N, 5.93. Rather low C value would be due to incomplete combustion during analysis run. Microcrystals suitable for the X-ray crystallographic analysis were prepared by recrystallization. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)(O-2,6-iPr2C6H3)2 (5) To an n-hexane solution (12 mL) containing Nb(NAr)(N=CtBu2)3 (3a) (80 mg, 0.13 mmol), an n-hexane solution (5 mL) containing 2,6-iPr2C6H3OH (46 mg, 0.26 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-hexane. The solution was placed in a freezer (−30 °C). The chilled solution afforded a yellow solid. Yield: 74 mg (83.2%).1H NMR (C6D6): δ 7.12 (d, 4H, J = 7.4 Hz, Ar), 6.99 (t, 2H, J = 7.4 Hz, Ar), 6.87 (d, 2H, J = 6.9 Hz, Ar), 6.71 (t, 1H, J = 7.8 Hz, Ar), 3.88 (m, 4H, CH(CH3)2), 2.38 (s, 6H, ArCH3), 1.24 (m, 24H, CHCH3), 1.12 (s, 18H, C(CH3)3). 13C NMR (C6D6): δ 203.9, 158.2, 156.2, 137.5, 131.6, 127.6, 123.6, 123.2, 122.2, 44.8, 30.3, 27.2, 23.8, 23.7, 19.2. Anal. Calcd. for C35H63N4Nb: C, 69.67; H, 8.70; N, 3.96. Found: C, 69.49; H, 9.00; N, 3.92. Synthesis of Nb(N-2,6-Me2C6H3)(O-2,6-iPr2C6H3)3 (6) Reaction with 2,6-iPr2C6H3OH To an n-hexane solution (18 mL) containing Nb(NAr)(N=CtBu2)3 (3a) (150 mg, 0.24 mmol), an n-hexane solution (8 mL) containing 2,6-iPr2C6H3OH (128 mg, 0.72 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-hexane. The solution was placed in a freezer (−30 °C), and the chilled solution afforded a yellow solid. Yield: 141 mg (79.0%). Reaction with Li(O-2,6-iPr2C6H3) To an Et2O solution (20 mL) containing Nb(N-2,6-Me2C6H3)Cl3(dme) (100 mg, 0.25 mmol), an Et2O solution (6 mL) containing Li(O-2,6-iPr2C6H3) (136 mg, 0.74 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-hexane. The solution was placed in a freezer (−30 °C), and the chilled solution afforded a yellow solid. Yield: 162 mg (87.0%). 1H NMR (CDCl3): δ 7.11 (d, 6H, J = 7.7 Hz, Ar), 7.00 (t, 3H, J = 7.7 Hz, Ar), 6.78 (d, 2H, J = 7.0 Hz, Ar), 6.67 (t, 1H, J = 7.4 Hz, Ar), 3.54 (m, 6, CH(CH3)2), 1.88 (s, 6H, ArCH3), 1.13 (d, 36H, J = 6.8 Hz, CH(CH3)2). 13C NMR (CDCl3): 158.1, 155.4, 137.3, 132.0, 127.1, 123.7, 123.4, 122.8, 27.3, 23.6, 18.4. Anal. Calcd. for C44H60NNbO3: C, 71.04; H, 8.13; N, 1.88. Found(1): 70.43; H, 7.91; N, 1.73. Found(2): 70.59; H, 7.90; N, 1.75. Rather low C value would be due to incomplete combustion during analysis run. Synthesis of Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7) Reaction of 3a with 1 equiv of C6F5OH To an n-hexane solution (12 mL) containing Nb(NAr)(N=CtBu2)3 (3a) (80 mg, 0.13 mmol), an n-hexane solution (5 mL) containing C6F5OH (24 mg, 0.13 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-hexane. The solution was placed in a freezer (−30 °C), and the chilled solution afforded an orange solid. Yield: 13 mg (9.6%). 1H NMR (C6D6): δ 9.67 (b, 1H, NH), 1.11 (s, 1H, C(CH3)3). 19F NMR (C6D6): δ −160.19 (d), −165.5 (t), −169.6 (t). 13C NMR (C6D6): δ 204.4, 192.4, 141.0, 139.4, 139.0, 137.5, 137.4, 136.0, 134.0, 128.2, 127.8, 47.9, 41.6, 31.1, 29.8, 29.7. Anal. Calcd. for C45H55F15N3NbO3: C, 50.81; H, 5.21; N, 3.95. Found: 50.94; H, 5.30; N, 4.07. Microcrystals suitable for X-ray crystallographic analysis were prepared by recrystallization from the chilled n-hexane solution. Reaction of 3a with 2 equiv of C6F5OH To an n-hexane solution (6 mL) containing Nb(NAr)(N=CtBu2)3 (3a) (44 mg, 0.07 mmol), an n-hexane solution (3 mL) containing C6F5OH (26 mg, 0.14 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-hexane. Then, the solution was placed in a freezer (−30 °C), and the chilled solution afforded an orange solid identified as 7. Yield: 23 mg (33.0%). Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) (8) To a sealed Schlenk glass tube containing Nb(NAr)(N=CtBu2)3 (3a) (150 mg, 0.24 mmol) and n-hexane (16 mL), an n-hexane solution (8 mL) containing (CF3)3COH (59 mg, 0.25 mmol) was added slowly at −30 °C. The reaction mixture was then stirred at 40 °C overnight. The volatiles were evaporated in vacuo, and the resultant residue was dissolved in minimum amount of n-hexane. Then, the solution was placed in a freezer (−30 °C), and the chilled solution afforded a yellow solid. Yield: 62 mg (30.0%), and conversion of 3a was 50%. 1H NMR (C6D6): δ 9.66 (b, 1H, NH), 6.94 (d, 2H, J = 7.5 Hz, Ar), 6.76 (t, 1H, J = 7.5 Hz, Ar), 2.53 (s, 6H, ArCH3), 1.35 (s, 9H, C(CH3)3), 1.20 (s, 36H, C(CH3)3), 0.94 (s, 9H, C(CH3)3). 19F NMR (C6D6): δ −73.88 (s). Anal. Calcd. for C40H67F9N4NbO: C, 53.91; H, 7.42; N, 6.45. Found(1): C, 52.90; H, 7.38; N, 6.28. Found(2): C, 52.27; H, 7.18; N, 6.12. Rather low C value would be due to incomplete combustion during analysis runs. Microcrystals suitable for the X-ray crystallographic analysis were prepared by recrystallization of the chilled n-hexane solution. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2) (9) To an n-hexane solution (31 mL) containing Nb(N-2,6-Me2C6H3)(N=CtBu2)3 (3a) (345 mg, 0.55 mmol), an n-hexane solution (9 mL) containing (CF3)2CHOH (184 mg, 1.09 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of dichloromethane. The solution was then placed in a freezer (−30 °C), and the chilled solution afforded an orange solid. Yield: 299 mg (66.0%). 1H NMR (C6D6): δ 9.77 (b, 1H, NH), 6.84 (d, 2H, J = 7.5 Hz, Ar), 6.70 (t, 1H, J = 7.8 Hz, Ar), 5.35 (m, 2H, CH(CF3)2), 2.52 (s, 6H, ArCH3), 1.27 (s, 9H, C(CH3)3), 1.16 (s, 18H, C(CH3)3), 0.98 (s, 9H, C(CH3)3). 19F NMR (C6D6): δ −75.1 (b). 13C NMR (C6D6): δ 199.6, 195.3, 124.6, 81.2, 44.4, 43.1, 41.4, 30.5, 30.3, 29.5, 26.9, 19.3. Anal. Calcd. for C32H48F12N3NbO2: C, 46.44; H, 5.85; N, 5.08. Found: C, 46.30; H, 5.83; N, 5.01. Microcrystals suitable for the X-ray crystallographic analysis were prepared by recrystallization. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)[OCH(CF3)2]2 (10) To a sealed Schlenk glass tube containing Nb(N-2,6-Me2C6H3)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2) (9) (54 mg, 0.07 mmol) and toluene (5 mL), NiBr2 (43 mg, 0.20 mmol) was added at low temperature (−30 °C). The reaction mixture was stirred for 2 h. The mixture was filtered through a filter paper for removal of the solid part. The solution was evaporated in vacuo, and the removal of HN=CtBu2 was observed by NMR spectrum. 1H NMR (C6D6): δ 6.84 (d, 2H, J = 7.6 Hz, Ar), 6.70 (t, 1H, J = 7.5 Hz, Ar), 5.35 (m, 2H, CH(CF3)2), 2.52 (s, 6H, ArCH3),1.16 (s, 18H, C(CH3)3). Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)2(OC6F5)(HN=CtBu2) (11) Reaction of 3a with 0.5 equiv of C6F5OH An n-hexane solution (6.0 mL) containing C6F5OH (15 mg, 0.08 mmol) was added to an n-hexane solution (15.0 mL) containing 3a (103 mg, 0.16 mmol) at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 1 h. The orange-yellow mixture was dried in vacuo, and the crude product was measured by 1H and 19F NMR spectra (conversion of 3a 45%). Microcrystals suitable for X-ray crystallographic analysis were collected (in small amount) from the chilled n-hexane solution. 1H NMR (C6D6): δ 9.70 (b, 1H, NH), 6.93 (d, 2H, J = 6.8 Hz, Ar), 6.74 (t, 1H, J = 7.6 Hz, Ar), 2.57 (s, 6H, ArCH3), 1.34 (s, 9H, C(CH3)3), 1.20 (s, 36H, C(CH3)3), 0.95 (s, 9H, C(CH3)3). 19F NMR (C6D6): δ −162.01 (d), −166.28 (t), −173.05 (t). 13C NMR (CDCl3): 133.2, 126.9, 122.5, 44.9, 30.6, 19.2. These chemical shifts were assigned from the mixture of 3a and 13 (different ratios shown below). Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)(O-2,4,6-Me3C6H2OH)2(HN=CtBu2) (12) To an n-hexane solution (15 mL) containing Nb(NAr)(N=CtBu2)3 (3a) (100 mg, 0.16 mmol), an n-hexane solution (5 mL) containing 2,4,6-Me3C6H2OH (44 mg, 0.32 mmol) was added slowly at −30 °C. The reaction mixture was warmed slowly to room temperature and stirred for 2 h. The volatiles were then evaporated in vacuo, and the resultant mixture was dissolved in minimum amount of n-pentane. The solution was placed in a freezer (−30 °C), and the chilled solution afforded a yellow solid. Yield: 36 mg (36.6%). 1H NMR (C6D6): δ 9.66 (b, 1H, NH), 6.90 (d, 2H, J = 7.6 Hz, Ar), 6.77 (s, 4H, Ar), 6.73 (t, 1H, J = 7.3 Hz, Ar), 2.43 (s, 12H, ArCH3), 2.41 (s, 6H, ArCH3), 2.15 (s, 6H, ArCH3), 1.35 (s, 9H, C(CH3)3), 1.12 (s, 18H, C(CH3)3), 0.94 (s, 9H, C(CH3)3). 13C NMR (C6D6): δ 202.6, 192.5, 159.2, 132.0, 129.9, 129.5, 128.4, 126.3, 123.1, 44.9, 41.7, 41.6, 34.5, 31.2, 30.4, 29.9, 22.7, 20.8, 19.3, 17.7, 14.3. Anal. Calcd. for C44H68N3NbO2: C, 69.18; H, 8.97; N, 5.50. Found: C, 68.72; H, 9.17; N, 5.26. Rather low C value would be due to incomplete combustion during analysis runs. Synthesis of Nb(N-2,6-Me2C6H3)(N=CtBu2)(O-2,4,6-Me3C6H2)2 (13) To a toluene solution (6 mL) containing Nb(N-2,6-Me2C6H3)(N=CtBu2)(O-2,4,6-Me3C6H2)2(HN=CtBu2) (12a) (36 mg, 0.05 mmol), a toluene solution (6 mL) containing NiBr2 (31 mg, 0.14 mmol) was added slowly at −30 °C, and the mixture was stirred for 3 days. Color of the mixture changed from orange to yellow, and the reaction profile was monitored by 1H NMR spectrum. The solution was placed in vacuo, and the residue was dissolved in minimum amount of n-pentane and placed in a freezer (−30 °C). The chilled solution afforded a yellow solid. Yield: 36 mg (68.0%). 1H NMR (C6D6): δ 6.90 (d, 2H, J = 7.5 Hz, Ar), 6.77 (s, 4H, Ar), 6.73 (t, 1H, J = 7. 7 Hz, Ar), 2.43 (s, 12H, ArCH3), 2.41 (s, 6H, ArCH3), 2.37 (s, 3H, ArCH3), 2.16 (s, 6H, ArCH3), 1.13 (s, 18H, C(CH3)3). Dissociation of HN=CtBu2 could be observed by 1H NMR spectrum. Crystallographic Analysis All measurements were made on a Rigaku XtaLAB P200 diffractometer using multilayer mirror monochromated Mo Kα radiation. The crystal collection parameters are listed in Table S1. The data were collected and processed using CrystalClear (Rigaku)62 or CrysAlisPro (Rigaku Oxford Diffraction),63 and the structure was solved by direct methods64 and expanded using Fourier techniques. The nonhydrogen atoms were refined anisotropically, and the hydrogen atoms were refined using the riding model. All calculations were performed using the Crystal Structure65 crystallographic software package, except for refinement, which was performed using SHELXL version 2014/7.66,67 Crystal data and the collection parameters are shown in Tables S1 and S2 in the Supporting Information, and structure reports and CIF and xyz files are also shown in the Supporting Information. CCDC numbers for complexes 1a, 1d, 4a, 4b, 7, 8, 9, and 11 are 1839646–1839653, respectively. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01065.Experimental procedure for some reactions with phenol, tables for crystal data and collection parameters of 1a, 1d, 4a, 4b, 7, 8, 9, 11, and NMR spectra for synthesized (arylimido)niobium complexes; NMR spectra monitoring some reactions with phenols; ORTEP drawings and selected bond distances and angles for 1a and 1d (PDF) Structure reports and CIF files for 1a, 1d, 4a, 4b, 7–9, and 11 (CIF) Structure reports and xyz files for 1a, 1d, 4a, 4b, 7–9, and 11 (xyz) Supplementary Material ao8b01065_si_001.pdf ao8b01065_si_002.cif ao8b01065_si_003.xyz The authors declare no competing financial interest. Acknowledgments This project was partly supported by Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS, Nos. 15H03812 and H1801982). N.S. and K.W. acknowledge the Tokyo Metropolitan government (Asian Human Resources Fund) for predoctoral fellowship. The authors thank Profs. S. Komiya, A. Inagaki, and Dr. S. Sueki (Tokyo Metropolitan University) for discussions. a Selected NMR spectra for (imido)niobium complexes, and experimental procedure for some reactions with phenol and the selected NMR spectra monitoring the reactions are shown in the Supporting Information. b Oak Ridge thermal ellipsoid plot (ORTEP) drawings for NbCl2(N-2,6-Me2C6H3)(O-2,6-iPr2C6H3)(dme) (1a, CCDC 1839646) and NbCl2(N-2,6-Me2C6H3)(O-2,6-Ph2C6H3)(dme) (1d, CCDC 1839647), selected bond distances and angles, and structure reports including CIF and xyz files are shown in the Supporting Information. c Activity by 1a: 23 kg PE/(mol Nb h) (25 °C), 11 kg PE/(mol Nb h) (50 °C). Activity by 1d: 12 kg PE/(mol Nb h) (25 °C). Conditions: catalyst (1a or 1d), 5.0 μmol; toluene, 30 mL; ethylene, 8 atm; dry-MAO (prepared by removing toluene and AlMe3 from the commercially available MAO),46−49 3.0 mmol; 1 h. d The structure reports including CIF and xyz files for Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-iPr2C6H3) (4a, CCDC 1839648) and Nb(N-2,6-Me2C6H3)(N=CtBu2)2(O-2,6-tBu2C6H3OH) (4b, CCDC 1839649), Nb(N=CtBu2)2(OC6F5)3(HN=CtBu2) (7, CCDC 1839650), Nb(N-2,6-Me2C6H3)(N=CtBu2)2[OC(CF3)3](HN=CtBu2) (8, CCDC 1839651), Nb(N-2,6-Me2C6H3)(N=CtBu2)[OCH(CF3)2]2(HN=CtBu2) (9, CCDC 1839652), and Nb(N-2,6-Me2C6H3)(N=CtBu2)2(OC6F5)(HN=CtBu2) (11, CCDC 1839653) are shown in the Supporting Information. ==== Refs References Wigley D. E. Organoimido Complexes of the Transition Metals . Prog. Inorg. Chem. 2007 , 42 , 239 –428 . 10.1002/9780470166437.ch4 . Nugent W. A. ; Mayer J. M. Metal–Ligand Multiple Bonds ; Wiley Interscience : New York , 1988 . For examples, synthesis of (imido)vanadium(V) complexes (refs 3–5), Devore D. 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PMC006xxxxxx/PMC6644437.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145890610.1021/acsomega.8b00362ArticleDepolying Tunable Metal-Shell/Dielectric Core Nanorod Arrays as the Virtually Perfect Absorber in the Near-Infrared Regime Chau Yuan-Fong Chou †Chou Chao Chung-Ting ‡Lim Chee Ming †Huang Hung Ji §Chiang Hai-Pang ∥⊥† Centre for Advanced Material and Energy Sciences, Universiti Brunei Darussalam, Tungku Link, Gadong BE1410, Negara Brunei Darussalam‡ Department of Physics, Fu Jen Catholic University, New Taipei City 242, Taiwan§ Instrument Technology Research Center, National Applied Research Laboratories, Hsinchu, Taiwan∥ Institute of Optoelectronic Sciences, National Taiwan Ocean University, No. 2 Pei-Ning Road, 202 Keelung, Taiwan⊥ Institute of Physics, Academia Sinica, Taipei 11529, Taiwan E-mail: hpchiang@mail.ntou.edu.tw.09 07 2018 31 07 2018 3 7 7508 7516 28 02 2018 05 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. In this paper, the coupled Ag-shell/dielectric-core nanorod for sensor application is investigated and the different dielectric core plasmonic metamaterial is adopted in our design. The operational principle is based on the concept of combining the lattice resonance, localized surface plasmon resonance (SPR), and cavity plasmon resonance modes within the nanostructure. The underlying mechanisms are investigated numerically by using the three-dimensional finite element method and the numerical results of coupled solid Ag nanorods are included for comparison. The characteristic absorptance/reflectance peaks/dips have been demonstrated to be induced by different plasmonic modes that could lead to different responses required for plasmonic sensors. A nearly perfect absorptance and an approximate zero reflectance with a sharp band linewidth are obtained from the proposed system, when operated as an SPR sensor with the sensitivity and figure of merit of 757.58 nm/RIU (RIU is the refractive index unit) and 50.51 (RIU–1), respectively. Our work provides a promising method for the future developments of more advanced metamaterial absorber for chemical sensing, thermal radiation tailoring, field enhanced spectroscopy, and general filtering applications. document-id-old-9ao8b00362document-id-new-14ao-2018-00362qccc-price ==== Body 1 Introduction Plasmonic perfect absorbers (PPAs) have fascinated increasing attention in the field of nanophotonic devices because of their unusual capacity of absorbing and enhancing electromagnetic (EM) waves to the nanometer-scale.1 It can be applied to different fields, such as resonators,2 refractive index (RI) sensors,3 nanoantennas,4 plasmonic solar cells,5 biosensing,6−10 and absorbers.11,12 Narrow-band plasmonic absorbers using surface plasmon resonance (SPR) effects are widely used in thermal radiation manipulation,13,14 detection, and sensors.15−18 On the basis of plasmonic nanostructures (PNSs) with the properties of gap plasmon resonance (GPR),19 cavity plasmon resonance (CPR),20 and metal–dielectric–metal structure,11,12 perfect plasmonic absorbers21,22 can be achieved in the visible, infrared regime, and other EM regimes. Recently, researchers have made many advancing efforts on PPAs to achieve narrow band absorption11 for plasmonic sensing applications. The narrow spectrum bandwidth is required in medical sensing applications, for example the monitoring of chemical reactions,23 the measurement of gas concentrations,24 as well as the detection of biomolecules.25 However, the general PPAs exhibit complicated structure, and the resonance wavelength is at a fixed wavelength, and this limits the diversification of their deployment. The various approaches which have been reported to narrow the band linewidth giving sharp spectrum feature12,26,27 are plasmonic gap resonance,28 surface lattice resonance,29 and magnetic dipole resonance.30 There are several metastructures which have been studied for the purpose of absorption and they are based on gap resonance;31,32 however, these literature studies did not report on the design of sensors having perfect absorptance and zero reflectance properties. PPAs need to be designed with simple structure and tunable in the infrared regime if they are to be successfully implemented in sensor applications. The large band linewidth of plasmon resonances causes strong radiative damping in metals, which is a major problem because it reduces the sensitivity and hence lowering the quality factor (Q-factor) of the sensor. An effective way to overcome this drawback is to couple the plasmonic effects to a system with a narrow resonance. There are several methods to narrow band tunability of PPAs, for example, the use of stacked graphene-dielectric sheet,31 applying phase-change material (Ge2Sb2Te5),32 and deploying microelectromechanical system.33 For practical applications, a large intensity variation of the absorbed or reflected light at a certain wavelength is desired, that is, sharp peaks/dips of absorptance/reflectance with a large modulation depth are required. In contrast to the above mentioned techniques, we proposed a dual band and tunability PPA consisting of coupled Ag-shell/dielectric-core (ASDC) nanorod array arranged in a square lattice. In our design, the PPA is made tunable by physically modifying the PPA’s material and geometry. Each resonance obtained from our proposed PPA has excellent correlation with respect to geometrical and material parameters, and furthermore, it shows excellent tunability for each resonant wavelength (λres). In the proposed structure, an open cavity is introduced in ASDC nanorods such that it is accessible to the surrounding medium, and this makes the ASDC nanorods an attractive RI sensor. The ASDC nanorods and the bottom Ag thin film layer provide an optical response of SPR and CPR, and the positive–negative charge pairs will induce an electromotive force on the metal surface, thus causing an effective coupled mode which supports a nearly perfect absorptance and an approximate zero reflectance. ASDC nanorods that are placed at a Bragg distance above a metal mirror (i.e., 100 nm Ag bottom layer) form a Fabry–Pérot nanocavity, and they constitute a coupled photonic–plasmonic system. The influences of structure and material parameters on the sensing performance are investigated by using the three-dimensional (3-D) finite element method (FEM). In addition, we put to use the strong localized enhancement of the EM wave in the gap and cavity regions in the ASDC nanorods, and we examine the RI infrared sensing performance. It is found that the absorptance bandwidth can be changed by varying the dielectric core in the Ag-shell nanorods, and a nearly perfect absorptance together with an approximate zero reflectance with a sharp band linewidth are obtained from the proposed system. The proposed structure can be operated as SPR sensors where the sensitivity and figure of merit (FOM) of 757.58 nm/RIU and 50.51 (RIU–1), respectively, are observed. This narrow band linewidth and perfect absorptance properties are required in sensing and filtering applications.34 2 Simulation Method and Models To analyze the proposed plasmonic system, 3-D FEM is performed using a commercially available software package (COMSOL multiphysics). Because of the symmetry of the structures, a plane wave polarized in x-axis is used as the incident light at normal incidence from the top surface. Periodic boundary conditions are considered in x- and y-directions, and perfectly matching layers are applied along the z direction. The Ag permittivity data cited in ref (35) is used. The absorptance (A) is calculated as 1 – reflectance (R) – transmittance (T), with transmittance being nearly zero in the infrared realm (i.e., the working region of the proposed PPA) because of the thickness of the bottom Ag film is thicker than the skin depth in the infrared region, the transmittance (T) channel is prevented, and the absorptance is reduced to 1 – R. The sensing capability of the SPR sensor is usually defined by the following definitions of sensitivity (S) and FOM.3,4 1 where Δλ is the corresponding central wavelength shift of the resonant dips and Δn is the difference of the RI. fwhm is the full width at half maximum of the SPR spectrum, and it is defined as the corresponding λres width at half percentage of the reflectance dip. The Q factor can be calculated as the ratio of peak resonance wavelength (λres) and the full width at half maximum, that is, Q = λres/fwhm.36 The coupled ASDC nanorod array is arranged in a square lattice. Figure 1 depicts a truncated view of the periodic arrays of the coupled ASDC nanorods. The proposed structure consists of periodic arrays of coupled ASDC nanorods placed directly on the surface of a uniform Ag film. The value of the incident EM wave is fixed at \|E0\| = 1 V/m. The closely spaced ASDC nanorods have an outer radius (R) and inner radius (r) of 80 and 70 nm, thickness (t = R – r) of 10 nm, and gap distance (g) of 20 nm. The filling medium in ASDC is set to air (ε = 1.00) and silica (SiO2). The lattice constant (a) of the arrays is 470 nm, and the bottom silver film has a thickness (s) of 100 nm. In addition, the whole structure is placed on a silica substrate (i.e., glass), and the surrounding material is assumed to be air or n, where n is the RI of the surrounding medium. The RI of silica is calculated through the Sellmeier equation.37 All materials are assumed to be nonmagnetic (i.e., μ = μ0). Figure 1 Truncated view of the periodic arrays of coupled ASDC nanorods placed directly on the surface of a uniform Ag film. The unit cell repeats in the x and y direction forming a square array with periodicity a. The origin [(x, y, z) = (0, 0, 0)] of the coordinate system is positioned in the middle plane of the simulation zone. The closely spaced ASDC nanorods have an outer radius (R) and an inner radius (r) of 80 and 70 nm, thickness (t = R – r) of 10 nm, and gap distance (g) of 20 nm. The filling relative permittivity (ε) in ASDC is set to air (ε = 1.00) and silica, respectively. The lattice constant (a) of the arrays is 470 nm, and the bottom silver film has a thickness (s) of 100 nm. In addition, the whole structure is placed on a silica substrate, and the surrounding material is assumed to be air or n, where n is the RI of the surrounding medium. Thanks to the rapid advances in the fabrication technique of nanophotonic structures, the proposed PNSs are compatible with the current fabrication technology such as a manufacturing based on secondary electron lithography generated by ion beam milling38−42 and other manufacturing processes.43 Superior shell-to-shell uniformity with a well-ordered feature is established.40,42 Spacer lithography can construct uniformly patterned nanoshell arrays with sub-10 nm thicknesses.40,44 3 Results and Discussion The EM wave coupling is mediated by diffraction in the plane of the ASDC nanorod arrays, which are composed of Ag-shell nanorods and dielectric-core nanorods in a single structure. Upon the illumination of the ASDC nanorod arrays with UV–visible–infrared light, the hybrid modes are excited and exhibited as sharp spectral peaks/dips in the optical absorptance/reflectance. These peaks/dips are associated with a manifestation of light trapping in the ASDC nanorod array system. The absorptance/reflectance spectra ascribe to the lattice resonance and the coupling from the nanochannel waveguide to the surface plasmon polariton (SPP) mode. Figure 2a,b shows the absorptance/reflectance spectra of the coupled ASDC nanorods with different dielectric core, that is, air and silica, where air and silica are the testing core materials for the purpose of comparison and they can be replaced by other materials. The top end of our proposed ASDC nanorod with air or silica core is opened to the surrounding ambience and it can be realized by using the fabrication processes as described in detail in ref (40). The solid Ag nanorods, as counterpart for a solid or nonshell case, are also investigated for the purpose of comparison. Because of the symmetry of the proposed structures, polarization-insensitive absorptance/reflectance can be easily realized. Figure 2 (a) Absorptance and (b) reflectance spectra of the coupled ASDC nanorods with different dielectric core (air and silica). The results include the data obtained for solid Ag nanorods which serves as a counterpart (solid case) for the purpose of comparison. As displayed in Figure 2a,b, there is one peak/dip with the maximum absorptance and minimum reflectance of 60.524 and 39.463% at λres of 955 nm for the solid case, and there are two peaks/dips with the maximum absorptance and minimum reflectance of 93.656 and 6.323% at λres of 855 nm for ASDC with air core and 98.489 and 1.496% at λres of 910 nm for ASDC with silica core for peak/dip 1, respectively. For cases involving ASDC nanorods, the absorptance/reflectance curves show two significant peaks/dips with extremely high/small values. They represent two distinct types of resonances, that is, the two narrow peaks/dips are result from the surface lattice resonance and gap and cavity plasmonic resonance, respectively.12 The number of absorptance/reflectance peak/dip is dependent on the resonant modes in the PNS, that is, only the SPR mode occurs in solid case, and for cases involving ASDC nanorods both the SPR and CPR modes occur simultaneously. It is noteworthy to point out that the corresponding peaks/dips of the absorptance/reflectance spectra have the same λres. As the dielectric core in the ASDC cases is changed from air to silica, the λres is red-shifted, which is in accord with previous literature studies.45 These resonances peaks and dips are attributed to (1) the vertical GPR mode among incident EM wave, Ag/ASDC nanorod arrays and the bottom Ag thin film (i.e., 100 nm thickness of Ag film on the silica substrate) and (2) the transverse CPR mode between the incident EM wave and the dielectric cores in the Ag shells. The vertical Fabry–Pérot cavities of the ASDC nanorods waveguide are formed by the dielectric cores, and the air gaps between metal nanorods behave as the dielectric interfaces.3 With the help of the Ag film of the ASDC nanorods and the bottom Ag thin film, the vertical GPR and transverse CPR modes can be well-excited. As is well-known, the λres of PNSs is dependent on the changing RI of the surrounding dielectric medium, a characteristic that has been widely used for sensing applications. The case for ASDC with ε = 1.00 (air) can be regarded as an SPR sensor, where the change of ε in the cavity of the ASDC nanorods (e.g., ASDC nanorods with silica core) causes a spectral shift and a large near-field intensity variation. To better understand the above-mentioned phenomena, we calculate the electric field intensity (\|E\|, V/m, Figure 3a), magnetic field intensity (\|H\|, A/m, Figure 3b), absorbed power density (Qe, W/m2, Figure 3c,d), and the surface charge density distributions (coulomb/m2, Figure 3e) at the corresponding λres extracted from peak 1 for the solid case and peak 1 and peak 2 for the ASDC case with silica core. From Figure 3, it is evident that the distribution profiles of each resonance have good correspondence with each PNS structure. It is obvious that the distribution profiles of \|E\|, \|H\|, and Qe are strongly confined in the gap region (i.e., gap enhancement) at peak 1 and peak 2 for all the cases, while only the case of ASDCs with silica core at peak 1 shows an enhanced distribution profile of \|E\|, \|H\|, and Qe around their outer sides (i.e., edge enhancement). The gap enhancement profiles indicate that the SPPs (i.e., GPR modes) were stimulated by the incident EM waves coupled with the Ag/ASDC nanorods and the bottom Ag thin film.46,47 The localized distribution profiles of \|E\|, \|H\|, and Qe around the Ag MNPs, show that Peak 1 was induced by the constructive interference, and this has enhanced the Ag MNP absorptance.48,49 The distribution profiles of \|E\|, \|H\|, and Qe for the case of ASDC with silica core at peak 2 is localized between the Ag MNPs and the Ag film, which indicates a stronger gap plasmon mode occurring between the gap of Ag MNPs and also between the Ag MNPs and bottom Ag film. They govern the absorptance and reflectance,33,46 hence resulting in stronger gap enhancement than that of the other cases. Figure 3 (a) Electric field intensity (\|E\|, V/m), (b) magnetic field intensity (\|H\|, A/m), absorbed power density (Qe, W/m2) in (c) x–y sectional plane and (d) x–z sectional plane, and (e) surface charge density (Coulomb/m2) distributions at the corresponding λres extracted from peak 1 and peak 2 of the solid case and the ASDC case with silica core. The mechanism of these distributions profiles in Figure 3a–d can be explained by the surface charge density distributions, as shown in Figure 3e. The surface current on the metal surface could be enhanced by the positive–negative charge pairs and they induced the electromotive force. The surface charge pairs of the solid case at peak 1 (λres = 955 nm) shows the same sign with an aggregation of (+ +) and (− −) charges at the opposite sides of the Ag nanorods, and there also is a weaker distribution of (+ +) (− −) on the surface of the bottom Ag thin film. These surface charges exhibit a typical dipole-like charge pattern, whose resonance is governed by the GPR mode. For the ASDC with silica core at peak 1 (λres = 910 nm), the charge pairs distribute strongly and uniformly in the form of (+ −) (+ −) on the rims and on the surface of the Ag-shell nanorods, and (− −) (+ +) distribution is observed on the surface of the bottom Ag thin film. There is also a strong dipole-like charge pattern on the surface of inner/outer rims and the bottom, which is governed by the vertical GPR mode. This gives rise to a stronger dipolar effect and enhances the field pattern around the gap and edge regions. Besides the GPR mode occurring on the MNP-dielectric interface, the SPP waves are also included in the light–electron interactions which happens between the incident EM waves and the dielectric cores (or cavities) region. This produces a strong coupling between the incident light and the electrons on the inner and outer Ag-shell walls, hence bringing about the transverse CPR mode in the nanocavities.48,50 A straightforward qualitative understanding to this is that the inner electric field in the ASDC is screened by the inner Ag-shell wall itself while the electric field skin effect makes its coupling to the outer Ag-shell wall dominant. As for the ASDC with silica core at peak 2 (λres = 1152 nm), the charge pairs distribute in the form of (− −) (+ +) at the opposite sides of Ag-shell nanorods and (+ +) (− −) on the surface of the bottom Ag thin film. In this case, the distribution of the GPR mode is larger than that of the CPR mode and this is due to the surface charge density in the gap region being denser than that of the lateral sides. This can be verified by the distribution profiles of \|E\|, \|H\|, and Qe, which show stronger field patterns in the gap region than those of the cases obtained from peak 1. This implies that the eigenmodes of peak 1 originate mainly from hybrid plasmon mode of the neighboring Ag-shell wall, which is caused by their strong mutual inductance and capacitive coupling but not from the individual ring resonator.16 Its near perfect absorption and approximately zero reflectance utilizes the Ohmic loss in the metal of the ASDC case. The resonance on the surface and in the cavity of the ASDC can be tuned by changing the geometric parameters of the structure, and this will be discussed latter. These lattice resonances can be tailored over a wide spectral range by changing the array lattice constant (i.e., period).11 The lattice constant, a, indicates the density of the ASDC nanorods in the period arrays along x- and y-axis, and it has a significant influence on the absorptance/reflectance spectra. To investigate the influence of the lattice constant, a, the absorptance/reflectance spectra for the case of ASDC (with silica core) nanorod arrays with a values in the range of [360, 370, 380, 400, 470, 500] nm were examined, see Figure 4a,b. As can be seen that the peak 1 has a noticeable blue shift with a stable magnitude of absorptance/reflectance as a is set in the range of [360, 370, 380, 400, and 470] nm, whereas the absorptance/reflectance have a slight blue shift and a decreasing (increasing) magnitude of absorptance (reflectance) as a is at 500 nm, indicating that the stronger coupling effect occurred as a in the range of [360, 370, 380, 400, and 470] nm. Note that the peak 2 and dip 2 of all cases possess the resonance wavelength around λres = 1150 nm. Figure 4 (a) Absorptance and (b) reflectance spectra for the case of ASDC (with silica core) nanorod arrays with different lattice constant, a = [360, 370, 380, 400, 470, and 500] nm. The other parameters are the same as the ASDC (with silica core) used in Figure 2. The absorptance and reflectance spectra can be also tuned by t, h, R, and r, respectively. To better understand both the characteristics of the GPR and CPR modes, the effects of the thickness (t = R – r) of the Ag-shell nanorods, outer radius (R), inner radius (r), and the height (h) of the ASDC nanorods on the absorptance/reflectance spectra are examined. The results of varying shell-thickness t in ASDC nanorods are shown in Figure 5a,b, respectively. The interaction between incident EM wave and ASDC nanorods could result in the splitting of SPR modes that are hybridized from an outer Ag-shell surface GPR mode and an inner Ag-shell surface CPR mode. The behavior of absorptance/reflectance spectra ascribed by varying Ag-shell thicknesses originates from capacitive coupling of the induced surface charges at the side wall of the ASDC nanorod gaps. As the thickness of the ASDCs increases from 8 to 13 nm, the λres is blue-shifted, which is consistent with previous studies.51 As the outer dimensions of ASDC nanorods remain intact, the λres is sensitive to the thickness of the Ag nanoshell (i.e., t = R – r), which blue-shifts from 940 to 820 nm for the case of peak/dip 1 and from 1220 to 1050 nm for the case of peak/dip 2, as the thickness is increased from 8 to 13 nm. The stronger CPR can be obtained from increasing the transverse cavity resonance using smaller t (e.g., t = 8, 9 and 10 nm) for peak/dip 1 while increasing the transverse cavity resonance using larger t (e.g., t = 11, 12 and 13 nm) for peak/dip 2. The λres is blue-shifted with decreasing cavity size in ASDC nanorods. This implies that, by adopting with a proper size of Ag shell-thickness, one can carve out a cavity region to generate a contour PPA with tailored absorptance/reflectance spectra at the desired λres. The key lies in the combination of the PNS with the photonic cavity in ASDC nanorods. The cavity in ASDC nanorods dramatically influences the resonance performance in PPA. Figure 5 (a) Absorptance and (b) reflectance spectra for the case of ASDC (with silica core) nanorod arrays with different shell-thickness of t = [8, 9, 10, 11, 12, and 13] nm. The other parameters are the same as the ASDC (with silica core) used in Figure 2. The absorptance/reflectance spectra for the case of ASDC (with silica core) nanorod arrays with varying outer radius (R), inner radius (r), and height (h) are investigated as shown in Figures 6 and 7, respectively. As it is shown in Figures 6 and 7, different cavity dimensions along transverse and vertical directions are demonstrated to be induced by different plasmonic modes that could lead to different responses required for plasmonic sensors. Being cavity in ASDC nanorods, the cavity channel can provide inner resonant modes with a localized electric field confinement far below the Abbe diffraction limit.40 The λres red-shifts with the increasing R, r (i.e., increasing the transverse cavity volume), and h (i.e., increasing the vertical cavity volume). The varying R, r, and h would yield a change of the cavity volume in ASDC and result in the change of the surface charge density on the Ag-shell surface, which is related to the number of positive–negative charge pairs, that is, varying electron density distributed on the inner and outer Ag-shell of the ASDC,51 thus forming a strong-coupled mode which favors the near perfect absorption with proper cavity volume in the ASDC nanorods. In particular, the strong-coupled modes induce efficient broad band absorptance and reflectance, whose spectral width and position can be manipulated by changing ASDC nanorods radius (R–r) and height (h). This suggests that the CPR with respect to the cavity volume of ASDC nanorods can be affected by the coupling from R, r, and h. As the Ag-shell thickness of ASDC nanorods remains intact, the λres red-shifts from 820 to 1020 nm with the increasing R(r) in the range of [60(50), 70(60), 80(70), 90(80), and 100(90)] (Figure 6a,b) and from 850 to 1000 nm with the increasing h in the range of [ 80, 90, 100, 110, 120, and 130] (Figure 7a,b) for peak/dip 1 cases. It is worth mentioning that the performance of absorptance/reflectance spectra are significantly affected by the size of R(r) for the peak/dip 1 cases and by the size of h for peak/dip 2 cases. In Figure 7a,b, the trend of absorptance/reflectance spectra is quite different between peak/dip 1 and 2. There is a maximum/minimum at h = 110 nm for the peak/dip 1, whereas the peak/dip 2 is monotone growing/declining with the increasing h. The discrepancies in the observed trends are attributable to the difference of coupling effect arising from the vertical CPR corresponding to the incident wavelength of EM wave and the length (i.e., h) of nanocavity channels in ASDC nanorods. Here, the band linewidth of the coupled photonic–plasmonic resonance is associated with the ASDC with cavity, which is linked to the Q-factor because of the modified photonic density of states and hence the modified radiative damping rate. From the Figures 4–7, we observe that the more the coupling effect on the SPRs and CPRs, the more absorptance and less reflectance of the ASDC PPA exhibits. This implies that the resonance arising from the ASDC PPA can be easily tuned by adjusting its geometrical parameters. Figure 6 (a) Absorptance and (b) reflectance spectra for the case of ASDC (with silica core) nanorod arrays with the outer radius of R = [60, 70, 80, 90, and 100] nm and the inner radius of r = [50, 60, 70, 80, and 90] nm. The thickness of ASDC is kept at t = 10 nm. The other parameters are the same as the ASDC (with silica core) used in Figure 2. Figure 7 (a) Absorptance and (b) reflectance spectra for the case of ASDC (with silica core) nanorod arrays with different height of h = [80, 90, 100, 110, 120, and 130] nm. The other parameters are the same as the ASDC (with silica core) used in Figure 2. For the nanoscale sensor applications, the ambient medium is referred to as the variation of RI. The testing sample surrounds the ASDC nanorods, and the testing medium is usually in liquid or gaseous states.48,49 Calculating the absorptance and reflectance spectra for the proposed structure, we investigated the ASDC (with silica core) nanorod arrays, to the environmental RI perturbations. The sensitivity of the sensor is dependent on the variation of the environmental RI surrounding the PNSs. To verify the sensitivity of the proposed ASDC (with silica core) nanorod array, the ambient RI of air (n = 1.00), water (n = 1.33), and phosphate buffer saline52 are used, and the corresponding absorptance/reflectance spectra are depicted in Figure 8a,b. A noticeable red shift in the position of the λres from 910 to 1160 nm is observed with the increasing RI of the surrounding ambience. These features in the absorptance/reflectance spectra indicate the superior sensing properties. The sensitivity of RI sensors depends critically on the local electric field intensity around the ASDC nanorods and the overlap of hot spots with the RI of the surrounding ambience. This is shown in Figure 8a,b, where the absorptance/reflectance spectra of the metasurface varies in response to the environmental perturbations. In Figure 8a,b, the two groups of resonant peaks and dips are both highly sensitive, although the different plasmonic modes produce the different λres responses. The physical inference for these red shifts is due to the effective increase in capacitance of the resonant structure attributed to the increase in the RI of the analyte.10 The trend of peak (dip) of the absorptance (reflectance) spectra shows a decreasing (increasing) with an increasing RI of the surrounding ambience. This is caused by the lesser SPR and CPR effects when higher RI of the surrounding medium is introduced into the PPA system. According to the absorptance and reflectance spectra at peak/dip 1, the calculated sensitivity, FOM, and Q factor can be achieved to the values of 757.58 nm/RIU, 50.51 (RIU–1), and 60.67, respectively. Noting that the near perfect absorptance and near zero reflectance resonant with a sharp band linewidth narrower corresponding to their SPR and CPR modes in Figure 8a,b are upright to each other and well-separated in wavelengths and in spatial distributions. Indeed, we find that the absorbing bands of peak 1 in Figure 8a displays good Lorentzian line shapes, and they are well-matched to the couple mode theory.10 Figure 8 (a) Absorptance and (b) reflectance spectra of ASDC (silica core) nanorod arrays with the surrounding RI of air (n = 1.00), water (n = 1.33) and phosphate buffer saline. A comparison of the (c) electric field intensity distributions (at cross-section across the ASDC nanorods center at z = 0 nm and y = 90 × 10–9 nm, respectively) and (d) surface charge density (including the 3-D profiles of electric force lines (pink lines) and energy flows (cyan arrows) of the proposed ASDC (silica core) nanorod arrays exposed to air (n = 1.00) and a surrounding medium water (n = 1.33). The other parameters are the same as the ASDC (with silica core) used in Figure 2. In an infrared absorptance (reflectance) spectra, the peaks (dips) correspond to the molecular groups which absorb (reflect) the infrared light at specific wavelengths, that is, it regulates the dipole moment by all (or some) number of its vibration normal coordinates, and it will surely result in some considerable infrared absorption bands. Therefore, all sensors have to be examined with respect to their sensitivity on marginal variations in the surrounding medium. The next sets of data are derived from the testing of the responsiveness of the proposed ASDC nanorod array under marginal conditions of the test sample (surrounding medium of the ASDC nanorod array). Figure 8c,d shows a comparison of the electric field intensity distributions (at the cross section across the ASDC nanorods center at z = 0 nm and y = 90 × 10–9 nm, respectively) and the surface charge density (including the 3-D profiles of electric force lines (pink lines) and the energy flows (cyan arrows) of the proposed ASDC (with silica core) nanorod arrays exposed to air (n = 1.00) and a surrounding medium of RI, n = 1.33 (water). After the ASDC nanorods adsorbed by the surrounding RI medium, a remarkable gap enhancement and localization of electric field intensity distributions can be found along the x axis because of the x-polarization of the incident EM wave. The electric force lines and energy flow arrows of n = 1.33 (Figure 8d) exhibit an irregular profile compared to that of n = 1.00 (Figure 8c), when the environmental RI perturbation influences the ASDC nanorods system. The proposed ASDC structure has a larger overspread capacity of the hot spots and the ambient media, which is approachable to the variation of surrounding medium, and would be applied not only for very sensitive RI sensing but also for improving most monolayer sensitivity, making it a greatly attractive PPA structure. More potentially, the localized electric field enhancement of the lattice resonance mode combined with the SPR and CPR modes is concentrated on the ASDC nanorod surface thus easily attainable for the measuring target biomolecules in the near infrared region. 4 Conclusions We have proposed a novel approach to the design of PPA which could support both tunability and sensitivity for highly sensitive RI sensor application. The influence of structure and material parameters, the SPRs and CPRs on the sensing performance have been investigated by 3-D FEM. The mechanisms of absorptance and reflectance spectra have been demonstrated to be induced by the different plasmonic modes generated on the periodic ASDC nanorods grating. In our design, the PPA is tuned by changing or modifying the device material and geometry. The proposed ASDC PNSs with a Fabry–Pérot nanocavity is shown to provide a means for reducing the band linewidth of the resonances, and therefore ameliorating the sensing properties of PNSs. The optical spectrum can be changed by varying the dielectric core of the ASDC, and a near perfect absorptance and an approximate zero reflectance having a sharp band linewidth can be obtained from the proposed system. The proposed ASDC PNS can be operated as SPR sensor with the sensitivity and FOM of 757.58 nm/RIU and 50.51 (RIU–1), respectively. In addition, the open cavity of our proposed ASDC nanorod system is accessible to the surrounding medium, and this makes it attractive for RI sensor and filtering applications. The authors declare no competing financial interest. Acknowledgments This work was supported by the University Research Grant of Universiti Brunei Darussalam (grant no. UBD/OVACRI/CRGWG (004) /170101) and Ministry of Science and Technology of Taiwan (MOST 106-2112-M-019-005-MY3, and 105-2221-E-492-036). ==== Refs References Landy N. I. ; Sajuyigbe S. ; Mock J. J. ; Smith D. R. ; Padilla W. J. Perfect metamaterial absorber . Phys. Rev. Lett. 2008 , 100 , 207402 10.1103/physrevlett.100.207402 .18518577 Kwon M.-S. ; Ku B. ; Kim Y. Plasmofluidic Disk Resonators . Sci. Rep. 2016 , 6 , 23149 10.1038/srep23149 .26979929 Yong Z. ; Zhang S. ; Gong C. ; He S. Narrow band perfect absorber for maximum localized magnetic and electric field enhancement and sensing applications . Sci. Rep. 2016 , 6 , 24063 10.1038/srep24063 .27046540 Lu X. ; Zhang L. ; Zhang T. Nanoslit-microcavity-based narrow band absorber for sensing applications . Opt. 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PMC006xxxxxx/PMC6644440.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145807510.1021/acsomega.8b02283ArticleEvaluating the Creaming of an Emulsion via Mass Spectrometry and UV–Vis Spectrophotometry Shinoda Ryo Uchimura Tomohiro Department of Materials Science and Engineering, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan E-mail: uchimura@u-fukui.ac.jp. Phone/Fax: +81-776-27-8610.22 10 2018 31 10 2018 3 10 13752 13756 05 09 2018 11 10 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. The creaming behavior of a turbid oil-in-water emulsion was observed via the processes of multiphoton ionization time-of-flight mass spectrometry (MPI-TOFMS) and ultraviolet–visible spectrophotometry (UV–vis), and the results were compared. The transmittance measurement by UV–vis showed that the turbidity of the toluene emulsion was decreased with time. However, non-negligible errors are common in the measurement of a sample with high turbidity. The online measurement by MPI-TOFMS detected many spikes in the time profile, which revealed the existence of toluene droplets in the emulsion. A smooth time profile suggested that the signal intensity had initially increased, and then decreased with time; the initial concentration of toluene was 3 g/L, which had decreased by half after 60 min. The signal behavior obtained using MPI-TOFMS differed only slightly from that obtained using UV–vis. Since a change in turbidity is not the same as a change in the local concentration of an oil component, MPI-TOFMS is useful for the analysis of a turbid emulsion and offers additional information concerning the creaming phenomenon of an emulsion. document-id-old-9ao8b02283document-id-new-14ao-2018-02283zccc-price ==== Body Introduction An emulsion is a system in which one liquid is dispersed with another and both are immiscible. Emulsions are applied in a wide variety of fields; many types of emulsion products are commercialized such as inks, paints, foods, cosmetics, and pesticide spraying agents. An emulsion has several specific characteristics that differentiate it from a so-called normal solution. For example, differences in the preparation conditions affect the properties even if the concentrations of the constituents are the same. Moreover, several collapse processes exist in an emulsion such as creaming, aggregation, coalescence, and Ostwald ripening. These processes are sensitive to the constituents and their concentrations, as well as to the preparation conditions. Among the collapse processes, creaming is a phenomenon wherein the emulsion droplets are moved to the upper or lower portions according to the differences in density between the dispersed phase and the continuous phase. The rate of creaming is affected by the droplet size, the viscosity of the dispersed phase, and so on. In any case, the condition of an emulsion changes with time. Also, collapse processes change the qualities of an emulsion product. Recent analytical studies of emulsions include evaluations of collapse processes,1,2 analysis of the interface of droplets,3−5 and analysis of emulsion products.6 Turbidity measured by ultraviolet–visible spectrophotometry (UV–vis) has normally been used for the evaluation of emulsion stability.7−10 For instance, the time profile of turbidity was studied to evaluate the coalescence and/or solubilization kinetics of oil in microemulsion droplets11,12 and for determining the phase-inversion temperature.13,14 However, when an incident light insufficiently passes through an emulsion with rather high turbidity, it is difficult to obtain reliable data. In the case of an oil-in-water (O/W) emulsion, a higher concentration of an oil constituent generally causes higher turbidity, although several other factors can also affect the turbidity. In a normal solution with higher absorbance due to a higher concentration, the solution should be normally diluted when measuring transmittance and absorbance. However, in the case of an emulsion, the size of droplets and the emulsion properties are changed by dilution. As a result, the original behavior of a collapse process cannot be evaluated. Multiphoton ionization time-of-flight mass spectrometry (MPI-TOFMS) has several notable and practical characteristics. The ionization method uses ultraviolet laser pulses with high optical selectivity and less contamination, although soot is often produced by electron ionization sources. Moreover, TOFMS can detect all of the induced ions in principle, and is a robust and reasonably easy way to accomplish design and handling.15−18 MPI-TOFMS is normally applied to atomic/molecular spectroscopy19−21 and to the trace analysis of compounds in real samples such as in an environmental setting.22−27 Although MPI is generally an ionization method for analytes in a gas phase, several vaporization methods for liquid samples have also been studied.28−30 Recently, we applied MPI-TOFMS to the online analysis of an emulsion.31−37 We developed a sample introduction technique for an emulsion, and the online mass analysis of an oil phase in an O/W emulsion without pretreatment was achieved.31 We reported the time profile of a peak area of an analyte, which was constructed by plotting the peak areas of the corresponding ions in a series of mass spectra. Spikes often appear in time profiles, particularly when measuring an emulsion with a white turbidity. Such spikes derive from oil droplets, and, in the case of a toluene O/W emulsion, we calculated the minimum diameter of a toluene droplet that could be detected as a spike.35,36 We also reported an online analysis of multiple components in an O/W emulsion34 and that of a multiple emulsion.33 We recently used a styrene O/W emulsion to achieve a quantitative analysis of the oil component in an O/W emulsion, and obtained a linear calibration curve of styrene in a concentration of as much as 5 g/L.37 Thus far, the possibility of using MPI-TOFMS to evaluate the creaming of an emulsion has only been suggested.31 In the present study, the time profiles of an O/W emulsion exhibiting a creaming phenomenon were measured by UV–vis and MPI-TOFMS, and the monitoring of an emulsion in a cuvette via these two types of measurement was aligned. Comparable results established the utility of MPI-TOFMS for the evaluation of creaming. Experimental Section Materials and Sample Preparation Toluene and sodium dodecyl sulfate (SDS), as an oil phase and an emulsifier, respectively, were purchased from WAKO Pure Chemical Industries (Osaka, Japan) and were used without further purification. Purified water was prepared in our laboratory. For the sample preparation, first, water (20 mL) and SDS were added to a vial that was shaken manually until the solid SDS was dissolved. Then, toluene was added and stirred with a homogenizer (AHG-160D, AS ONE, Osaka) at a speed of 5000 rpm for 10 min. The concentrations of both toluene and SDS were 3 g/L with respect to the water. After stirring, 3.5 mL of the prepared sample was transferred to a 5 mL cuvette. UV–Vis To establish the degree of turbidity, UV–vis transmittance was measured with a spectrophotometer (Shimadzu, UV-160, Japan). To suppress the vaporization of toluene, a cuvette with a lid was placed on the holder of the spectrophotometer. As will be described later, a local region of the sample in a cuvette was monitored by MPI-TOFMS. Therefore, when measuring turbidity, a horizontal slit (2 mm in width) was placed in the front of the cuvette, as shown in Figure 1a, in a region where the transmittance measurement was limited in terms of height. As a result, the incident light passed through the region 1.8–2.0 cm from the bottom of the cuvette. Figure 1 Experimental setup. (a) Horizontal slit (2 mm in width) was placed in the front of a cuvette when using UV–vis. The incident light was passed through a region 1.8–2.0 cm from the bottom of the cuvette. (b) Emulsion in a cuvette with a lid that had a small hole (φ 1 mm) was monitored by MPI-TOFMS. The tip of the capillary column was set at the same height as that of the transmittance measurement, i.e., ca. 2 cm from the bottom of the cuvette. The wavelengths of the incident light were set to 600 and 266 nm. While each constituent in an emulsion had no absorption in terms of its former wavelength, the absorption for toluene was in the latter wavelength, which approximated the wavelength of the laser pulse for MPI. In the present study, the turbidity was expressed by −log I/I0, where I and I0 were the incident light intensity and the transmitted light intensity, respectively. MPI-TOFMS MPI-TOFMS used in the present study is described in detail elsewhere,31 and only briefly discussed here. The sample introduction port was composed of a pair of concentric capillary columns. An emulsion was placed in a cuvette with a lid that had a small hole (1 mm in diameter) to suppress the vaporization of toluene as much as possible. An inner capillary column was inserted through the hole to introduce an emulsion. The tip of the capillary column was set at the same height as the region of the transmittance measurement described previously, i.e., ca. 2 cm from the bottom of the cuvette. Ambient air flowed through an outer capillary column, and the flow rate was adjusted to 2 mL/min via a flow meter (RK-1250, Kofloc, Kyoto, Japan). The pressure of the vacuum chamber was ca. 1 × 10–2 Pa. The linear-type TOFMS used in the present study was developed at Kyushu University, Japan. The fourth-harmonic of a Nd:YAG laser (GAIA II, 266 nm, 4 ns, 10 Hz, Rayture Systems, Tokyo, Japan) was used for ionization. The laser pulse, which was adjusted to ca. 20 μJ, was focused with a plano-convex lens (f = 200 mm). The ion signals were acquired without averaging every 0.1 s via a 1 GHz digitizer (AP240, 1 GS/s, Acqiris/Agilent Technologies, Tokyo, Japan). The mass resolution was typically 220 at m/z 92 when measuring a toluene O/W emulsion with a white turbidity (see Figure S1 in the Supporting Information). The time profile for toluene shown in Figure 4a was constructed by plotting the sum of the peak areas of m/z 92 (a molecular ion) and m/z 91 (a fragment ion) on a series of mass spectra. Figure 4b shows the time profile for the average of every 1200 plots, which corresponded to every 120 s, applied in the three experiments including that in Figure 4a. It should be noted that the flow of ambient air did not influence the time profile of the peak area of toluene, as shown in Figure 4, but was used simply to stably introduce an emulsion into TOFMS. Results and Discussion Features of an Emulsion The features of the emulsion prepared in a cuvette with time are shown in Figure 2. Just after the preparation, the emulsion developed a white turbidity, the lower portion of which turned slightly transparent with time, while the turbidity in the middle portion, monitored using UV–vis and MPI-TOFMS in the present study, seemed unchanged. Figure 2 Photograph of an O/W emulsion with time. Time Profile of Turbidity by UV–Vis Figure 3 shows the results of transmittance measurement obtained by UV–vis where the wavelength of the incident light was 600 nm. This indicates the turbidity of an emulsion because the three constituents of the present emulsion, i.e., toluene, water, and SDS, each had no absorption. In this study, the turbidity of the first 5 min was seldom unchanged or was slightly decreased, and was then clearly decreased with time at the height of the monitoring position, although the change in turbidity could not be confirmed by the naked eye (Figure 2). In this manner, the UV–vis time profile measurement of turbidity was useful for evaluating the creaming in an emulsion. However, as shown in Figure 3, the value of turbidity (−log I/I0) was more than 1.0 during the first 35 min. That is, the transmittance was less than 10%, which means that the incident light was seldom transmitted through the emulsion. In conventional UV–vis spectroscopy, such an experimental condition often results in an increase in the incidence of errors. That is, non-negligible errors easily occur in the measurement of a sample with turbidity such as the one in this study. Figure 3 Time profile of the turbidity of an O/W emulsion measured by UV–vis at 600 nm. The error indicates the standard deviation (n = 3). Incidentally, the value of −log I/I0 obtained at 266 nm was saturated at 2.1 throughout the measurement time because, in addition to the presence of turbidity, toluene has absorption at this wavelength. As a result, the creaming of the present emulsion could not be evaluated using the transmittance at this wavelength. Therefore, it is quite difficult to use UV–vis to explain the relationship between the local concentration of a constituent of a dispersed phase and the creaming phenomenon. The proper absorbance might have been obtained by adding water to dilute toluene and also to reduce the turbidity, but the properties of the resultant diluted emulsion, including any creaming behavior, would surely be changed. A shortening of the light path is one possible experimental condition that could enhance transmittance, which could allow an estimation of the concentration. However, the use of a thinner cuvette would be impractical and would likely influence the creaming phenomenon of the emulsion because situations such as convection differ between a thin cuvette and the rather large storage containers that are used in practical situations. Time Profile of the Concentration of the Oil Phase by MPI-TOFMS A time profile of the peak area of toluene obtained by online MPI-TOFMS is shown in Figure 4. Figure 4a shows the results of a time profile without averaging. In this measurement, the recording began at the same time that the capillary column was inserted into an emulsion. In Figure 4a, although the signal for toluene was very small, it was surely found after ca. 100–113 s in three measurements and was then adopted as the time required for sample introduction. After that, many spikes appeared on the time profile, which decreased both in number and intensity with time. The appearance of spikes suggested the existence of highly concentrated toluene, i.e., toluene droplets.31,35 In the present study, the number and size of the toluene droplets seemed to decrease at the monitoring position in the cuvette with the occurrence of the creaming of an emulsion. Figure 4 Time profile of the peak area of toluene in an O/W emulsion measured by MPI-TOFMS. (a) Time profile recorded without averaging (by every 0.1 s). (b) Time profile of an average of every 1200 plots, which corresponds to every 120 s, was applied in three experiments including that in (a). The first plot (corresponding to the average of the first 120 s) was ignored because a time from 0 to ca. 120 s was required to pass the sample through the capillary column (see the text). The intensity of the second plot was set to 100%, and the right-hand scale indicates the concentration of toluene where an intensity of 100% is compatible with 3 g/L of toluene (see the text). The error indicates the standard deviation (n = 3). Next, an average of every 1200 plots, which corresponded to every 120 s, was re-plotted. The time profile is shown in Figure 4b. The first plot (corresponding to the average value of the first 120 s) was ignored (masked) because a time that ranged from 0 to ca. 120 s was required for the sample to pass through the capillary column. In addition, the average intensity of the second plot was set to 100% in Figure 4b. Previously, we reported the quantitative analysis of an O/W emulsion wherein a linear calibration curve of the oil phase of styrene in an emulsion with up to 5 g/L was obtained.37 Although toluene was used in the present study, the results in Figure 4b suggest a change in the concentration of toluene at the sampling position. The concentration of toluene at the beginning of the measurement was 3 g/L. In this figure, the right-hand scale indicates the concentration of toluene where a signal intensity of 100% is compatible with 3 g/L of toluene. Interestingly, the concentration increased ca. 25% during the first few minutes, which could have been due to the introduction of a large number of, and/or a large size of, toluene droplets. After that, the concentration of toluene decreased at a rate of ca. 0.2 g/(L min) from 8 to 16 min. Finally, the signal intensity was decreased by half compared with the initial (at 2 min) signal intensity, which amounted to a decrease of ca. 1.5 g/L after 60 min. These toluene concentrations were still rather high and not suitable for the measurement of absorbance by UV–vis at 266 nm, and, of course, the decrease in transmittance that was caused by turbidity should also be taken into account. In this manner, the creaming behavior can be quantitatively evaluated using MPI-TOFMS, which can demonstrate the advantage of the present method. The changes in the signal intensities obtained by UV–vis (Figure 3) and MPI-TOFMS (Figure 4b) differed slightly. That is, the turbidity was either almost constant or slightly decreased for the first few minutes when using UV–vis, while the concentration was somewhat increased when using MPI-TOFMS. Although further studies are needed, one conceivable reason is as follows. The present emulsion had a white turbidity and was polydispersed, and at the monitoring point in the middle part of the cuvette, relatively large oil droplets, which were moved from a lower position, were first detected by MPI-TOFMS. Therefore, the local concentration in the middle portion was increased first. This concentration change, however, only scarcely contributed to the turbidity. In addition, assuming that the latter part of the results (from 14 min or later) of Figures 3 and 4b were a single exponential decay, the decay time constants were calculated to be 34 and 10 min, respectively. In this manner, the differences in behavior between the turbidity and the concentration could be detected. MPI-TOFMS directly provides information concerning the concentration of an oil component in an emulsion. This method can also be used to monitor the multiple constituents in an emulsion.33,34 Therefore, the influence of creaming on each constituent would be evaluated. We are now studying the formulation for the signal behavior obtained by MPI-TOFMS for a quantitative evaluation of the creaming of an emulsion. Conclusions In the present study, an O/W emulsion that showed creaming was measured via UV–vis and MPI-TOFMS. Transmittance measurement provided information concerning turbidity, but the measurement of emulsions with higher levels of turbidity is difficult in principle. On the other hand, with online mass analysis using MPI-TOFMS, the change in the concentration of an oil phase accompanied by creaming could be evaluated. The tendency of the change in the signal intensities obtained from both methods was slightly different, which suggested that the two methods provided different information, i.e., the turbidity and the local concentration of an oil phase. The information for a local concentration that was obtained by MPI-TOFMS would be difficult to obtain via conventional UV–vis, which makes MPI-TOFMS a useful tool for the evaluation of the collapse process of an emulsion. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02283.Mass spectrum of toluene of a toluene O/W emulsion (Figure S1) (PDF) Supplementary Material ao8b02283_si_001.pdf The authors declare no competing financial interest. Acknowledgments T.U. thanks the University of Fukui for the financial support. ==== Refs References Scheuble N. ; Iles A. ; Wootton R. C. R. ; Windhab E. J. ; Fischer P. ; Elvira K. S. Microfluidic technique for the simultaneous quantification of emulsion instabilities and lipid digestion kinetics . Anal. Chem. 2017 , 89 , 9116 –9123 . 10.1021/acs.analchem.7b01853 .28770989 Mitsunobu M. ; Kobayashi S. ; Takeyasu N. ; Kaneta T. 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PMC006xxxxxx/PMC6644441.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145811610.1021/acsomega.8b02298ArticleMicrowave-Assisted aza-Friedel–Crafts Arylation of N-Acyliminium Ions: Expedient Access to 4-Aryl 3,4-Dihydroquinazolinones Sawant Rajiv T. †Stevens Marc Y. ‡Odell Luke R. Department of Medicinal Chemistry, Uppsala Biomedical Center, Uppsala University, P.O. Box 574, SE-751 23 Uppsala, Sweden E-mail: luke.odell@ilk.uu.se.29 10 2018 31 10 2018 3 10 14258 14265 06 09 2018 12 10 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A one-pot microwave-assisted aza-Friedel–Crafts arylation of N-acyliminium ions, generated in situ from o-formyl carbamates and different amines, is reported. This metal-free protocol provides rapid access to diverse 4-aryl 3,4-dihydroquinazolinones in excellent yield without any aqueous workup. A solvent-directed process for the selective aza-Friedel–Crafts arylation of electron-rich aryl/heteroaryl/butenyl-tethered N-acyliminium ions is also described. document-id-old-9ao8b02298document-id-new-14ao-2018-02298xccc-price ==== Body Introduction N-Acyliminium ions1−5 are versatile electrophiles that provide direct access to α-substituted amino derivatives via the intra- or intermolecular addition of various nucleophiles. In particular, in situ-generated N-acyliminium ions have been widely exploited in the synthesis of bioactive nitrogen-containing heterocycles, especially in the preparation of alkaloid natural products.1,2,6,7 Accordingly, the development of rapid, convenient, and high-yielding protocols for the selective intra- or intermolecular nucleophilic addition to cyclic N-acyliminium ions remains a field of considerable interest.8−11 The C4-substituted quinazolinone framework is known to exhibit a wide range of biological properties. For example, SM-15811 is a potent Na+/Ca2+ exchanger inhibitor,12−14 proquazone is an anti-inflammatory drug,15,16 and 4-disubstituted 3,4-dihydroquinazolinones are T-type channel selective calcium blockers with in vivo central nervous system efficacy in epilepsy and tremor models17,18 (Figure 1). Finally, the 3,4-dihydroquinazolinones DPC 961 and DPC 083 and related analogs are potent human immunodeficiency virus non-nucleoside reverse transcriptase inhibitors.19,20 Figure 1 Structures of pharmaceutically important 4-aryl quinazolinones. The known methods for the synthesis of 4-aryl substituted 3,4-dihydroquinazolinones include a two-step condensation of aldehyde, urea, and carboxylic acid,21 and a three-step synthesis from o-amino acetophenones12−14,17,18 and the organocatalytic asymmetric synthesis of trifluoromethyl 3,4-dihydroquinazolinones based on the aza-Friedel–Crafts reaction of indoles with cyclic N-acylketimines using a chiral phosphoric acid catalyst.22 Xie and co-workers reported the enantioselective aza-Friedel–Crafts reaction of naphthols/phenols with cyclic N-acylketimines using a chiral quinine-squaramide catalyst.23 Despite these elegant approaches, the existing methods require either lengthy reaction sequences or isolation of cyclic N-acylketimines, which limits their utility in the generation of diverse 3,4-dihydroquinazolinone libraries. During the preparation of this manuscript, Chandrasekharam and co-workers reported a water-mediated multicomponent synthesis of 4-aryl substituted 3,4-dihydroquinazolinones under conventional heating.24 However, it is important to highlight that the use of water as a reaction medium demanded long reaction times, and, in many cases, chromatographic purification was required, both of which detract from its appeal as a green protocol. Moreover, the scope of this method is restricted to indole and mono-functional amine nucleophiles, limiting its utility in library generation. In this context, an environmentally benign and expedient method for the rapid synthesis of 4-aryl 3,4-dihydroquinazolinone libraries is highly desirable. As part of our ongoing research program, we recently reported a highly efficient solvent-directed diversity-oriented synthesis of skeletally diverse 3,4-dihydroquinazolinones scaffold libraries based on N-acyliminium ion chemistry under environmentally benign reaction conditions.25−29 Our recent findings demonstrated that the intramolecular cyclization of aryl/heteroaryl tethered nucleophiles with N-acyliminium ions27 leads to the formation of 3,4-dihydroquinazolinone-embedded polyheterocycles (Scheme 1). With the aim of developing an expedient approach to 4-aryl/heteroaryl 3,4-dihydroquinazolinones, we were encouraged to investigate the intermolecular functionalization of N-acyliminium ions (I) with indoles and arenes to produce 4-aryl 3,4-dihydroquinazolinone scaffold libraries based on a cascade imine/cyclization/aza-Friedel–Crafts reaction sequence. Herein, we present one-pot microwave-assisted metal-free sequential N-acyliminium ion/aza-Friedel–Crafts arylation that provides rapid access to 4-aryl/heteroaryl 3,4-dihydroquinazolinones from readily available precursors (Scheme 1). Scheme 1 Proposed Reaction Sequence for the aza-Friedel–Crafts Arylation of N-Acyliminium Ions Results and Discussion We started our investigation by optimization of the reaction conditions for the aza-Friedel–Crafts arylation of the N-acyliminium ion generated in situ from o-formyl carbamate 1a and NH4OAc 2a (Scheme 2). We were pleased to observe that the reaction proceeded in either AcOH or EtOH/AcOH as the solvent. The reaction between o-formyl carbamate 1a and NH4OAc 2a (2 equiv) in AcOH under microwave heating at 130 °C for 10 min provided N-acyliminium ion intermediate Ia (confirmed by liquid chromatography/mass spectrometry (LC/MS)), which upon subsequent treatment with indole 3a (1.3 equiv) and an additional 20 min of heating at 130 °C produced 4-indolyl 3,4-dihydroquinazolinone 4a in excellent yield (95%). Similarly, the two-step reaction sequence in EtOH/AcOH (9:1) afforded dihydroquinazolinone 4a in a slightly reduced yield (91%) under optimal protic solvent/Brønsted acid combinations (Scheme 2). Scheme 2 Optimization Studies for the aza-Friedel–Crafts Arylation of an N-Acyliminium Ion With the optimized reaction conditions in hand, we first explored the scope of the aza-Friedel–Crafts arylation of different cyclic N-acyliminium ions generated in situ from o-formyl carbamates 1b–1h and NH4OAc 2a with indole 3a in AcOH (Scheme 3). N-Acyliminium ions 1b–1h derived from aldehydes 1b–1h containing electron-donating/withdrawing and halogen substituents reacted smoothly with indole to afford the corresponding 4-indolyl 3,4-dihydroquinazolinones 4b–4f in good to excellent yields (86–92%). The introduction of an o-substituent and N-1-benzyl substituent was also well-tolerated, giving 3,4-dihydroquinazolinones 4g and 4h in good yield. Next, the scope of the indole nucleophile was explored and indole derivatives containing electron-donating/withdrawing, and halogen substituents furnished the corresponding 4-indolyl 3,4-dihydroquinazolinones 4i, 4j, and 4n in good to excellent yield (83–92%). Sterically hindered o-substituted indoles reacted smoothly, producing 3,4-dihydroquinazolinones 4k–4m in good to excellent yield. The protocol also worked well with N-methylindole and 7-azaindole to afford 3,4-dihydroquinazolinones 4o and 4p in 84 and 90% yield, respectively (Scheme 3). Scheme 3 Scope of aza-Friedel–Crafts Arylation of N-Acyliminium Ions with Indoles Isolated yield. All reactions were performed with 1 equiv o-formyl carbamate (1b–1h), 2 equiv NH4OAc (2a) in 1 mL AcOH, 130 °C, MW, 10 min, and then 1.3 equiv indole (3a–3i) 130 °C, MW, 20–30 min. Next, to further expand the scope and applicability of microwave-assisted aza-Friedel–Crafts arylation of N-acyliminium ions, we investigated the effect of varying the arene and amine components (Scheme 4). Our protocol tolerated a wide range of amine nucleophiles, affording the corresponding 3,4-dihydroquinazolinone in up to 95% yield. Primary alkyl amines such as benzylamine 2a and 2-thiophenemethylamine 2b worked well to afford N-3-functionalized 3,4-dihydroquinazolinones 6a, 6b in excellent yields (>94%). A one-step three-component reaction between aldehyde 1a, benzylamine 2b, and indole 3a afforded 6a in reduced yield (85%) confirming that the two-step sequential approach is preferable. N-Acyliminium ions also reacted smoothly with 1,3-dimethoxybenzene 5a to produce 4-aryl 3,4-dihydroquinazolinones 6c–6e in moderate to good yields. It is important to highlight that the branched amine N-methyl 4-amino piperidine 2d was efficiently transformed into 6e, an analog of SM-15811 in satisfactory yield. Finally, m-cresol 5b underwent chemoselective C-functionalization to produce 6f in 52% yield (Scheme 4). Scheme 4 Scope of aza-Friedel–Crafts Arylation of N-Acyliminium Ions with Arenes, Isolated yield. Unless otherwise stated, reactions were performed with 1 equiv o-formyl carbamate (1b–1h), 2 equiv NH4OAc (2a) and 1.3 equiv amine (2b–2d) in 1 mL AcOH, 130 °C, MW, 10 min, and then 1.3 equiv Nu (3a/5a/5b) 130 °C, MW, 20–30 min. One-step reaction in AcOH, MW, 130 °C, 20 min. Next, we sought to expand the scope of the selective cascade arylation reaction using challenging amine nucleophiles bearing pendant electron-rich aryl/alkenyl moieties (2e–2j, Scheme 5). Gratifyingly, electron-rich aryl/heteroaryl/butenyl-tethered N-acyliminium ions were generated in situ from o-formyl carbamate and amines 2e–2h in EtOH/AcOH (9:1) and reacted smoothly with indole 3a to afford N-3-aryl/heteroaryl/butenyl-tethered 4-aryl 3,4-dihydroquinazolinones 7a–7d in excellent yield (Scheme 5). However, with indole tethered N-acyliminium ions, intramolecular aza-Friedel–Crafts cyclization was more favored under the optimized reaction conditions. The N-acyliminium ion derived from 4-aminomethyl indole 2i gave 7e in 36% yield along with the polycyclic product 7e′, whereas the tryptamine derivative gave only traces of 7f (Scheme 5). It is important to highlight that by changing the solvent composition the reaction can be paused at the N-acyliminium ion stage, followed by selective functionalization at the C-4 position with an external indole nucleophile, despite the presence of a pendant electron-rich aryl, thiophene, indole, or alkene nucleophile. Thus, the two-step protocol using a minimum of acetic acid can effectively suppress competing intramolecular aza-Friedel–Crafts27 and aza-Prins cyclization28 reactions, allowing the selective C-4 functionalization by an indole nucleophile. Scheme 5 Solvent-Directed Selective 4-Arylation of Aryl/Alkenyl Tethered N-Acyliminium Ions, Isolated yield. All reactions were performed with 1 equiv o-formyl carbamate (1a), 1.3 equiv amine (2e–2j) in 1 mL EtOH/AcOH (9:1), 130 °C, MW, 10 min, and then 1.5 equiv Nu (3a) 130 °C, MW, 20 min. Product not isolated. Conclusions In conclusion, we have developed a highly efficient, metal-free microwave-assisted aza-Friedel–Crafts arylation of N-acyliminium ions. The solvent-directed selective aza-Friedel–Crafts arylation of challenging aryl/heteroaryl/butenyl-tethered N-acyliminium ions was achieved to produce 4-aryl 3,4-dihydroquinazolinones. This protocol offers a rapid and direct approach to generate polyfunctionalized 4-aryl 3,4-dihydroquinazolinone libraries in excellent yields under environmentally benign reaction conditions and in a short reaction time. Moreover, the protocols utilize readily available and stable o-formyl carbamate precursors and are compatible with a broad scope of amine and aryl/heteroaryl nucleophiles. Further investigations to expand the scope of this approach and explore the biological activity of these compounds are underway in our laboratory. Experimental Section All reagents and solvents were obtained from commercial suppliers and used without further purification. The yields stated refer to homogenous and spectroscopically pure isolated material. Thin layer chromatography (TLC, 0.25 mm E. Merck silica plates, 60F-254) was used to assess reaction progress and the plates were visualized with 254 nm UV light. Silica gel chromatography was performed using E. Merck silica gel (60 Å pore size, particle size 40–63 nm). 1H NMR spectra were recorded at 400 MHz and 13C NMR spectra at 100 MHz. The chemical shifts for 1H NMR and 13C NMR were referenced to tetramethylsilane via residual solvent signals (1H, CDCl3 at 7.26 ppm; 13C, CDCl3 at 77.16 ppm; 1H, DMSO-d6 at 2.45 ppm; 13C, DMSO-d6 at 39.43 ppm; 1H, CD3OD at 3.31 ppm; and 13C, CD3OD at 49.0 ppm). Microwave reactions were performed in an Initiator single mode reactor producing controlled irradiation at 2450 MHz and the temperature was monitored using the built-in online IR sensor. LC/MS was performed on an instrument equipped with a CP-Sil 8 CB capillary column (50 × 3.0 mm2, particle size 2.6 μm, pore size 100 Å) operating at an ionization potential of 70 eV using a CH3CN/H2O gradient (0.05% HCOOH). High-resolution mass values were determined using a 7-T hybrid ion trap and a time of flight detector and an electrospray ionization source. All reactions were performed in sealed Pyrex microwave-transparent process vials designed for 0.5–2 mL reaction volumes, unless otherwise stated. Preparation of o-Formyl Carbamates The required known compounds 1a–1h were prepared from the corresponding amino alcohols following the literature procedure.25,26 General Procedure A One-Pot, Two-Step Preparation of 4-Aryl 3,4-Dihydroquinazolinones (4a–4p and 6a–6f) Exemplified by 4a A 0.5–2 mL Pyrex process vial was charged with aldehyde 1a (40 mg, 224 μmol), NH4OAc 2a (34 mg, 448 μmol), and acetic acid (1 mL). The vial was sealed and subjected to microwave irradiation at 130 °C for 10 min, after which indole (3a, 34 mg, 290 μmol) was added. The vial was re-sealed and heated by microwave at 130 °C for 20 min, and thereafter the reaction mixture was concentrated in vacuo. Silica gel chromatography (2–5% MeOH in dichloromethane (DCM) or 30–85% EtOAc in n-pentane) provided the title compound as a white solid (56 mg, 95%). General Procedure B One-Pot, Two-Step Preparation of 4-Aryl 3,4-Dihydroquinazolinones (7a–7e) Exemplified by 7b A 0.5–2 mL Pyrex process vial was charged with aldehyde 1a (40 mg, 224 μmol), amine 2f (53 mg, 290 μmol), and ethanol/acetic acid (9:1, 1 mL). The vial was sealed and subjected to microwave irradiation at 130 °C for 10 min, after which indole (3a, 39 mg, 334 μmol) was added. The vial was re-sealed and heated by microwave at 130 °C for 20 min, and thereafter the reaction mixture was concentrated in vacuo. Silica gel chromatography (2–5% MeOH in DCM or 55–70% EtOAc in n-pentane) provided the title compound as a white solid (85 mg, 90%). 4-(1H-Indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4a)24 Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 448 μmol), and nucleophile 3a (34 mg, 290 μmol). Yield: 56 mg (95%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 10.99–10.90 (m, 1H), 9.27–9.22 (m, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.18–7.14 (m, 2H), 7.11–7.02 (m, 2H), 6.96–6.87 (m, 2H), 6.86–6.81 (m, 1H), 6.79–6.73 (m, 1H), 5.80 (d, J = 2.2 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 154.1, 137.6, 137.1, 127.9, 127.0, 125.3, 123.5, 122.2, 121.6, 121.2, 119.6, 119.0, 118.7, 114.0, 112.0, 50.9. High resolution mass spectrometry HRMS (electrospray ionization, ESI): calcd for C18H17N4O [M + MeCN + H]+ 305.1402; found 305.1418. TLC (SiO2): Rf = 0.06 (60% EtOAc in n-pentane). 4-(5-Methoxy-2-methyl-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4b) Prepared following the general procedure (A), starting from aldehyde 1b (40 mg, 191 μmol), amine 2a (29 mg, 376 μmol), and nucleophile 3a (29 mg, 248 μmol). Yield: 52 mg (92%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 10.98 (d, J = 2.5 Hz, 1H), 9.12 (d, J = 1.9 Hz, 1H), 7.66–7.48 (m, 1H), 7.37 (m, 1H), 7.18 (d, J = 2.5 Hz, 1H), 7.11 (m 1H), 7.07 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.94 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.73 (dd, J = 8.7, 2.7 Hz, 1H), 6.56 (d, J = 2.7 Hz, 1H), 5.79 (d, J = 2.3 Hz, 1H), 3.58 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 153.9, 153.7, 136.7, 130.8, 124.9, 123.0, 122.9, 121.2, 119.2, 118.6, 118.2, 114.4, 112.9, 112.2, 111.6, 55.2, 50.6. HRMS (ESI): calcd for C19H19N4O2 [M + MeCN + H]+ 335.1508; found 335.1517. TLC (SiO2): Rf = 0.13 (5% MeOH in DCM). 6-Bromo-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4c) Prepared following the general procedure (A), starting from aldehyde 1c (40 mg, 155 μmol), amine 2a (24 mg, 311 μmol), and nucleophile 3a (47 mg, 292 μmol). Yield: 48 mg (91%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.19–10.92 (m, 1H), 9.56–9.44 (m, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.35–7.31 (m, 1H), 7.28 (dd, J = 8.5, 2.3 Hz, 1H), 7.25 (d, J = 2.5 Hz, 1H), 7.13–7.05 (m, 2H), 7.00–6.93 (m, 1H), 6.81 (d, J = 8.5 Hz, 1H), 5.92–5.82 (m, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 153.3, 136.7, 136.6, 130.3, 129.0, 124.7, 124.3, 123.3, 121.3, 119.0, 118.8, 117.7, 115.7, 112.0, 111.8, 50.1. HRMS (ESI): calcd for C18H16BrN4O [M + MeCN + H]+ 383.0507; found m/z 383.0516. TLC (SiO2): Rf = 0.15 (5% MeOH in DCM). 6-Fluoro-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4d) Prepared following the general procedure (A), starting from aldehyde 1d (40 mg, 203 μmol), amine 2a (31 mg, 402 μmol), and nucleophile 4a (31 mg, 265 μmol). Yield: 50 mg (88%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.01 (s, 1H), 9.31 (s, 1H), 7.65–7.48 (m, 1H), 7.48–7.31 (m, 1H), 7.24 (s, 1H), 7.22–7.19 (m, 1H), 7.13–7.04 (m, 1H), 7.01–6.92 (m, 2H), 6.91–6.82 (m, 1H), 6.82–6.75 (m, 1H), 5.84 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 156.4 (d, 1JCF = 236.1 Hz), 153.2, 136.3, 133.3 (d, 4JCF = 1.9 Hz), 124.3, 123.1 (d, 3JCF = 7.0 Hz), 122.8, 120.8, 118.7, 118.3, 117.1, 114.4 (d, 3JCF = 7.7 Hz), 113.9 (d, 2JCF = 22.7 Hz), 112.50 (d, 2JCF = 23.6 Hz), 111.3, 49.9. HRMS (ESI): calcd for C16H13FN4O [M + H]+ 282.1043; found 282.1054. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). 7-Chloro-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4e) Prepared following the general procedure (A), starting from aldehyde 1e (40 mg, 187 μmol), amine 2a (29 mg, 376 μmol), and nucleophile 3a (29 mg, 248 μmol). Yield: 49 mg (88%); white solid. 1H NMR (CD3OD, 400 MHz): δ 7.41 (m, 1H), 7.35 (m, 1H), 7.19 (s, 1H), 7.09 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.94 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.88 (d, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.3, 0.9 Hz, 1H), 6.76 (dd, J = 8.3, 2.0 Hz, 1H), 5.92 (s, 1H). 13C NMR (CD3OD, 100 MHz): δ 153.8, 139.4, 137.4, 132.3, 128.9, 125.4, 123.9, 121.8, 121.4, 121.1, 119.7, 119.3, 118.2, 113.6, 112.3, 50.7. HRMS (ESI): calcd for C18H16ClN4O [M + MeCN + H]+ 339.1013; found 339.1029. TLC (SiO2): Rf = 0.09 (5% MeOH in DCM). 4-(1H-Indol-3-yl)-7-(trifluoromethyl)-3,4-dihydroquinazolin-2(1H)-one (4f) Prepared following the general procedure (A), starting from aldehyde 1f (40 mg, 162 μmol), amine 2a (25 mg, 324 μmol), and nucleophile 3a (25 mg, 213 μmol). Yield: 46 mg (86%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.17–10.91 (m, 1H), 9.85–9.48 (m, 1H), 7.54–7.48 (m, 1H), 7.45–7.41 (m, 1H), 7.41–7.36 (m, 1H), 7.28–7.22 (m, 1H), 7.20–7.14 (m, 2H), 7.14–7.05 (m, 2H), 6.99–6.93 (m, 1H), 5.94 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 152.7, 137.5, 136.3, 127.9 (q, 2JCF = 31.8 Hz), 127.2, 125.7 (q, 4JCF = 1.2 Hz), 123.6 (q, 1JCF = 272.1 Hz), 124.3, 123.0, 120.9, 118.6, 118.4, 116.9, 115.95 (q, 3JCF = 4.5 Hz), 111.3, 109.5 (q, 3JCF = 4.2 Hz), 49.9. HRMS (ESI): calcd for C19H16F3N4O [M + MeCN + H]+ 373.1276; found 373.1281. TLC (SiO2): Rf = 0.15 (5% MeOH in DCM). 4-(1H-Indol-3-yl)-8-methoxy-3,4-dihydroquinazolin-2(1H)-one (4g) Prepared following the general procedure (A), starting from aldehyde 1g (40 mg, 191 μmol), amine 2a (29 mg, 376 μmol), and nucleophile 3a (29 mg, 248 μmol). Yield: 45 mg (80%); white solid. 1H NMR (CDCl3/CD3OD, 400 MHz): δ 7.31–7.24 (m, 1H), 7.23–7.16 (m, 1H), 7.03 (s, 1H), 6.97–6.90 (m, 1H), 6.82–6.76 (m, 1H), 6.68–6.64 (m, 2H), 6.46–6.37 (m, 1H), 5.82 (s, 1H), 3.76 (s, 3H). 13C NMR (CDCl3/CD3OD, 100 MHz): δ 156.2, 146.5, 138.3, 126.1, 126.0, 124.2, 123.1, 122.9, 122.5, 120.0, 119.7, 117.8, 56.3, 52.5. HRMS (ESI): calcd for C19H19N4O2 [M + MeCN + H]+ 335.1508; found 335.1524. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). 1-Benzyl-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4h) Prepared following the general procedure (A), starting from aldehyde 1h (40 mg, 149 μmol), amine 2a (23 mg, 298 μmol), and nucleophile 3a (23 mg, 196 μmol). Yield: 45 mg (86%); white solid. 1H NMR (CDCl3, 400 MHz): δ 8.34 (s, 1H), 7.66–7.49 (m, 1H), 7.42–7.29 (m, 6H), 7.25–7.17 (m, 1H), 7.15–7.05 (m, 3H), 6.98–6.90 (m, 1H), 6.88–6.81 (m, 2H), 6.00 (s, 1H), 5.55 (s, 1H), 5.29 (d, J = 16.6 Hz, 1H), 5.21 (d, J = 16.6 Hz, 1H). 13C NMR (CDCl3, 100 MHz): δ 155.5, 137.8, 137.6, 137.0, 128.8, 128.3, 127.1, 126.9, 126.6, 125.4, 124.0, 123.4, 122.7, 122.4, 120.2, 119.7, 117.0, 114.3, 111.7, 51.0, 46.2. HRMS (ESI): calcd for C23H20N3O [M + MeCN + H]+ 354.1606; found 354.1606. TLC (SiO2): Rf = 0.13 (5% MeOH in DCM). 4-(5-Methyl-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4i) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3b (38 mg, 290 μmol). Yield: 57 mg (92%); yellow solid. 1H NMR (DMSO-d6, 400 MHz): δ 10.87 (s, 1H), 9.29 (s, 1H), 7.36–7.33 (m, 1H), 7.31–7.26 (m, 1H), 7.20–7.17 (m, 1H), 7.16–7.10 (m, 2H), 7.01–6.87 (m, 3H), 6.84–6.78 (m, 1H), 6.10–5.68 (m, 1H), 2.35 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 153.8, 137.2, 135.1, 127.5, 126.9, 126.5, 125.2, 123.2, 122.8, 121.9, 120.8, 118.8, 117.5, 113.6, 111.3, 50.5, 21.4. HRMS (ESI): calcd for C19H19N4O [M + MeCN + H]+ 319.1559; found m/z 319.1575. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). 4-(5-Bromo-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4j) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3c (57 mg, 291 μmol). Yield: 70 mg (91%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.17 (br s, 1H), 9.29 (br s, 1H), 7.67 (d, J = 1.9 Hz, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.21–7.14 (m, 2H), 7.11–7.08 (m, 1H), 6.93 (d, J = 7.5 Hz, 1H), 6.85 (dd, J = 8.0, 1.2 Hz, 1H), 6.79 (ddd, J = 7.5, 7.5, 1.2 Hz, 1H), 5.80 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 153.7, 137.2, 135.4, 127.7, 126.7, 126.5, 124.8, 123.7, 121.5, 121.4, 121.0, 118.1, 113.7, 111.4, 50.1. HRMS (ESI): calcd for C18H16BrN4O [M + MeCN + H]+ 383.0507; found 383.0502. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). 4-(2-Methyl-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4k) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3d (38 mg, 290 μmol). Yield: 58 mg (93%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 10.93 (s, 1H), 9.28 (s, 1H), 7.34–7.26 (m, 1H), 7.26–7.20 (m, 1H), 7.11 (m, 1H), 7.01–6.96 (m, 1H), 6.89–6.81 (m, 2H), 6.77–6.72 (m, 2H), 5.95 (s, 1H), 2.46 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 153.7, 137.7, 135.8, 133.2, 127.9, 127.0, 126.7, 121.9, 121.2, 120.5, 118.8, 113.9, 113.7, 110.9, 49.9, 11.8. HRMS (ESI): calcd for C19H19N4O [M + MeCN + H]+ 319.1551; found 319.1559. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). 4-(2-Phenyl-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4l) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3e (56 mg, 290 μmol). Yield: 69 mg (91%); yellow solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.43 (s, 1H), 9.35 (d, J = 1.9 Hz, 1H), 7.81–7.73 (m, 2H), 7.63–7.55 (m, 2H), 7.54–7.47 (m, 1H), 7.45–7.41 (m, 1H), 7.37–7.32 (m, 1H), 7.29–7.24 (m, 1H), 7.17–7.05 (m, 2H), 6.98–6.91 (m, 1H), 6.89 (dd, J = 8.0, 1.1 Hz, 1H), 6.70 (m, 1H), 6.61–6.54 (m, 1H), 5.99 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 153.8, 137.6, 136.8, 136.8, 132.6, 129.3, 129.2, 128.5, 128.1, 126.7, 126.5, 122.0, 121.6, 121.3, 120.3, 119.3, 114.0, 113.5, 111.8, 50.5. HRMS (ESI): calcd for C24H21N4O [M + MeCN + H]+ 381.1715; found 381.1730. TLC (SiO2): Rf = 0.20 (5% MeOH in DCM). 4-(5-Methoxy-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4m) Prepared following the general procedure (A) starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3f (47 mg, 292 μmol). Yield: 52 mg (76%); pink solid. 1H NMR (DMSO-d6, 400 MHz): δ 10.76 (s, 1H), 9.34 (d, J = 1.9 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.15–7.09 (m, 1H), 7.01–6.96 (m, 1H), 6.90–6.85 (m, 1H), 6.81–6.74 (m, 3H), 6.65 (dd, J = 8.7, 2.5 Hz, 1H), 5.90 (d, J = 1.7 Hz, 1H), 3.64 (s, 3H), 2.43 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 153.8, 153.2, 137.8, 133.8, 130.8, 127.9, 127.2, 127.1, 121.8, 121.3, 114.0, 113.8, 111.4, 109.5, 101.7, 55.5, 49.9, 11.9. HRMS (ESI): calcd for C20H21N4O2 [M + MeCN + H]+ 349.1665; found 349.1669. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). Methyl 3-(2-oxo-1,2,3,4-Tetrahydroquinazolin-4-yl)-1H-indole-4-carboxylate (4n) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3g (51 mg, 291 μmol). Yield: 60 mg (83%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.46 (s, 1H), 9.28 (s, 1H), 7.75–7.66 (m, 2H), 7.27–7.20 (m, 1H), 7.19–7.14 (m, 1H), 7.03–6.97 (m, 1H), 6.93–6.88 (m, 1H), 6.87–6.83 (m, 1H), 6.84–6.73 (m, 2H), 6.27 (d, J = 2.5 Hz, 1H), 3.93 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 168.7, 154.1, 137.8, 137.6, 127.6, 126.9, 126.4, 123.1, 122.8, 122.6, 122.3, 121.1, 120.3, 118.8, 117.0, 113.8, 52.2, 49.8. HRMS (ESI): calcd for C18H16N3O3 [M + H]+ 322.1192; found 322.1202. TLC (SiO2): Rf = 0.17 (5% MeOH in DCM). 4-(1-Methyl-1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4o)24 Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3h (38 mg, 290 μmol). Yield: 52 mg (84%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 9.27 (d, J = 1.9 Hz, 1H), 7.56 (m, 1H), 7.40 (m, 1H), 7.21 (t, J = 2.2 Hz, 1H), 7.15 (s, 1H), 7.17–7.07 (m, 1H), 7.13–7.06 (m, 1H), 6.99 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 7.00–6.93 (m, 1H), 6.86 (dd, J = 8.0, 1.2 Hz, 1H), 6.78 (m, 1H), 5.82 (d, J = 2.2 Hz, 1H), 3.74 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 153.7, 137.1, 127.6, 127.2, 126.5, 125.3, 121.7, 121.3, 120.8, 119.4, 118.8, 117.6, 113.7, 109.8, 50.2, 32.3. HRMS (ESI): calcd for C19H19N4O [M + MeCN + H]+ 319.1559; found m/z 319.1567. TLC (SiO2): Rf = 0.08 (60% EtOAc in n-pentane). 4-(1H-Pyrrolo[2,3-b]pyridin-3-yl)-3,4-dihydroquinazolin-2(1H)-one (4p) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 3i (34 mg, 288 μmol). Yield: 53 mg (90%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.53 (s, 1H), 9.31 (s, 1H), 8.19 (d, J = 4.6 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.33–7.26 (m, 2H), 7.17–7.08 (m, 1H), 7.05–6.95 (m, 2H), 6.91–6.84 (m, 1H), 6.83–6.78 (m, 1H), 6.07–5.61 (m, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 153.7, 148.9, 142.7, 137.1, 127.7, 127.3, 126.6, 123.3, 121.3, 121.0, 117.2, 117.2, 115.2, 113.7, 50.5. HRMS (ESI): calcd for C15H13N4O [M + H]+ 265.1089; found 265.1076. TLC (SiO2): Rf = 0.09 (5% MeOH in DCM). 3-Benzyl-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (6a)24 Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2b (36 mg, 336 μmol), and nucleophile 3a (34 mg, 290 μmol). Yield: 75 mg (95%); yellow solid. 1H NMR (CDCl3/CD3OD, 400 MHz): δ 11.14 (d, J = 2.5 Hz, 1H), 9.70 (s, 1H), 7.53–7.48 (m, 1H), 7.47–7.45 (m, 1H), 7.44–7.35 (m, 3H), 7.34–7.27 (m, 3H), 7.15–7.07 (m, 2H), 7.04–6.94 (m, 2H), 6.92 (dd, J = 8.0, 1.1 Hz, 1H), 6.78 (m, 1H), 5.75 (s, 1H), 5.17 (d, J = 15.5 Hz, 1H), 3.77 (d, J = 15.4 Hz, 1H). 13C NMR (CDCl3/CD3OD, 100 MHz): δ 155.8, 138.5, 138.3, 136.6, 129.5, 128.9, 128.7, 128.3, 128.1, 126.1, 124.7, 123.0, 122.8, 122.5, 120.3, 119.9, 117.0, 114.7, 112.6, 56.5, 47.8. HRMS (ESI): calcd for C23H20N3O [M + H]+ 354.1606; found 354.1613. TLC (SiO2): Rf = 0.20 (40% EtOAc in n-pentane). 4-(1H-Indol-3-yl)-3-(thiophen-2-ylmethyl)-3,4-dihydroquinazolin-2(1H)-one (6b) Prepared following the general procedure (A), starting from aldehyde 1a (40 mg, 224 μmol), amine 2c (33 mg, 292 μmol), and nucleophile 3a (34 mg, 290 μmol). Yield: 76 mg (94%); white solid. 1H NMR (CDCl3/CD3OD, 400 MHz): δ 7.44 (m, 1H), 7.38 (m, 1H), 7.35 (s, 1H), 7.31 (dd, J = 4.8, 1.6 Hz, 1H), 7.14–7.07 (m, 2H), 6.99–6.95 (m, 2H), 6.95–6.91 (m, 1H), 6.91–6.86 (m, 2H), 6.79 (m, 1H), 5.86 (s, 1H), 5.29 (dd, J = 15.5, 0.9 Hz, 1H), 4.07 (d, J = 15.5 Hz, 1H). 13C NMR (CDCl3/CD3OD, 100 MHz): δ 156.1, 141.8, 139.4, 137.3, 129.8, 129.1, 128.7, 128.4, 127.2, 127.1, 126.0, 123.9, 123.8, 123.3, 121.3, 120.9, 117.5, 115.6, 113.6, 57.1, 43.7. HRMS (ESI): calcd for C21H18N3OS [M + H]+ 360.1171; found 360.1168. TLC (SiO2): Rf = 0.16 (5% MeOH in DCM). 3-Benzyl-4-(2,4-dimethoxyphenyl)-3,4-dihydroquinazolin-2(1H)-one (6c) Prepared following the general procedure (A) but with 30 min of heating in the second step, starting from aldehyde 1a (40 mg, 224 μmol), amine 2b (36 mg, 336 μmol), and nucleophile 5a (40 mg, 289 μmol). Yield: 72 mg (86%); yellow solid. 1H NMR (CDCl3, 400 MHz): δ 8.96 (s, 1H), 7.51 (m, 5H), 7.45–7.40 (m, 1H), 7.30 (m, 1H), 7.22–7.15 (m, 1H), 7.08–6.93 (m, 2H), 6.77–6.57 (m, 2H), 6.05 (s, 1H), 5.62 (d, J = 15.1 Hz, 1H), 4.01 (s, 6H), 3.93 (d, J = 15.1 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 160.1, 157.0, 153.4, 137.8, 136.4, 128.4, 127.9, 127.7, 127.4, 127.1, 125.9, 122.9, 121.6, 121.1, 113.6, 105.7, 98.6, 55.6, 55.2, 54.6, 46.8. HRMS (ESI): calcd for C23H23N2O3 [M + H]+ 375.1709; found 375.1711. TLC (SiO2): Rf = 0.26 (40% EtOAc in n-pentane). 4-(2,4-Dimethoxyphenyl)-3,4-dihydroquinazolin-2(1H)-one (6d) Prepared following the general procedure (A) but with 30 min of heating in the second step, starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (34 mg, 441 μmol), and nucleophile 5a (40 mg, 289 μmol). Yield: 35 mg (55%); white solid. 1H NMR (DMSO-d6, 400 MHz,): δ 9.21 (s, 1H), 7.17–7.07 (m, 1H), 7.05–6.95 (m, 3H), 6.86–6.79 (m, 2H), 6.64–6.59 (m, 1H), 6.54–6.48 (m, 1H), 5.80 (d, J = 2.4 Hz, 1H), 3.86 (s, 3H), 3.76 (s, 3H). 13C NMR (DMSO-d6, 400 MHz,): δ 159.4, 156.2, 153.6, 136.7, 127.3, 127.2, 125.8, 124.9, 121.2, 120.6, 113.3, 104.6, 98.1, 55.2, 54.8, 50.2. HRMS (ESI): calcd for C18H20N3O3 [M + MeCN + H]+ 326.1505; found 326.1508. TLC (SiO2): Rf = 0.21 (5% MeOH in DCM). 4-(2,4-Dimethoxyphenyl)-3-(1-methylpiperidin-4-yl)-3,4-dihydroquinazolin-2(1H)-one (6e) Prepared following the general procedure (A) but with 30 min of heating the second step, starting from aldehyde 1a (40 mg, 224 μmol), amine 2d (33 mg, 289 μmol), and nucleophile 5a (68 mg, 492 μmol). Yield: 43 mg (50%); white solid. 1H NMR (CDCl3, 400 MHz): δ 7.47 (s, 1H), 7.24–7.20 (m, 2H), 7.08–6.96 (m, 1H), 6.86–6.76 (m, 1H), 6.65–6.58 (m, 1H), 6.40–6.31 (m, 1H), 6.29 (dd, J = 8.5, 2.4 Hz, 1H), 5.95 (s, 1H), 4.41–4.14 (m, 1H), 3.84 (s, 3H), 3.67 (s, 3H), 2.95 (d, J = 10.7 Hz, 1H), 2.79 (d, J = 11.7 Hz, 1H), 2.24 (s, 3H), 2.21–1.97 (m, 4H), 1.59 (d, J = 12.1 Hz, 1H), 1.45 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ 160.0, 155.3, 154.9, 135.0, 127.5, 127.0, 125.3, 125.0, 123.2, 121.8, 113.3, 104.4, 98.2, 55.2, 55.0, 54.9, 54.8, 52.2, 51.4, 45.2, 29.4, 28.7, 21.8. HRMS (ESI): calcd for C22H28N3O3 [M + H]+ 382.2131; found 382.2132. TLC (SiO2): Rf = 0.23 (10% MeOH in DCM). 4-(4-Hydroxy-2-methylphenyl)-3,4-dihydroquinazolin-2(1H)-one (6f) Prepared following the general procedure (A) but with 30 min in heating the second step, starting from aldehyde 1a (40 mg, 224 μmol), amine 2a (33 mg, μmol), and nucleophile 5b (31 mg, 289 μmol). Yield: 30 mg (52%); white solid. 1H NMR (DMSO-d6, 400 MHz): δ 9.62 (s, 1H), 9.15 (s, 1H), 7.07 (t, J = 7.9 Hz, 2H), 6.95 (s, 1H), 6.89 (d, J = 7.8 Hz, 1H), 6.81–6.74 (m, 2H), 6.63 (s, 1H), 6.55 (d, J = 7.8 Hz, 1H), 5.79 (d, J = 2.3 Hz, 1H), 2.17 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 154.0, 153.4, 137.5, 137.0, 128.5, 127.5, 126.9, 126.3, 121.9, 120.9, 119.9, 115.9, 113.6, 50.7, 20.7. HRMS (ESI): calcd for C15H15N2O2 [M + H]+ 255.1143; found 255.1134. TLC (SiO2): Rf = 0.22 (5% MeOH in DCM). 4-(1H-Indol-3-yl)-3-(3-methoxyphenethyl)-3,4-dihydroquinazolin-2(1H)-one (7a) Prepared following the general procedure (B), starting from aldehyde 1a (40 mg, 224 μmol), amine 2e (43 mg, 290 μmol), and nucleophile 3a (39 mg, 334 μmol). Yield: 82 mg (92%); off-white solid. 1H NMR (CDCl3, 400 MHz): δ 8.33–8.20 (m, 1H), 7.99 (s, 1H), 7.56 (m, 1H), 7.34 (m, 1H), 7.20–7.13 (m, 3H), 7.13–7.04 (m, 2H), 6.91–6.86 (m, 1H), 6.79 (ddd, J = 14.3, 7.7, 1.1 Hz, 2H), 6.74 (dd, J = 7.9, 2.1 Hz, 2H), 6.67–6.63 (m, 1H), 5.59 (s, 1H), 4.01 (ddd, J = 14.0, 9.0, 5.0 Hz, 1H), 3.66 (s, 3H), 3.22 (ddd, J = 13.8, 8.7, 7.1 Hz, 1H), 2.95 (ddd, J = 13.2, 9.0, 7.1 Hz, 1H), 2.71 (ddd, J = 13.4, 8.6, 5.0 Hz, 1H). δ 13C NMR (CDCl3, 100 MHz): δ 159.7, 154.1, 141.3, 136.7, 135.7, 129.6, 128.2, 126.8, 125.4, 122.8, 122.6, 122.1, 121.6, 121.3, 120.3, 119.4, 117.5, 114.3, 113.8, 112.2, 111.6, 56.9, 55.2, 47.3, 34.6. HRMS (ESI): calcd for [M + H]+ 398.1869; found 398.1873. TLC (SiO2): Rf = 0.13 (50% EtOAc in n-pentane). 3-(3,4-Dimethoxyphenethyl)-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (7b) Prepared following the general procedure (B), starting from aldehyde 1a (40 mg, 224 μmol), amine 2f (53 mg, 290 μmol), and nucleophile 3a (39 mg, 334 μmol). Yield: 85 mg (90%); white solid. 1H NMR (CDCl3/CD3OD, 400 MHz): δ 7.33–7.24 (m, 2H), 7.21–7.13 (m, 1H), 7.03–6.95 (m, 1H), 6.96–6.87 (m, 2H), 6.85–6.76 (m, 1H), 6.71–6.53 (m, 4H), 6.50–6.36 (m, 2H), 5.40 (s, 1H), 3.70–3.56 (m, 4H), 3.47 (s, 3H), 3.07–2.87 (m, 1H), 2.73–2.61 (m, 1H), 2.52–2.37 (m, 1H). 13C NMR (CDCl3/CD3OD, 100 MHz): δ 154.6, 148.9, 147.6, 137.1, 135.4, 132.4, 128.2, 127.0, 125.4, 123.6, 122.5, 122.2, 121.8, 121.0, 119.8, 119.0, 116.5, 113.9, 112.3, 111.9, 111.7, 57.2, 56.0, 55.7, 47.6, 33.9. HRMS (ESI): calcd for C26H26N3O3 [M + H]+ 428.1974; found 428.1977. TLC (SiO2): Rf = 0.10 (50% EtOAc in n-pentane). 4-(1H-Indol-3-yl)-3-(2-(thiophen-2-yl)ethyl)-3,4-dihydroquinazolin-2(1H)-one (7c) Prepared following the general procedure (B), starting from aldehyde 1a (40 mg, 224 μmol), amine 2g (37 mg, 290 μmol), and nucleophile 3a (39 mg, 334 μmol). Yield: 80 mg (96%); light yellow solid. 1H NMR (CDCl3, 400 MHz): δ 8.32–8.24 (m, 1H), 8.15 (s, 1H), 7.58 (m, 1H), 7.35 (m, 1H), 7.21–7.13 (m, 2H), 7.14–7.03 (m, 3H), 6.94–6.85 (m, 2H), 6.83–6.70 (m, 3H), 5.67 (s, 1H), 4.01 (ddd, J = 13.3, 8.4, 5.2 Hz, 1H), 3.34–3.15 (m, 2H), 3.00–2.84 (m, 1H). 13C NMR (CDCl3, 100 MHz): δ 154.1, 141.8, 136.7, 135.6, 128.2, 127.1, 126.8, 125.4, 125.3, 123.8, 122.9, 122.7, 122.2, 121.5, 120.4, 119.4, 117.5, 113.9, 111.6, 57.2, 47.5, 28.5. HRMS (ESI): calcd for C22H29N3OS [M + H]+ 374.1327; found 374.1324. TLC (SiO2): Rf = 0.15 (50% EtOAc in pentane). 3-(But-3-en-1-yl)-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (7d) Prepared following the general procedure (B), starting from aldehyde 1a (40 mg, 224 μmol), amine 2h (21 mg, 290 μmol), and nucleophile 3a (39 mg, 334 μmol). Yield: 68 mg (96%); off-white solid. 1H NMR (CDCl3, 400 MHz): δ 8.32 (s, 1H), 8.05 (s, 1H), 7.63 (dd, J = 7.9, 1.1 Hz, 1H), 7.35 (m, 1H), 7.22–7.14 (m, 2H), 7.09 (m, 2H), 7.01 (ddd, J = 7.7, 1.5, 0.7 Hz, 1H), 6.85–6.72 (m, 2H), 5.88 (s, 1H), 5.78 (m, 1H), 5.15–4.88 (m, 2H), 3.91 (ddd, J = 13.9, 8.7, 6.4 Hz, 1H), 3.04 (ddd, J = 14.2, 8.6, 5.9 Hz, 1H), 2.47–2.35 (m, 1H), 2.33–2.20 (m, 1H). 13C NMR (CDCl3, 100 MHz): δ 154.2, 136.7, 135.7, 135.6, 128.2, 126.8, 125.4, 122.7, 122.6, 122.1, 121.5, 120.3, 119.4, 117.6, 116.7, 113.9, 111.6, 56.4, 44.7, 32.2. HRMS (ESI): calcd for [M + H]+ 318.1606; found 318.1606. TLC (SiO2): Rf = 0.2 (50% EtOAc in n-pentane). 3-((1H-Indol-4-yl)methyl)-4-(1H-indol-3-yl)-3,4-dihydroquinazolin-2(1H)-one (7e) Prepared following the general procedure (B), starting from aldehyde 1a (40 mg, 224 μmol), amine 2i (42 mg, 290 μmol), and nucleophile 3a (39 mg, 334 μmol). Yield: 32 mg (36%); off-white solid. 1H NMR (DMSO-d6, 400 MHz): δ 11.13 (d, J = 13.2 Hz, 2H), 9.66 (s, 1H), 8.28 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.42–7.23 (m, 4H), 7.05 (m, 3H), 6.90 (m, 4H), 6.68 (m, 1H), 6.45 (s, 1H), 5.60 (s, 1H), 5.50 (d, J = 15.0 Hz, 1H), 3.83 (d, J = 15.0 Hz, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 152.9, 137.1, 136.3, 136.2, 128.4, 127.8, 127.1, 126.7, 125.3, 124.7, 124.0, 121.6, 121.3, 121.2, 121.0, 119.2, 119.0, 118.5, 116.3, 113.6, 112.1, 111.0, 99.8, 54.6, 44.7. HRMS (ESI): calcd for [M + H]+ 393.1715; found 393.1729. TLC (SiO2): Rf = 0.1 (50% EtOAc in n-pentane). Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02298.Copies of 1H and 13C NMR spectra for all compounds (PDF) Supplementary Material ao8b02298_si_001.pdf Author Present Address ‡ Department of Radiology, Stanford University, Stanford, California 94306, United States (M.Y.S.). Author Present Address † Beactica AB, Uppsala Business Park, Virdings allé 2, 75450 Uppsala, Sweden (R.T.S.). The authors declare no competing financial interest. Acknowledgments This research was supported by Uppsala University. The authors thank Dr Lisa Haigh (Imperial College London) for assistance with HRMS determination. ==== Refs References Speckamp W. N. ; Moolenaar M. J. New Developments in the Chemistry of N -Acyliminium Ions and Related Intermediates . Tetrahedron 2000 , 56 , 3817 –3856 . 10.1016/S0040-4020(00)00159-9 . Maryanoff B. E. ; Zhang H. ; Cohen J. H. ; Turchi I. J. ; Maryanoff C. A. 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PMC006xxxxxx/PMC6644442.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145894110.1021/acsomega.8b00985ArticleHexagon Flower Quantum Dot-like Cu Pattern Formation during Low-Pressure Chemical Vapor Deposited Graphene Growth on a Liquid Cu/W Substrate Pham Phuong V. SKKU Advanced Institute of Nano Technology (SAINT), SKKU, Suwon, Gyeonggi-do 440-746, Republic of KoreaCenter for Multidimensional Carbon Materials, Institute for Basic Science, Ulsan 44919, Republic of Korea E-mail: pvphuong@skku.edu.18 07 2018 31 07 2018 3 7 8036 8041 12 05 2018 09 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. The H2-induced etching of low-dimensional materials is of significant interest for controlled architecture design of crystalline materials at the micro- and nanoscale. This principle is applied to the thinnest crystalline etchant, graphene. In this study, by using a high H2 concentration, the etched hexagonal holes of copper quantum dots (Cu QDs) were formed and embedded into the large-scale graphene region by low-pressure chemical vapor deposition on a liquid Cu/W surface. With this procedure, the hexagon flower-etched Cu patterns were formed in a H2 environment at a higher melting temperature of Cu foil (1090 °C). The etching into the large-scale graphene was confirmed by optical microscopy, atomic force microscopy, scanning electron microscopy, and Raman analysis. This first observation could be an intriguing case for the fundamental study of low-dimensional material etching during chemical vapor deposition growth; moreover, it may supply a simple approach for the controlled etching/growth. In addition, it could be significant in the fabrication of controllable etched structures based on Cu QD patterns for nanoelectronic devices as well as in-plane heterostructures on other low-dimensional materials in the near future. document-id-old-9ao8b00985document-id-new-14ao-2018-00985accc-price ==== Body Introduction Graphene, a honeycomb crystal carbon lattice, has attracted huge research interest during the past few years due to its anomalous properties, including very high carrier mobility, extremely high mechanical strength and optical transparency, electrical conductivity, etc.1−25 As a result, graphene is considered to be an ideal nanomaterial for next-generation semiconductors to replace silicon. The etching of material is the block removal from a material matrix by chemical or physical methods—the reverse of the growth process. Understanding the etching mechanism is necessary for material design as well as the realization of its capabilities. Material growth/etching requires a high-energy barrier nucleation process. Controlling the etching/growing parameters may originate from the formation of a thermodynamic and stably ordered structure with the ability to form a different kinetically controlled metastable structures with high-energy crystal facets and edges. The material growth systems have been controlled with different scales (nanometer to micrometer).26−28 The family of snow-crystal-like graphene with patterns is formed by a nonlinear process in nature.29 In contrast, the highly crystalline materials (e.g., Si) are etched via an anisotropic rule,30 leading to stable etched patterns with Euclidean geometries. The underlying mechanism is attributed to various etching rates on various crystalline directions and surfaces related to various free energies. However, the etched structures of low-dimensional materials beyond Euclidean geometries have not been investigated. The high crystal C atom single-layer supplies a simple model to study the fundamental growth and/or etching process via the chemical vapor deposition (CVD) method. Significant efforts have been created to develop graphene growth strategies with controlled size, crystallinity, and edge structures via CVD.31−34 In addition, several reports on graphene etching have been carried out utilizing various etchants, such as plasma H2,35−38 H2,39,40 and metallic nanoparticles.41−43 Recently, graphene crystal patterns were well-controlled through inert gases and a H2 source.44 Those discoveries inspired us to additionally examine the fundamental issue of the graphene etching mode. In this work, we report the first observation of a new kind of etched geometry of a large-scale grown graphene region that is beyond known Euclidean geometries to date. The hexagon quantum dot (QD)-like etched Cu pattern is a new morphology that has not yet been revealed. Results and Discussion For the etched graphene growth procedure on a liquid Cu/W substrate, a schematic of the hexagon QD-like Cu pattern formation during low-pressure CVD graphene growth onto a liquid Cu/W substrate is illustrated in Figure 1. Low-pressure chemical vapor deposition (LPCVD) is investigated at low pressure (6 Torr) using Cu (thickness of 250 μm) as a catalytic substrate located on a W foil (thickness of 80 μm). First, a Cu/W substrate was heated in the H2 environment (100 sccm) for 30 min to the melting point of Cu (1090 °C) and then annealed in H2 (100 sccm) for 30 min. The integrated etching/growth proceeded via the LPCVD approach with hexagon flower-etched Cu patterns, embedded into the large-scale graphene located on a liquid Cu/W substrate using a CH4/H2 ratio of 6/100 sccm in 30 min. Finally, the furnace was opened and CH4 gas was turned off; H2 flow was continued for fast cooling to 100 °C. Figure 1 (a) Schematic of the formation of a hexagon flower QD-like Cu pattern during low-pressure CVD graphene growth on a liquid Cu/W substrate. There have been several investigations on the controlled diffusive etching modes for crystal growth of low-dimensional materials that are responsible for etched graphene pattern formation,1−4,39 generating an effective engineering method for etched patterns on other 2D and 3D material systems. However, etching to form flower QD-like Cu patterns embedded into the large-scale graphene region is quite new, strange, and intriguing in the field of etching at the micro- and nanoscale. To date, there is no scientific report in terms of theory and simulation investigations that detail the relationship of the hexagonal-shaped etching mode and the underneath graphene structure. For morphology investigations of solid Cu, liquid Cu, and graphene before and after the CVD process, scanning electron microscopy (SEM), atomic force microscopy (AFM), and optical microscopy (OM) were carried out, as shown in Figure 2a–f. Here, Figure 2a exhibits the morphology of bare Cu foil (250 μm) through the SEM image before the CVD process. Figure 2b reveals the SEM image of liquid Cu at 1090 °C after being resolidified. Figure 2c is the micrograph photo of etched large-scale graphene on the liquid Cu/W substrate. Figure 2d reveals the AFM image of the surface liquid Cu after being resolidified on W foil with a liquid Cu thickness of 150 nm after 1090 °C and resolidified as shown in Figure 2e, corresponding to the blue line in Figure 2d. Figure 2f is the OM images of liquid Cu located on W foil after being resolidified, corresponding to the AFM image in Figure 2d. Figure 2 (a) OM image of bare Cu foil (thick 250 μm). (b) SEM image of liquid Cu at 1090 °C after being resolidified. (c) Micrograph photo of etched large-scale graphene on liquid Cu/W. (d) AFM of liquid Cu on W foil after being resolidified. (e) Thickness of liquid Cu at 1090 °C and resolidified at about 150 nm, corresponding to the blue line in (d). (f) OM images of liquid Cu on W foil after being resolidified, corresponding to the AFM image in (d). Figure 3 and Figures S1 and S2 in the Supporting Information reveal a new Euclidean geometry of the QD-like Cu patterns which are partially etched Cu hexagons at the center of six edge sites and embedded into the large-scale graphene located on the liquid Cu/W surface after a higher melting point of Cu foil (1090 °C). Because the melting temperature for W foil was 3422 °C, its morphology did not change after treatment at 1090 °C. Similarly, the SEM images at various magnifications were taken for hexagon flower-etched Cu patterns embedded into large-scale graphene located on a liquid Cu/W substrate, as shown in Figure 4 and Figures S3 and S4 in the Supporting Information. Figure 3 OM images of hexagon flower-etched Cu patterns embedded into the large-scale graphene located on a liquid Cu/W substrate. Figure 4 SEM images of hexagon flower-etched Cu patterns embedded into the large-scale graphene located on a liquid Cu/W substrate at different magnifications. For further demonstrations, the AFM data were applied to observe the hexagon flower-etched Cu patterns embedded into large-scale graphene located on a liquid Cu/W substrate, as shown in Figure 5 and Figure S5 in the Supporting Information for two-dimensional (2D) and three-dimensional (3D) images at different corners. In addition, their phase images, which are characterized for softness, stiffness, and the adhesion between the AFM cantilever tip with the specimen surface, are shown in Figure 5d and Figure 5Sd. Figure 5 AFM images of hexagon flower-etched Cu patterns embedded into large-scale graphene located on a liquid Cu/W substrate, (a,b) 3D AFM mapping image, (c) 3D AFM mapping image at different views of (a), and (d) phase image of (a). To investigate the existence of graphene on liquid Cu/W and the Cu pattern etching effect, Raman spectroscopy was carried out at two regions: graphene and Cu pattern (see Figure 6). At the Cu pattern regions, the Raman peaks in Figure 6c,d correspond to the OM images that are directly captured by the Raman analysis instrument, with the red cross in Figure 6a and the black cross in Figure 6b. They showed the presence of some peaks of CuO, Cu2O, and Cu(OH)2, which are already well-known from a previous report.45 On the other hand, in the blue and green graphene regions in Figure 6c and the red region in Figure 6d, corresponding blue and green crosses in Figure 6a and red cross in Figure 6b show clearly the D, G, and 2D peaks of Raman data as the fingerprints of the graphene structure on liquid Cu/W substrates. The Raman mapping of Figure 6b was also obtained for the etched Cu pattern at a wavelength scan range from 0 to 800 nm for the typical existence of CuO, Cu2O, and Cu(OH)2 peaks (Figure 6g), which was proven in a previous report.45Figure 6a,f shows the zoomed-in images of Figure 6c,d, respectively. Figure 6 (a,b) OM images captured from Raman spectroscopy of hexagon flower-etched Cu patterns embedded into the large-scale graphene located on a liquid Cu/W substrate. (c,d) Raman data of different positions marked as blue, green, and red crosses in (a,b). (e,f) Zoomed-in image of graphene in (c,d), respectively. (g) Raman mapping with a scan range from 0 to 800 nm of region (b). For an explanation about the mechanism of etched Cu pattern formation embedded into large-area graphene film, see Figure 7. It is believed that the etching mode occurred due to the diffusion of etchants (H2 molecules or H radicals) at the graphene/liquid Cu interface and the diffusion on the graphene surface. Consequently, the etching effect formed at the defects sites or grain boundaries was embedded into the large-scale graphene to create the hexagon QD-like Cu patterns. Here, the diffusion at the graphene/liquid Cu interface might be the key role for formation of the etched Cu pattern. The high etching rate is because of high H2 etchant concentration as well as hindered diffusion at the graphene/liquid Cu interface. H2 diffusion on isotropic liquid Cu indicates the physical origin of the high symmetry of etched Cu patterns. Also, it is a key factor for visualizing the etched line mode because controlled diffusive etching varies with nanoparticle etching from previous reports.41−43 Figure 7 Mechanism of etched Cu patterns embedded into the large-area graphene film with diffusion of etchants (H2 molecules or H radicals) underneath and above the liquid Cu surface, which could be mostly responsible for the formation of etched Cu patterns. Conclusions The resulting hexagon flower QD-like etched Cu patterns have revealed a new kind of unknown Euclidean geometry during large-scale CVD graphene growth. Here, we have established the first observation of etched Cu patterns on large-scale graphene that can form a new etched hexagon shape. The experimental results provide clear proof of this etching mode. Etching to form the hexagon QD-like Cu pattern was induced at high H2 concentration. This study is expected to be further computationally and experimentally examined for in situ observation of integrated graphene etching/growth in the future. This new integrated growth/etching effect shows an unknown graphene etching mode that enables other nanomaterial structures to be formed. In addition, further investigation of the graphene etching mode on new substrates (e.g., Ni/W) is another intriguing phenomenon which will be discovered in the near future. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00985.OM, SEM, and AFM images of hexagon flower QD-etched patterns embedded into the large-scale graphene located on the liquid Cu/W foil (PDF) Supplementary Material ao8b00985_si_001.pdf The author declares no competing financial interest. 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PMC006xxxxxx/PMC6644443.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145899510.1021/acsomega.8b01331ArticleLocal Domain Size in Single-Chain Polymer Nanoparticles Pomposo José A. †‡§Moreno Angel J. †∥Arbe Arantxa †Colmenero Juan †‡∥† Centro de Física de Materiales (CSIC, UPV/EHU) and Materials Physics Center MPC, Paseo Manuel de Lardizabal 5, E-20018 San Sebastián, Spain‡ Departamento de Física de Materiales, Universidad del País Vasco (UPV/EHU), Apartado 1072, E-20800 San Sebastián, Spain§ IKERBASQUE—Basque Foundation for Science, María Díaz de Haro 3, E-48013 Bilbao, Spain∥ Donostia International Physics Center (DIPC), Paseo Manuel de Lardizabal 4, E-20018 San Sebastián, Spain E-mail: Josetxo.pomposo@ehu.eus.02 08 2018 31 08 2018 3 8 8648 8654 13 06 2018 20 07 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Single-chain polymer nanoparticles (SCNPs) obtained through chain collapse via intramolecular cross-linking are attracting significant interest for nanomedicine and biomimetic catalysis applications, among other fields. This interest arises from the possibility to bind active species (e.g., drugs and catalysts)—either temporally or permanently—to the SCNP local pockets formed upon chain collapse. However, direct quantification of the size and number of such local domains in solution—even if highly desirable—is currently highly demanding from an experimental point of view because of the small size involved (<5 nm). On the basis of a scaling analysis, we establish herein a connection between the global compaction degree (R/R0) and the size (ξ) and number (n) of the “collapsed domains” generated upon SCNP formation at high dilution from a linear semiflexible precursor polymer. Results from molecular dynamics simulations and experimental data are used to validate this scaling analysis and to estimate the size and number of local domains in polystyrene SCNPs synthesized through a “click” chemistry procedure, as a representative system, as well as for relevant catalytic SCNPs containing Cu, Pt, and Ni atoms. Remarkably, the present work is a first step toward tuning the local domain size of the next generation of SCNPs for nanomedicine and bioinspired catalysis applications. document-id-old-9ao8b01331document-id-new-14ao-2018-01331cccc-price ==== Body 1 Introduction Intramolecular cross-linking of individual polymer chains in a good solvent at high dilution produces single-chain polymer nanoparticles (SCNPs) via intrachain collapse.1−5 The compaction of a single chain to a SCNP resembles—in some way—the conformational rearrangement needed by some biomacromolecules to reach its functional state.6−8 Depending on the specific nature of the intramolecular cross-links formed, dynamic or permanent SCNPs result by involving reversible or irreversible intrachain interactions, respectively.9,10 These soft nano-objects have attracted significant interest for a variety of applications, including nanomedicine and biomimetic catalysis uses.3 The possibility to bind active species (e.g., drugs and catalysts)—either temporally or permanently—to the SCNP local pockets formed upon intrachain chain collapse remains as a driving force toward bioinspired applications.6,8 To understand the possibilities that SCNPs offer for nanomedicine and biomimetic catalysis, the reader is referred to several illustrative works11−25 and a recent book.26 Significant theoretical effort27−32 has been recently devoted to understand how the nature of the intramolecular cross-links (both reversible and irreversible interactions), the amount of functional reactive groups, x, and the precursor molecular weight, Mw, determine the global collapse degree upon intramolecular cross-linking, R/R0, where R0 and R are the linear precursor polymer size and the SCNP size, respectively. However, to our best knowledge, no attempt has been yet carried out to establish a connection between the global compaction degree (R/R0) and the size (ξ) and number (n) of local pockets (domains) generated upon SCNP formation at high dilution. It is worthy of mention that a direct visualization/quantification of such local domains in solution—even if highly desirable—is currently highly demanding from an experimental point of view.26 We use herein the analogy of chain confinement into a spherical cavity33 to the collapse of a linear chain to a SCNP to perform a scaling analysis34,35 allowing to establish a connection between the global compaction degree (R/R0) and the size (ξ) and number (n) of “collapsed domains” generated upon SCNP formation at high dilution. It is worthy of mention that the case of confinement of a linear semiflexible polymer chain into a cavity under good solvent conditions has recently attracted significant theoretical and computer simulations efforts.36−39 Remarkably, the present work provides a solid basis to understand the effect of (i) the strength of the intramolecular interactions, (ii) the amount of functional groups, (iii) the precursor molecular weight, and (iv) the precursor chain stiffness on the size and number of local domains of SCNPs in solution. The article is organized as follows. In Section 2, we first introduce two expressions for the change in free energy upon collapse based, respectively, on the analogy of SCNP formation to the case of chain confinement into a spherical cavity under good solvent conditions and on the number of new intramolecular bonds formed and the change in free energy per each new bond formed. Next, useful scaling expressions for the global compaction degree and the size and number of local domains are derived. Validation of the scaling analysis with results from molecular dynamics (MD) simulations and reliable experimental data, as well as an estimation of the size and number of local domains for relevant catalytic SCNPs in solution containing Cu, Pt, and Ni atoms, is provided in Section 3 and, finally, the conclusions of the work are given in Section 4. 2 Scaling Analysis 2.1 Free Energy of Collapse Let us assume that the change in free energy, ΔFc, upon collapse of a linear precursor polymer of size R0 to a SCNP of size R in a good solvent under high dilution conditions follows an expression similar to that corresponding to chain confinement into a spherical cavity under good solvent conditions35−39 1 where kB is the Boltzmann’s constant, T the absolute temperature, and α is the scaling exponent of the resulting collapsed domains upon SCNP formation (see below). It is reasonable to assume that SCNPs with different chain stiffness will show different values of the exponent in eq 1, as observed in computer simulations of semiflexible chains confined in a spherical cavity.36 Because the free energy of collapse comes from all of the new intramolecular bonds formed, we can additionally write 2 where xN/2 is the number of new intramolecular bonds formed, x is the fraction of reactive groups in the linear precursor polymer of total number of monomers N, and \|Δfc\| is the absolute value of the change in free energy per each new bond formed. Implicit in eq 2 is the assumption that by means of dimerization of the corresponding reactive monomers the maximum number of intrachain bonds (xN/2) is obtained. Experimentally, this is expected to be the case for SCNPs prepared through highly reactive cross-linking procedures (e.g., “click” chemistry reactions10) but not for SCNPs prepared by means of weak, dynamic interactions9 (e.g., hydrogen bonds). For real systems, Δfc is expected to contain a favorable enthalpic contribution, Δhc < 0, because of (exothermic) bond formation and an unfavorable entropic contribution, Δsc < 0, because of severe configurational restriction upon chain collapse. 2.2 Global Compaction Degree By combining eqs 1 and 2, we obtain an analytical expression for the compaction degree (R/R0) as a function of x and N such as 3a 3b Taking into account that the size of a semiflexible linear chain in a good solvent is given37 (to a first approximation) by R0 ≃ bC∞1/5Nν0, where b is the monomer size, C∞ is the characteristic ratio,34 and ν0 is the Flory exponent (ν0 ≈ 3/5), eq 3a can be rewritten as 4a 4b Consequently, according to the present scaling analysis, the exponents of the dependence of SCNP size R on x and N are actually connected through eq 4b. 2.3 Local Domain Size Similar to the case of a linear chain confined in a spherical cavity,35−39 we introduce herein the concept of“cross-linked blobs” or collapsed domains upon SCNP formation via chain collapse. The average number of such domains (n) each one having associated a free energy around kBT and a size ξ can be estimated from the free energy of collapse such as37,39 5 Therefore, by using eqs 1 and 2, we obtain the following expressions for the domain size 6a 6b whereas the number of monomers in each collapsed blob (g) is just given by37,39 7 It is worthy of mention that α in eq 6a is related to the scaling exponent of the cross-linked blobs, so its value gives an idea of compaction at local level (i.e., at domain size ξ). Conversely, the value of ν in eq 4a gives an indication of chain compaction at global scale (i.e., at SCNP size R). In general, one expects 1/3 ≤ (α, ν) ≤ 1 where the lower value is expected for globule-like compaction, whereas the upper value corresponds to rod-like compaction.34 It is instructive to rewrite eqs 5 and 7 by using eq 6b, such as 8 and 9 Hence, according to the present scaling analysis n depends linearly on x, N, and \|Δfc\|, whereas g depends inversely on x and \|Δfc\| but it is independent on N. 3 Results and Discussion 3.1 Scaling Analysis Validation with Data from MD Simulations Analysis of R/R0 and xN/2 data from MD simulations of semiflexible SCNPs in terms of eq 3a allows one to determine the scaling exponent β (and hence α from eq 3b) as well as the absolute value of the change in free energy per each new bond formed, \|Δfc\|. Figure 1 shows such an analysis for SCNPs having different chain stiffness, as characterized by the value of the bending constant k employed in the coarse-grained MD simulations.40 Note that k is related to the well-known characteristic ratio, C∞, and it was previously reported that C∞ ≈ 1.7, 5, 9, and 15 for k = 0 (fully flexible case), 3, 5, and 8, respectively.40 The dependences of β, the exponent of the free energy of collapse 3/(3α – 1), and \|Δfc\| on chain stiffness are illustrated in Figure 2 for semiflexible SCNPs. Upon increasing the SCNP characteristic ratio, the value of β was found to decrease nearly linearly with C∞ (see Figure 2A). Concerning the exponent of the free energy of collapse, it decreases abruptly on increasing C∞ toward a plateau value for the stiffer SCNPs (Figure 2B). This behavior is in agreement with that reported by Cifra and Bleha36 in computer simulations of the confinement of a semiflexible linear chain in a closed spherical cavity reaching a limiting value of 2.6 upon increasing chain stiffness. Interestingly, \|Δfc\| also reaches a plateau value upon increasing SCNP stiffness (Figure 2C). As noted by Cifra and Bleha,36 for semiflexible linear chains, the free-energy penalty on chain compaction is maximum for the fully flexible case when compared with the case of stiffer systems. According to the data in Figure 2C, there is a difference in \|Δfc\| between the fully flexible and stiffer SCNPs around 3.7 kBT. An estimation of the free energy of collapse as a function of SCNP stiffness at identical compaction degree can be obtained from eq 1. As an example, at a compaction degree R/R0 = 0.65, we obtain = 218.0, 14.8, 7.1, and 4.4 for SCNPs with C∞ = 1.7, 5, 9, and 15, respectively. Figure 1 MD simulations data of compaction degree (R/R0) as a function of new intramolecular bonds formed (xN/2) for SCNPs having different chain stiffness:40 (A) C∞ = 1.7, (B) C∞ = 5, (C) C∞ = 9, and (D) C∞ = 15 (see text for details). Figure 2 Dependence of (A) scaling exponent β, (B) scaling exponent 3/(3α – 1), and (C) change in free energy per new bond formed, \|Δfc\|, on chain stiffness for semiflexible SCNPs with different values of characteristic ratio, C∞, according to MD simulations (see eqs 1–3a and text for details). Table 1 shows a comparison of the values of ν = ν0 + β and , as derived from the present scaling analysis, to those previously reported for semiflexible SCNPs with different C∞ values as estimated directly from MD simulations data.40 As stated previously in Section 2, ν gives an indication of chain compaction at global scale (i.e., at SCNP size R) whereas the value of α is indicative of chain compaction at local level (i.e., at domain size ξ). In general, a good agreement is observed between the values of ν from the present scaling analysis and MD simulations. Concerning the values of α, the MD simulations provide values that are systematically larger than those derived from the scaling analysis, although both data sets follow the general trend of a higher value of α on increasing C∞. Table 1 Comparison of ν and α Values from the Scaling Analysis of This Work to Estimations from MD Simulations of SCNPs with Different C∞ Values40 C∞ β ν α νMDsim. αMDsim. 1.7 –0.08 0.52 0.41 0.50 0.63 5 –0.16 0.44 0.49 0.47 0.74 9 –0.22 0.38 0.55 0.42 0.81 15 –0.29 0.31 0.62 0.30 0.84 Figure 3 shows the evolution of the collapsed domain size, ξ (in σ units, where σ is the bead size) and the number of beads in a domain, g, as a function of SCNP stiffness, for SCNPs with a fraction of reacting monomers of x = 0.2 and N = 400, as calculated from eqs 4a–5 with data from Figure 1 and Table 1. Inspection of Figure 3 reveals that a minimum SCNP stiffness is required for the scaling analysis to provide consistent values of ξ and g. As a matter of fact, the domain size of fully flexible SCNPs in Figure 3A approaches the bead size which invalidates the scaling analysis for this case. Inasmuch C∞ ≥ 5, consistent values of ξ, n, and g are obtained showing a collapsed domain size around 3.7σ, about 33 collapsed blobs per SCNP and ∼12 beads per blob for semiflexible SCNPs (x = 0.2, N = 400) with C∞ = 5, 9, and 15 because of the similar values of \|Δfc\| (refer to eqs 6bb, 8, and 9). Figure 3 Dependence of (A) collapsed domain size, ξ (in σ units, where σ is the bead size) and (B) number of beads per collapsed domain, g, on chain stiffness for semiflexible SCNPs (x = 0.2, N = 400) as calculated from eqs 6bb and 9 with data from Figure 1 and Table 1. Figure 4 illustrates the dependence of ξ and g on the fraction of reacting monomers, x, for SCNPs with N = 400 and C∞ = 9. Chain collapse from a linear precursor with low amount of reacting monomers (e.g., x = 0.05) produces a few collapsed domains (∼8) of relatively large size (∼8.4σ) containing a large amount of beads per blob (∼51). Upon increasing x, both collapsed domain size and number of beads per blob reduce notably (refer to eqs 6bb and 9), whereas the number of blobs is found to increase linearly with x (refer to eq 8). Figure 4 Dependence of (A) collapsed domain size, ξ, and (B) number of beads in a collapsed domain, g, on the fraction of reacting monomers x for SCNPs with N = 400 and C∞ = 9, as calculated from eqs 6bb and 9 with data from Figure 1 and Table 1. 3.2 Scaling Analysis Validation with Experimental Data Complementary to the analysis based on MD simulations data, we have also analyzed data for real SCNPs in terms of eq 3a. In this sense, compaction data from well-defined polystyrene (PS)-SCNPs synthesized via “click” chemistry,41 covering from x = 0.025 to x = 0.3 and three different values of weight-average molecular weight (Mw ≈ 44, 111, and 232 kDa) were found to merge into a single master curve with β ≈ −0.20 and \|Δfc\| ≈ 0.16, when plotted according to eq 3a (see Figure 5). Figure 5 Compaction degree (R/R0) as a function of new intramolecular bonds formed (xN/2) for PS-SCNPs (C∞ ≈ 9.5 for PS34) of different molecular weight:41Mw = 44 kDa (solid blue points), Mw = 111 kDa (solid red points), and Mw = 232 kDa (solid green points). Apparently, the experimental data best follow the scaling behavior at large values of N. From ν = ν0 + β and , we obtain ν = 0.4 and α = 0.53 based on the value of β ≈ −0.20. Note that the values of β and ν for PS-SCNPs (C∞ ≈ 9.5 for PS34) are in excellent agreement with those predicted by a model of elastic SCNPs recently developed by our group27 (β = −1/5, ν = 2/5). When compared with MD simulations of SCNPs prepared from a precursor polymer of C∞ = 9, the value of \|Δfc\| was found to be about fivefold lower for real PS-SCNPs. However, it is worth stressing that similar values should not be expected even at a qualitative level. Thus, though one finds similar scaling properties for the molecular size and may expect similar entropy loss per monomer because of network formation in the coarse-grained and in the real SCNPs, this is not the case for the associated enthalpy change, Δhc, because the bonded and nonbonded interactions in the simplified bead-spring model of the simulations40 are of different nature than that in the real polymers.10 Figure 6 illustrates the dependence of ξ and g on the fraction of reacting monomers, x, for PS-SCNPs of Mw ≈ 44, 111, and 232 kDa, as calculated form eqs 6bb and 9 with data from Figure 5. On increasing the amount of functional monomers from x = 0.1 to x = 0.3, a decrease in the domain size is observed from ξ ≈ 4 nm to ξ ≈ 2 nm. According to the results displayed in Figure 6A, the domain size in PS-SCNPs shows a weak dependence on Mw. Remarkably, on the basis of the present scaling analysis, the number of monomers in each local domain is expected to be independent of Mw, as illustrated in Figure 6B. By taking into account that for real systems σ ≈ 0.64 nm and each bead in the MD simulations comprises ∼5 monomers, the results shown in Figure 6 are in good agreement with those displayed in Figure 4. Figure 6 Dependence of (A) collapsed domain size, ξ, and (B) number of monomers in a collapsed domain, g, on the fraction of reacting monomers, x, for PS-SCNPs (C∞≈ 9.5 for PS34) of Mw = 44 kDa (solid blue points), Mw = 111 kDa (solid red points) and Mw = 232 kDa (solid green points) as calculated from eqs 6bb and 9 with data from Figure 5. 3.3 Estimation of Local Domain Size in Pt-, Cu- and Ni-Containing Catalytic SCNPs in Solution Data about the local domain size, number of domains, and number of monomers per domain for relevant metal-containing catalytic SCNPs20,22,42 in solution are given in Table 2. The precursor polymers were based on either PS or poly(methyl methacrylate) (PMMA), both having a similar value of chain stiffness34 (C∞(PS) = 9.5, C∞(PMMA) = 9.0). Estimations were carried out based on experimental R/R0 data by assuming α ≈ 0.53 in eq 6aa, for PtII-, CuII-, and NiII-containing SCNPs employed with success in amination of allyl alcohol,20 selective alkyne homo-coupling reactions,22 and photoreduction of carbon dioxide,42 respectively. Table 2 Local Domain Size (ξ), Number of Domains (n), and Number of Monomers per Domain (g) in Relevant Metal-Containing Catalytic SCNPs (See Text) catalyst reaction R0 (nm) R (nm) ξ (nm) n g refs PtII@PS-SCNPs amination of allyl alcohol 7.4 4.9 2.4 9 40 (20) CuII@PMMA SCNPs selective alkyne homo-coupling 26 15 5.9 16 125 (22) NiII@PMMA SCNPs photoreduction of carbon dioxide 4.2 3.7 3.0 1.9 241 (42) As stated previously, direct quantification of the size and number of SCNP local domains in solution is currently highly demanding from an experimental point of view, so there are no experimental data available. Nevertheless, experimental evidence of the presence of local compact domains in isolated SCNPs is provided in Figure 7, as revealed by transmission electron microscopy (TEM) measurements performed to Cu-containing SCNPs upon solvent removal and deposition onto a carbon grid.22 Obviously, upon solvent removal the dimensions of the SCNPs change and often a pancake morphology is observed for isolated SCNPs deposited onto solid substrates. Moreover, the SCNP size is found to depend to a large extent on the surface energy of the substrate.27 Figure 7 TEM image showing the presence of local compact domains in a Cu-containing SCNP in the dry state.22 4 Conclusions Quantification of the size and number of local domains of SCNPs in solution is currently of great interest because of the possibilities that offer such local pockets to bind—either temporally or permanently—active species to them (e.g., drugs and catalysts) rendering the resulting SCNPs highly attractive for applications in nanomedicine and catalysis, among other fields. A scaling analysis was performed to establish a connection between the global compaction degree (R/R0) and the size (ξ) and number (n) of collapsed domains generated upon SCNP formation at high dilution from a linear semiflexible precursor polymer. The scaling analysis was validated with results from MD simulations and reliable experimental data. On the basis of the scaling equations here proposed, we have estimated—as an example of application—the size and number of local domains for relevant catalytic SCNPs in solution containing Cu, Pt, and Ni atoms. Remarkably, this work is a first step toward tuning the local domain size of the next generation of SCNPs for nanomedicine and bioinspired catalysis applications. The authors declare no competing financial interest. Acknowledgments The financial support by the Spanish Ministry “Ministerio de Economia y Competitividad”, MAT2015-63704-P (MINECO/FEDER, UE), the Basque Government, IT-654-13, and the Gipuzkoako Foru Aldundia, RED 101/17, is acknowledged. ==== Refs References Mavila S. ; Eivgi O. ; Berkovich I. ; Lemcoff N. G. Intramolecular Cross-Linking Methodologies for the Synthesis of Polymer Nanoparticles . Chem. Rev. 2016 , 116 , 878 –961 . 10.1021/acs.chemrev.5b00290 .26270435 Altintas O. ; Barner-Kowollik C. Single-Chain Folding of Synthetic Polymers: A Critical Update . 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PMC006xxxxxx/PMC6644444.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145799310.1021/acsomega.8b01680ArticleSimple Preparation of Porous Carbon-Supported Ruthenium: Propitious Catalytic Activity in the Reduction of Ferrocyanate(III) and a Cationic Dye Veerakumar Pitchaimani †‡Salamalai Kamaraj §Thanasekaran Pounraj ∥Lin King-Chuen †‡† Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan‡Institute of Atomic and Molecular Sciences and ∥Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan§ Department of Mechanical Engineering, PSN Institute of Technology and Science, Tamil Nadu, Tirunelveli 627152, India* E-mail: spveerakumar@gmail.com (P.V.).* E-mail: kclin@ntu.edu.tw. Phone: +866-2-33661162 (K.-C.L.).04 10 2018 31 10 2018 3 10 12609 12621 17 07 2018 20 09 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. The present study involves the synthesis, characterization, and catalytic application of ruthenium nanoparticles (Ru NPs) supported on plastic-derived carbons (PDCs) synthesized from plastic wastes (soft drink bottles) as an alternative carbon source. PDCs have been further activated with CO2 and characterized by various analytical techniques. The catalytic activity of Ru@PDC for the reduction of potassium hexacyanoferrate(III), (K3[Fe(CN)6]), and new fuchsin (NF) dye by NaBH4 was performed under mild conditions. The PDCs had spherical morphology with an average size of 0.5 μm, and the Ru NP (5 ± 0.2 nm) loading (4.01 wt %) into the PDC provided high catalytic performance for catalytic reduction of ferrocyanate(III) and NF dye. This catalyst can be recycled more than six times with only a minor loss of its catalytic activity. In addition, the stability and reusability of the Ru@PDC catalyst are also discussed. document-id-old-9ao8b01680document-id-new-14ao-2018-016802ccc-price ==== Body Introduction Over the past few years, biowastes and non-biodegradable plastic waste materials as low-cost feedstock have been utilized for the production of value-added carbon nanomaterials with a wide range of applications.1 Until now, enormous amounts of plastic wastes were generated because of increased demand of plastic-related production.2 These plastics are not biodegradable and thus generate extremely troublesome components for landfilling. Handling and managing these plastic wastes is a huge task, because a large amount of these wastes are generally dumped into landfill or disposed in the ocean, thereby causing a very serious environmental issue.3 Therefore, a large number of methods have been investigated to convert plastic wastes into useful products.4 For instance, they have been transformed into different kinds of carbon-based nanostructures including nanotubes,5 spheres,6 hollow spheres,7 nanosheets,8 activated carbons,9 and graphene flake/foil10 for sustainable energy applications.11 Carbon-based nanostructure materials, particularly biowaste activated carbon spheres (CSs), have gained a wide range of interests because of their excellent energy storage ability, good electrical conductivity, biocompatibility, and electrical properties, and thus, they have received a wide attention.12 In recent years, active metals supported on the porous carbon substrate such as Rh,13 Re,14 Ru,15 Os,16 Ir,17 and Pt18 have been used as active catalysts for organic/inorganic transformations. Among them, Ru-based nanomaterials are widely explored as nanocatalysts19 because of their low cost and attractive feature of superior catalytic activity and stability. Generally, potassium hexacyanoferrate(III), (K3[Fe(CN)6]), is well-known as one of the most common pollutants, which is found in contaminated air, water, and soil in the environment. It can easily accumulate inside humans, aquatic animals, and other living organisms through food chains20 and has been proved to have mutagenicity, acute toxicity, carcinogenicity, and high environmental mobility, even in a trace level.21 In contrast, Fe(II) is considered as an essential nutrient required in metabolic pathways for humans and animals. The most common type of anemia is caused by the iron deficiency whereas some diseases such as hemochromatosis can be due to iron overload; the United States Recommended Daily Allowance (USRDA) for iron is 18 mg.22 Besides, the conversion of Fe(III) into Fe(II) offers several important advantages and attractive applications including possibilities for (i) tin purification, (ii) separation of copper out of molybdenum ore, (iii) wine and citric acid in large-scale preparation, (iv) serving as a benchmark for electron transfer reaction, and (v) medical diagnosis for diabetic patients and designing of amperometric biosensor for electrochemical applications.23 Recently, Pastoriza-Santos et al.24 carried out the reduction of Fe(CN)63– with NaBH4, using gold nanorods containing metallodielectric hollow shells of SiO2 or TiO2. However, these catalytic supports are expensive compared to the carbon nanostructure. Remarkably, these supports are difficult to remove from the active phase, but the carbon support can be easily burnt to separate the active metallic phase. Carregal-Romero’s group fabricated spherical Au NP heterostructures25 and have used them for catalytic reduction of Fe(CN)63– by NaBH4 in aqueous solution; however, this catalyst rate was 4 times higher than that of the uncatalyzed reaction. Despite their successful results, low stability and inconvenient recovery restrict practical application of the materials. Jana et al.26 prepared mesoporous Au-boehmite film catalyst for reduction Fe(CN)63–. However, this catalyst achieved large rate constant and was four times recycled but required high loadings, harsh conditions, and high-boiling-point aprotic solvents for the catalytic methods. Likewise, Miao et al.27 used Fe3O4@GNSs as the catalyst for the reduction of Fe(CN)63– in aqueous media. This magnetic composite was used as an ideal catalyst and shows recyclability along with persistent catalytic activity even after being recycled six times. Chen et al.28 developed the submicron-sized PEGDMA@Au NP microsphere catalyst for the reduction of Fe(CN)63– by NaBH4 in aqueous solution, but they failed to report recyclability and leaching experiments. Likewise, Yang et al.29 used 2,6-pyridinedicarboxylic acid-protected Au NP as a catalyst for the same reduction. However, the catalyst exhibits poor rate constant and lack of recyclability and stability. Wu et al.30 have demonstrated an ionic liquid-based synthesis of hollow and porous platinum nanotubes as a new catalyst, however required harsh preparation conditions, exhibited poor stability, and produced silica waste after etching. Recently, Jiang demonstrated a new approach for the one-pot synthesis of gold hollow nanospheres exhibiting excellent catalytic activity toward the reduction of Fe(CN)63– by NaBH4 in water.31 Being stable in air and water, this catalyst could be reused 10 times. However, a long time period was required for completion of reduction. Therefore, the above-mentioned catalysts are less advantageous with limited practical applications in catalysis, owing to some unique natures, such as great chemical stability, low corrosive capability, high thermal stability, hydrophobic feature, easy recovery, and low price.32 On the other hand, clean water is the major topic of the current research because it is an important source for humans and the environment.33 Discharge of effluents containing toxic dyes and heavy metal ions from manufacturing industries such as cosmetic, leather, paper, textile, pharmaceuticals, and so on into nearby water bodies is highly detrimental to the human health and environment.34 Hence, the development of robust and smart functionalized nanomaterials with practical feasibility and biocompatibility is necessitated to act as an effective adsorbent for removal of toxic dyes.35 In particular, metals or metal oxide-containing carbon nanomaterials have been reported as inexpensive nano-adsorbents for the adsorption/removal of heavy metals and dyes.34,35 Thus, utilization of inexpensive nano-adsorbents for the treatment of industrial dye effluents could be helpful in resolving the human and environment problems.36,37 In this work, small-sized Ru NPs were decorated on the PDC support by the microwave-assisted (MW) reduction method, which has been popularly employed over the past few years for its advantageous nature, such as easy control of particle size and surface area, purity and high production yield, quick operation time, desirable temperature regulation, and tenability for the carbonaceous structure. Herein, we report a novel preparation to fabricate Ru NPs supported on porous PDC (Ru@PDC) by converting waste plastics into porous carbons. We adopted plastic wastes as the primary carbon rich precursor and ruthenium(III) acetylacetonate [Ru(acac)3] as a metal precursor. A more detailed description for the preparation procedure of Ru@PDC catalyst is shown in Scheme 1. Scheme 1 Schematic Diagram for the Preparation and Application of the Ru@PDC Catalyst Results and Discussion Phase Structure The phase structures of the as-prepared samples were examined using powder X-ray diffraction (PXRD) and Raman spectroscopy. Analyzing the XRD of carbon samples exhibits two broad peaks with low intensities at 2θ ≈ 23.5° and 43.6° corresponding to the (002) and (100) diffraction planes ascribed to the graphitic and amorphous carbon structure, respectively (Figure 1a). Upon increasing the temperature, the diffraction peaks of PDC-600, PDC-700, and PDC-800 samples become broader slightly with larger intensities, indicating that the carbon structure is characteristic of a more extent of graphitic nature implying a trend of polymer aggregation into large polyaromatic structures. Aggregation reactions proceed progressively with temperature at 600 °C and higher until the carbonaceous materials are formed showing nanometer-scale morphology containing highly organized carbons.38 On the other hand, additional sharp diffraction peaks were observed for the Ru@PDC composite at 2θ ≈ 38.4°, 42.3°, 44.1°, 58.5°, 69.6°, and 78.2° corresponding to the (100), (002), (101), (102), (110), and (103) planes of the hexagonal close-packed Ru (JCPDS 06-0663), in excellent agreement with the reported values.31 The XRD pattern of Ru@PDC shows a broad peak located at 44.1° composed of overlap between both C(100) and Ru(101) diffractions, thus suggesting that the catalyst contains smaller size ruthenium particles with diameters ≈5 nm. Additionally, the low C(002) peak intensity in the catalyst was observed, because of general lack of graphitic ordering. Scherrer formula (eq 1) in the following was adopted to calculate the apparent crystallite size for a given reflection. 1 where D denotes the mean size of the crystallite perpendicular to the planes (hkl) and K is a Scherrer parameter adopted as 1.84 for (100) and 0.94 for (002) for half-widths. λ is equal to 0.15406 nm, the used wavelength of the X-ray radiation, β is the breadth at half maximum intensity in radians, and θ is the Bragg angle for the reflection concerned. The average particle size of Ru NP was evaluated to be 5–6 nm, consistent with the high resolution transmission electron microscopy (HR-TEM) results (vide infra). Figure 1 (a) PXRD patterns, (b) Raman spectra, (c) N2 sorption-isotherms, and (d) TGA curves of the as-prepared PDC and Ru@PDC materials. Raman Analysis Raman spectroscopy is used to inspect the structure defects and disorder nature of carbonaceous materials. Raman spectra for all the samples (Figure 1b) exhibited two marked peaks at around 1344 and 1601 cm–1, which were ascribed to the D band and G band, respectively.39 The G band at 1592–1601 cm–1 is due to the E2g C–C stretching mode of sp2-bonded two-dimensional hexagonal lattice of graphite layers, while the D-band at 1323–1344 cm–1 is attributed to the A1g vibrational mode and is characteristic of the disorder nature. The intensity ratio of ID/IG is used to evaluate the defects of carbon-based samples; a smaller ratio suggests more significant defects on graphitic carbons. As such, the band intensity ratios (ID/IG) for PDC-600 and PDC-700 are 0.48 and 0.51, respectively, both indicative of low graphitization.40 However, the peak area ratio of ID/IG for pristine PDC and Ru@PDC increased to 0.78 and 0.83, respectively, indicating graphitization enhancement of the PDC-800 after the high-temperature treatment; the fact agrees with the XRD measurements (see Figure 1a). The resulting ID/IG ratios of all samples were calculated and are listed in Table 1. This consequence is in agreement with those by the XPS and HR-TEM analyses (vide infra). Table 1 Textural Properties of the As-Prepared PDC and Ru@PDC Materials sample STota (m2 g–1) SMicrob (m2 g–1) SMesoc (m2 g–1) VTota (cm3 g–1) DPd (nm) Dme (%) ID/IG PDC-600 97.6 31.2 66.4 0.032 7.8 0.48 PDC-700 294.2 103.1 191.1 0.071 7.5 0.51 PDC-800 466.7 217.2 249.5 0.086 7.5 0.78 Ru@PDC 396.5 184.6 211.9 0.082 7.3 6.01 0.83 a Surface area (STot) from the BET method and total pore volume (VTot) calculated at P/P0 = 0.99. b Microporous surface areas (SMicro) obtained from t-plot analyses. c SMeso (SMeso = STot – SMicro). d Average pore size (DP) derived by BJH adsorption branches of isotherms. e Metal dispersion measured by H2 chemisorption at 323 K. Textural Property The nitrogen adsorption/desorption isotherms at 77 K of PDC and Ru@PDC samples are shown in Figure 1c. The isotherms exhibited a type-IV curve with hysteresis loop associated with capillary condensation in the range of P/P0 from 0.45 to 0.99. This finding indicated that the porosity of the obtained PDC and Ru@PDC was essentially made up of micropores/mesopores, and it may be generated by the CO2 activation. The textural properties including BET total surface area (STot), micropore surface area (SMicro), mesopore surface area (SMeso), total pore volume (VTot), and average pore diameter (DP) are summarized in Table 1. The BET surface area of Ru@PDC (SBET = 396.5 m2 g–1) sample significantly decreased compared to PDC-800 (466.7 m2 g–1), indicating that Ru NPs blocked the pores of the CSs and thus diminished the surface area and the total pore volume of the Ru@PDC.41 These results verified that the Ru NPs were impregnated on the surface of the PDC matrix. The Ru NPs were well dispersed on the surface and no obvious aggregation was observed, whereas unsupported Ru NPs were likely to aggregate immediately.42a The Ru@PDC catalysts were characterized and are listed in Table 1. It seems that a higher BET specific surface area tends to favor a higher Ru dispersion. Masthan et al. have reported the H2 dispersion measurement for Ru/γ-Al2O3, the H2 absorption equilibrium was much faster at a higher temperature (373 K) rather than ambient temperature.42b The value of Ru dispersion and average crystallite size for Ru-based catalysts based on H2 chemisorption method is provided in Table S1, (Supporting Information). The Barrett–Joyner–Halenda (BJH) model was adopted to evaluate the pore size distributions in terms of the adsorption branches of the isotherms (Figure S1 of the Supporting Information). Thermal Stability Thermal properties of the samples thus prepared were further examined by thermogravimetric analysis (TGA), as displayed in Figure 1d. The weight loss below 200 °C (12–18% for all samples) can be attributed to the adsorbed water evaporation. The other weight losses began at around 558 °C, which are mainly due to the burning of the carbon structures. It indicated the high purity of the prepared PDC. However, a trace amount of residues was also observed after complete decomposition of carbon samples. Inductively coupled plasma–atomic emission spectroscopy (ICP–AES) was further employed to analyze the elemental contents, obtaining the Zn species present in the carbon material with a content of 1017 ppm (i.e., ca. 0.10 wt %). The impregnation of ZnCl2 during the process tended to cause dehydration of the carbon substrate and subsequently to result in charring and aromatization along with the creation of porosities. The mobile liquid ZnCl2 (mp ≈ 283 °C) was expected to occur in the earlier stage of the activation. Sometimes, the Zn ions are expected to be strongly intercalated between the carbon interlayers, when the activation temperature is increased beyond 700 °C (bp of ZnCl2 ca. 730 °C), and a strong interaction between carbon atoms and Zn species between the carbon interlayers might leave trace Zn residue unvaporized.21 The burning of PDC in the Ru@PDC sample began at around 561 °C because of the interaction between Ru NP and carbon atoms inducing defects in the graphitic carbon structure of PDC.43 While further heating, no weight loss was found significantly, verifying that the particle crystallinity took place at 800 °C. Moreover, according to the data from TG measurements, the content of Ru in Ru@PDC was evaluated to be 4.01 wt %. This experiment evidences additionally that Ru NPs are successfully incorporated onto the PDC support. Morphology and Microstructure The CSs were prepared by using plastic wastes as carbon sources without any catalysts under hydrothermal conditions, as reported earlier.44 According to this method, the SEM images of PDC-800 spheres exhibited uniform spherical shapes ranging from 300 to 500 nm in diameter, as displayed in the Figure S2, Supporting Information. As can be seen, a minor agglomeration bonding between spheres can be ascribed to polymerization interruption or structure collapse. Additionally, the microstructures of PDC-600 and PDC-700 were further examined using HR-TEM as displayed in Figure S3 Supporting Information, which shows appearance of smooth surface with perfect spheres. HR-TEM observation at different magnifications (Figure 2a,b) and enlarged portions (Figure 2c–g) shows the shape of the PDC-800 particle, which has sizes of few hundred nanometers along with porous microstructure. Moreover, the selected area electron diffraction (SAED) reveals that the PDC-800 has a typical amorphous carbon microstructure (Figure 2h), which preserves the structural integrity and spherical morphology. Figure 2 (a–g) HR-TEM images of the as-prepared bare PDC-800 with different magnifications and (h) electron diffraction pattern of the representative image of PDC-800. The bars represent (a) 0.5 μm, (b) 200 nm, (c) 100 nm, (d) 50 nm, (e) 20 nm, (f) 10 nm, and (g) 5 nm. As shown in Figure 3, HR-TEM images of the Ru@PDC catalyst showed that Ru NP had an average size of 5 ± 0.2 nm, which are apparently distributed smoothly on the surface of PDC. A representative histogram of particle size distribution of Ru NP in Ru@PDC catalyst is shown in Figure S4 (Supporting Information). Energy-dispersive spectrometry analysis evidenced the presence of Ru, C, and O elements in the Ru@PDC catalyst (Figure S5, Supporting Information). Again, it is believed that Ru NPs have been successfully planted on the surface of PDC matrix. Figure 3 (a–f) Typical HR-TEM images of the Ru@PDC catalyst with different magnifications. As displayed in Figure 4a–c, the additional filed emission TEM (FE-TEM) images of Ru@PDC verify a uniform distribution of the Ru NP on the surface of PDC carbon matrix. The Ru presence was confirmed from SAED pattern of Ru NPs (Figure 4d). It was found that some Ru NPs aggregate to form small clusters with a maximum size of 6 nm. The images also indicate that some Ru NPs are successfully planted in the PDC carbon matrix. Figure 4 (a–c) Additional HR-TEM images of the Ru@PDC catalyst and (d) SAED pattern. Surface Element Composition Analysis The surface element compositions of PDC-800 and Ru@PDC samples were characterized using XPS. Figure 5a shows the XPS survey spectra of PDC-800 and Ru@PDC, containing C, O, and Ru elements. The C 1s XPS spectrum (Figure 5b) exhibits a strong peak at 284.3 eV, ascribed to the C–C/C=C bonds, and two relatively weaker peaks with the binding energies (B.E) at about 285.1 and 288.7 eV, attributed to the C–H and C=O species, respectively. It is notable that the two bands with B.E at 284.3 and 280.4 eV can be readily assigned to Ru 3d3/2 and 3d5/2, respectively, in the nanoparticles by referring to the values of Ru metal at 285 and 280 eV, respectively.45 As shown in Figure 5c for the O 1s spectrum, a broad band is found and deconvoluted into four peaks with a B.E ca. 529.9 eV (C=O), 530.7 (COOH), 532.6 (O–C–O), and 534.2 eV (C–OH). The Ru 3p signal of Ru@PDC (Figure 5d) is fitted into a pair with the B.E of ca. 461.1 (Ru 3p3/2) and 483.2 eV (Ru 3p1/2), corresponding to the photoemission of metallic Ru.46 Additionally, the elemental analysis by the ICP–AES technique evidenced the presence of the Ru element in the Ru@PDC catalyst material with a content of 4.01 wt % (see Table S4, Supporting Information). Figure 5 (a) XPS survey spectra of the pristine PDC-800 and the Ru@PDC samples, and the corresponding core-level spectrum of (b) C 1s + Ru 3d, (c) O 1s, and (d) Ru 3p. FT-IR Study FT-IR spectra were recorded for PDC and Ru@PDC samples, and the results are shown in Figure S6, Supporting Information. The appearance of a weakly broad band at ∼3345 cm–1 is attributed to the hydroxyl group (−OH), and a weak band at 1638 cm–1 is attributed to the skeleton vibration of aromatic (−C=C−) rings. The band at 2926 cm–1 is assigned to CH2 asymmetric stretching, while the band at 1442 cm–1 is ascribed to the C–H bending mode. Other bands at 1110–1258 cm–1 are due to the C–O stretching mode.11a After carbonization, most peaks disappear with increase of temperature from 600 to 800 °C (Figure S6, Supporting Information). However, a small peak at 1594 cm–1 remains, implying that some aromatic rings in the carbonized samples still exist. Meanwhile, after the carbonization process in conjunction with Ru NP immobilization, the bands of −OH groups in the Ru@PDC nanocomposite decrease in intensity. This is due to a strong interaction of the functional group with Ru metal.44 In addition, different types of plastic materials were utilized as carbon sources, including high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polyacrylate (PC). The produced solid CSs had smooth surfaces and the size of CSs is in the range from 3 to 8 μm (Figure S7, Supporting Information). In terms of TEM analysis (Figure S8, Supporting Information), the CSs produced with various plastic precursors are solid in nature, and their spherical images of CSs can be clearly seen in all the cases of carbon sources at 800 °C (Figure S8, Supporting Information). All samples are carbon-rich materials (∼68–69%) as revealed by the elemental analysis and XRD patterns (Figure S9, Supporting Information), showing carbon content on weight basis, and possession of trace amounts of heteroatoms (such as N, S, and Cl Table S2, Supporting Information), whose presence should be beneficial to the preparation of active carbons. Controlling of the carbonization conditions and activation process is not the only key factor to determine porous structure of CSs, which may also be affected by the structure and nature of the precursors. It is important for starting plastics to possess high content in hydrocarbons for the preparation of porous carbon by the self-assembly approach with which hydrocarbons are aggregated into higher poly-hydrocarbons resulting in the formation of carbon materials. The elemental analysis (Table S3, Supporting Information) reveals that the hydrogen content is much greater in LDPE than in HDPE and PC. As reported, it is well-known processes for the generation of CSs from aromatic hydrocarbons and from degradation of plastic materials to the mixture of hydrocarbons.44 Catalytic Study Ruthenium-supported carbon materials have been vastly envisaged for catalytic applications over the last years.45,46 Because carbon-supported Ru NPs containing mesoporous structures and large surface areas have been explored as superior catalysts in the inorganic and organic fields.47 Therefore, the designed Ru@PDC catalysts can serve as a novel catalyst for inorganic reduction reactions with a highly efficient performance. The dispersibility of heterogeneous catalysts in solution should play a key role in order to enhance the catalytic activity. In this manner, the presence of functional groups on the surface renders the catalyst to disperse fabulously in the solution, as displayed in Figure S6b (Supporting Information). The catalytic activities of Ru@PDC have been tested for the reduction of [Fe(CN)6]3– using NaBH448 as a reducing agent. Initially, we tested the blank experiment in the absence of either reducing agent or Ru@PDC catalyst and found that the concentration of [Fe(CN)6]3– did not change. It is a fact that the reaction does not occur significantly in the presence of either the reducing agent or Ru@PDC catalyst alone (Figures S10a,b, Supporting Information). Therefore, the combination of all these reagents is required for the reduction reaction. The catalytic activity of Ru@PDC with excess NaBH4 was performed, showing that the characteristic yellow color (i.e., high catalytic activity) in aqueous solution disappeared in inorganic reaction within seconds, as shown in Figure 6a. We verified such an efficient reaction through analysis of the catalyst effect on the kinetic reduction of K3[Fe(CN)6] in the presence of NaBH4, which is essentially based on an electron-transfer process, and the kinetic change can be easily monitored using UV–vis absorption spectroscopy. The K3[Fe(CN)6] aqueous solution in light yellow showed its absorption band at 420 nm. When NaBH4 was added, the absorption intensity at 420 nm decreased gradually because of the [Fe(CN)6]4– formation, indicating that Fe(III) ions were reduced to Fe(II), along with the solution color changed to colorless, as displayed in Figure 6b,c. Under the given conditions, we tested the same reaction by NaBH4 in the presence of either the Ru NP catalyst (Figure 5d) or available commercial Ru/C catalyst (Figure 6e); but both of them take a long period of 30 s (reaction was incomplete). These results indicated that both Ru NP and Ru/C catalysts possess a less catalytic activity, when compared with the Ru@PDC catalyst. This is due to insufficient porous features to allow diffusion of reactants and products. When the concentration of NaBH4 far exceeds [Fe(CN)6]3–, the kinetic reduction can be treated as a pseudo-first-order reaction, and then the kinetic rate R can be expressed as eq 2(49) 2 where t is the reaction time; k is the pseudo-first-order reaction rate constant; and Ct and C0 denote the concentration of [Fe(CN)6]3– at time t and the initial time t0, respectively. The Ct/C0 is proportional to the relative intensity of At/A0, where At and A0 are the peak absorbance at time t and t0, respectively. Hence, the pseudo-first-order kinetic model is described as eq 3 3 Figure 6 UV–vis spectra for the reduction of K3[Fe(CN)6] by NaBH4 in the presence of (a) Ru@PDC recorded within 30 s, (b)1.0 mg, (c) 2.0 mg of Ru@PDC, (d) Ru NP, (e) Ru/C, and (f) conversions vs catalysts. The pseudo-first-order rate constant (k) can be estimated from the linear plot of ln(At/A0) versus the reduction time (t). Accordingly, the rate constant (k) is calculated to be 0.0942, 0.1011, 0.0612, and 0.021 s–1, for Ru@PDC (1.0 mg; 2.0 mg), Ru NP, and ruthenium black (Ru/C) catalysts, respectively. This plot ln(At/A0) versus time reveals that the [Fe(CN)6]3– can be converted completely into [Fe(CN)6]4–, as displayed in Figure 6f. Conversion of the catalytic products is determined by eq 4(50) 4 The calculated results demonstrated that a good conversion (98%) was observed for the Ru@PDC catalyst, while lower conversions were obtained by unsupported Ru NP (65%) and commercial Ru/C (23%) catalysts; however, both the Ru NP and Ru/C catalysts show slightly a lower catalytic activity due to its lower surface area. The Ru@PDC was found to exhibit a much better catalytic activity for [Fe(CN)6]3– reduction than that of ruthenium black (kRu@PDCs: 0.1011 s–1 vs kRublack: 0.021 s–1). Meanwhile, the k value of Ru@PDC (0.1011 s–1) is also higher than those of Pd/GPDAP (kPd/GPDAP = 2.330 × 10–2 s–1),48a Au@IFMC-100 (kAu@IFMC-100 = 3.07 × 10–2 s–1),49 ce-MoS2 (kce-MoS2 = 53 ± 7 × 10–2 s–1),51 and other unsupported noble metal nanoparticles (see Table S7, Supporting Information) under the same reaction conditions, further demonstrating that the Ru@PDC have excellent reactivity for the [Fe(CN)6]3– reduction. In addition, we examined the catalytic activity of the Ru@PDC catalyst using Na2S2O3 as an alternative reducing agent. At first, we tested the catalytic reduction reaction in the absence of a catalyst, but the result showed that the reduction did not proceed significantly in Na2S2O3 solution (Figure S11a, Supporting Information). Then, we added different dosages of catalyst into the reaction mixture and found the depletion of [Fe(CN)6]3– peaking at 420 nm (Figure S11b,c, Supporting Information), revealing that the [Fe(CN)6]3– is converted into [Fe(CN)6]4–; the corresponding kinetic plot is given in inset of Figure S11 Supporting Information. The linear plot of ln(At/A0) against time yielded the apparent rate constant to be 0.0932 s–1 (1.0 mg) and 0.1154 s–1 (2.0 mg) at ambient temperature (Figure S8, Supporting Information). A few important studies reported previously based on the Na2S2O3 reducing agent are selected for comparison with our results, as summarized in Table S7. For instance, Ajit et al.52 have reported a porous platinum nanostructured catalyst for the catalytic reduction of [Fe(CN)6]3–, yielding a smaller rate constant, kPt NNs = 1.52 × 10–4 s–1 for 60 min. Likewise, the Au/boehmite26 catalyst yielded the reduction rate constant, kAu/boehmite, = 5.16 × 10–5 s–1 and NiWO4 NP53 yielded kNiWO4 NP = 1.06 × 10–4 s–1. These catalytic results are less efficient than those obtained with the Ru@PDC catalyst. The reaction mechanism invoked for the catalytic reduction of [Fe(CN)6]3– in the presence of reducing agents over the Ru@PDC catalyst comprises two steps based on the earlier reports.24 They are (i) rapid polarization of the metal NP by NaBH4 (fast) and (ii) transfer of excess surface electrons to [Fe(CN)6]3– complex ions (slow). That is, an electron-transfer process must be involved to form the reduced [Fe(CN)6]4– ions. Hence, the reduction reactions in an aqueous solution can be written as eq 5(48b) 5 To test the catalytic activity of Ru@PDC, [Fe(CN)6]3– reduction by Na2S2O3 was investigated involving the following reaction eq 6(54) 6 Catalytic Activity Catalytic activity depends significantly on particle size and shape, and thus, how to synthesize colloidal nanoparticles with well-controlled size and shape is urgent and challenging.31 Actually, the nanomaterial owns several catalytic merits including crystal plane, crystal phase, and small size. Among them, the size effect is an important parameter for both homogenous and heterogeneous catalysis. The particle size effect of metal nanoparticles on the catalysis has been thoroughly investigated.55 Regarding the particle size effect, the Ru@PDC catalyst yielded higher activity because of the smaller size of Ru embedded, resulting in a faster reaction (k = 0.1011 s–1), probably due to the larger surface area (particle size 5 ± 0.2 nm), in contrast to the boehmite supported Au NP (15–40 nm),26 which led to the k was 0.103 min–1 for the reduction of K3[Fe(CN)6]. Likewise, the cases of Fe3O4@Au hollow sphere20 (Au NP: 25–30 nm, k: 36.55 × 10–3 s–1), graphene/Pd47b (Pd NP: 18.8 nm, k: 36.55 × 10–3 s–1) catalysts appear to have larger particle size than our catalyst systems. The detailed data of various catalysts with different sizes of metal nanoparticles and their rate constants are listed (Table S6, Supporting Information). The results indicated that the smaller particle size may lead to a larger surface area and subsequently was favorable for a good catalyst with high efficiency. In other words, the small nanoparticles are more effective catalysts than the larger NP, because an increase in the electron density for the small metal atoms leads to an increased reactivity on the surface of the catalysts. Moreover, PDC-supported metal (Mn, Fe, Co, Cu, and Ni) catalysts for the reduction of K3[Fe(CN)6] were investigated, and the results given in Table S6 (Supporting Information) shows that the reaction is sensitive to the change of metals. Turnover frequency (TOF) is used to quantify the catalytic activity of Ru@PDC and is defined as the number of [Fe(CN)6]3– molecules converted to [Fe(CN)6]4– with 1.0 mg of catalyst per s. Typically, 3.0 mmol of [Fe(CN)6]3– solution was completely reduced in the presence of Ru@PDC in 30 s, while Ru NPs and Ru/C exhibited lower catalytic activity within the same reaction time. We can simply calculate TOF values for a catalytic reaction using eq 7:56 7 The TOF of Ru@PDC was up to 5.0 × 10–5 s–1 for the [Fe(CN)6]3– reduction reaction, which was much larger than those of other nanocatalysts reported previously and was comparable with that of Ru NP and commercial Ru/C (see Table S7, Supporting Information). Effect of Catalyst Dosage Figure S12 Supporting Information shows the k result as a function of different amounts of catalyst (0.25–3.0 mg mL–1). The k value is proportion to the catalyst amount because of an increase in the number of reaction sites.57 The obtained slope can be used to evaluate the pseudo-first-order of kinetic rate constant. It becomes feasible to understand why a large catalyst amount in the reaction may result in a rapid reduction of [Fe(CN)6]3–. For instance, Reddy et al.58 have demonstrated that gum acacia-stabilized gold nanoparticles (GA/Au NPs) for catalytic reduction of [Fe(CN)6]3– showed a similar dependence of the k value on catalyst dosages. Moreover, the reaction orders and the rate constants were displayed in Table S8 (Supporting Information). Hence, the reaction constant and the order of the reaction are increasing linearly with increase of Ru@PDC catalyst dosage. Stability and Reusability Stability and reusability should be considered for the practical applications of catalysts.59 The Ru@PDC catalyst may be facilely recovered by ultracentrifugation (10 000 rpm) after the reaction. As a result, the catalyst was inspected up to six consecutive cycles for the reduction of [Fe(CN)6]3– and monitored by UV–vis spectroscopy. As shown in Figure 7a, the apparent rate constants reveal that the catalyst preserves more than 85% of the initial catalytic ability after six consecutive cycles. The reaction mixtures were also analyzed by ICP–optical emission spectrometry to check if any Ru was leached, but no significant leaching was found. Hence, the reaction rate change cannot be caused by the loss of Ru from the catalyst. The rational decrease of catalytic ability might be due to the trace loss of catalyst after several times of use, as confirmed by HRTEM images (Figure 7b,c). The powder XRD pattern of the reused Ru@PDC catalyst demonstrates to understand retention of the crystallinity after the catalytic reaction as shown in Figure 7d. The energy-dispersive X-ray spectroscopy (EDX) analysis of the Ru NP proved the existence of an elemental ruthenium signal. Other EDX signals emitting from C, O, and Cu atoms were also noticed (Figure 7e). This result indicated that the Ru species were successfully immobilized on the nanocomposite, even after being recycled six times. Figure 7 (a) Recycling test of the Ru@PDC catalyst toward the [Fe(CN)6]3– reduction, (b,c) FE-TEM images, (d) XRD pattern, (e) EDX spectrum, (f) N2 sorption-isotherm, and (g) corresponding pore size distribution of the reused Ru@PDC catalyst. Figure 7f shows the nitrogen adsorption/desorption isotherms of the reused Ru@PDC catalyst and pore-size distributions (Figure 7g) derived from the adsorption branch of the isotherms according to the BJH method. We also noted that the surface area of the spent Ru@PDC catalyst had shown slightly lower surface area (SBET = 388.3 m2 g–1) than the fresh catalyst (SBET = 396.5 m2 g–1, Table 1) after six uses. The catalytic activity decreased from the fourth to sixth cycles, probably because of the loss of catalyst during the recycling process. Notably, the reused catalyst exhibits a type-IV curve corresponding to a mesoporous structure with pore volume (VTot = 0.080 cm3 g–1); hence, these results demonstrated that the reused Ru@PDC catalyst remained stable without any change in its porous structure after six runs. Reduction of New Fuchsin (NF) In addition, we performed the reduction of a cationic triarylmethane dye (new fuchsin, NF); the chemical structure and characteristics of dye used in the study is shown in Table S9, (Supporting Information). The reduction process of NF dye was monitored using UV–vis spectrophotometry, as shown in Figure 8. Note that the reaction does not proceed significantly in the absence of catalyst, indicating the indispensable role of the catalyst for the NF reduction. As shown in Figure 8b–f, the absorbance peak of NF at 543 nm decreases to a different extent depending on the added amount of 0.5, 1.0, 1.5, 2.0, and 3.0 mg of Ru@PDC catalyst. The NF solution changes red color to colorless when the reduced NF is formed, confirming the catalytic activities (inset of Figure 8b–f). As revealed in Figure 8g, the absorbance at 543 nm disappears within 9 min after the introduction of 3.0 mg of Ru@PDC catalyst. In contrast, 30, 18, 16, and 13 min are required to complete the reduction by adding 0.5, 1.0, 1.5, and 2.0 mg of catalysts, respectively (see Figure 8h). It suggests that an increased catalyst dosage should result in fast diffusion of dye molecules/reagents on the catalyst surface and thus enhance the catalytic activity which may be reflected in the apparent rate constant kapp measurements (Figure 8h). The kapp value is evaluated to be 0.1911, 0.3061, 0.4019, 0.6903, and 0.7601 min–1 for addition of 0.5, 1.0, 1.5, 2.0, and 3.0 mg of catalyst, respectively (see Figure 8i). These results indicate that the reaction follows a pseudo-first-order kinetics because of the presence of excessive NaBH4. The kapp obtained for Ru@PDC (0.7601 min–1) turns out to be much larger than that of graphene quantum dots (kGQDs = 0.0263 min–1).60 The fact may be caused by (i) a large surface area of Ru@PDC to effectively adsorb a more amount of NF molecules and (ii) fast electron transfer from NaBH4 to the adsorbed NF dye molecules via the catalyst. Thus, it facilitates the reduction of organic pollutants absorbed on the catalyst surface. Figure 8 UV–vis spectra for the reduction of NF dye in aqueous medium in the (a) absence of catalyst, in contrast to the presence of (b) 0.5, (c) 1.0, (d) 1.5, (e) 2.0, and (f) 3.0 mg of Ru@PDC catalyst, (g) plot of ln(At/A0) vs time for catalytic reduction of NF at different catalyst dosages, (h) linear region of plot of ln(At/A0) vs time of catalytic reduction of NF used for calculation of kapp, and (i) dependence of kapp on catalyst dosage for reduction of NF. Conclusions In this work, a porous and heterogeneous stable catalyst, Ru@PDC, was successfully prepared and characterized. The catalyst exhibit enhanced catalytic activity for the reduction of inorganic complex and cationic dye with higher reaction kinetics as compared with other catalysts. Furthermore, the as-prepared nanocatalyst possesses several advantages: (i) it can be easily separated from the reaction mixture, (ii) not much loss of catalytic activity is found even after several cycles of reuse, (iii) it takes short time (∼30 s) to complete the reaction showing superior activity, (iv) the catalyst was fabricated using plastic as solid waste feedstock, and (v) moreover, the catalyst has been used for perspective applications. The catalysts were found to be stable and effective for more than 6 runs, with a conversion efficiency of ∼98%. The decrease of conversion can be probably attributed to a reduction in the surface active sites of the catalyst. Experimental Section Materials Ruthenium(III) acetylacetonate (Ru(acac)3, ≥99.9%), ruthenium black (Ru/C, ≥98%), potassium ferricyanide (K3[Fe(CN)6], 99%), New Fuchsin (NF), sodium borohydride (NaBH4, 99.99%), and sodium thiosulphate (Na2S2O3, ≥98%) were purchased from Sigma-Aldrich. All other chemicals belonged to analytical grade, and all solutions were freshly prepared using Milli-Q water. Preparation of Porous Carbon Plastic-derived carbon (PDC) have been prepared from soft drink plastics collected from a local market at Taipei, which were utilized as carbon sources according to the methods reported previously.7−9 Typically, waste plastic bottles shredded into small size of 30–50 mm pieces (∼2.0 g) were added in the 100 mL capacity Teflon-lined stainless steel autoclave. The sealed autoclave was then heated to the temperature (ramp at 20 °C min–1) in the muffle furnace at ∼300 °C for 6 h. Subsequently, the carbonaceous material was cooled slowly back to room temperature (RT), followed by thorough washing with copious amounts of benzene and ethanol, and then air-dried at 100 °C overnight. The PDC samples thus obtained were represented to be PDC-x, where x denotes the final carbonization temperature (in °C) used. To enhance their porosities, the PDC substrates were pyrolyzed again under the condition of flowing CO2 (flow rate 30 mL min–1) at 400 °C for 30 min. In comparison, the carbon sources generated by other plastic materials such as HDPE, LDPE, PC, and polypropylene showed the maximum extent of oil formation containing aromatic hydrocarbons to form CSs. The mechanism of char formation in the thermal process of polymer degradation was interpreted appreciably elsewhere.3,4 Furthermore, we have analyzed the chemical composition of plastic wastes and their elemental composition of plastic-derived char (before activation), PDC (after activation), and Ru@PDC as displayed in Tables S2–S4, Supporting Information. In addition, the yields of carbon after carbonization and activation under different conditions were presented in Table S5, Supporting Information. Physical Activation In general, the endothermic reactions for physical activation of the carbonaceous material by using water vapor steam and CO2 are shown in eqs 8 and 9(61) 8 9 10 11 Besides, the C + H2O reaction in eq 8 is accompanied by the formation of CO2 + H2, while catalyzed on the carbon surface as shown in eq 10. Because of endothermic reactions for eqs 8 and 9, the activation process may be controlled accurately in the heating furnace. External heating to remain high temperature is required to drive. In contrast to the reactions 8 and 9 which can be driven accurately at high temperature, the reaction 11 is extremely exothermic and its progression is difficult to control. A decrease in the average particle size and the product yield may happen as a result of over-heating the external carbon surface.62 Preparation of the Ru@PDC Catalyst Ru@PDC nanocomposite was obtained by immobilization of Ru NPs on the PDC-800 support. In brief, 0.2 g of the as-prepared PDC-800 powdered sample was mixed with Ru(acac)3 for 4.01 wt % loading in 5.0 mL tetrahydrofuran. Then, the mixture was removed into a 50 mL Teflon-coated microwave reactor for microwave irradiation at power of 300 W for 1 h. The Ru0 state was reduced effectively from the Ru3+ ions upon irradiation and was dispersive on the mesoporous PDC carbon support. Catalyzed Reduction of Ferrocyanate(III) To conduct the reduction reaction, we prepared 3.0 mL of 3 × 10–3 M [K3Fe(CN)6] into 1.0 mg mL–1 Ru@PDC catalyst and then added rapidly 0.2 mL of 0.04 M ice-cold fresh NaBH4 or Na2S2O3. As the reaction proceeded, the solution in yellow faded to be colorless. Subsequently, the kinetic measurements were carried out for K3Fe(CN)6 by using UV–vis spectroscopy fixed at 420 nm to monitor the reduction reaction in a quartz cuvette. When the reaction was complete, the catalyst was removed by using an ultracentrifuge for further investigation of its reusability. Catalyzed Reduction of the Cationic Dye Experimental procedure for the cationic dye reduction is based on an earlier report.63 Typically, 1.0 mg mL–1 of solid catalyst (Ru@PDC) was added into 3.0 mL of NF (5 mM) dye solution at RT under vigorous magnetic stirring in dark condition for 5 min to allow the dye molecules adsorbed physically onto the Ru@PDC composite. Then, 0.1 mL of 0.04 M ice-cold NaBH4 was added and the mixture was kept stirring under ambient conditions. Approximately, 3.0 mL of sample was taken from the mixture and the dye solution was analyzed by UV–vis spectrophotometric measurements (at 553 nm) to follow the catalytic reduction reaction. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01680.Additional experimental details including catalyst characterization and ruthenium dispersion measurements; results obtained from pore size distribution; SEM, TEM, EDX, XRD, FT-IR, and UV–vis studies of assorted samples; carbon yield calculation; chemical and elemental composition analyses; comparison of catalytic activity reduction of K3[Fe(CN)6] over the Ru@PDCs and other reported catalyst; kinetic parameters for the Ru@PDC catalyst at different dosages; and chemical structure and some properties of NF dye (PDF) Supplementary Material ao8b01680_si_001.pdf The authors declare no competing financial interest. Acknowledgments The authors are grateful for the financial supports (NSC 102-2113-M-002-009-MY3 to KCL) from the Ministry of Science and Technology (MOST), Taiwan. ==== Refs References a Zhuo C. ; Levendis Y. A. 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PMC006xxxxxx/PMC6644445.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 3145888310.1021/acsomega.8b00596ArticleTwo-Dimensional Double Hydroxide Nanoarchitecture with High Areal and Volumetric Capacitance Deshmukh Abhay D. †Urade Akanksha R. †Nanwani Alisha P. †Deshmukh Kavita A. §Peshwe Dilip R. §Sivaraman Patchaiyappan ∥Dhoble Sanjay J. ‡Gupta Bipin Kumar ⊥†Energy Materials and Devices Laboratory, Department of Physics and ‡Nanomaterials Research Laboratory, Department of Physics, RTM Nagpur University, Nagpur 440033, India§ Department of MME, Visvesvaraya National Institute of Technology, Nagpur 440010, India∥ Polymer Science and Technology Centre, Naval Materials Research Laboratory (DRDO), Ambernath (E) 421506, India⊥ CSIR-National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, India* E-mail: abhay.d07@gmail.com (A.D.D.).* E-mail: bipinbhu@yahoo.com (B.K.G.).02 07 2018 31 07 2018 3 7 7204 7213 29 03 2018 18 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. The development of high volumetric or areal capacitance energy storage devices is critical for the future electronic devices. Hence, the hunting for next-generation electrode materials and their design is of current interest. The recent work in the two-dimensional metal hydroxide nanomaterials demonstrates its ability as a promising candidate for supercapacitor due to its unique structure and additional redox sites. This study reports a design of freestanding high-mass-loaded copper-cobalt hydroxide interconnected nanosheets for high-volumetric/areal-performance electrode. The unique combination of hydroxide electrode with high mass loading (26 mg/cm2) exhibits high areal and volumetric capacitance of 20.86 F/cm2 (1032 F/cm3) at a current density of 10 mA/cm2. This attributes to the direct growth of hydroxides on porous foam and conductivity of copper, which benefits the electron transport. The asymmetric supercapacitor device exhibits a high energy density of 21.9 mWh/cm3, with superior capacitance retention of 96.55% over 3500 cycles. document-id-old-9ao8b00596document-id-new-14ao-2018-00596qccc-price ==== Body Introduction The proliferating demand for green and clean energy has excited tremendous research efforts in the design and fabrication of the energy storage devices, in particular, supercapacitor. Recent research has focused on improving the specific capacitance of supercapacitor, but a typical challenge for such a system is to increase the volumetric capacitance.1−3 The only way to increase the volumetric capacitance is higher mass loading with three-dimensional (3D) porous network. Nevertheless, the crucial disadvantage of higher mass loading per cubic centimeter is increasing the dead mass of an electrode.4 The highest volumetric capacitance achieved for hydrated ruthenium oxide5 is around 1000–1500 F/cm3, activated graphene6,7 can reach the capacitance of 200–350 F/cm3, activated carbon8,9 can reach 60–100 F/cm3, and Mxene10,11 can reach 1000 F/cm3 but only with low mass loading or in thin film. The two-dimensional solids are gaining tremendous attention, in particular, layered double hydroxide (LDH) with a general formula of [M12+–x M3+x(OH)2]12−22 (M2+and M3+, the bivalent and trivalent metal cations, respectively), due to the double benefit of their large active surface area23−25 and high density of redox active species. Metal hydroxides had been studied and proved to be a promising candidate for supercapacitors exhibiting gravimetric capacitance exceeds that of reported metal oxide materials. However, volumetric capacitance is limited due to lower mass loading of active material. With increasing mass loading per centimeter cubic, metal oxide/hydroxides show decrease in cyclability as well as capacitance owing to lower surface active sites and etching of active material. To date, all oxide/hydroxides have been studied in thin film with lower mass loading.26−30 Layered double hydroxide (LDH) nanosheets as electrode for supercapacitor application have raised particular interest.30,31 Several LDHs have been studied, including Ni–Co LDHs, Ni–Mn LDHs, Ni–Fe LDHs, and Cu–Co LDHs.31−38 Although there are some publications on preparations39−43 and applications of Cu–Co LDHs, to our knowledge, the higher mass loading of Cu–Co LDHs for high areal/volumetric supercapacitor application has not been reported so far. The higher stability, good conductivity, excellent electrochemical activity, and multiple functionality over other metal hydroxides have let the Cu–Co LDHs to be an ideal electrode for supercapacitor application. The copper-cobalt double hydroxides reported in this study were prepared by growing copper-cobalt double hydroxide higher mass loading on the nickel foam with porous and layered nanostructures for supercapacitors. Here, we report a two-dimensional copper-cobalt double hydroxide with different mass loadings per centimeter square, demonstrating the improved electrochemical properties when used as electrode material in a supercapacitor. The unique combination of double hydroxide electrode with a high mass loading of 26 mg/cm2 exhibits a high volumetric capacitance of 1032 F/cm3 at a current density of 10 mA, which will open up new opportunities to develop a high volumetric supercapacitor and a new field in double hydroxide materials. Results and Discussion A schematic representation of the deposition process of high-density layered copper-cobalt hydroxide (L-CCH) architecture is shown in Figure 1. The electrodeposited L-CCH electrode results in the formation of a porous 3D-interconnected two-dimensional layered framework with a large oxygen-containing functional group, which was beneficial for increasing the electrochemical sites within the network. The mass loading of the L-CCH can easily be tuned with a variation of concentration of solute in the electrodeposition process. The change in concentration of solute has been preferred over time to have higher density of L-CCH electrode instead of growth in the layered structure (Figure 1a). The growth mechanism and morphology of L-CCH network is well understood from the field emission scanning electron microscopy (FESEM) images that reveal the growth of 2D layered like interconnected structure (Figure 1b). The insightful observation indicates the dense network of L-CCH nanosheets that are approximately 100 nm in width and few microns in length (Figure 1c). This interconnected structure can play an important role in increasing the surface area and electrochemical sites for absorption. Further energy-dispersive spectroscopy (EDS) analysis was carried out, which confirms the presence of cobalt, copper, and oxygen, as shown in Figure 1f. The detailed spectroscopic analysis of L-CCH structure was explored by Fourier transform infrared spectroscopy (FTIR) (Figure 1d). The peaks at 3134 and 3438 cm–1 are assigned to O–H stretching vibration of water molecules in the interlayer and hydrogen-bonded hydroxyl group of copper-cobalt hydroxide. The other absorption bands observed below 700 cm–1 are associated with metal oxygen stretching and bending modes. The spectrum shows a distinct peak at 524 and 541 cm–1, indicating the absorptions associated with Cu–O stretching and Cu–OH bending vibrations, whereas that at 511 cm–1 corresponds to the Co–O stretching vibration.44Figure 1e shows the X-ray diffraction (XRD) pattern of the L-CCH electrode, which shows the 2θ values at 13°, 26°, 33°, and 36°, corresponding to (001), (002), (120), and (121) lattice plane, corresponding to copper hydroxide (JCPDS 42-0746), and the peaks observed at 43°, 51°, 59°, and 74° 2θ values, corresponding to (200), (102), (003), and (311) lattice plane, which are in good agreement with the XRD data of cobalt hydroxide of JCPDS 300443. The average grain size was estimated by using the Debye–Scherer formula: D = 0.9 λ/β cos(θ), where λ is 1.54A° for the wavelength of Cu Kα radiation, “β” is full width at half-maximum, and “θ” is the Bragg angle. The average grain size was found to be in the range of 100 nm, which is in agreement with the SEM results. Figure 1 (a) Schematic representation of the synthesis procedure of L-CCH electrode showing different mass loadings. (b, c) Microscopic images of L-CCH electrode confirm the high mass loading with interconnected layered growth of double hydroxide. (d) Fourier transform infrared spectra of L-CCH electrode shed the light on types of bonding and surface groups. (e) X-ray diffraction spectra of L-CCH, which is a combination of copper and cobalt hydroxide results in double hydroxide electrode. (f) Energy-dispersive spectrum for L-CCH, which shows the pure formation of double hydroxide. Layered double hydroxides (LDHs) have been extensively studied as pseudocapacitive material and acknowledged as superior to metal oxides due to higher electrochemical activity. However, the performance of hydroxides/oxides completely depends on the electrode film thickness, which limits their use in requisite high volumetric or areal capacitance devices. Moreover, thick-film electrode without insulating binder deteriorates the performance of metal oxide/hydroxide due to collapse of 3D architecture as well as limited ionic transport in the inner volume of the thicker electrode. To enhance the volumetric capacitance of L-CCH electrode, a higher mass loading of 26 mg/cm2 electrode was examined. In the initial experiments, we found that the volumetric capacitance of lower mass loading L-CCH1 is negligible compared to volumetric capacitance of high mass loading of L-CCH3. First, the electrochemical properties of different areal density L-CCH electrodes were tested in 2 M KOH electrolyte. The electrically conductive layer allows the thicker film with the high mass loading formation without any binder. The obtained cyclic voltammetry (CV) curves in 2 M KOH from 0.0 to 0.45 V potential versus Ag/AgCl are shown in Figure 2a. It is clearly seen from the typical CV curves of L-CCH electrode that the capacitance increases by a factor of 10 in the higher-areal-density electrode. Galvanostatic charge/discharge measurement (Figure 2b) was carried out for the L-CCH1, L-CCH2, and L-CCH3 electrode at a current density of 20 mA/cm2, which shows the discharge time of 12.63, 32.72, and 192 s, respectively, suggesting the formation of more redox sites in higher mass loading, which is in agreement with CV results. Figure 2c shows the CVs of L-CCH3 electrode at a scan rate ranging from 5 to 100 mV/s showing the semirectangular shape retained even at a very high scan rate of 100 mV/s. The semirectangular shape may be explained by the existence of double-layer capacitance as well as pseudocapacitive behavior. The possible electrochemical faradic process of L-CCH electrodes in 2 M KOH as electrolyte is 1 2 To study the areal and volumetric capacitance of the L-CCH3, electrode galvanostatic charge/discharge measurements were performed at 10, 20, 30, 40, 50, 60, 80, 102, 122, 204, and 306 mA/cm2 in 2 M KOH, shown in Figure 2d. A plot of current density verses areal/volumetric capacitance for the high mass loading (L-CCH3) electrode is shown in Figure 2e. The excellent volumetric capacitance of 1032 F/cm3 and areal capacitance of 20.86 F/cm2 were measured at a current density of 10 mA/cm2. The capacitance of L-CCH3 electrode was found to be 18.04 F/cm2, which is 18 times higher than that of L-CCH1 electrode (1.06 F/cm2) at the current density of 20 mA/cm2 and retains 50% of capacitance even at a high current density of 100 mA/cm2 (shown in Supporting Information Tables S1 and S2). This high capacitance value at a high scan rate is assumed due to the more porous nature of L-CCH3 electrode, which increases the rate of intercalation/deintercalation of an electrolyte ion in the L-CCH 2D-interconnected layered framework. In addition, it also reveals that the volumetric/areal capacitance is dependent on the mass loading of L-CCH electrode. The cyclic stability of the electrode was examined for >2000 charge/discharge cycles at a very high current density of 60 mA/cm2 (Figure 2f). Usually, at higher current density, the electrode stability decreases rapidly. However, in this study, the capacity retention of 80% was found after 2000 cycles at 60 mA/cm2. Figure 2 (a) Cyclic voltammetry curve at 5 mV/s for different mass loadings. (b) Galvanostatic charge/discharge curves of different mass loadings at 20 mA/cm2 current density. (c) Cyclic voltammetry curves recorded at various scan rates for higher areal density electrode. (d) Galvanostatic charge/discharge curve of higher areal density electrode at different current densities. (e) Variation of areal and volumetric capacitance with current densities calculated from galvanostatic charge/discharge curve. (f) Cyclic stability performance for 2000 charge/discharge cycles; the inset figure shows last 10 charge/discharge cycles. (g) Ragone plot of the estimated volumetric energy density and volumetric power density at various charge/discharge rates. To elucidate the charge/discharge kinetics and semirectangular behavior of L-CCH electrode for lower and higher mass loading, the method given by Trasatti45,46 et al. was employed. According to this method, the total charge storage of an electrode is a sum of contribution due to an inner surface, where faradic reaction occurs from the slow H+ ion donating species, and the outer surface, where redox reaction can occur from fast charge transfer. The capacitive charge (including both pseudocapacitance and electric double-layer capacitance) depends on the potential scan rate given by Trasatti in a way as follows 3 4 where Q* is the total charge obtained by integrating the CV curve at different scan rates. The capacitive charge contribution and the total charge contribution can be calculated from the graphs, as shown in Figure 3a,b. In this process, the total charge storage can be calculated from the y-axis intercept of Q* verses V1/2 and the charge stored on the outer surface of the electrode can be calculated from the plot of Q* against V−1/2. Moreover, the difference between the total charge and the charge stored on the outer surface will give the value of the charge stored in the inner surface. As shown in Figure 3a, the value of the maximum total charge stored is found to be 8.3486 C/cm2, which is equivalent to the maximum areal capacitance ∼20.86 F/cm2 for the potential window 0.45 V. Furthermore, the charge stored at the outer surface, (Figure 3b) was found to be 1.606 C/cm2 (∼3.56 F/cm2) and the charge stored in the inner surface (diffusion-controlled redox capacity) was found to be 6.7426 C/cm2 (∼14.98 F/cm2). From Figure 3c, it is clear that the diffusion-controlled redox capacity increases on increasing the mass loading. The L-CCH3 electrode exhibited 80.77% diffusion-controlled redox capacity, whereas L-CCH2 and L-CCH1 exhibited 45.94 and 43.66%, respectively. Increase in the total charge might be the reason for the corresponding higher areal and volumetric capacitance for L-CCH3 electrode compared to that of L-CCH2 and L-CCH1. It is speculated that the surface Grotthuss mechanism18 is responsible for the diffusion of H+ ions to the inner region surface. Figure 3 (a) Plot of 1/Q* against V1/2 to find the total charge (Q) stored by the electrode material. (b) Plot of Q against V–1/2 to find the charge stored only on the outer surface of the electrode material (Q*outer). (c) Contributions of double-layer capacity and diffusion-controlled redox capacity to the total capacity of the electrode. (d) Plot of log I and log V to find the value of b (b = 0.53, 0.75, and 0.71 for L-CCH1, L-CCH2, and L-CCH3) The total electrochemical charge storage can be obtained by integrating the CV curve, and this charge originates from two contributions: the faradic contribution limited by ion diffusion together with fast charge-transfer process at the surface and the non-faradic contribution from the fast electric double-layer effect. This effect can be analyzed by the CV curve at the different scan rates using the power law47−49i = aVb, where “a” and “b” are adjustable parameters. b value can be determined by the slope of log i against log V. Ideally, if b value is ∼0.5, then the capacity response is diffusion controlled and it satisfies Cottrell’s equation9i = V1/2 and b = 1 shows purely capacitive response (including both double-layer capacitance and diffusion-controlled redox capacitance). From Figure 3d, the b value of L-CCH3 is found to be 0.53, which demonstrates the dominancy of diffusion-controlled redox capacitance, which agrees with our results. For L-CCH1 and L-CCH2, b values were 0.75 and 0.71, respectively, which shows the capacitive response. This confirms the higher mass of our L-CCH3 electrode increases the diffusion-controlled redox activity and is dominant in this study. The electrochemical impedance spectroscopy (EIS) is an important characterization tool to evaluate the resistive behavior of the electrode. Figure 4a shows the respective Nyquist plots for three different electrodes from f = 0.01 to 10 000 Hz, where Z′ is the real part and Z″ is the imaginary part of the impedance, respectively. The Nyquist plots consist of three characteristic regions:50,51 (1) a low-frequency region that is represented by an inclined line along the imaginary axis which shows capacitive behavior also known as double-layer capacitive region; (2) a high-frequency region represented by a partial semicircle which shows the blocking behavior of the supercapacitor (in this region, the supercapacitor behaves as a pure resistor and the resistance values determines the charge-transfer and series resistance of the electrode); and (3) a middle-frequency range that shows the effect of electrode thickness and the porosity on the diffusion of ions from electrolyte to electrode. The inset of the Figure 4g shows the equivalent circuit [R(C{RQ})W] used for the fitting curve of L-CCH3. Rs is the equivalent series resistance of an electrolyte, Rct is the charge-transfer resistance during the faradic reaction, Cd is the electric double-layer resistance, Zw is the Warburg impedance that represents the impedance of the diffusion controlling process in the electrolyte, and CPE is the constant-phase element. Electrochemical impedance is defined as51,52ZCPE = [Y0(jω)n]−1, where the unit of Y0 is 1 S/cm2 and is independent of frequency and n is the exponent whose values varies between −1 and +1; when n = −1, the CPE is pure inductor; when n = 0, the CPE behaves as a pure resistor; and when n = 1, CPE is pure capacitor. In our experiment, the CPE value for L-CCH3 is found to be n = 1.0, which shows that CPE behaves like a pure capacitor, which is in good agreement with our results. Nyquist plots clearly show that in the high-frequency region, L-CCH3 has a lower series resistance value (Rs = 0.59 Ω) than that of L-CCH1 and L-CCH2 (Supporting Information Figure S1), which implies that the resistance of an electrolyte solution is lesser in L-CCH3 electrode. Moreover, the impedance spectrum of L-CCH3 shows a smaller semicircle (Rct = 0.11 Ω) than that that of the L-CCH1 and L-CCH2, which suggests that the L-CCH3 has a good electrical conductivity between the prepared electrode and current collecting electrode, as well as lower charge-transfer resistance, which is attributed to the significant increase in the capacitance value of L-CCH3. Additionally, in the low-frequency region, the ideal straight line implies a higher rate of electrolyte diffusion and mass transfer. Figure 4c shows the good access to the electrolyte and ionic diffusion even after 2000 cycles attributed to the excellent electrical conductivity and very low internal resistance (Figure 4b). The Bode plots show phase angle values obtained for higher mass loading electrode is close to 74, which is almost near to that of the ideal capacitor, suggesting that the prepared material is suitable for the fabrication of low-leakage capacitor. Moreover, no significant change occurs in the phase angle after 2000 cycles. The EIS data strongly supports the excellent electrochemical activity and high rate capability. Figure 4 (a) Nyquist plot (−Z″ vs Z′ plot) for the different areal density electrodes within the frequency range from 0.01 to 100 000 Hz. (b) Bode phase angle plot (phase angle against frequency for different areal density electrodes). (c) Plot of the real part of capacitance against frequency for different areal density electrodes. (d) Plot of the imaginary part of capacitance against frequency for different areal density electrodes. (e) Normalized reactive power \|Q\|/\|S\| and reactive power \|P\|/\|S\| vs frequency plots (f) Nyquist plot (−Z″ vs Z′ plot) for L-CCH3 before and after 2000 cycles; the inset shows the equivalent circuit used for fitting the data. (g) Bode phase angle plot before and after cycles for L-CCH3 The relaxation time constant τ is a measure of how fast the device can discharge and obtained from the analysis of the complex plots. The complex capacitance is expressed as C(ω) = C′(ω) + jC″(ω), where C′(ω) is the real part of the capacitance that shows the amount of stored energy in farad and C″(ω) is the imaginary part of the capacitance that shows the energy dissipation as a function of the angular frequency and they are given by22 5 6 where Z′ and Z″ are the real and imaginary part of the impedance, ω = 2πf, and Z(ω) = Z′(ω) + jZ″(ω), where ω is the angular frequency. The complex power is defined as S(ω) = P(ω) + jQ(ω), where P(ω) is the real part of complex power called active power and Q(ω) is the imaginary part of the complex power called reactive power, and they are given by 7 8 where ΔVrms = ΔVmax/√2 and ΔVmax is the maximum value of the potential (here, 0.45 V). From Figure 4e, the relaxation time constant (τ = 1/f) can be calculated from either the frequency corresponding to the half of the maximum value of the real part of capacitance, that is, from C′(ω) against the frequency plot or peak frequency corresponding to the C″(ω) against frequency plots. As shown in Figure 4c, the real part of the capacitance decreases as the frequency increases, which is a characteristic of the electrode material and the electrode/electrolyte interface. The more detailed characteristics of relaxation time constant can be obtained from the normalized power against frequency plot. Figure 4f shows the plot of the real part of power P/S and imaginary part Q/S against frequency plots. The power dissipated into the systems can be analyzed from the active power P/S. The impedance behavior of the supercapacitor varies from the ideal capacitive behavior at a low frequency where no power is dissipated to the pure resistive behavior at a high frequency where P = 100%. In fact, P/S and Q/S show the opposite trend with respect to the frequency. The crossing of the two plots P/S and Q/S appears at ϕ = 45° and will give the value of the frequency corresponding to the relaxation time. It defines the capacitive behavior at frequencies below 1/τ and resistive behavior at frequencies above 1/τ. From the crossing point of the two plots, the relaxation time has been calculated for higher and lower mass loading. The obtained relaxation times for L-CH1, L-CCH2, and L-CCH3 were 1, 2, and 12 s, respectively. Figure 5 (a) Cyclic voltammogram of carbon cloth as a negative electrode and L-CCH3 as positive electrode at 5 mV/sec. (b) Cyclic voltammetry curves of an asymmetric L-CCH3//CC electrode at different scan rates. (c) Galvanostatic charge/discharge curves at different current densities from 3 to 10.5 mA/cm2. (d) Plot of areal and volumetric capacitance as a function of current densities. (e) Cyclic performance during 3500 charge/discharge cycles. It is clearly seen from the figure that only 0.1% loss is observed during cyclic performance; inset figure shows the last 10 charge/discharge cycles. (f) Nyquist plot (−Z″ vs Z′ plot) of L-CCH3//activated carbon cloth (ACC) electrode within the frequency range from 0.01 to 100 000 Hz. (g) Plot of real and imaginary capacitance against frequency. (h) Normalized power against frequency. (i) Ragone plot showing the relationship between the volumetric energy density and power density of a typical electrolytic capacitor, supercapacitor, Li thin-film batteries, and our prepared L-CCH3//ACC electrode. To study the practical application of high mass loading L-CCH3 architecture, an asymmetric supercapacitor is fabricated by using L-CCH3 as a positive electrode and the activated carbon cloth (ACC) as a negative electrode. Figure 5a shows the CV curves of carbon cloth within a 0.0 to 1.0 V and L-CCH3 electrode within a 0.0 to 0.45 V voltage window. The prepared L-CCH3//ACC asymmetric device operates at a higher voltage window from 0.0 to 1.2 V at different scan rates ranging from 10 to 200 mV/s, exhibiting the combined behavior of carbon cloth and L-CCH electrode (Figure 5b). The CV curves show obvious reduction oxidation peak within a 0 to 1.2 V potential window. Interestingly, with the increasing scan rate, no major shifts are observed in the oxidation reduction peak, which shows the device ability to sustain a high charge/discharge rate and an excellent ability of ion diffusion into the electrode surface. Figure 5c shows the galvanostatic charge/discharge curve of L-CCH3//ACC at various current densities from 3 to 10.5 mA/cm2. The areal capacitance values calculated from charge-discharge (CD) curves are 2.43, 2.32, 2.24, 2.18, 2.14, 2.04, and 1.98 F/cm2 and 109.73, 104.38, 100.81, 98.14, 96.35, 92.11, and 89.22 F/cm3 at the current densities of 3, 4.5, 6, 7.5, 9, 10.5, and 12 mA, respectively (shown in Supporting Information Table S4). The comparative study of different areal capacitance values and their energy densities is shown in Supporting Information Table S5, which shows the highest performance of L-CCH electrode in terms of area/volumetric capacitance as well as energy density. The electrochemical impedance spectroscopy of an asymmetric device is also studied to gain the understanding of electrochemical behavior. Figure 5d represents plot of areal and volumetric capacitance as a function of current densities and Figure 5e shows the cyclic performance during 3500 charge/discharge cycles. Figure 5f shows the Nyquist plot and the corresponding equivalent circuit. The asymmetric device shows a very low series resistance Rs = 0.9 Ω and a charge-transfer resistance of 2.11 Ω. The relatively low values of Rs and Rct represent the higher accessibility in the diffusion of ions in the electrolyte to the electrode material during charge/discharge cycles, which is responsible for an excellent electrochemical performance of an asymmetric device. To study the stability of an asymmetric device, the cycle stability tests were carried out at 8.33 mA/cm2 current density, shown in Figure 5e. The asymmetric device exhibits an excellent cyclic stability with capacitive retention of 96.55% after 3500 cycles. Figure 5i shows the Ragone plot of several commercially available energy storage devices. It can be seen from the plot that the energy density of L-CCH3//ACC supercapacitor reached close to the energy density of Li thin-film batteries and power density approached close to the power density of the 25 mF supercapacitor. The imperative feature of this device is a very small characteristic relaxation time constant τ0 (the minimum time required to discharge all energy from the device), which is only 12 s. Thus, the areal/volumetric capacitance values described above are probably near to the maximum values possible for LDH materials in general. Thus, the fact is that high volumetric and areal capacitance of L-CCH material may also enable LDHs use in supercapacitors. Thus, this work opens up exciting possibilities of developing higher mass loading of LDH electrode supercapacitor devices using a large variety of combinations of metals and their chemistries. Conclusions In summary, a simple and reproducible method has been adopted to demonstrate the electrochemical performance of copper-cobalt hydroxide with higher mass loading in aqueous electrolyte. Benefits of the double metal hydroxide and 2D structure contribute to the ultra-high volumetric/areal pseudocapacitive performance. The 2D hydroxide structure reveals an enhanced volumetric capacitance response of 1032 F/cm3. The enhanced capacitance was due to the high mass loading layered structure with porous structure leading to more electrochemical sites for redox response, as well as high ionic diffusion and better electron conductivity of copper in copper-cobalt hydroxide electrode. The prepared asymmetric supercapacitor showed a high areal and volumetric capacitance of 2.43 F/cm2 (109 F/cm3) at 2.41 mA/cm2 and exhibited an excellent cyclic stability with 96.55% capacitive retention after 3500 cycles at the current density of 8.33 mA/cm2. Thus, this effective method of fabricating a freestanding metal hydroxide/carbon asymmetric device paves the way for practical application of an energy storage device. Methods Synthesis of L-CCH/Ni-Foam Electrode All chemicals were of analytical grade and used without further purification. Before depositing copper-cobalt LDHs, nickel foam was washed with 0.1 M HCl solution for 3 min and cleaned with deionized water to remove NiO layer from its surface. The cleaned nickel foam was dried in a vacuum oven overnight at 60 °C and used as the working electrode (1.5 cm × 1 cm). Platinum and Ag/AgCl were used as the counter and reference electrode, respectively. The Cu–Co LDHs were electrochemically deposited by dissolving 4 mM Cu(NO3)2·3H2O and 8 mM of Co(NO3)2·6H2O in 30 mL of distilled water at a constant voltage of −1 V for 300 s. Further, the electrodeposited Cu–Co LHDs were cleaned with distilled water and dried in vacuum oven overnight at 80 °C. The mass loading of the electrode was measured before and after electrodeposition. The same procedure is repeated by taking different concentrations of copper nitrate trihydrate and cobalt nitrate hexahydrate to prepare a different mass loading electrode to compare the results. The different mass loading electrode of 4.69, 8.77, and 26.53 mg/cm2 are denoted as L-CCH1, L-CCH2, and L-CCH3, respectively. Characterization Powder X-ray diffraction (PXRD) patterns were recorded with a PAN-analytical diffractometer equipped with Cu Kα (λ = 1.5405 Å) X-ray source in geometry and 0.5 s dwelling time with a proportional detector. An anti-scattering incident slit (2 mm) and nickel filter were used. The tube voltage and current were 40 kV and 40 mA, respectively. Scanning electron microscopy (SEM) was performed on a JEOL JSM 7610F FESEM equipped with energy-dispersive spectroscopy (EDS) Oxford instruments, X-Max with coating unit (make: JEOL, model: JEC-3000FC). The EDS mapping was obtained at low magnification at random points of the electrodeposited film. Fourier transform infrared spectra were obtained from the Perkin Elmer Spectrum one instrument. All electrochemical measurements were carried out using a Metrohm Autolab 128N potentiostat (Netherland) with 2 M KOH aqueous electrolyte in electrochemical cell. Ag/AgCl and platinum foil were used as pseudo reference electrode and counter electrode, respectively, whereas electrodeposited L-CCH was the working electrode. Electrochemical Measurement Electrochemical performance of L-CCH electrodes was studied by using a three-electrode configuration and 2 M KOH as an electrolyte. Platinum foil and silver/silver chloride electrode are used as a counter and a reference electrode, respectively. Cyclic voltammetry was performed on a Metrohm Autolab-128N potentiostat. The electrodeposited L-CCH electrodes were used as a working electrode in a three-electrode system. CV measurement was carried out at different scan rates in the potential window 0 to 0.45 V. The charge/discharge study was performed at different current densities of 10, 20, 30, 40, 50, 60, 80, 102, 122, 142, 204, and 306 mA/cm2 in a potential window of 0 to 0.45 V. Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00596.Performance data for three independent CCH pseudocapacitive electrodes, areal and volumetric capacitance at different current densities from 10 to 122 mA/cm2, performance comparison of copper-cobalt hydroxide pseudocapacitor with oxides, areal and volumetric capacitance of an asymmetric device at different current densities, performance comparison of L-CCH//ACC two-electrode pseudocapacitor, equivalent circuit of L-CCH-1 and L-CCH-2, CV curves at different cell voltages at a scan rate of 100 mV/s, CD curves at different cell voltages, plot of the bode modulus and phase angle against frequency for two-electrode, CV curve before and after 3500 cycles at a scan rate of 10 mV/s (PDF) Supplementary Material ao8b00596_si_001.pdf Author Contributions All authors contributed to the experimental design and data analyses. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript The authors declare no competing financial interest. Acknowledgments A.D.D. acknowledges the financial support from Naval Research Board (NRB), DRDO New Delhi (NRB Sanctioned no: DRDO/NRB/4003/PG/338). A.D.D. acknowledges ENVIRON Care Product for providing the activated carbon cloth (ACC) for this research work. A.D.D. also acknowledges Celgard, LLC, North Carolina for their support in making available the material for our research. ==== Refs References Simon P. ; Gogotsi Y. Materials for electrochemical capacitors . Nat. Mater. 2008 , 7 , 845 –854 . 10.1038/nmat2297 .18956000 Augustyn V. ; et al. High-rate electrochemical energy storage through Li-intercalation pseudocapacitance . Nat. Mater. 2013 , 12 , 518 –522 . 10.1038/nmat3601 .23584143 Gogotsi Y. ; Simon P. True performance metrics in electrochemical energy storage . 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PMC006xxxxxx/PMC6644446.txt	==== Front ACS OmegaACS OmegaaoacsodfACS Omega2470-1343American Chemical Society 10.1021/acsomega.8b00488ArticleRegulating the Microstructure of Intumescent Flame-Retardant Linear Low-Density Polyethylene/Nylon Six Blends for Simultaneously Improving the Flame Retardancy, Mechanical Properties, and Water Resistance Zhao Pan Lu Chang Gao Xi-ping Yao Da-Hu Cao Cheng-Lin Luo Yu-Jing Chemical Engineering & Pharmaceutics School, Henan University of Science and Technology, 263, Kaiyuan Avenue, Luoyang 471023, China E-mail: luchang139@126.com (C.L.).27 06 2018 30 06 2018 3 6 6962 6970 15 03 2018 14 06 2018 Copyright © 2018 American Chemical Society2018American Chemical SocietyThis is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. A compatibilizer was melt-blended with intumescent flame-retardant linear low-density polyethylene/nylon six blends (LLDPE/PA6/IFR) by different methods, and the effect of microstructure on the flame retardancy, mechanical properties, and water resistance was investigated. Melt-blending compatibilizers with LLDPE/PA6/IFR above the polyamide-6 (PA6) melt temperature formed the microstructure with IFR dispersion in the LLDPE matrix and good interphase adhesion between the PA6 phase and the matrix. Compared with the blends with the lack of compatibilizers, although good interphase adhesion improved the mechanical properties and water resistance, IFR dispersion in the LLDPE matrix reduced the flame retardancy sharply. To obtain the microstructure with IFR dispersion in the PA6 phase and strong interface adhesion of the PA6 phase with a matrix, a novel method in which a compatibilizer was melt-blended with LLDPE/PA6/IFR between the melt temperatures of LLDPE and PA6 was employed. The results showed that the flame retardancy, mechanical properties, and water resistance were improved simultaneously. document-id-old-9ao8b00488document-id-new-14ao-2018-00488tccc-price ==== Body 1 Introduction Intumescent flame retardants are now being used more and more in polymer flame retardants because they are more environmentally friendly than traditional halogen-containing flame retardants. The characteristic of intumescent flame retardants is that the combustion chamber can form a honeycomb expansion carbon layer, which reduces the mass transfer and heat transfer between the gas phase and the solidification phase.1−3 Acid source, carbon source, and blowing agent are the three elements of chemically intumescent flame retardants.4−6 In general, ammonium polyphosphate (APP as the acid source and blowing agent), pentaerythritol (PER as the charring agent) and melamine (as the blowing agent) form a traditional IFR system together. However, IFR has poor water resistance and compatibility with many polymer matrices, resulting in the decrease of flame retardancy and the damage to the mechanical properties of polymer composites. These drawbacks will restrict their wide industrial applications. Microencapsulation of IFR is regarded as an efficient method to get rid of these drawbacks mentioned above.7−11 The microencapsulated IFR particles have a core–shell structure, which allows the isolation of encapsulated substances from the surrounding and thus improves their compatibility with the polymer matrix and the water resistance. In general, the encapsulating shell, such as polyurethane (PU), melamine–formaldehyde, melamine, silicon resin, or urea–melamine–formaldehyde, was prepared by the in situ polymerization method.12−17 Physics encapsulation technology was also employed to prepare microencapsulated IFR particles. Wang18,19 provided a novel method of preparing microencapsulated IFR by the extrusion of melamine phosphate and PER, together with a polypropylene (PP) carrier. The results showed that the IFR encapsulated by the PP carrier improved flame retardancy and water resistance. Zeng20 used PU as a carrier resin to encapsulate melamine pyrophosphate/PER through melt blending, and encapsulated IFR was adopted to flame-retard PP. The results showed that encapsulated IFR endowed PP with better flame retardancy, water resistance, and mechanical properties. Compared with the in situ polymerization method, physics encapsulation technology has the advantages of a simple process and less environmental problems. Polymer blending has gained considerable interest as a suitable way to tailor the properties of polymeric materials without investing in new chemistry.21 However, the flame retardancy of polymer blends is more complex than that of pure polymers because of the complicated microstructure caused by the dispersion of IFR and their synergists in the multiphase.22−24 Lu25 reported that APP had a tendency to disperse in the polyamide-6 (PA6) phase of polystyrene (PS)/PA6 blends. The blends with cocontinuous phase structures have better flame retardancy than that of the blends with the sea-island structure. For the blends of PS/PA6 and acrylonitrile–butadiene–styrene/PA6 with sea-island morphology, Lu26,27 found that blends with organic montmorillonite and APP, respectively, distributed in the phase interface and PA6 phase have better flame retardancy than that of the blends with clay and APP dispersed in the PA6 phase. Jin28 employed the compatibilizer to adjust the microstructure for improving the mechanical properties and flame retardancy of PP/ethylene-octene copolymer/IFR. Along with the increase of compatibilizer content, the blends exhibited different microstructures. The addition of compatibilizers in a proper range caused the simultaneous improvement on mechanical properties and flame retardancy. IFR for flame-retardant polymer blends also encounters the challenges of poor water resistance and damage on mechanical properties. Although the microstructure of polymer blends contributes to the augment of the flame retardancy to a great extent, improving IFR water resistance and decreasing its damage on mechanical properties with resorting to regulating the microstructure have not been studied systematically. In this paper, we attempted to regulate the microstructure of polymer blends for the purpose of overcoming these disadvantages. The microstructure in which IFR localizes in the dispersed phase can be regarded as the microencapsulated IFR in which dispersed phase as the shell encapsulates IFR to improve water resistance. Compatibilizers can be employed to improve the compatibility. Moreover, char-forming polymers chosen as dispersed phase to encapsulate IFR particles could endow the blends with better flame retardancy. The flammability of linear low-density polyethylene (LLDPE)/PA6 blends has limited its applications in packaging and automotive fields. In the LLDPE/PA6 blends, higher affinity of IFR for PA6 than for LLDPE should exhibit the dispersion of IFR in the PA6 phase. Furthermore, it was reported that PA6 acted as the charring agent of APP to enhance the charring performance, resulting in better flame retardancy.29−31 Therefore, the use of IFR as flame retardants was investigated for their potential in improving the fire-retardant behavior of LLDPE/PA6. The IFR system used consists of APP and PER. Meanwhile, maleic anhydride grafted with LLDPE (LLDPE-g-MAH) was chosen as a compatibilizer for enhancing the compatibility of LLDPE/PA6 blends. The compatibility of LLDPE-g-MAH may prohibit the dispersion of IFR in the PA6 phase, while LLDPE, PA6, and IFR are melt-blended with LLDPE-g-MAH simultaneously. In order to prepare the blends with IFR dispersion in the PA6 phase, a two-step processing method was employed: PA6, LLDPE, and IFR were melted above the melting temperatures of LLDPE and PA6 to first prepare blends of LLDPE/PA6/IFR, and then LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR between the melt temperatures of LLDPE and PA6. The effects of the microstructure on the flame retardancy, mechanical properties, and water resistance were investigated. 2 Results and Discussion 2.1 Dispersion of APP and PER in LLDPE/PA6 Blends and Their Microstructure In order to investigate the spontaneous dispersion of APP or PER in the blends of LLDPE/PA6, PA6/LLDPE/APP or PA6/LLDPE/PER prepared by method one was investigated by Fourier transform infrared (FTIR) spectra, respectively. Figure 1 shows the FTIR spectra of the remaining parts of the PA6/LLDPE/IFR or PA6/LLDPE/PER extracted by formic acid or formic acid and ethyl alcohol, respectively. The −CH2– asymmetric stretching (2919 cm–1), symmetric stretching (2849.5 cm–1), bending vibrations (1469 cm–1), and the wagging vibration of C–H (719.4 cm–1) were observed, indicating that the remaining parts consisted of PE. In these blends, PA6 formed a continuous phase because of high PA6 content. The absence of PA6 in the remaining parts indicated that continuous PA6 phase was completely extracted by formic acid. The absence of APP or PER in the remaining parts showed that APP or PER was also completely extracted by formic acid or ethyl alcohol, respectively. If APP or PER was localized in the LLDPE phase of blends, they could not be extracted by formic acid or ethyl alcohol, with the result that APP or PER could be detected in the remaining parts. Therefore, the results that APP or PER was not detected in the remaining parts indicated that APP or PER is localized in the PA6 phase. In the blends tested by FTIR, APP or PER should disperse spontaneously in the polymer with high affinity, due to which the processing temperature was higher than the melting temperature of LLDPE and PA6. Therefore, the localization of APP or PER in the PA6 phase indicated that the affinity of APP or PER is higher for PA6 than for LLDPE because of similar polarity between PA6 and IFR. Figure 1 FTIR spectra of the (a) remaining part of LLDPE/PA6/APP extracted by formic acid and (b) remaining part of LLDPE/PA6/PER extracted by formic acid and ethyl alcohol. Scanning electron microscopy–energy-dispersive spectroscopy (SEM–EDS) was employed to characterize the dispersion of IFR in the blends and the microstructure of the blends, as shown in Figure 2. SEM results showed that APP and PER were irregular particles. The spherical particles with a large dimension were observed in the matrix of C-0. The results of FTIR showed that IFR is spontaneously distributed in the PA6 phase. Therefore, IFR particles were coated by the PA6 phase to form the spherical particles. The EDS results showed that the P content was 1.83 wt %. Generally, the detected depth for the EDS measurement is about 1000 nm, so small amounts of elemental P dispersed in the PA6 phase were detected. Some cavities were seen on the surface of C-0, and there were clear interfaces between PA6 particles and LLDPE, indicating poor compatibility between PA6 and LLDPE, which is due to the different polarity between PA6 and LLDPE, exhibiting a weak interfacial adhesion. The results showed that C-0 formed sea-island morphology in which IFR localized in the dispersed PA6 phase and PA6 phase and the LLDPE matrix exhibited poor interface adhesion. Figure 2 SEM–EDS of IFR and blends prepared by different methods. For blends of C2-M1 and C8-M1, irregular cavities with lengths of about some 10 μm and spherical particles with a dimension of about 3–5 μm were observed. The exfoliation of IFR particles from the LLDPE matrix formed larger irregular cavities, while the PA6 phase formed the small spherical particles in the LLDPE matrix. The results indicated that PA6 and IFR particles were both dispersed in the LLDPE matrix. The EDS results of C2-M1 and C8-M1 also indicated that IFR was dispersed in the LLDPE matrix rather than in the PA6 phase, due to which the percentages of elemental P and O in C2-M1 and C8-M1, respectively, were higher than that of C-0. The cavities formed by the exfoliation of IFR from the LLDPE matrix and clear interfaces between IFR particles and the LLDPE matrix indicated poor interfacial adhesion. Meanwhile, strong interfacial adhesion between PA6 particles and the LLDPE matrix was observed because of vague interface. The results indicated that the compatibilizer improved the interfacial adhesion between the PA6 phase and the LLDPE matrix. The different polarity between IFR and LLDPE caused a weak interfacial adhesion. The SEM–EDS results showed that LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method one formed the sea-island morphology such that both IFR and PA6 localized in the LLDPE matrix and LLDPE-g-MAH improved the interfacial adhesion of the PA6 phase and the LLDPE matrix. Interfacial tensions between the filler and each polymer determine the filler distribution in the filler-filled polymer blends.32,33 In the uncompatibilized blends, IFR dispersion in the PA6 phase rather than in the LLDPE phase indicated that the interfacial tension between IFR and PA6 was lower than that of IFR and LLDPE. When LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR, LLDPE-g-MAH was reacted with PA6 to form a copolymer. The copolymer should migrate to the interface of PA6 and LLDPE to reduce the interfacial tension and phase size, indicating that the interfacial tension between LLDPE and PA6 was lower than that of IFR and PA6. As a result, IFR particles were found to localize in the LLDPE matrix rather than in the PA6 phase. The blends of C2-M2 and C8-M2 were prepared via a two-step process. At the first step, IFR should disperse in the PA6 phase because of similar polarity between PA6 and IFR. When LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR at the second step, LLDPE-g-MAH should not change the dispersion of IFR, due to which the PA6 phase remained solid, exhibiting IFR dispersion in the PA6 phase. The typical empty holes and the exfoliation phenomenon that appeared in C-0, C2-M1, and C8-M1 almost vanished, which suggested good interfacial adhesion. The results indicated that LLDPE-g-MAH improved the compatibility between PA6 and LLDPE. The PA6 phase remained solid when LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR. Therefore, the improvement of interfacial adhesion and compatibility between PA6 particles and LLDPE should be attributed to the reaction between the maleic anhydride group in the LLDPE-g-MAH melt and the amidogen on the surface of solid PA6 particles. The EDS results showed that the percentages of elemental P and O in C2-M2 and C8-M2, respectively, were lower than that of C-0, indicating that LLDPE-g-MAH encapsulated the PA6 phase, demonstrating that IFR dispersed in the PA6 phase was hard to be detected by EDS. As the content of LLDPE-g-MAH increased from 2 to 8 wt %, the coating thickness of LLDPE-g-MAH on the surface was also increased, causing the percentages of P and O to decrease from 14.88 and 1.03 to 14.12 and 0.6%, respectively. Therefore, the sea-island morphology in which IFR localized in the dispersed PA6 phase and LLDPE-g-MAH improved the compatibility of the PA6 phase and the LLDPE matrix was formed in LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method 2. In order to confirm the reaction between LLDPE-g-MAH and PA6 in the blends prepared by method 2, the blends of LLDPE/PA6 (85/15) or LLDPE/PA6/LLDPE-g-MAH (80/15/5) were extracted by dimethylbenzene to remove LLDPE and LLDPE-g-MAH and the remaining parts were tested by FTIR, as shown in Figure 3. The blend of LLDPE/PA6 was melt-blended above the melting temperature of LLDPE and PA6. LLDPE/PA6/LLDPE-g-MAH blend was prepared by method 2. The typical infrared spectra of PA6 were shown, and no characteristic peaks of LLDPE were found in the remaining part of LLDPE/PA6, indicating that whole LLDPE was dissolved by dimethylbenzene and the remaining part consisted of PA6. For the remaining part of LLDPE/PA6/LLDPE-g-MAH prepared by method 2, the peaks at 2934 and 2968 cm–1 shown in LLDPE/PA6 were replaced by 2918 and 2850 cm–1, respectively. The peaks at 2934, 2968 cm–1 in the remaining part of LLDPE/PA6 were the −CH2– asymmetric stretching and symmetric stretching of PA6, respectively. From Figure 1, it was observed that the peaks at 2918 and 2850 cm–1 were the −CH2– asymmetric stretching and symmetric stretching of LLDPE. The results indicated that the remaining part of LLDPE/PA6/LLDPE-g-MAH contained LLDPE or LLDPE-g-MAH. LLDPE or unreacted LLDPE-g-MAH can be extracted by dimethylbenzene, but LLDPE-g-MAH reacted with PA6 should not dissolve in dimethylbenzene. Therefore, it can be concluded that the maleic anhydride group in the LLDPE-g-MAH melt can react with the amidogen on the surface of solid PA6 particles in the blends prepared by method 2. Figure 3 FTIR spectra of the remaining part of LLDPE/PA6 (a) or LLDPE/PA6/LLDPE-g-MAH (b) extracted by dimethylbenzene. 2.2 Flame Retardancy The flame retardancy of LLDPE/PA6/IFR and LLDPE/PA6/IFR/LLDPE-g-MAH prepared by different methods was investigated by limiting oxygen index (LOI) and UL-94 test, as shown in Table 1. For C-0, the LOI value was 29.7, and the samples achieved a V-2 rating in UL94 testing because of the melt dripping during combustion. Processing methods showed a remarkable influence on the flame retardancy. Blends of LLDPE/PA6/IFR/LLDPE-g-MAH prepared by method 1 exhibited poor flame retardancy, and the LLDPE-g-MAH content showed weak influence on the flame retardancy. The LOI values were only 24–24.7% and had no vertical rating in the UL-94 test. However, the flame retardancy of the blends prepared by method 2 was significantly improved, compared with the blends prepared by method 1. LOI values of C2-M2 and C5-M2 were both 28.6 and the samples passed the UL-94 V-2 rating, indicating that their flame retardancy was close to C-0. The best flame retardancy was exhibited in C8-M2. The LOI value reached 30.0%. The samples passed the UL-94 V-0 rating, indicating that the dripping properties exhibited in C-0, C2-M2, and C5-M2 were restrained in C8-M2. Table 1 Flammability Characteristics of Blends sample code LOI (%) UL-94 rating C-0 29.7 V-2 C2-M1 24.0 no rating C5-M1 24.0 no rating C8-M1 24.7 no rating C2-M2 28.6 V-2 C5-M2 28.6 V-2 C8-M2 30.0 V-0 The results showed that blends with IFR dispersion in the PA6 phase (C-0, C2-M2, C5-M2, and C8-M2) exhibited better flame retardancy than the blends with IFR dispersion in the LLDPE phase (C2-M1, C5-M1, and C8-M1). It was reported that PA6 can act as the charring agent of APP to enhance the charring performance and flame retardancy.29−31 Therefore, IFR dispersion in the PA6 phase was more beneficial for the reaction between APP and PA6, exhibiting better flame retardancy, compared with the blends with IFR dispersion in the LLDPE phase. C8-M2 samples had no melt dripping during combustion. The reason for this may be the case that LLDPE-g-MAH increases the viscosity of blends and enhances the antidripping property. Melt flow index (MFI) can relate to viscosity indirectly, which indicates the dripping properties of flame-retardant LLDPE/PA6 blends. MFI experiments were used to test the flow rate of flame-retardant LLDPE/PA6 blends at different LLDPE-g-MAH contents, as shown in Table 2. The highest MFI value observed in C-0 indicated the lowest melt viscosity. Obviously, the melt dripping of C-0 should be attributed to the low melt viscosity. MFI values of the LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method 1 were lower than that of C-0. Moreover, the increase of LLDPE-g-MAH contents caused a sharp reduction of MFI values. The results indicated that LLDPE-g-MAH increased the melt viscosity. The copolymer formed by the reaction of LLDPE-g-MAH and PA6 can entangle with PA6 and LLDPE molecular chains at the interface to increase the flow resistance of melt, resulting in high melt viscosity. The increase of LLDPE-g-MAH contents in the blends produced more copolymers, resulting in higher melt viscosity. For the LLDPE/PA6/IFR/LLDPE-g-MAH blends prepared by method 2, the MFI values were also lower than that of C-0 and the increase of LLDPE-g-MAH contents caused the reduction of MFI values. The MFI value of C8-M2 was about one-third of C-0, indicating a sharp increase of melt viscosity, resulting in the enhancement of antidripping property. The increase of melt viscosity can also be attributed to the copolymer formed by the reaction of LLDPE-g-MAH and PA6. Table 2 Effect of LLDPE-g-MAH Contents on the MFI of the Blends Prepared by Different Methods sample code C-0 C2-M1 C5-M1 C8-M1 C2-M2 C5-M2 C8-M2 MFI (g/10 min) 105 71 52 25 67 43 34 2.3 Cone Calorimeter Analysis The cone calorimeter was also employed to evaluate the flame retardancy. Samples of C-0, C8-M1, and C8-M2 were chosen for testing by cone calorimetry. The heat release rate (HRR), total heat released (THR), and mass loss curves recorded during cone calorimeter tests are presented in Figure 4. The related data are presented in Table 3. Figure 4 Relationship between (a) HRR, (b) THR, and (c) mass loss and time of blends prepared by different methods. Table 3 Cone Calorimeter Test Data of Blends sample code PHHR (kW/m2) THR (kJ/m2) char residues (wt %) MAHRE (kW/m2 s) FPI (m2 s/kW) FGI (kW/m2 s) C-0 223 99 32 153 0.35 0.44 C8-M1 296 113 25 209 0.23 1.04 C8-M2 246 88 29 139 0.31 0.56 The peak HRR (PHRR) value of C8-M1 was higher than that of C-0 and C8-M2, indicating that the flame retardancy of C8-M1 was poorer than that of C-0 and C8-M2. The results showed that IFR dispersion in the PA6 phase enhanced flame retardancy in comparison with IFR dispersion in the LLDPE phase. The HRR curves of C-0 and C8-M2 displayed two peaks: the first peak was assigned to the formation of expandable char layers and the second peak was assigned to the further decomposition of a carbonaceous residue.34 The first PHHR and the HRR values for the initial 250 s of C8-M2 were lower than that of C-0, indicating that the char layer of C8-M2 formed in the initial period can provide better protection against the combustion of the matrix than that of C-0. Combustion experiments performed in the UL94 or LOI test were similar to the scenario of cone calorimetry at the ignition stage.35−37 Therefore, the results that the LOI and the vertical combustion level of C8-M2 were better than that of C-0 can be due to reduced combustion intensity in the initial period. The second PHHR value of C8-M2 was higher than that of C-0, indicating that the stability of the carbonaceous residue in C8-M2 was poorer than that of C-0. The THR of C-0 and C8-M2 was lower than that of C8-M1, and the mass loss curve showed that the char residue of C-0 and C8-M2 was higher than that of C-0, indicating that the dispersion of IFR in the PA6 phase was more favorable to the formation of a carbonaceous residue, resulting in the decrease of THR. Fire performance index (FPI) is defined as the proportion of TTI and PHRR; fire growth index (FGI) is defined as the proportion of PHRR and time to PHRR; MARHE is the maximum of the average rate of heat emission. The values of MARHE, FPI, and FGI can evaluate the fire hazard. It can be seen from Table 1 that the highest MAHRE and FGI values or the lowest FPI was observed in C8-M1, suggesting that C8-M1 possessed the strongest fire hazard in comparison with C-0 and C8-M2. 2.4 Characterizations of Residue Char Figure 5 shows the residue of the blends at the end of the cone calorimeter test. Broken char residue was observed in C8-M1. C-0 and C8-M2 formed a coherent and dense char residue with high intumescentia. The formation of highly intumescent, coherent, and dense char layer could provide a better protective shield; thus, the heat and mass transfer between the gas and condensed phase could be slowed, and the underlying materials are protected from further burning. For this reason, C-0 and C8-M2 exhibited much lower PHRR values than C8-M1. Figure 5 Photos of the aspect of the crust of blends after the cone calorimeter test. Char residues of LLDPE/PA6/IFR and LLDPE/PA6/IFR/LLDPE-g-MAH prepared by different methods were examined by SEM, as shown in Figure 6. The char residue of C2-M1 and C8-M1 was completely a loose structure; many voids can be observed. A continuous and compact char residue was formed in C-0, C2-M2, and C8-M2. Compared with the loose structure displayed in C2-M1 and C8-M1, a continuous and compact char residue can accumulate flammable gas and improve the blocking ability of heat and gas, resulting in the improvement of flame retardancy. Figure 6 SEM of intumescent char residues for blends prepared by different methods. The different morphologies of char residues should be attributed to the different dispersion of IFR in polymer blends. Compared with IFR dispersion in the LLDPE phase, IFR dispersion in the PA6 phase was beneficial for the reaction between APP and PA6, forming a high char residue, resulting in the formation of a continuous and compact char residue. However, IFR dispersion in the LLDPE phase was against the charring performance, forming loose char residues. 2.5 Water Resistance of Flame-Retardant LLDPE/PA6 Table 5 shows the mass loss percentages, LOI, and the UL-94 test results of flame-retardant LLDPE/PA6 after water immersion. The highest mass loss percentage was exhibited in C-0. Mass loss percentages of LLDPE/PA6/LLDPE-g-MAH/IFR blends prepared by method 1 were higher than that of blends prepared by method 2. The results indicated that LLDPE/PA6/LLDPE-g-MAH/IFR prepared by method 2 exhibited the best water resistance. The LOI and UL-94 test results were in accordance with the mass loss results. After water immersion, the LOI value of C-0 reduced from 29.7 to 24%, and the samples failed to pass the UL-94 test, indicating remarkable reduction in flame retardancy because of poor water resistance. Blends of C2-M1, C5-M1, and C8-M1 had poor flame retardancy before water immersion. Therefore, little change in flame retardancy was observed after water immersion. Good water resistance was exhibited in C2-M2, C5-M2, and C8-M2. After water immersion, the LOI values were reduced slightly, and UL-94 rating grades were unchanged. The different water resistance exhibited in Table 4 should be attributed to different microstructures of the blends. The sample of C-0 had the microstructure in which IFR dispersed in the dispersed PA6 phase and the interfacial adhesion between the PA6 phase and the LLDPE matrix was poor. Poor interfacial adhesion contained a number of microgaps, through which the water entered to dissolve IFR, resulting in poor water resistance. Blends of LLDPE/PA6/LLDPE-g-MAH/IFR prepared by method 1 possessed the microstructure in which IFR and PA6 phase both dispersed in the LLDPE matrix and the interfacial adhesion between the PA6 phase and the LLDPE matrix or IFR particles and the LLDPE matrix was strong or poor, respectively. Although the compatibilization of LLDPE-g-MAH on LLDPE/PA6 decreased the microgaps, the dispersion of IFR in the LLDPE matrix and the poor interfacial adhesion between IFR particles and the LLDPE matrix were both against the improvement of water resistance. In blends of LLDPE/PA6/LLDPE-g-MAH/IFR prepared by method 2, the microstructure in which IFR dispersed in the dispersed PA6 phase and LLDPE-g-MAH improved the interfacial adhesion between the PA6 phase and the LLDPE matrix was formed. Good interfacial adhesion reduced the microgaps, indicating that the water was hard to diffuse into the matrix. The PA6 phase can protect the IFR component from attack by water. Accordingly, good water resistance was observed in these blends. Table 4 Mass Loss, UL-94, and LOI Results of Blends after Water Treatment at 70 °C for 168 h sample code mass loss percentage (%) LOI (%) UL-94 rating C-0 3.2 ± 0.1 24 failed C2-M1 1.7 ± 0.2 24 failed C5-M1 1.9 ± 0.1 24.7 failed C8-M1 1.6 ± 0.1 24 failed C2-M2 0.9 ± 0.1 26.3 V-2 C5-M2 0.9 ± 0.1 27.1 V-2 C8-M2 0.7 ± 0.1 28.6 V-0 2.6 Mechanical Properties The mechanical properties of flame-retardant LLDPE/PA6 are compiled in Table 5. The tensile strength, elongation at break, and impact strength of C-0 were 7.1 MPa, 9.4%, and 3.0 kJ/m2, respectively. Poor compatibility between PA6 and LLDPE should be responsible for the deteriorated mechanical properties. For the blends prepared by method 1, compatibilizer LLDPE-g-MAH improved the tensile strength and elongation at break. Introduction of LLDPE-g-MAH to C-0 through method 1 also improved the impact strength except for C2-M1. The mechanical properties of the blends prepared by method 2 were higher than that of C-0 and the blends prepared by method 1. This phenomenon should be attributed to different morphologies. For the blends prepared by method 1, PA6 phase and IFR were both dispersed in continuous LLDPE phase. The compatibilizer elevated the interphase adhesion of PA6 and LLDPE phases and decreased PA6 phase size. Therefore, the tensile strength and elongation at break were increased from 7.1 MPa and 9.4% for C-0 to 9.0 MPa and 23.4% for C5-M1, respectively. For the blends prepared by method 2, IFR was dispersed in the PA6 phase and the compatibilizer elevated the interphase adhesion between the PA6 phase and the matrix. Therefore, the tensile strength, elongation at break, and impact strength were increased about 60, 300, and 50% in comparison with C-0, respectively. Table 5 Mechanical Properties of the Blends Prepared by Different Methods sample code tensile stress (MPa) elongation at break (%) impact strength (kJ/m2) C-0 7.1 ± 0.9 9.6 ± 3.5 3.0 ± 0.08 C2-M1 8.3 ± 1.2 27.3 ± 8.3 2.0 ± 0.01 C5-M1 9.0 ± 1.5 23.4 ± 9.3 3.3 ± 0.98 C8-M1 9.0 ± 0.9 23.3 ± 10.2 3.5 ± 0.78 C2-M2 11.9 ± 0.5 28.9 ± 4.9 3.7 ± 0.06 C5-M2 11.7 ± 0.6 24.2 ± 8.2 4.3 ± 0.08 C8-M2 11.9 ± 0.6 29.4 ± 7.1 4.4 ± 0.02 It was observed that the compatibilizer contents exhibited a weak effect on the mechanical properties of LLDPE/PA6/LLDPE-g-MAH/IFR prepared by different methods, due to which the increase of compatibilizer contents caused little change of the mechanical properties. For the blends prepared by method 1, although the increase of LLDPE-g-MAH content reduced the PA6 phase size and contributed to the augment of the mechanical properties to a certain extent, the poor interphase adhesion between IFR and the LLDPE matrix showed a greatly negative impact on mechanical properties, indicating that the mechanical properties were not influenced remarkably along with the increase of LLDPE-g-MAH content. In the blends prepared by method 2, the PA6 phase remained solid when LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR. Therefore, LLDPE-g-MAH cannot reduce the PA6 phase size. The increase of LLDPE-g-MAH content increased the coating thickness of LLDPE-g-MAH on the PA6 particles surface rather than increasing the interfacial strength. Therefore, the poor effect of the compatibilizer contents on mechanical properties was exhibited. 3 Conclusions Different processing methods were employed to prepare LLDPE/PA6/IFR blends with different microstructures, and the results showed that the microstructure affected the flame retardancy, mechanical properties, and water resistance greatly. Melt-blending IFR with LLDPE/PA6 simultaneously formed the microstructure in which IFR was selectively dispersed in the PA6 phase of LLDPE/PA6/IFR and the interphase adhesion between PA6 and the LLDPE matrix was poor. Although IFR dispersion in the PA6 phase was beneficial for the increase of flame retardancy, poor interphase adhesion deteriorated the mechanical properties and water resistance. When the compatibilizer LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR simultaneously, a microstructure was formed, indicating that IFR and PA6 particles were respectively dispersed in the LLDPE matrix and that LLDPE-g-MAH strengthened the interphase adhesion between the PA6 phase and the LLDPE matrix rather than IFR particles and the LLDPE matrix. The improvement of the interphase adhesion increased the mechanical properties and water resistance, but IFR dispersion in the LLDPE matrix decreased the flame retardancy sharply. A novel processing method that the IFR was first melt-blended with LLDPE/PA6 and then LLDPE-g-MAH was melt-blended with LLDPE/PA6/IFR between the melt temperatures of LLDPE and PA6 was employed to obtain the microstructure with the dispersion of IFR in the PA6 phase and strong interface adhesion of the PA6 phase with the matrix. The flame retardancy, mechanical properties, and water resistance were improved simultaneously. IFR dispersion in the PA6 phase and high viscosity caused by the compatibilization of LLDPE-g-MAH should be responsible for the improvement of flame retardancy. Strong interface adhesion of the PA6 phase with the matrix and IFR dispersion in the PA6 phase caused good water resistance. Moreover, good mechanical properties were attributed to the strong interface adhesion between the PA6 phase and the LLDPE matrix. 4 Experimental Section 4.1 Materials The materials used in this study were LLDPE (LL6201XR, MI = 50 g/10 min, d = 0.926 g/cm3) supplied by ExxonMobil Corp. and PA6 (33500, relative viscosity of 3.50, d = 1.14 g/cm3) supplied by Xinhui Meida-DSM Nylon Chips Co., Ltd. The IFR system consists of APP and PER, and the weight ratio of APP to PER is 4:1. APP [(NH4PO3)n, purity level > 90%] was supplied by Zhejiang Longyou Gede Chemical Factory (China). PER was supplied by Jinan Taixing Fine Chemicals Co., Ltd. LLDPE-g-MAH (TRD200L, MI = 2 g/10 min, d = 0.92 g/cm3) was supplied by Wujiang Siruda Plastic Industry Co., Ltd. The amount of maleic anhydride in LLDPE-g-MAH was 1 wt %. 4.2 Preparation of Composites All the materials were oven-dried for 12 h at 85 °C before extrusion and injection. The blends were extruded via a corotating twin-screw extruder with a barrel diameter of 20 mm and a barrel-length-to-diameter ratio of 25. Then the extruded blends were molded into sheets of suitable thickness at the injection pressure of 50 MPa via an injection molding machine. The formula of the blend is listed in Table 6. Table 6 Formulation of Blends sample code LLDPE PA6 IFR LLDPE-g-MAH processing method C-0 64 16 20 0 method 1 C2-M1 62 16 20 2 method 1 C5-M1 59 16 20 5 method 1 C8-M1 56 16 20 8 method 1 C2-M2 62 16 20 2 method 2 C5-M2 59 16 20 5 method 2 C8-M2 56 16 20 8 method 2 Two processing methods were employed for preparing the blends. Method 1: all the materials were melt-blended above the melt temperature of LLDPE and PA6 for preparing the blends. The temperatures from hopper to die were 140, 160, 190, 220, and 240 °C. Method 2: two steps were employed to prepare the blends. In the first step, PA6 and LLDPE were melt-blended with IFR to prepare LLDPE/PA6/IFR, and the processing temperature was higher than the melt temperature of LLDPE and PA6. The temperatures from hopper to die were 140, 160, 190, 220, and 240 °C. In the second step, LLDPE/PA6/IFR was melted with LLDPE-g-MAH, and the processing temperature was between the melt temperatures of LLDPE and PA6. The temperatures from hopper to die were 140, 150, 160, 160, and 160 °C. 4.3 Measurement and Characterization LOI was measured according to ASTM D2863-77. The apparatus used was a JF-3 instrument (Chengde, China). The specimens used for the test were of dimensions 120 × 6 × 3 mm3. The vertical test was carried out according to the UL94 test standard. The specimens used for the test were of dimensions 127 × 12.7 × 3 mm3. The sample flammability performed on the cone calorimeter (FTT, UK) test according to ISO 5660 standard procedures. The specimens used for the test were of dimensions 100 × 100 × 3 mm3. The specimen was exposed horizontally at an incident flux of 35 kW/m2. FTIR spectra were recorded on a Bruker Vector 33 spectrometer. LLDPE/PA6/APP (24/56/20) or LLDPE/PA6/PER (27/63/10) in which LLDPE was used as the matrix and PA6 formed dispersed phase was pressed into disks with KBr. Before being pressed, the samples of LLDPE/PA6/APP were extracted by formic acid for 48 h. The samples of LLDPE/PA6/PER were extracted first by formic acid and later by alcohol. The sample of LLDPE/PA6 (85/15) or LLDPE/PA6/LLDPE-g-MAH (80/15/5) was extracted by dimethylbenzene at 120 °C to remove LLDPE and LLDPE-g-MAH, and the residuum was pressed into disks with KBr for the FTIR test. A JEOL 6301F scanning electron microscope was used to investigate the morphology of residue char and molded specimens at an acceleration voltage of 20 kV. The residue char was obtained from the specimen left after the vertical test. The molded specimens were fractured in liquid nitrogen. To determine the water resistance of polymer blends, specimens, of the same size as used for the UL-94 test, were put in distilled water at 70 °C and kept at this temperature for 168 h. The water was replaced every 24 h, according to UL746C. The treated specimens were subsequently dried in a vacuum oven at 80 °C for 72 h, and the weight of the specimens was measured before water immersion and after drying. The mass loss percentages were calculated in the following equation18 where W0 is the initial weight of the specimens before water immersion and W is the remaining weight of the specimens after water immersion and drying. The tensile strength was measured by a tensile tester (LJ1000, Guangzhou Test Instrument Factory, China) according to ASTM D638. The MFI of melting polymer blends was measured by a melt index instrument, and the measurement temperature was 225 °C; load was 2.16 kg. The authors declare no competing financial interest. Acknowledgments The authors gratefully acknowledge the financial support of this work by the National Natural Science Foundation of China (contract number: 51673059), Natural Science Foundation of Education Department of Henan Province (contract number: 17A150009), and Student Research Training Program of HAUST. ==== Refs References Chen M. ; Xu Y. ; Chen X. ; Ma Y. ; He W. ; Yu J. ; Zhang Z. Thermal stability and combustion behaviors of flame retardant polypropylene with thermoplastic polyurethane encapsulated ammonium polyphosphate . High Perform. Polym. 2014 , 26 , 445 –454 10.1177/0954008313517910 . 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