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Article Cite This: ACS Omega 2019, 4, 3700−3707
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Complementarity of Raman and Infrared Spectroscopy for Structural Characterization of Plant Epicuticular Waxes Charles Farber,† Jingbai Li,§ Elizabeth Hager,† Robert Chemelewski,† John Mullet,† Andrey Yu. Rogachev,*,§ and Dmitry Kurouski*,†,‡ †
Department of Biochemistry and Biophysics and ‡The Institute for Quantum Science and Engineering, Texas A&M University, College Station, Texas 77843, United States § Department of Chemistry, Illinois Institute of Technology, Chicago, Illinois 60616, United States
ACS Omega 2019.4:3700-3707. Downloaded from pubs.acs.org by 185.89.100.95 on 02/25/19. For personal use only.
S Supporting Information *
ABSTRACT: Raman and infrared (IR) are two complementary vibrational spectroscopy techniques that enable labelfree, noninvasive, and nondestructive structural characterization of analyzed specimens. IR spectroscopy is broadly utilized in various research areas ranging from food chemistry and agriculture to geology and medicine. Recently, Raman spectroscopy (RS) has attracted the interest of researchers from these fields because of its minimal interference from water and significant reduction of equipment costs. In this study, we evaluated the complementarity of RS and IR for structural characterization of epicuticular waxes, sophisticated chemical mixtures of long fatty acids, and their derivatives excreted by plants. We show that IR can sense R−O−H vibrations, which are characteristic for alcohols and sugars, as well as carbonyl groups, which can be assigned to aldehydes, ketones, acids, and esters in the chloroform-extracted waxes of Sorghum bicolor. At the same time, RS can detect only C−C, C−H, and CH2 vibrations of these molecules in the same wax extracts. Using theoretical calculations, we were able to elucidate the origin of this phenomenon. It was found that an increase in the aliphatic chain length in carboxylic acids resulted in a quadratic and linear increase in the intensity of aliphatic vibrations in the corresponding Raman and IR spectra, respectively. Thus, the complementary of RS and IR that holds for small molecules may not be always observed for large biological molecules.
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INTRODUCTION Infrared (IR) spectroscopy, including near- and mid-IR, is broadly used for analyzing food, grains, animal feeds, minerals, and soils. It is based on the absorption of electromagnetic radiation by vibrational states of molecules. Consequently, the corresponding IR absorption spectrum will reflect the chemical or structural organization of the sample.1 Similar information about a chemical structure can be obtained by Raman spectroscopy (RS), which is based on inelastic light scattering. In Raman scattering, incident photons interact with molecules bringing them to virtual energy states. Next, photons scatter back with either higher or lower energies relative to the intent light. The shift in energy corresponds to the chemical structure of molecules in the sample.2,3 Unlike in IR, water does not generally interfere with Raman analyses of samples. This advantage of RS makes it attractive for practical applications in various research areas ranging from agriculture to medicine.4 For instance, it has been recently demonstrated that RS can be used to conduct forensic analysis of body fluids5 and gun-shot residues,6 monitor changes in the protein secondary structure,7 and detect and identify fungal spores,8 bacteria,9 and cancer cells,10 as well as reveal sophisticated subcellular organization.11 © 2019 American Chemical Society
Development of portable Raman instrumentation has expanded in-field application of the method in areas including forensics,4,12 minerology,13,14 and plant pathology. Our group demonstrated that RS can be used for highly accurate diagnostics of fungal diseases of maize, wheat, and sorghum.15,16 Invasion of such fungal pathogens occurs through a plant cuticle, a sophisticated barrier that protects the plant against agricultural pests and abiotic stresses such as drought and heat. The chemical composition of a cuticle also determines canopy reflectance, photosynthesis, and water use efficiency.17−19 The plant cuticle is primarily composed of long-chain chemically modified hydrocarbons which are secreted by epidermal cells. They form an insoluble cuticle membrane and soluble epicuticular wax layers.20 The composition and structure of waxes and other chemicals that accumulate on the surfaces of plants vary by organ, cell type, developmental stage, and environmental conditions. For instance, Sorghum bicolor, which originally evolved in Africa, accumulates large amounts of wax on its leaves and stems to Received: December 30, 2018 Accepted: February 5, 2019 Published: February 19, 2019 3700
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minimize water loss. This adaptation lends to its worldwide distribution.17,18 Plant epicuticular waxes in general and sorghum wax in particular are enriched in long aliphatic chains and consequently can be a source of valuable bioproducts such as degradable biopolymers and biofuel.21,22 Biodegradable polymers are a new generation of environmental-friendly materials that are based on petroleum, plant, or animal sources. They are expected to be a solution for the massive amounts of oil-based nonbiodegradable plastic waste.23 From a chemical perspective, biodegradability is based on the presence of ester, ether, and amide groups in alkyl chains, which make the polymer easily accessible to bacteria.24 Additionally, the crystallinity of polymers is highly important for biodegradable properties of polymers.25 Chemical analyses of plant epicuticular waxes, conducted by chemical extraction and chromatographic analysis, revealed large amounts of all these chemical groups.26−29 Considering the high amount of wax on the plant surface, ease of harvesting, and plant accessibility to genetic modifications, which can be done to increase the wax production, it is expected that plant wax-based biopolymers will be highly desired in the nearest future. Evaluation of the commercial value and biodegradable properties of such a wax material will require fast and labelfree analytical techniques that will be able to reveal the amount and spatial distribution of ester, ether, and amide groups. Our group has recently demonstrated the potential of atomic force microscopy−IR (AFM−IR) to study the distribution of chemical groups in intact plant waxes.30 As both Raman and IR spectroscopy meet these criteria, it is extremely important to develop these analytical techniques for the structural characterization of plant epicuticular waxes. In this study, we utilize both Raman and IR to investigate the structure and composition of sorghum epicuticular waxes. We demonstrate that these spectroscopic techniques provide completely different but complementary information about the wax. We also employ theoretical calculations to elucidate the origin of observed differences between collected Raman and IR spectra.
Figure 1. Raman spectra of an intact sorghum stem (blue) and mechanically scratched waxes (red) acquired using Rigaku (top) and CBEx (bottom) hand-held Raman spectrometers.
Table 1. Wax IR Bands and Their Assignments wavenumber (cm−1)
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RESULTS AND DISCUSSION Raman-Based Structural Characterization of Intact Sorghum Wax. We investigated whether the chemical composition of epicuticular waxes could be probed using hand-held Raman spectrometers. Raman spectra were collected from wax located on different parts of the sorghum plant using the Rigaku and CBEx spectrometers (see Experimental Section). Results indicated that reliable Raman readings of epicuticular waxes were possible only from the sorghum stem (internodes), where a substantial amount of this material was accumulated. Spectral acquisition from leaves, sheathes, and other plant organs did not allow for noninvasive and nondestructive characterization of sorghum epicuticular waxes. It has been found that the Rigaku spectrometer allowed for a much better sensing of intact wax on the plant surface, whereas no clear wax signature could be obtained with the CBEx spectrometer (Figure 1). This could be due to the tighter focus of the Rigaku spectrometer, which enabled better light focusing on the thin film of wax on the plant surface. We found that the collected Raman spectra from both intact and mechanically scratched waxes exhibited only peaks that could be assigned to C−C (1061, 1117, and 1130 cm−1), CH2 (1170, 1292 and 1418 cm−1), and CH2/CH3 (1438 and 1462 cm−1) vibrations (vibrational bands at 1603 and 1630 cm−1
vibration1
assignment
668 720 730 890 947 958 1027 1093 1155 1292 1305 1378
out of plane ring bending CH2 in-plane rocking CH2 in-plane rocking CH2 wagging C−O stretching C−O stretching C−O stretching ring stretching C−O stretching CH2 twisting C−O stretching CH3 symmetric deformation;OH deformation of carboxyl monomer
1438 1463 1472 1696
CH2 stretching CH2 scissoring CH2 scissoring CO stretching
1710
CO stretching
aromatic ring alkanes alkanes alkanes carbohydrates carbohydrates alcohols aromatic ring alcohol alkane alcohol alkane or carboxylic acid alkane alkane alkane carbonyl compound carbonyl compound
originate from lignin of plant cell walls, Table 1). Surprisingly, we did not observe any alcohol (R−O−H) or carboxyl vibrations in the collected spectra. At the same time, the presence of alcohols and carbonyl-containing compounds (aldehydes, ketones, acids, and esters) was confirmed by high-performance liquid chromatography (HPLC) analyses of these waxes. Such an analysis is typically conducted through solvent extraction of plant waxes with hexanes, diethyl ether, chloroform, or mixtures of these, with subsequent separation of wax components by HPLC.22 Raman and IR Structural Characterization of Extracted Wax. We extracted epicuticular waxes from all major 3701
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Figure 2. Raman (left) and IR (right) spectra of chloroform-extracted epicuticular waxes from the (a) MI (stem) (green), (b) sheaths (whorl dark red; fully expandedblue), (c) nodes (elongatingpurple; expandingdark green), and (d) leaves (senescentlight blue; expanded black; whorlred) of sorghum.
cm−1) was not observed. Nevertheless, these results show that RS can be used to distinct wax from different plant parts based on the unique vibrational signature of the corresponding Raman spectra. Following RS analysis, the extracted wax samples were reanalyzed using Fourier transform infrared (FTIR) spectroscopy. It was found that the IR spectra of the waxes featured many more peaks than Raman spectra. Two IR bands at 720 and 730 cm−1 can be assigned to the in-plane rocking mode of CH2. This pair of peaks indicates crystallinity in the sample: in molten or disordered samples, a single peak would appear at approximately 725 cm−1, whereas in crystalline samples, this peak splits into 720 and 730 cm−1.1 This suggests that after resolidifying post-extraction, the wax undergoes crystallization.31,32 In all IR spectra, except for those for leaves, we found a set of five relatively distinct peaks between 850 and 1100: 890, 947, 958, 1027, and 1093 cm−1. Within the spectra in which they appear, the 947/958 and 1027 peaks are the most intense of the set. These peaks correspond to C−O stretching vibrations which are commonly found in carbohydrates. Interestingly, the only C−O stretch vibration observed in the Raman spectra occurs at 1156 cm−1; the other C−O stretching bands at 1027 and 1305 cm−1 are not observed. The band at
parts of sorghum and investigated them by RS using the Rigaku spectrometer (Figure 2). It was found that Raman spectra of wax extracts had vibrational bands at 1061, 1117, 1130, 1292, 1418, 1438, and 1462 cm−1, previously observed in the spectrum of intact wax on sorghum. We also found that the vibrational band at 1170 cm−1, which was previously observed in the Raman spectrum of intact wax, shifted to 1156 cm−1. This band has high intensity in the spectra of senescent and expanded leaves and lower intensity in the spectra of the elongating and expanding node as well as in the middle internode (MI) spectra. We also found that relative intensities of C−C peaks (1061, 1117, and 1130 cm−1) in all Raman spectra remain nearly constant to CH2 vibration at 1292 cm−1. At the same time, relative intensities of a triplet ∼1440 cm−1 to CH2 vibration at 1292 cm−1 directly depend on the plant part where the wax was extracted from. For instance, the ratio between peaks at 1292 and 1438 cm−1 in the Raman spectrum of the MI was found to be 1:1, whereas that in the spectra of whorl and fully expanded sheaths and the expanding node was observed to be ∼1:1.6. This ratio is 1.8 for the elongating node, 1.9 for the whorl leaf, and 2.5 for senescent and expanded leaves. Similar to what was found in the case of intact measurements of wax, the vibrational band of carbonyl (∼1630 3702
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1093 cm−1 corresponds to substituted benzene ring vibrations, providing further evidence of the presence of aromatic compounds in the wax. The peak at 1378 cm−1 can be assigned to either CH3 symmetric deformations or OH deformation in carboxylic acid monomers. The absence of this peak in the Raman spectra suggests that this may be an OH-associated peak. The peak doublet at 1463 and 1472 corresponds to CH2 scissoring vibrations. In wax, it is expected that there would be many carbonyl-containing compounds, as fatty acids contain these groups. In the IR spectrum of the wax, two peaks are observed at 1696 and 1710, both corresponding to the CO stretch of carbonyls. These bands are markedly absent from the Raman spectra, despite being obtained from the same samples. Therefore, we can conclude that IR spectroscopy can probe different chemical properties from RS. Specifically, it enables detection and quantitative evaluation of carbohydrates, carbonyl-containing compounds, and alcohols in wax extracts from different parts of sorghum. Evaluation of Wax Esterification. Detection and identification of carbonyl vibrational bands in the vibrational spectra of plant waxes are very important for evaluation of their biodegradability properties. This is because ester and amide functionalities in long hydrocarbon chains make them more accessible for biodegradation. Therefore, ester- and amide-rich plastic materials are highly desirable for the modern polymer industry. We demonstrated that IR spectroscopy allows not only for the detection of carbonyl vibration in plant waxes but also for the correct assignment of the chemical nature of the carbonyl moiety in these natural polymeric materials. Specifically, we observed carbonyl bands in all wax extracts to be centered around 1710 cm−1, which indicates the presence of ketones, aldehydes, and carboxylic acids. Despite evidence suggesting that some unsaturated and aromatic esters could be present, there is no evidence of saturated esters. We currently work on gas chromatography−mass spectrometry analysis of field-grown TX08001 sorghum wax to determine the exact percentages of the unsaturated, aromatic, and saturated waxes present in sorghum wax. Nevertheless, our results suggest that there is very little ester and even less amide groups present in the wax extracted from sorghum. At the same time, carnauba wax exhibits carbonyl vibrations around 1735 cm−1 (Figure S1), which indicates the presence of saturated waxes in this natural polymer. Indeed, HPLC analysis of this wax revealed the ester load to be approximately 81%.33 Probing Wax Composition with AFM−IR Spectroscopy. One can suspect that wax extraction, which was utilized in this study, as well as other solvent-based extraction methods commonly used for characterization of wax composition, could be potentially destructive to esters. Consequently, this may obscure accurate determination of their presence in wax. To verify this hypothesis, we utilized a noninvasive and nondestructive technique known as AFM−IR to probe the chemical composition of wax of sorghum. In AFM−IR, a sample is irradiated by pulsed IR radiation with an AFM tip in proximity to the sample surface.34−36 The sample expands as the laser sweeps through its tuning range. This expansion is directly correlated with how well its components absorb at different wavelengths. These expansions cause the tip to oscillate. These oscillations, following Fourier transformation, correspond to the macroscopic IR spectrum of the sample. This means that AFM−IR can acquire IR data below the diffraction limit.37 This method is label free, noninvasive, and
nondestructive. The AFM probe can be removed from the surface after analysis and, with proper settings, will not damage the surface. Recent applications of AFM−IR include investigation of malaria-infected blood cells,38 CH3NH3PbI3 polycrystalline perovskite films,39 and bacteria.40 Using AFM−IR, we investigated the external surface of wax from a MI of sorghum (Figure 3, top). The point spectra
Figure 3. AFM−IR spectra collected from the external surface of sorghum wax (top) and chemical maps of intact MI wax of sorghum (bottom). 1450 cm−1: left, max V = 0.14; 1725 cm−1: right, max V = 0.23. Scale bar: 1 μm. Map dimensions: 10 × 10 μm.
confirmed the presence of carbohydrates (980−1004 cm−1) and different alcohols (1060−1200 cm−1). We also observed C−H bending vibrational bands at 1384 and 1408 cm−1, as well as the CH2 scissoring band at 1468 cm−1. We observed carbonyl vibration bands in a narrow peak from 1712 to 1720 cm−1, suggesting the presence of compounds such as aldehydes, aromatic and unsaturated esters, and carboxylic acids. Aliphatic esters have a carbonyl band at around 1735− 1750 cm−1 and are thus unlikely to be detectable in this sample. In the chemical maps of the wax surface (Figure 3 bottom), it is observed that the distributions of the 1450 cm−1 (CH2) and 1725 cm−1 (carbonyl) peaks vary in similar ways across the surface. The intensities of these two groups appear to correspond to each other: each is high or low intensity in the same regions of the wax. Our results also demonstrate that AFM−IR could be used not only for the detection and identification of esters in wax material but also for the evaluation of their distribution across the polymer surface. It is important to note that such analysis can be performed in less than square millimeter of wax, which makes AFM−IR unique for fast and accurate evaluation of biodegradability of polymeric materials. Theoretical Calculations of IR and Raman Spectra. To further shed light on trends observed, we performed a comprehensive theoretical investigation of Raman and IR spectra of the target acid with the help of density functional theory. To make our conclusions method-independent, several 3703
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Raman spectra shows a dramatically different behavior. While in the former, both bands are clearly visible, in the case of Raman spectra, the high intensity of the ν(C−H)2 peak in systems with a long aliphatic chain makes the ν(CO) band almost invisible. Because of the long aliphatic chain, triacontanoic acid might be present in the sample as a set of conformers, each with its own spectroscopic signature. Thus, we decided to also use theoretical calculations to better understand experimentally acquired Raman spectra of sorghum waxes and evaluate contributions from different possible conformers (Table 3).
functional/basis sets combination were tested. They all have shown consistent qualitative and even quantitative agreement (see the Supporting Information for details). The question to answer iswhy carbonyl group vibrations are visible in the IR spectra of plant waxes and invisible in the corresponding Raman spectra? We hypothesized that a carbonyl (CO) peak in the Raman spectra could be obscured by a very strong activity of ν(C−H) of long-chain carbonyl-containing hydrocarbons. To verify this hypothesis, we calculated IR and Raman spectra for a set of aliphatic acids from smallest acetic acid (CH3COOH) to longer acids, such as CH3(CH2)2COOH, CH3(CH2)8COOH, and CH3(CH2)18COOH. In all molecules, the CO stretching, O−H stretching, and C−H stretching vibrations were found at the similar position as in triacontanoic acid (Table 2), which is abundant in sorghum plant waxes, thus allowing us to perform a direct comparison.41
Table 3. Relative Energy of Conformer 1−5 in Different Phasesa gas
Table 2. Selected Vibrational Modes (cm−1) of Aliphatic Acid CH3(CH2)n−1COOH Calculated at the PBEh-3c/Def2mSVP Level of Theory n ν(O−H) ν(C−H)1a ν(C−H)2a ν(CO)
1 3865.27 3257.42 3133.28 1949.16
3 3860.73 3130.88 3120.21 1935.79
9 3859.59 3159.26 3116.67 1934.45
19 3859.44 3153.84 3117.66 1934.47
hexane
THF
conformer
ΔE
p
ΔE
p
ΔE
P
1 2 3 4 5
4.51 0.00 0.54 0.89 1.12
0.03 56.31 22.65 12.48 8.52
4.12 0.00 0.14 0.59 0.73
0.04 40.72 32.28 15.16 11.80
4.06 0.13 0.00 0.50 0.64
0.04 31.05 38.94 16.68 13.29
a
The ratio of Boltzmann probabilities was calculated in accordance with the lowest conformer in each case. Relative energies (ΔE) are in kcal/mol. The ratios of Boltzmann distribution p are in %.
ν(C−H)1 represents the asymmetric aliphatic C−H stretching associated with an IR peak. ν(C−H)2 denotes the symmetric vibration of C−H stretches related with a notable Raman activity. a
The five lowest energy conformations of triacontanoic acid are presented in Figure 5. The most stable conformer 2 was
Analysis of the IR and Raman intensities revealed that in both cases, the stretching mode of the carboxyl group (ν(C O)) exhibits a constant magnitude for all aliphatic acid, regardless of their chain length. In contrast, while in the IR spectra, the C−H vibrations show a linear correlation with the number of carbon atoms in the backbone (Figure 4), those in the Raman spectra were found to exhibit a quadratic correlation. The longer chain of the aliphatic acid resulted in higher intensity of the asymmetric mode ν(C−H)1. Consequently, the activity ratio between ν(C−H)2 and ν(CO) in IR and
Figure 5. Equilibrium geometry of the linear and the four lowest conformer of triacontanoic acid. The stability in terms of electronic energy is 2 > 3 > 4 > 5 > 1.
found to exhibit a twisted structure instead of being linear as 1. For conformer 4, part of the chain remained linear. It is worth noting that that these distinct conformations were found to be less symmetric than conformer 1. Consequently, the response of the molecular vibrations, especially for those from C−H
Figure 4. Computed Raman activity (in Å4/AMU) and IR intensity (in kM/mole) as a function of the length of the aliphatic acid CH3− (CH2)n−1−COOH, where n = 1, 3, 9, 19, and 29. 3704
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peaks were found to be different in the conformers. For example, in 2−5, the 1150 cm−1 peak became at least as twice higher as that in 1. Further inspection of this peak revealed that 2, 4, and 5 have similar behavior but 3 exhibits notably stronger activity. The next important peak near the 1400 cm−1 is dramatically reduced (∼50%) when the linear conformation transforms to nonlinear. These findings suggest that RS can be used to probe the conformational composition of aliphatic hydrocarbons in wax. Specifically, it indicates that MI and sheath waxes primarily contain linear hydrocarbons, whereas twisted aliphatic chains dominate in the waxes of leaves, likely due to contaminants such as insecticides or other excreted molecules. The 1550 cm−1 peak corresponds to in-plane bending of C−H bonds. Such vibrations were found at certain positions of the chain with local symmetry and thus are Raman-active. Moreover, the Raman response superficially depends on the entire structure of the molecule. In other words, this is independent on the out-of-plane vibrating and thus the activity ratio between inand out-of-plane vibrations has no clear correlation. For instance, one can clearly note a shoulder peak (Figure 6) in 1 corresponding to the CH2 group adjacent to the carboxylate group and a sharp peak for a delocalized CH2 bending over the aliphatic chain (1577 cm−1, Figure 6). Both peaks were weaker than the previous one at 1393 cm−1. In contrast, the shoulder peak disappeared in other conformers, in which only the chain vibration is detectable, and the activity became stronger compared to the corresponding peaks near 1400 cm−1. Additionally, the variation of the 1550 cm−1 peak was found to be relatively less pronounced.
bonds, notably depends on the specific conformation of the molecule. The relative energies of these conformers were calculated in gas phase as well as solution phase. All conformers are in the same order in all considered phases, which indicates minimal influence of the solvent of conformer composition of the sample. The linear conformer is ∼4 kcal/mol less stable than the lowest one. To evaluate the population of each conformer, we used the Boltzmann distribution to simulate their contributions to the triacontanoic acid sample. We noted relatively high abundance of conformer 2−5. In contrast, the linear conformer shows the contribution of less than 1%. Moreover, as the solvent molecule becomes more polar, the energy gaps between conformers were found to be smaller and, consequently, their populations increase. Solution effect on the Raman spectrum was found to be systematic. According to the computed Raman spectra for conformer 1 in several phases, the Raman activity decreased along with the increase of the solvent polarity. At the same time, the positions of characteristic peaks at 1400 and 1500 cm−1 were slightly shifted toward a lower wavenumber. The ratio between these peaks was also found to the same, thus meaning that polarity of the solvent has almost no influence on the position and ratio of the peaks. When comparing the Raman spectra for the lowest 20 conformers of triacontanoic acid, we found in all cases that the peaks at 1150, 1400, and 1550 cm−1 show strong activities (Figure S5a). Notable deviations in the ratio between these peaks suggested that the changes in the Raman spectrum mainly rely on the conformational composition of the sample. Moreover, a comparison of the Raman spectra of 1−5 (Figure 6) clearly showed the lower Raman activity near 1150 cm−1 but higher activity near 1400 and 1550 cm−1.
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CONCLUSIONS The use of two complementary methods, IR and RS, in the characterization of epicuticular plant wax was demonstrated. Despite acquisition on the self-same samples, spectra acquired by IR and RS showed striking differences in peak composition. While IR was able to detect the CO vibrations associated with esters, RS was much more sensitive to the various CH vibrations. By AFM−IR, we confirmed our observations from macroscale IR and described the spatial distributions of CH2 and carbonyl groups across the wax surface. Following density functional theory calculations, it was revealed that the intensity of CH vibrations in Raman and IR scales differently with chain length: CH peak vibrations in Raman scale quadratically, whereas in IR, they scale linearly. Thus, in Raman, the signal for expected bands such as the carbonyl is overwhelmed by the increasing signal from long-chain alkanes. While Raman and IR are comparable for smaller molecules, they become more complementary as the molecule becomes larger. These differences will be notable should either (or both) be used in the characterization of biodegradable polymers.
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Figure 6. Calculated Raman spectra for conformer 1−5.
EXPERIMENTAL SECTION Wax Extraction for Raman and ATR−FTIR. Wax from the surface of different parts of Sorghum bicolor TX080001 mutant was extracted through reflowing heated chloroform over the surface of the tissue until all visible wax (bloom) was removed [combining methods of Harron42 and Hwang.22 Chloroform extracts were condensed under continuous N2 flow, resolubilized by small volumes of chloroform, and transferred onto aluminum foil where they were dried under continuous air flow.
On the basis of the calculated vibrational modes, peaks at 1150 and 1400 cm−1 belong to the out-of-plane vibration of C−H bonds, where the former corresponds to a concerted wagging of C−H bonds and the latter represents twisting of C−H bonds. Interestingly, these two types of modes are always associated with the same CH2 groups in the aliphatic chain in different conformations (Figure S5b). Thus, it resulted in the same activity ratio between these two peaks, although the 3705
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Wax Extraction for AFM−IR. All wax analyzed by AFM− IR was taken from the MI of Sorghum bicolor BTx398 plants. A razor blade was used to peel up a section of stem tissue, cracking the wax on its surface. Wax fragments were then removed from the surface using tweezers and adhered to 12 mm AFM discs (Ted Pella, Inc. Redding, CA) using doublesided scotch tape and stored at 4 °C before analysis. Spectroscopy. Raman spectra were collected with a handheld portable Rigaku Progeny ResQ spectrometer (Rigaku Analytical Devices, Inc. Wilmington, MA), equipped with a 1064 nm Nd:YAG laser. The following experimental parameters were used for all collected Raman spectra: 80 s acquisition time, 200 mW power, and baseline spectral correction by device software. In parallel, Raman spectra were collected with a CBEx Snowy Range spectrometer (Snowy Range, Laramie, WY) equipped with a 785 nm laser. The following experimental parameters were used for all collected Raman spectra: 80 s acquisition time and 200−250 mW power. IR spectra were collected with a PerkinElmer spectrum 100 FTIR spectrometer in attenuated total reflectance (ATR) mode. Spectra were recorded with a resolution of 4 cm−1 in the range of 4000−400 cm−1. A background spectrum was acquired immediately before the measurement. Wax was extracted as previously described and deposited onto the crystal. The wax was not intact for these readings. ATR correction was conducted using OMNIC software (Thermo Fisher Scientific). AFM−IR analysis was conducted using a Nano-IR2-s system (Anasys Instruments Inc., Santa Barbara, USA) with a 1 kHz pulse rate, 10 ns pulse length optical parametric oscillator laser as the source. Contact mode AFM tips (Anasys Instruments Inc., Santa Barbara, USA) with a resonance frequency of 13 ± 4 kHz and a spring constant of 0.007−0.4 N/m were used to obtain all spectra and maps. The system was purged with N2 to control humidity. Single point spectra were obtained in the ranges of 900−2000 with 4 cm−1 resolution. IR maps at different wavenumbers were obtained to probe the wax surface. Point spectra were smoothed with Analysis Studio’s (Anasys Instruments Inc., Santa Barbara, USA) built-in smoothing function using two points. The spectra were then imported into the MATLAB R2017b (Mathworks, Natick, USA) addon PLS_toolbox 8.6.1 (Eigenvector Research Inc, Manson, USA) for averaging. IR map images were processed using built-in Analysis Studio functions. Spectral Processing. Processing and averaging for the Raman spectra were done with GRAMS/AI 7.0 software (Thermo Galactic, Salem, NH). Calculations. All systems under consideration were initially optimized at the PBEh-3c/Def2-mSVP level of theory (socalled PBEh-3c approach), that consists of a hybrid Perdew, Burke, and Ernzerhof functional with 42% of Hartree−Fock exchange term. The basis set superposition errors and London dispersion effects were accounted by the implementation of geometrical counterpart and dispersion correction (D3) schemes, respectively. On the basis of the optimized geometry, IR and Raman spectra were computed at the same level of theory. The “resolution-of-identity” approximation to the Coulomb term and the auxiliary basis set for Coulomb fitting was used to speed up the computation. To evaluate the accuracy of the approach used, test calculations of geometry and spectroscopic characteristics of the parent triacontanoic acid were computed with help of different approaches (PBE0/
cc-pVDZ and PBE0/cc-pVTZ). Surprisingly, all techniques show very high similarity of results obtained, thus making our conclusions method-independent (see Supporting Information for details). PBEh-3c and PBE0/cc-pVDZ calculations were performed by ORCA 4.0.0 suit of program, whereas PBE0/ccpVTZ calculations were carried out by Firefly 8.1.0. Analysis of the conformational landscape of the triacontanoic acid was carried out using molecular dynamics with help of GFN-xTB approximation. One hundred and twenty five conformers were generated and optimized at the PBEh-3c/ Def2-mSVP level of theory. The lowest 20 conformers then were taken to compute the corresponding Raman spectrum at the same level of theory.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03675.
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Detailed description of calculations; IR Spectrum of carnauba wax; computed Raman spectrum for triacontanoic acid; computed Raman activity and IR intensity; computed Raman spectra for linear triacontanoic acid; calculated Raman spectra for the lowest 20 conformers; selected vibration modes for linear triacontanoic (1) acid; peak assignment of triacontanoic acid and peak shift of vibrational modes; selected vibrational modes of aliphatic acid CH3(CH2)n‑1COOH; absolute and relative energy for the linear (1) and the lowest 20 conformers (2-21) of triacontanoic acid; vibration modes for conformer 2-5; vibration modes for C 29H 59COOH; vibration modes for CH3 COOH; vibration modes for C3H7COOH; vibration modes for C9H19COOH; and vibration modes for C19H39COOH (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Phone: 312-567-3151 (A.Y.R.). *E-mail:
[email protected]. Phone: 979-458-3778 (D.K.). ORCID
Dmitry Kurouski: 0000-0002-6040-4213 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to AgriLife Research of Texas A&M for the provided financial support. We also acknowledge the Governor’s University Research Initiative (GURI) grant program of Texas A&M University, GURI Grant agreement no. 12-2016, M1700437.
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REFERENCES
(1) Colthup, N. B.; Daly, L. H.; Wiberley, S. E. Introduction to Infrared and Raman Spectroscopy, 3rd ed.; Academic Press: San Diego, CA, USA, 1990. (2) McCreery, R. Raman Spectroscopy for Chemical Analysis; Wiley: New York, 2000. (3) Long, D. A. The Raman Effect: A Unified Treatment of the Theory of Raman Scattering by Molecules; Wiley: West Sussex: England, 2002. 3706
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DOI: 10.1021/acsomega.8b03675 ACS Omega 2019, 4, 3700−3707
