High-Field Electron Paramagnetic Resonance and Density Functional

Article pubs.acs.org/JPCA

High-Field Electron Paramagnetic Resonance and Density Functional Theory Study of Stable Organic Radicals in Lignin: Influence of the Extraction Process, Botanical Origin, and Protonation Reactions on the Radical g Tensor Christian Baḧ rle,†,∥ Thomas U. Nick,‡,⊥ Marina Bennati,‡,# Gunnar Jeschke,§,¶ and Frédéric Vogel*,† †

Paul Scherrer Institut, Research Department General Energy, 5232 Villigen PSI, Switzerland Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany § EPR Research Group, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland ‡

S Supporting Information *

ABSTRACT: The radical concentrations and g factors of stable organic radicals in different lignin preparations were determined by X-band EPR at 9 GHz. We observed that the g factors of these radicals are largely determined by the extraction process and not by the botanical origin of the lignin. The parameter mostly influencing the g factor is the pH value during lignin extraction. This effect was studied in depth using high-field EPR spectroscopy at 263 GHz. We were able to determine the gxx, gyy, and gzz components of the g tensor of the stable organic radicals in lignin. With the enhanced resolution of high-field EPR, distinct radical species could be found in this complex polymer. The radical species are assigned to substituted o-semiquinone radicals and can exist in different protonation states SH3+, SH2, SH1-, and S2-. The proposed model structures are supported by DFT calculations. The g principal values of the proposed structure were all in reasonable agreement with the experiments.

■

INTRODUCTION Lignin isolated from biomass contains a significant amount of stable organic radicals. These radicals can be detected using electron paramagnetic resonance (EPR) spectroscopy as first reported by Rex et al.1 They were studied extensively by Steelink et al.2−5 and others.6−8 It was shown that typical lignin preparations exhibit a radical concentration in the order of 1017 spins g−1 and that the radical species are stable under ambient conditions. The radical species are assumed to be semiquinone radicals stabilized in the polyphenolic lignin matrix.2−5 This hypothesis was supported by mainly three observations. First, the lignin macromolecule contains structural elements that make the formation of semiquinone-like species through oxidation reactions very likely (Figure 1). Second, the radical concentration in lignin increases significantly upon basification, which can be explained by a comproportionation reaction of hydroquinones HQ and quinones Q to semiquinone radicals (SQ) and semiquinone radical anions SQA (Figure 1). Third, the isotropic g factor giso of lignin is similar to the giso factors of model semiquinone radicals.2−5 Radicals in lignin are closely related to those in humic acids (HAs). Both substances exhibit similar structural elements as HAs are formed by the degradation of lignin containing © XXXX American Chemical Society

Figure 1. Subunits in lignin and the formation of semiquinone radicals (SQ) that can be deprotonated to the semiquinone radical anion (SQA) upon basification or can disproportionate to a hydroquinone (HQ) and a quinone (Q). R = OMe for syringyl subunits and R = H for guaiacyl subunits.

biomass.2,9−11 The radical content in HAs is also assigned to semiquinone-like species. However, conventional X-band EPR Received: March 6, 2015 Revised: May 15, 2015

A

DOI: 10.1021/acs.jpca.5b02200 J. Phys. Chem. A XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry A investigations yield only the giso factor, the anisotropy of the g tensor cannot be resolved. Using high-field EPR (HFEPR) spectroscopy, it is possible to resolve the three principal values gxx, gyy, and gzz of the g tensor, as was recently demonstrated for radicals in natural HAs by Christoforidis et al.12 They found that two radical species are present in HAs. The first species prevails at pH 5 and is characterized by gxx = 2.0032, gyy = 2.0032, and gzz = 2.0023, whereas the second species, prevailing at pH 12, is characterized by gxx = 2.0057, gyy = 2.0055, and gzz = 2.0023. Both species are assumed to be π-type radicals because they show similarities to phenolic model compounds such as gallic acid. The dependence of g tensor principal values of semiquinone and tyrosyl radicals on polarity and proticity of the radical environment has been studied in detail by EPR,13−21 density functional theory (DFT),22−27 and Car−Parrinello molecular dynamics computations.28 These studies show that the gxx and gyy tensor components are most sensitive to the radical environment, i.e., protons from H bonds and cations near the phenoxy oxygen nuclei. The work of Christoforidis et al.12,29,30 demonstrated the potential of HFEPR in the investigation of organic radicals in biomass. Corresponding data for radicals in lignin are still missing. In this paper we report on the first HFEPR (263 GHz) measurements of radicals in lignin. Furthermore, we investigate the influence of (a) the botanical origin and (b) the lignin isolation method on the radical structure and compare the results with DFT calculations for the proposed radicals.

cylindrical brass probe (E9501310, Bruker) with a polished bronze mirror at the end of a corrugated waveguide. The field values during the sweeps were calibrated by an internal spectrometer linearization procedure. A power attenuation of 0 dB was used corresponding to the maximum output power of the bridge (about 15 mW), whereas 5 mW was incident at the sample. A field modulation amplitude of 3 G and a conversion time of 1000 ms were used. The number of scans varied from 4 to 27 depending on the radical concentration of the sample. The CW-EPR spectra were recorded with quadrature detection. The spectra were post processed by phase correction to account for phase shifts. The samples were measured at room temperature. A 1,3-bisdiphenylene-2-phenylallyl (BDPA giso = 2.006033) radical sample was used as reference for the g factor determination. X-Band CW-EPR. All spectra were recorded using a MiniScope MS 400 table top EPR spectrometer from Magnettech. A microwave attenuation of 29 dB (Pmax = 20 mW) was used to avoid saturation effects. A modulation amplitude of 2 G and a receiver gain of 2−100 (linear factor) were applied. The radical concentration as well as the g factor were determined using a 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL giso = 2.0062334) radical standard for calibration. DFT Calculations. All calculations were performed using the ORCA 3.0.2 program package.35 The geometry optimizations were carried out using the unrestricted B3LYP36−38 functional. A combination of the triple-ζ basis set def2TZVPP39 and the auxiliary basis set def2-TZVPP/JK40 was used. The RIJCOSX41 approximation was applied. The conductor like screening model COSMO42,43 was used to simulate the polymer matrix influences assuming a polarity of ε = 24.3 (ethanol). The g tensors were calculated44,45 using the BP8636,46 functional and the DZVP47 basis set. The hyperfine interactions were calculated using the B3LYP functional and the EPR-II48 basis set. This combination was proven to be effective in literature.49 The energy was converged to 10−9 Eh. All optimized structures were checked by computation of the vibrational frequencies.

■

EXPERIMENTAL SECTION Sample Preparation. All lignin samples were prepared and measured in powder form. The wood pretreatment and Klason lignin isolation was performed as described elsewhere.31 Dioxane lignin was extracted as described in literature.32 Organosolv lignins were prepared as follows: The extractive free wood samples were treated with aqueous ethanol solution (60 vol %) containing sulfuric acid (5 wt %) in a microwave reactor at 200 °C for 55 min. After reaction, the vessel was cooled to room temperature and the contents were filtrated. The residue was washed with about 30 mL of ethanol (same concentration as for the reaction) and then washed with acetone until no color remained in the filtrate. The filtrate was collected into a flask and concentrated using a rotary evaporator. The concentrated filtrate was poured into about 400 mL of water under stirring. The precipitated Organosolv lignin was collected by filtration, washed thoroughly with water, and air-dried. pH Manipulation of Lignin. To manipulate the proton concentration present in the solid lignin samples a sample of pine Klason lignin was dispersed in a solution that contained either sulfuric acid for the low pH values or sodium hydroxide for the high pH values, respectively. The pH value was measured using a Handylab pH/LF 12 pH meter from Schott Instruments. Afterward, the lignin was separated by filtration and dried at 50 °C in a vacuum oven. High-Frequency CW-EPR. Continuous-wave (CW) EPR spectra were recorded at 263 GHz on a Bruker Elexsys E780 quasi-optical spectrometer using induction mode operation. A Faraday rotator separates the perpendicular component, with respect to the linearly polarized incident microwave beam, of the elliptically polarized microwave signal. The sample is placed in the PTFE cup (o.d. 4 mm, height 3 mm) of a nonresonant

■

RESULTS AND DISCUSSION The extraction process and raw material used for the isolation of lignin influences the chemical structure of lignin and the electronic structure of the radicals contained in the polymer. To study this influence, we measured the EPR spectra of several lignin samples using CW- X-band and HFEPR. Sample Preparation. The samples were prepared from two hardwood (beech, poplar) and two softwood (pine, spruce) trees, which served as raw material for the lignin extraction. The following samples were prepared: (I) Klason lignin: The lignin is separated from the cellulose by a two-step hydrolysis of the carbohydrates in concentrated and 3 wt % (∼0.3 mol L−1; pH ≈ 0.2) sulfuric acid at 100 °C. (II) Dioxane Lignin: The isolation takes place under the exclusion of oxygen in a water/dioxane mixture, containing 0.1 mol L−1 (pH ≈ 1.0) hydrochloric acid, at 90 °C. (III) Organosolv lignin: The lignin is extracted using a water/ ethanol mixture, containing 5 wt % (∼0.5 mol L−1; pH ≈ 0.0) sulfuric acid, at 200 °C. (IV) Wood: Chopped, but untreated wood from the trees. B


Article

The Journal of Physical Chemistry A

concentration of ∼1.9 × 1018 spins g−1. The most likely cause for this increase is the higher temperature of 200 °C in the Organosolv process, compared to only 90 and 100 °C in the Dioxane and Klason processes, respectively. At 200 °C thermal energy appears to be sufficient for homolytic bond scission that results in formation of additional radicals. Alternatively, the additional radicals may have been formed by oxidation of hydroquinone anions. The highest mean g factor of 2.0049 was detected for the untreated wood samples (Class IV), with the values of all woods exceeding 2.0045. For all samples obtained by extraction in the presence of acids, we find g values below 2.0045. For the Dioxane lignins (Class II), the mean g value is 2.0043, for the Klason lignins (Class I) it is 2.0039, and for the Organosolv lignins (Class III) it is 2.0037. There is no significant correlation between the radical concentrations and the g factors. The variation of the g factors is possibly a consequence of the different acidity in the extraction mixtures, which may influence protonation states of the semiquinone radicals or lead to predominance of different radical species. In particular, it cannot be excluded at this stage that the additional radicals generated in the Organosolv process are different from the radicals in the other samples. To check this hypothesis, the proton concentration in a pine Klason lignin sample was changed by rinsing the sample with solutions of different pH values. After drying, the X-band EPR spectra were recorded (Figure 3b). In all cases we observed a single broad signal with no resolved hyperfine coupling. Table 1 shows the g factors, which increase with increasing pH value and cover the range of g values observed in the class IV wood samples and the class II Dioxane lignins. The g values of the Klason and Organosolv lignins produced in this study are similar to the ones observed for the rinsed sample at pH 1.0. This can be explained by the low pH during the extraction, which is below 1.0. More detailed information on the structure of these radicals cannot be obtained from X-band EPR due to the limited g resolution and the absence of resolved hyperfine couplings. In particular, it is not possible to deduce from the g factors whether the differences in the spectra are caused by protonation/deprotonation of one radical species or whether completely different radical species prevail at different pH values or after different extraction processes. High-Field EPR. The electronic structure of the radicals contained in the acid- and base-treated lignin was studied in depth by HFEPR at ∼9.4 T/263 GHz, where the g tensor principal values could be resolved. The three components gxx, gyy, and gzz were determined by fitting the HFEPR spectra using the EasySpin toolbox developed by Stoll et al.51 We estimate an error of 0.5 × 10−4 for the reported gii (ii = x, y, z)values.52 A free electron exhibits a g factor of ge = 2.002319. If the electron is located in a molecule, the g factor shifts away from ge. This g shift is related to spin−orbit coupling when the external magnetic field is parallel to the corresponding principal axis. For π radicals, such as semiquinone-type radicals, the principal axis of gzz is oriented perpendicular to the aromatic system and is only very weakly influenced by spin−orbit coupling.53,54 Hence, the corresponding gzz shift is small and depends only weakly on radical structure and environment. In contrast, gxx and gyy correspond to in-plane orientations and exhibit large shifts due to spin−orbit coupling with contributions mainly from the oxygen atoms.53,54 This was also confirmed by Kaupp et al. at the DFT level.55 For these values we observe strong variation with pH. The gxx and gyy values increase significantly

X-Band EPR. All samples yielded X-band EPR spectra containing one single broad signal with no resolved hyperfine interactions (Figure 3b). Only the g factor and the radical concentration of the powders could be determined from those measurements. Figure 2 shows a plot of the radical concentration in spins per gram sample mass vs the g factor of the detected radicals.

Figure 2. Plot of the g factor vs the radical concentration in several lignin preparations.

According to this plot, the samples can be grouped into four classes (I−IV), with each class corresponding to samples from all four different woods subjected to the same preparation method. Within each class g and the radical concentration vary only moderately, and this variation among the different wood samples does not correlate between classes. The variations are due to limited accuracy of the measurements and heterogeneity of the material. We estimate a 5% error in the radical concentration mainly from sample positioning in the cavity and an error of g of about 2 × 10−4 that is related to the line width. These error estimates suggest that inhomogeneity of lignin is the dominating effect. The lignin structure varies significantly, even within the same tree.50 The spectra arise from a mixture of different species in different environments (vide inf ra). Also the fractions of the species and the mean radical environment are likely to be influenced in slightly different ways for the different woods. Some variations may also arise because the extraction process is not perfectly reproducible. However, from these data we can safely conclude that the botanical origin of the lignin has smaller impact on radical concentration and the g factor than the extraction method. The lowest radical concentration was detected in the untreated wood samples (Class IV). These samples showed a mean radical concentration of 6 × 1016 spins g−1. Because the vast majority of the radicals in wood are located in the lignin fraction, which makes up only ca. 30 wt % of the softwoods and ca. 22 wt % of the hardwoods, we can estimate a radical concentration in the range ∼1 × 1017 to ∼5 × 1017 spins g−1 in the lignin fraction. Within experimental uncertainty these radical concentrations are the same as in Klason (Class I) and Dioxane (Class II) lignin, containing ∼1.7 × 1017 and ∼2.1 × 1017 spins g−1, respectively. Hence, these two extraction methods do not cause significant changes in the radical concentration of the lignin polymer. In contrast, Organosolv lignin (Class III) has an about 10-fold increased radical C


Article


(Figure 3a). Absorption along the three components differs significantly in signal width, which is large along gxx and gyy and small along gzz. There are two major contributions to the broadening. First there is a distribution of the principal g values that is most likely related to different numbers and geometries of hydrogen bonds to the radical28 as well as different substituents on the phenolic rings. This g strain shows a directional dependence, it is large along gxx and gyy and small along gzz. Second there are unresolved proton hyperfine couplings. We quantify the g strain by the full width at half-maximum of a Gaussian distribution function (σi) of the gxx, gzz and gyy value respectively. Unresolved hyperfine couplings along the principal axes directions of the g tensor lead to further broadening. Although this broadening is small compared to g broadening along gxx and gyy, it is the dominant effect along gzz. The spectra of three pH preparations of Klason lignin at 9 GHz (Figure 3a) and 263 GHz (Figure 3b) are compared. The overall set of spectra can be fitted satisfactorily by assuming three different sets of principal g values and hence three radical species R1, R2, and R3 (Table 2), respectively. Table 2. Contribution of the Different Radical Species R1, R2, and R3 to the EPR Spectra between pH = 1 and pH = 13.3, As Determined by Fitting the HF EPR Spectra pH pH pH pH pH

= = = = =

1.0 3.7 6.9 8.6 13.3

R1

R2

R3

1.00 0.27 0.21 0.15 0.00

0.00 0.64 0.69 0.74 0.00

0.00 0.09 0.10 0.11 1.00

To quantify both broadening contributions, we fitted X-band and HFEPR data of the R1 (pH 1) and the R3 (pH 13.3) samples iteratively. In the initial step, the HFEPR spectra, for which g broadening dominates at least along gxx and gyy, were fitted with only g broadening. In the second step, the hyperfine broadening parameters were determined by fitting the X-band EPR spectrum, for which g broadening is a minor contribution, while the g broadening parameters were held constant. In the third step, the HFEPR spectrum was fitted again by varying the g broadening factors while the hyperfine broadening parameters from the previous step were kept constant. Steps 2 and 3 were repeated until the resulting g and hyperfine broadening parameters led to a satisfying fit of both spectra (Figure 3). The spectra of the R2 (pH 3.7−pH 8.6) species contained also contributions of the R1 and the R3 species. Therefore, an admixture of both spectra was used to fit the R2 spectra using the procedure mentioned above (Table 3, cf. Figures 3 and S5, Supporting Information). For the hyperfine broadening we cannot resolve any anisotropy or differences between the three species. An isotropic broadening of 15 MHz fits all spectra. The g principal values of radicals R2 and R3 are within the range expected for semiquinone radical anions and neutral semiquinone radicals in

Figure 3. (a) HFEPR spectra of Klason lignin at pH = 1.0, pH = 8.6, and pH = 13.3 with the corresponding fits. (b) X-band EPR spectra of Klason lignin at pH = 1.0, pH = 6.9, and pH = 13.3 with the corresponding fits. (c) Structures of the proposed radicals species in different protonation states SH3+, SH2, SH1-, and S2-; R = CH3 for all DFT calculations.

upon basification while the gzz value stays constant. There is no clearly resolved hyperfine interaction in any of the spectra

Table 1. g Factors (Determined by X-Band EPR) of Pine Klason Lignin Subjected to Solutions with Different pH Values (pH Variation by Addition of H2SO4 or NaOH)

g

pH = 1.0

pH = 3.7

pH = 6.8

pH = 8.6

pH = 13.3

2.0034 ± 2×0.00−4

2.0039 ± 2×10−4

2.0042 ± 2×10−4

2.0042 ± 2×10−4

2.0049 ± 2×10−4

D


Article


Table 3. g Factors and g Strain (σi) of the Different Radical Species R1, R2, and R3, As Determined by Fitting of the HF- and XBand EPR Spectra and g Factors of the Different Protonation States of the Proposed Structures SH3+, SH2, SH1-, and S2-, As Determined by DFT (BP86/DVPZ/COSMO) gxx R1exp R2exp R3exp SH3+DFT SH2DFT SH1-DFT S2-DFT a

2.0042 2.0053 2.0060 2.00477 2.00457a 2.00640 2.00607a 2.00668 2.00633a 2.00684 2.00648a

gyy 2.0033 2.0044 2.0056 2.00395 2.00382a 2.00474 2.00455a 2.00625 2.00594a 2.00663 2.00628a

σxx

gzz

σyy −4

7.8 × 10 7.9 × 10−4 6.9 × 10−4

2.0025 2.0024 2.0024 2.00221 2.00222a 2.00213 2.00220a 2.00213 2.00214a 2.00215 2.00216a

σzz −4

giso −4

6.9 × 10 4.7 × 10−4 4.4 × 10−4

1.9 × 10 0.1 × 10−4 0.9 × 10−4

2.0034 2.0041 2.0047 2.00361 2.00354a 2.00444 2.00427a 2.00502 2.00480a 2.00521 2.00498a

g factor after correction as reported by Kaupp et al.22

environments of different polarity.24−27,56 The isotropic g values for R1, R2, and R3 are 2.0034, 2.0041, and 2.0047, respectively. A decrease of giso with decreasing pH has been observed for semiquinones13,14,16 and has been attributed to conversion from the radical anion via the neutral species to the radical cation.56 Similar effects have been observed in synthetic dopa-melanin, using Q-band EPR.57 However, the g resolution of Q-band EPR is lower compared to HFEPR. Considering in addition the structure of lignin, we tentatively assign the three species to o-semiquinone radicals derived from syringyl units (Figure 3c). Among those, R1 is assigned to the radical cation SH3+, R2 to the neutral radical SH2, and R3 to radical anions SH1- and/or S2-. To determine if similar species are also present in the lignin extracted by the different methods (Classes I−IV), HFEPR spectra of these samples were recorded as well (Figure 4). The spectra of the Klason and the Dioxane lignin contained small signals of a Mn(II) species. These traces of manganese

probably arise from minerals within the wood samples that were not completely removed during wood pretreatment and lignin extraction. The g anisotropy of all samples could be resolved and some general qualitative trends could be observed. For none of the spectra hyperfine interactions are resolved. The position of the edge of the signal corresponding to the gzz component was identical in all spectra. However, the gxx and gyy values depend significantly on the extraction method. The gxx and gyy signals are broader than the one of the gzz component in all spectra. The gii values were determined by fitting the HFEPR spectra (Table 4). An admixture of a Mn(II) species was taken Table 4. g Principal Factors and giso of the Different Radical Species in Organosolv, Dioxane, and Klason Lignin As Determined by Fitting the HFEPR Spectra Organosolv ligninbeech Organosolv ligninpine Dioxane ligninbeech Klason ligninpine

gxx

gyy

gzz

giso

2.0046 2.0045 2.0056 2.0042

2.0033 2.0032 2.0048 2.0035

2.0024 2.0024 2.0024 2.0024

2.0034 2.0034 2.0042 2.0033

into account for fitting the Klason and the Dioxane lignin spectra. The isotropic g values are 2.0034, 2.0042, and 2.0033 for the Organosolv lignins of both woods, the Dioxane lignin, and the Klason lignin, respectively. For the comparison of the different extraction methods we simplified the fitting procedure described above. Only g strains were considered and an admixture of different species was neglected. The influence of the botanical origin is negligible compared to the influence of the lignin isolation method. The g values are similar for hardwood and softwood based Organosolv lignin radicals. The radical species in Klason lignin also have similar g values. We assume that the dominant radical species in Organosolv as well as Klason lignin is the SH3+ species because its g principal values roughly match those of the R1 species (pH = 1; Figure 3). For the Dioxane lignin the dominant species is assigned to the SH2 radical, probably with an admixture of SH3+ and SH1-/S2- as it is also the case for the HFEPR spectrum of the R2 species. These observations support the assumption that the acid concentration present during extraction method is accountable for the differences in the EPR spectra of lignin preparations. DFT Calculations. To assign the species DFT calculations of the suggested structures SH3+, SH2, SH1-, and S2- were

Figure 4. (a) HFEPR spectrum of hardwood (beech) and softwood (pine) Organosolv lignin. (b) HFEPR spectrum of softwood (pine) Klason lignin. (c) HFEPR spectrum of hardwood (beech) Dioxane lignin. Mn(II) traces contained in the lignin samples are marked with an asterisk (*). E


Article


origin of the lignin. The parameter influencing the g value most is the pH value during lignin extraction. This effect was studied in depth using high-field EPR spectroscopy at 263 GHz. Here, sharp spectral features along gzz could be resolved from broad spectral features along gxx and gyy. We were able to determine the gxx, gyy, and gzz components of the g tensor of the stable organic radicals in lignin. Distinct radical species could be distinguished in this complex polymer. The radical species are assigned to substituted o-semiquinone radicals and can exist in different protonation states SH3+, SH2, SH1-, and S2-. The protonated form SH3+ is dominant below pH 1. The neutral form SH2 is dominant at pH 3.7−pH 8.9. However, also the SH3+, SH1-, and S2- species are detectable at those pH values. The deprotonated species SH1- and S2- are dominant at pH 13. We supported our model by DFT calculations. The g principal values of the proposed structure were all in reasonable agreement with the experiments. However, the gxx components were overestimated due to neglect of matrix interactions. Protonation of the o-semiquinoid radicals in the solid lignin matrix increased with decreasing pH during extraction. The protonated form SH3+ appeared to be dominant in Klason and Organosolv lignin, whereas the neutral form SH2 dominated in Dioxane lignin and anionic species appeared to contribute most strongly in untreated wood samples.

performed. The connections to the polymeric lignin network were mimicked by methyl groups. The influences of larger substituents at the aliphatic backbone have a negligible influence on the g tensor due to low spin density population on the sp3 carbons (Tables S2, S4, and S6, Supporting Information). Table 3 summarizes the calculated and the experimentally determined gxx, gyy, and gzz values. In general, the calculated gxx value is by far larger than the experimentally observed values. Such an overestimate in the g factors has been found before for p-benzosemiquinone by Kacprzak and Kaupp.22 They assume that this deviation is caused by an underestimation of the energy of certain exited states, leading to an overestimated g factor. If we apply their empirical correction factor for the calculated g factors, we obtain a rather good agreement of R3 with SH1- and S2- and reasonable agreement of R2 with SH2, although no explicit interactions with the lignin matrix have been considered, i.e., H bonds. Note that the gxx and gyy values for R2 have a larger uncertainty caused by significant contributions of R1 and R3 to the spectrum, which are difficult to disentangle due to the limited resolution of the g strain. This uncertainty is hard to estimate but is probably smaller than 5 × 10−4. The larger gxx value obtained by DFT can be explained by the fact that the gxx value is extremely dependent on the polarity of the radicals surroundings.24−27,43 The simulation of the polarity by the COSMO model is not sufficient to achieve an accurate model, because the DFT calculations are performed on a single molecule in gas phase with artificial modified dielectric constant, and bimolecular interactions such as hydrogen bonds with the polymer matrix are not simulated. The deviation between the theoretical and experimental gxx and gyy values are consistent with the expectation that the lignin’s polymeric network interacts with the radical species SH2, SH1-, and S2-. To obtain better agreement, an adequate model of such matrix interactions would be required. Matrix interactions, in particular dipolar hyperfine couplings to protons in neighboring diamagnetic side groups and structural variation in the linker between the radical side group and the polymer network, also influence the accuracy of predictions of line broadening by unresolved hyperfine couplings by the DFT computations. Therefore, we refrain from reporting simulated spectra including DFT-computed hyperfine couplings. Such simulations are in good agreement with the experimental high-field EPR spectra if g strain is also included but overestimate the width of the X-band EPR spectrum of the pH = 1.0 sample. This problem can be traced back to a large isotropic coupling of the β-proton in the geometry-optimized structure and can be alleviated by rotating this proton into the ring plane. Taking into account that hydrogen bonding interactions of the radical with the matrix may further reduce this coupling, we believe that the DFTcomputed hyperfine couplings are consistent with our assignment of the radicals.

■

ASSOCIATED CONTENT

S Supporting Information *

X-band and HFEPR spectra of all lignin samples. Optimized structures (DFT), geometries, and Loewdin spin populations. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.5b02200.

■

AUTHOR INFORMATION

Corresponding Author

*F. Vogel. Telephone: +41 56 310-2135. E-mail: frederic. [email protected]. Notes

The authors declare no competing financial interest. ∥ C. Bährle. E-mail: [email protected]. ⊥ T. U. Nick. E-mail: [email protected]. # M. Bennati. E-mail: [email protected]. ¶ G. Jeschke. E-mail: [email protected].

■

ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (SNF) as part of NRP66 “Resource Wood” under grant number 406640136892. We thank Rene Tschaggelar and Igor Tkach for technical support during the EPR measurements. We also thank Zhiqiang Ma, Victoria Custodis, and Jeroen Anton van Bokhoven for supplying the Organosolv lignin samples and Alexander Wokaun for the fruitful discussions.

■

■

CONCLUSION Radical concentrations and g factors of different lignin preparations were determined. Whereas the Dioxane and Klason extraction methods did not lead to significant changes in radical concentration compared to the wood samples from which the lignin was extracted, an about 10-fold increase in radical concentration was observed with the Organosolv process. We observed that the g factor of a lignin is largely determined by the extraction process and not by the botanical

REFERENCES

(1) Rex, R. W. Electron Paramagnetic Resonance Studies of Stable Free Radicals in Lignins and Humic Acids. Nature 1960, 188, 1185− 1186. (2) Steelink, C. Free Radical Studies of Lignin, Lignin Degradation Products and Soil Humic Acids. Geochim. Cosmochim. Acta 1964, 28, 1615−1622. (3) Steelink, C. Stable Phenoxy Radicals Derived from Phenols Related to Lignin. J. Am. Chem. Soc. 1965, 87, 2056. F


Article

The Journal of Physical Chemistry A (4) Steelink, C.; Hansen, R. E. A Solid Phenoxy Radical from Disyringylmethane−a Model for Lignin Radicals. Tetrahedron Lett. 1966, 7, 105−111. (5) Fitzpatrick, J. D.; Steelink, C. Benzosemiquinone Radicals in Alkaline Solutions of Hardwood Lignins. Tetrahedron Lett. 1969, 10, 5041−5044. (6) Oniki, T. Origin of Free Radicals Produced From the Syringyl end Groups in Lignins. J. Wood Sci. 1998, 44, 314−319. (7) Dizhbite, T.; Telysheva, G.; Jurkjane, V.; Viesturs, U. Characterization of the Radical Scavenging Activity of Lignins - Natural Antioxidants. Bioresour. Technol. 2004, 95, 309−317. (8) Nsimba, R. Y.; West, N.; Boateng, A. A. Structure and Radical Scavenging Activity Relationships of Pyrolytic Lignins. J. Agric. Food Chem. 2012, 60, 12525−12530. (9) Steelink, C.; Tollin, G. Stable Free Radicals in Soil Humic Acid. Biochim. Biophys. Acta 1962, 59, 25−34. (10) Cornelius, S., Electron Paramagnetic Resonance Studies of Humic Acid and Related Model Compounds. In Coal Science; American Chemical Society: Washington, DC, 1966; Vol. 55, pp 80−90. (11) Riffaldi, R.; Schnitzer, M. Electron Spin Resonance Spectrometry of Humic Substances1. Soil Sci. Soc. Am. J. 1972, 36, 301−305. (12) Christoforidis, K. C.; Un, S.; Deligiannakis, Y. High-Field 285 GHz Electron Paramagnetic Resonance Study of Indigenous Radicals of Humic Acids. J. Phys. Chem. A 2007, 111, 11860−11866. (13) Burghaus, O.; Plato, M.; Rohrer, M.; Moebius, K.; MacMillan, F.; Lubitz, W. 3-mm High-field EPR on Semiquinone Radical Anions Q.cntdot.- Related to Photosynthesis and on the Primary Donor P.cntdot.+ and Acceptor QA.cntdot.- in Reaction Centers of Rhodobacter Sphaeroides R-26. J. Phys. Chem. 1993, 97, 7639−7647. (14) MacMillan, F.; Lendzian, F.; Lubitz, W. EPR and ENDOR Characterization of Semiquinone Anion Radicals Related to Photosynthesis. Magn. Reson. Chem. 1995, 33, S81−S93. (15) Rohrer, M.; Plato, M.; Macmillan, F.; Grishin, Y.; Lubitz, W.; Mobius, K. Orientation-Selected 95 GHz High-Field ENDOR Spectroscopy of Randomly Oriented Plastoquinone Anion Radicals. J. Magn. Reson., Ser. A 1995, 116, 59−66. (16) Knüpling, M.; Törring, J. T.; Un, S. The Relationship Between the Molecular Structure of Semiquinone Radicals and Their g-Values. Chem. Phys. 1997, 219, 291−304. (17) MacMillan, F.; Hanley, J.; van der Weerd, L.; Knüpling, M.; Un, S.; Rutherford, A. W. Orientation of the Phylloquinone Electron Acceptor Anion Radical in Photosystem I. Biochemistry 1997, 36, 9297−9303. (18) Dorlet, P.; Boussac, A.; Rutherford, A. W.; Un, S. Multifrequency High-Field EPR Study of the Interaction between the Tyrosyl Z Radical and the Manganese Cluster in Plant Photosystem II. J. Phys. Chem. B 1999, 103, 10945−10954. (19) Lubitz, W.; Feher, G. The Primary and Secondary Acceptors in Bacterial Photosynthesis III. Characterization of the Quinone Radicals Q A − · and Q B − · by EPR and ENDOR. Appl. Magn. Reson. 1999, 17, 1−48. (20) Dorlet, P.; Seibold, S. A.; Babcock, G. T.; Gerfen, G. J.; Smith, W. L.; Tsai, A.-l.; Un, S. High-Field EPR Study of Tyrosyl Radicals in Prostaglandin H2 Synthase-1†. Biochemistry 2002, 41, 6107−6114. (21) Flores, M.; Okamura, M. Y.; Niklas, J.; Pandelia, M.-E.; Lubitz, W. Pulse Q-Band EPR and ENDOR Spectroscopies of the Photochemically Generated Monoprotonated Benzosemiquinone Radical in Frozen Alcoholic Solution. J. Phys. Chem. B 2012, 116, 8890−8900. (22) Kacprzak, S.; Kaupp, M. Electronic g-Tensors of Semiquinones in Photosynthetic Reaction Centers. A Density Functional Study. J. Phys. Chem. B 2004, 108, 2464−2469. (23) Solc, R.; Gerzabek, M. H.; Lischka, H.; Tunega, D. Radical Sites in Humic Acids: A Theoretical Study on Protocatechuic and Gallic Acids. Comput. Theor. Chem. 2014, 1032, 42−49. (24) Witwicki, M.; Jaszewski, A. R.; Jezierska, J.; Jerzykiewicz, M.; Jezierski, A. The pH-Induced Shift in the g-Tensor Components of

Semiquinone-Type Radicals in Humic Acids - DFT and EPR Studies. Chem. Phys. Lett. 2008, 462, 300−306. (25) Witwicki, M.; Jezierska, J. Protic and Aprotic Solvent Effect on Molecular Properties and g-Tensors of o-Semiquinones with Various Aromacity and Heteroatoms: A DFT Study. Chem. Phys. Lett. 2010, 493, 364−370. (26) Witwicki, M.; Jezierska, J. Protonated o-Semiquinone Radical as a Mimetic of the Humic Acids Native Radicals: A DFT Approach to the Molecular Structure and EPR Properties. Geochim. Cosmochim. Acta 2012, 86, 384−391. (27) Witwicki, M.; Jezierska, J.; Ozarowski, A. Solvent Effect on EPR, Molecular and Electronic Properties of Semiquinone Radical derived from 3,4-Dihydroxybenzoic Acid as Model for Humic acid Transient Radicals: High-field EPR and DFT Studies. Chem. Phys. Lett. 2009, 473, 160−166. (28) Asher, J. R.; Doltsinis, N. L.; Kaupp, M. Extended Car-Parrinello Molecular Dynamics and Electronic g-Tensors Study of Benzosemiquinone Radical Anion. Magn. Reson. Chem. 2005, 43, S237−S247. (29) Christoforidis, K. C.; Un, S.; Deligiannakis, Y. Effect of Metal Ions on the Indigenous Radicals of Humic Acids: High Field Electron Paramagnetic Resonance Study. Environ. Sci. Technol. 2010, 44, 7011− 7016. (30) Giannakopoulos, E.; Christoforidis, K. C.; Tsipis, A.; Jerzykiewicz, M.; Deligiannakis, Y. Influence of Pb(II) on the Radical Properties of Humic Substances and Model Compounds. J. Phys. Chem. A 2005, 109, 2223−2232. (31) Bährle, C.; Custodis, V.; Jeschke, G.; van Bokhoven, J. A.; Vogel, F. In situ Observation of Radicals and Molecular Products During Lignin Pyrolysis. ChemSusChem 2014, 7, 2022−9. (32) Evtuguin, D. V.; Neto, C. P.; Silva, A. M. S.; Domingues, P. M.; Amado, F. M. L.; Robert, D.; Faix, O. Comprehensive Study on the Chemical Structure of Dioxane Lignin from Plantation Eucalyptus Globulus Wood. J. Agric. Food Chem. 2001, 49, 4252−4261. (33) Bennati, M.; Farrar, C. T.; Bryant, J. A.; Inati, S. J.; Weis, V.; Gerfen, G. J.; Riggs-Gelasco, P.; Stubbe, J.; Griffin, R. G. Pulsed Electron-Nuclear Double Resonance (ENDOR) at 140 GHz. J. Magn. Reson. 1999, 138, 232−243. (34) Capiomont, A.; Chion, B. Lajzerowicz-Bonneteau, J.; Lemaire, H. Interpretation and Utilization for Crystal Stucture Determination of ESR Spectra of Single Crystals of Nitroxide Free Radicals. J. Chem. Phys. 1974, 60, 2530−2535. (35) Neese, F. The ORCA Program System. Wiley Interdiscip. Rev.Comput. Mol. Sci. 2012, 2, 73−78. (36) Becke, A. D. Density-Functional Exchange-Energy Approximation with Correct Asymptotic Behavior. Phys. Rev. A: At., Mol., Opt. Phys. 1988, 38, 3098−3100. (37) Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648−5652. (38) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron Density. Phys. Rev. B: Condens. Matter Mater. Phys. 1988, 37, 785−789. (39) Weigend, F.; Ahlrichs, R. Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys. Chem. Chem. Phys. 2005, 7, 3297−3305. (40) Weigend, F. A Fully Direct RI-HF Algorithm: Implementation, Optimised Auxiliary Basis Sets, Demonstration of Accuracy and Efficiency. Phys. Chem. Chem. Phys. 2002, 4, 4285−4291. (41) Neese, F.; Wennmohs, F.; Hansen, A.; Becker, U. Efficient, Approximate and Parallel Hartree-Fock and Hybrid DFT Calculations. A ’Chain-of-Spheres’ Algorithm for the Hartree-Fock Exchange. Chem. Phys. 2009, 356, 98−109. (42) Klamt, A.; Schuurmann, G. COSMO: A New Approach to Dielectric Screening in Solvents with Explicit Expressions for the Screening Energy and its Gradient. J. Chem. Soc., Perkin Trans. 2 1993, 799−805. (43) Sinnecker, S.; Rajendran, A.; Klamt, A.; Diedenhofen, M.; Neese, F. Calculation of Solvent Shifts on Electronic g-Tensors with the Conductor-Like Screening Model (COSMO) and its SelfG


Article

The Journal of Physical Chemistry A Consistent Generalization to Real Solvents (Direct COSMO-RS). J. Phys. Chem. A 2006, 110, 2235−2245. (44) Neese, F. Prediction of Electron Paramagnetic Resonance g Values using Coupled Perturbed Hartree−Fock and Kohn−Sham Theory. J. Chem. Phys. 2001, 115, 11080−11096. (45) Neese, F. Efficient and Accurate Approximations to the Molecular Spin-Orbit Coupling Operator and their Use in Molecular g-Tensor Calculations. J. Chem. Phys. 2005, 122, 034107. (46) Perdew, J. P. Density-Functional Approximation for the Correlation Energy of the Inhomogeneous Electron Gas. Phys. Rev. B: Condens. Matter Mater. Phys. 1986, 33, 8822−8824. (47) Godbout, N.; Salahub, D. R.; Andzelm, J.; Wimmer, E. Optimization of Gaussian-Type Basis Sets for Local Spin Density Functional Calculations. Part I. Boron through Neon, Optimization Technique and Validation. Can. J. Chem. 1992, 70, 560−571. (48) Barone, V. Structure, Magnetic Properties and Reactivities of Open-Shell Species From Density Functional and Self-Consistent Hybrid Methods. In Recent Advances in Density Functional Methods; Chong, D. P., Ed.; World Scientific: Singapore, 1996; pp 287−334. (49) Asher, J.; Kaupp, M. Car-Parrinello Molecular Dynamics Simulations and EPR Property Calculations on Aqueous Ubisemiquinone Radical Anion. Theor. Chem. Acc. 2008, 119, 477−487. (50) Vanholme, R.; Demedts, B.; Morreel, K.; Ralph, J.; Boerjan, W. Lignin Biosynthesis and Structure. Plant Physiol. 2010, 153, 895−905. (51) Stoll, S.; Schweiger, A. EasySpin, a Comprehensive Software Package for Spectral Simulation and Analysis in EPR. J. Magn. Reson. 2006, 178, 42−55. (52) Nick, T. U.; Lee, W.; Kossmann, S.; Neese, F.; Stubbe, J.; Bennati, M. Hydrogen Bond Network between Amino Acid Radical Intermediates on the Proton-Coupled Electron Transfer Pathway of E. coli alpha2 Ribonucleotide Reductase. J. Am. Chem. Soc. 2015, 137, 289−98. (53) Stone, A. In Gauge Invariance of the g Tensor; Proceedings of the Royal Society of London A: Mathematical, Physical and Engineering Sciences, London; The Royal Society: London, 1963; pp 424−434. (54) Stone, A. J. g Factors of Aromatic Free Radicals. Mol. Phys. 1963, 6, 509−515. (55) Kaupp, M.; Remenyi, C.; Vaara, J.; Malkina, O. L.; Malkin, V. G. Density Functional Calculations of Electronic g-Tensors for Semiquinone Radical Anions. The Role of Hydrogen Bonding and Substituent Effects. J. Am. Chem. Soc. 2002, 124, 2709−2722. (56) Pedersen, J. A. CRC Handbook of EPR Spectra from Quinones and Quinols; CRC Press: Boca Raton, FL, 1985. (57) Pasenkiewicz-Gierula, M.; Sealy, R. C. Analysis of the ESR Spectrum of Synthetic DOPA Melanin. Biochim. Biophys. Acta, Gen. Subj. 1986, 884, 510−516.

H


High-Field Electron Paramagnetic Resonance and Density Functional

Recommend Documents