Qualitative and quantitative analysis of solid lignin samples by carbon

172

Anal. Chem. 1907, 59, 172-179

Qualitative and Quantitative Analysis of Solid Lignin Samples by Carbon- 13 Nuclear Magnetic Resonance Spectrometry Galen R. Hatfield, Gary E. Maciel,* Oktay Erbatur,' a n d Gaye E r b a t u r ' Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 The solid llgnln preparations from two common woods, red oak and kdsepole plne, have been methylated and acetylated In order to examtne the relatlonsMps between the lacnuclear magnetlc resonance chemlcal shift and molecular structure In solid Hgnln samples. Comparlson of the untreated and chemlcatly modified Hgnlns results In a detailed set of chemical shift assignments, many of whlch directly refled prevlously reported Sdutkm-state studies on model cOmpOLmls and ilgnin extracts. The present study also demonstrates the abiltty of solid state ''C cross polarlzatlon magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectrometry to Identify many of the key functlonalttles In llgnln and to probe llgnln chemistry. Flnaily, the ablllty of I3C CP/MAS NMR spectrometry to yleld quantttative resutts In llgnln and wood spectra Is dlscussed.

Of the three major components of wood-cellulose, lignin, and hemicellulose-lignin is perhaps the least well understood. Its structure, chemistry, and utilization have been the topic of extensive studies, and yet many questions remain to be answered. The need to remove lignin in the production of paper products and the role of lignin in forages for ruminant animals have prompted much of this study. Recently, new potential has been seen for use of lignin in engineering plastics and other polymeric materials (1). NMR spectrometry has become one of the most successful structural tools available for the study of lignin chemistry. Extensive 13C NMR studies of lignin have been carried out in the liquid state (2-6), but solid-state studies have been extremely limited (7-10). Solid-state 13C NMR with cross polarization (CP) and magic angle spinning (MAS) (11-13) has proven to be invaluable in the study of complex organic solids. This approach renders the question of solubility irrelevant and eliminates the structural uncertainties associated with the dissolution process. As such, solid-state '3c CP/MAS NMR spectrometry permits detailed analysis of a complex organic solid in its natural state. The chemical shift in an NMR experiment is an extremely powerful tool for structural elucidation in macromoleculessuch as lignin. Extensive chemical shift tables have appeared in the literature (2-6) for lignin model compounds and for lignin extracts in the solution state. However, to date few chemical shift assignment studies have been carried out in the solid state. Direct application of solution-state assignments to the unaltered solid has been shown to be sometimes hazardous (14) (e.g., because of conformational differences), and the need to examine the chemical shift/structure relationship in the solid state is clear. In addition to examination of this relationship, the relative quantitativeness of 13C NMR spectra for lignin and similar samples needs to be ascertained. In this study, lignin preparations from two common woods, Quercus rubra (red oak) and Pinus contortu (lodgepole pine), have been chemically modified in order to better examine these relationships. The changes that are observed in the NMR spectra of the modified lignins permit a straightforward

analysis of some of the key functionalities observable in the solid state and demonstrate the ability of solid-state 13C CP/MAS NMR spectrometry to probe lignin chemistry. This study also addresses the ability of 13C CP/MAS NMR to quantitate the structural changes and differences in lignin that are observable by this technique. EXPERIMENTAL SECTION Preparation of (HC1-Hydrolysis) Lignins. Many types of lignin isolation procedures have been described, and there is no compelling evidence for choosing any one of these as representing lignin most faithfully. We have chosen, more or less arbitrarily, an HC1-hydrolysis procedure (15). Wood samples (red oak and lodgepole pine) were ground to 100 mesh (USA) and then dried under vacuum torr) at 80 "C overnight. The wood was then extracted with a benzene/ethanol mixture (2:1, v:v) in a Soxhlet apparatus for 48 h to remove any resin and was again dried under vacuum at 80 "C Overnight. The resin-free wood was then treated with fuming HC1 at a ratio of 13 cm3 of HCl/g of wood in a stoppered flask at -50 "C while being stirred by a magnetic stirrer for 1h (15). Stirring continued at room temperature for 8 h more. Two volumes of water were added and the precipitated lignin was fiitered out by suction, washed with two more volumes of distilled water, and finally dried under vacuum at 80 OC. 0-Methylation of Lignins. Methylation of lignins was carried out via the procedure of Liotta (16,17)for coals. This procedure involves the treatment of lignin with tetrabutylammonium hydroxide and subsequent alkylation by CH31. Two separate samples were prepared in this method, one with natural-abundance CH31 and one with CH31labeled with 13Cto 11%. Acetylation of Lignins. A 3-g sample of lignin was refluxed at 80 "C with 50 mL of a pyridine/acetic anhydride mixture (2:1, v:v) for 48 h while being stirred by a magnetic stirrer. The reaction mixture was then diluted with 500 mL of distilled water and filtered by suction. The solid was then washed several times with 5% aqueous tetrahydrofuran (THF) to release any residual pyridine and then finally washed with distilled water. The solid was then dried under vacuum at 80 "C overnight. NMR Spectrometry. 13CCP/MAS NMR spectra were obtained on a home-built spectrometer at a carbon frequency of 25.3 MHz (2.3 T field strength). Samples were spun at approximately 3.5 lrHz in a modified windmill-type spinner (18). The magic angle waa adjusted to within 0.1" by using the '9Br spectrum obtained by placing KBr in a spinner (19). The decoupler field strength was 12 G. The details of repetition time and cross polarization time are discussed in the text. RESULTS AND DISCUSSION Lignin consists of a variety of phenylpropane repeat units, most significantly those of syringyl (1) and guaiacyl (2) moieties. A great deal of work has gone into the development

On leave from the Chemistry Department,Cukurova University, PK 171, Adana, Turkey. 0003-2700/87/0359-0172$01.50/00 1986 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987

173

n

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Flgure 1. 13C CP/MAS spectra of red oak samples: (A) ground wood, (B) lignin preparation, (C) cellulose, (D) 0.85C 4- 0.15B.

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Figwe 2. 13C CP/MAS spectra of lodgepole pine samples: (A) ground wood, (B) lignin preparation, (C) cellulose, (D) 0.82C -t 0.18B.

of structural models for lignin. Such models have often drawn upon the results of solution-state NMR studies (2-6)and will serve as the basis of discussion here. The 13CCP/MAS NMR spectra of red oak and lodgepole pine woods are shown in Figures 1A and 24, respectively. The spectra of the HC1-hydrolysis lignin preparations from each of these woods are given in Figures 1B and 2B. The details of these spectra will be the focus of most of the discussion below. However, it is visually instructive at this point to note the results obtained when adding the spectrum of each lignin preparation to a spectrum of cellulose (Figure 1C or Figure

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Figure 3. 13C CP/MAS spectra of red oak lignin samples: (A) acetylated untreated ROL, (B) %-methylated untreated ROL, (C) %-en-

riched methylated, (D) acetylated. 2C). The result of this spectral addition is given in Figure 1D for red oak and Figure 2D for lodgepole pine. Note that these spectra very closely resemble those of the wood samples themselves, revealing the large contributions that lignin and cellulose make to wood. Differences between the calculated (summed) spectra and the spectra of the actual corresponding woods can be assigned to various other fractions in wood, such as hemicellulose. The basic features of the whole wood spectra have been discussed elsewhere (20,21) and will not be repeated here. In order to make direct spectral comparisons convenient, the spectra of the chemically modified lignins presented are plotted in such a manner that the amplitude of the peak due to alkylated aromatic carbons (G1 and Sl) is kept constant. Neither 0-methylation nor acetylation should affect the intensity of this resonance, and therefore intensity changes will be based relative to this peak as a reference. The method of isolating lignin used in this study is one that is expected to yield lignin samples that closely resemble native lignin and are relatively free from carbohydrates such as cellulose. It is possible that some carbohydrate residue may still be present in the samples, contributing to spectral intensities around 105, 89-84, 75-72, and 66 ppm. However, the preparation of these lignins is designed to greatly minimize carbohydrate residue. The contributions from carbohydrate functionalities to the intensities found in the lignin spectra should therefore be minimal and consequently would not interfere with any of the chemical shift/structure relationships discussed below. Furthermore, such small contributions should not jeopardize the integrity of any quantitative results within the context of the error limits given below. 0-Methylation of Lignins. In the 0-methylation reaction we expect a "capping" of acidic hydroxyls to form the corresponding methoxy groups. Figure 3A-C shows the result of methylation on red oak lignin (ROL), with part C showing the effect of using 13C-labeledCHJ. The intense, sharp peak at 56 ppm in these spectra has been previously assigned to aromatic methoxy groups ( 2 ) . If methylation occurs on the aromatic ring, we expect to see an increase in the relative

174


intensity of this peak in the spectrum of the methylated sample, compared to the unaltered lignin spectrum shown in Figure 3A. This is, in fact, the case and can be seen dramatically in the spectrum of the I3C-enriched sample. At the same time, if Ar-OH groups are transformed into Ar-OCH, in this process, then we would expect to see a decrease in the relative intensity of phenol-type C-OH carbons. These carbons have been previously shown to give signals at about 148 ppm (Z),and one can see that intensity is dramaticallyreduced there upon methylation. We also notice an increase in the relative intensity at around 154 ppm in Figure 3, parts B and C, relative to part A and thus assign this peak to aromatic C-0-R carbons with methoxy groups attached. In the transition from a phenolic structure to the corresponding methoxy-substituted aromatic moiety, the phenolic C-OH carbon of similar compounds has been reported to shift to lower shielding by about 5 ppm (22),lending support to the present assignments. One can also see in Figure 3 that, upon methylation, intensity at 61 ppm is increased. This is dramatically seen in the spectrum of the 13C-enrichedsample, showing that this resonance must be due to another type of methoxy "cap". Experiments on a series of model compounds revealed that this method of methylation did not affect hydroxyl groups at any site on a saturated aliphatic side chain. However, all hydroxyl sites on the aromatic ring were easily methylated, whether present as phenol, guaiacyl, or syringyl moieties. For both the phenol and guaiacyl-type models, the resulting methoxy "cap" gave rise to intensity at 55-56 ppm, the same chemical shift as the methoxy carbon present in unmodified syringyl and guaiacyl moieties. However, methylation of a syringyl-type hydroxy site also yielded methoxy intensity of 61 ppm. The 13CNMR spectrum of 1,2,3-trimethoxybenzne, for example, contains intensity in the methyl region at both 56 and 61 ppm. Thus, intensity at 61 ppm in the spectrum of methylated ROL can be assigned to methoxy carbons of this type. While intensity at 61 ppm can be clearly assigned to 1,2,3-trimethoxybenne-typemoieties, it is unlikely that lignin in its unaltered state contains any of this functionality (2-6). Intensity found in this region for nonmethylatd lignin is most likely due to overlapping signals from various Cy-OH and CP-OAr carbons ( 2 ) . Methoxy carbons present in unaltered lignin, then, all appear at 56 ppm, and intensity in this region for unaltered lignin arises from both syringyl and quaiacyl methoxy carbons. In addition to the spectral changes discussed above, intensity changes also occurred a t 63 and 75 ppm upon methylation. These changes suggest that the methylation procedure also affects the aliphatic side chain, as well as the aromatic ring. While methylation did not affect hydroxyl groups at any site on a saturated aliphatic side chain, it was found to occur at Cy (in cinnamyl alcohol) when a double bond was present between the Ca and CP carbons. A peak at 63 ppm is clearly present in the spectrum of untreated lignin but virtually absent in the spectrum of the methylated material. If methylation occurs a t the Cy-OH site in structures of the cinnamyl alcohol type, then it is reasonable to assign the resonance a t 63 ppm in untreated lignin to Cy-OH carbons. This coincides exactly with the chemical shift of the C r carbon in cinnamyl alcohol. After methylation, the methoxy carbons of Cr-OCH, appear at 56 ppm and overlap with intensity from Ar-OCH3 methoxy carbons. The chemical shift of the Cy carbon in cinnamyl alcohol is moved to 73 ppm upon methylation,where a relative increase in intensity is also observed in the spectrum of Figure 3B. In the transformation from alcohol to the corresponding methyl ether, the a-carbon signals of primary and secondary

lk

I " " I " " I " " I " " 1 " " 150 100

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Figure 4. 13C CPIMAS difference spectra of red oak lignin samples: (A) untreated minus acetylated, (B) untreated minus 13C acetylated, (C) ROL for comparison.

alcohols in solution typically shift to lower shielding by 9-11 ppm (23). Thus, the observed change in the lignin resonance at 63 for Cy-OH to 75 ppm for Cy of Cy-OCH, is entirely reasonable. Resonances due to Ca and CP in this type of moiety have been assigned previously to roughly 130 and 125 ppm (2),respectively, and were found in the cinnamyl alcohol study to remain relatively constant upon methylation at the Cy position. Other resonances, which remain essentially constant upon methylation, are readily interpreted on the basis of previous liquid-state assignments (2). Intensity in the region of 85-90 ppm in Figure 3 can be assigned to Ca and CP carbons attached to oxygen. The peak at 106 ppm is assigned to hydrogen-bearing aromatic carbons ortho to methoxy functionalities (G2, S2, S5). The intensity at 137 ppm has been assigned to aromatic carbons bearing alkyl groups (Gl, SI), and the intensity at 116 ppm to aromatic carbons ortho to phenolic C-OH moieties (G5).Intensity around 125 ppm has been assigned to various hydrogen-bearing aromatic carbons not adjacent to oxygen functionalities (G6).Upon methylation of a phenolic moiety in model compounds similar to guiacyl and syringyl structures, the chemical shifts of all aromatic carbons except G4 and S4 remain virtually constant (Z), as observed. One means of focusing on spectral changes is through the use of difference spectra. In most applications, including the present work, the results obtained in this type of spectral analysis are to be regarded as visual aids, not as quantitative differences. Small changes in chemical shifts, changes in line width, and intensity changes on shoulders all contribute to the complexity and meaning of difference spectra. Nevertheless, valuable information can be discerned from this method of spectral analysis. The pattern obtained by subtracting the spectrum of unmodified ROL (Figure 3A) from the spectrum of I3C-rnethylatedROL (Figure 3C) is given in Figure 4B. This difference spectrum, like those discussed below, was obtained by optimizing the nulling of the intensity at 137 ppm, which was chosen as the constant in comparing spectra for reasons discussed earlier. Positive peaks represent intensity that was greater after methylation and negative peaks indicate a decrease in intensity after methylation. The loss in intensity a t 148 ppm due to phenolic C-OH carbons is clearly seen as a negative peak, while the increases at 154,75, 61, and 56 ppm, discussed above, are prominent positive peaks in the difference spectrum shown in Figure 4B.


175

i " " I ' " ' i " " i " " i " "

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Flgure 5. '% CPlMAS interrupted decoupllng spectra of red oak lignin samples: (A) untreated, (B) methylated, (C) 13C-enriched methylated, (D) acetylated.

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Figure 7. I3C CP/MAS difference spectra of lodgepole pine lignin samples: (A) acetylated untreated LPL, (B) 13C-methylateduntreated LPL, (C) LPL for comparison.

I

1

D

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Figure 8. '% CP/MAS interrupted decoupling spectra of lodgepde pine lignin samples: (A) untreated, (B) methylated, (C) 13C-enrichedmethacetylated. ylated, (0) 200

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Figure 8. 13C CP/MAS spectra of Lodgepole Pine Lignin samples: (A) untreated, (B) methylated, (C) 13C-enrlchedmethylated, (D) acetylated.

An interrupted decoupling experiment (24) was also carried out on each of the modified and unmodified ROL lignin samples, and the results are shown in Figure 5A-C. In this experiment, a delay period of typically 50 ps is inserted between the CP contact period and data acquisition. During this delay period, the 'H decoupler is turned off, and 13C magnetization due to 13C's with strong dipolar interactions with protons is attenuated by dephasing. The result is that 13C signal intensity persists for non-hydrogen-bearing and methyl carbons (for which dipolar interactions with protons are strongly attenuated by rapid rotation), while signals due to methylene and methine carbons are dramatically reduced. This provides an excellent aid for chemical shift assignment and structural elucidation. The results shown in Figure 5A-C are entirely consistent with the analysis of chemical shift

assignments presented above. Intensity due to various substituted aromatic carbons and methyl carbons can be seen to persist in the interrupted decoupling experiment, while intensity due to methylene and methine type carbons is virtually eliminated. For example, intensity at 154 and 56 ppm, due to non-hydrogen-bearing and methyl carbons, respectively, is seen to persist after interrupted decoupling. On the other hand, intensity at 113 and 63 ppm, due to methine and methylene carbons, respectively, can be seen to vanish, as expected. The methylation treatment was also applied to lodgepole pine lignin (LPL), and the results are shown in Figure 6B,C, with the corresponding difference spectrum given in Figure 7B and interrupted decoupling results shown in Figure 8B,C for comparison. Many of the spectral changes noted above for red oak lignin are also observed in the methylation of lodgepole pine lignin. Aside from the changes that occur in the lignin spectra upon modification of the samples, the differences in the overall appearance of the ROL and LPL samples themselves will be discussed later, along with any

176


significant differences in intensity changes which occur upon modification. In general, changes in relative 13C NMR intensities upon methylation are smaller for LPL than for ROL, making confirmation of some assignments discussed above difficult. Nevertheless, in LPL we again see increases in intensities at 56 and 61 ppm, corresponding to the various methoxy groups discussed above for ROL. And once again, intensities due to hdyrogen-bearing aromatic carbons ortho to methoxy functionalities (106 ppm; G2,S2, S5), hydrogen-bearing aromatic carbons ortho to phenolic OH groups (116 ppm; G5), alkylbearing aromatic carbons (137 ppm; G1, Sl), etherified a and @ carbons (85-90 ppm; Ca, Cs), and other hydrogen-bearing aromatic carbons (125 ppm; G6) remain constant. Spectral changes such as these should be essentially the same for any wood-derived lignin sample, given that the main features of the molecular nature of lignin are similar for all woods. Differences in the magnitude of these changes reflect the differences in the relative amounts of certain moieties in lignin. Some of these differences will be discussed below. The fact that we observe the same spectral changes for methylation of ROL and LPL lends increased validity to the assignments given above. Figure 7B shows the difference spectrum obtained by subtracting the spectrum of unmodified LPL (Figure 6A) from the spectrum of 13C-methylatedLPL (Figure 6C). As in Figure 5B for ROL, the loss in intensity at 148 ppm is clearly seen as a negative peak, while the increases at 154,75, 61, and 56 ppm are observed as prominent positive peaks. All of the assignments discussed above are again verified in the interrupted decoupling results shown in Figure 8A-C. Acetylation of Lignins. The result of acetylation of red oak lignin is shown in Figure 3D, and the corresponding interrupted decoupling result is shown in Figure 5D. The two most notable changes upon acetylation are the appearance of intensities at 21 and 171 ppm. These two resonance positions are easily assigned as the methyl and carbonyl carbons of the resulting acetoxy esters, respectively. Other spectral changes that occur upon acetylation include significant decreases in intensity at 148 and 106 ppm. These changes can be explained easily on the basis of acetylation of phenolic OH groups. Our analysis of lignin methylation led to the assignment of the phenolic C-OH carbon to 148 ppm. Acetylation of hydroxy groups on aromatic rings would eliminate intensity due to this type of carbon, and a corresponding intensity decrease at 148 ppm is clear in Figures 3D and 4A. Acetylation of a phenolic OH group is known to cause an increase in shielding of 4-5 ppm, for the attached ring carbon (22),and an increase in intensity in the form of broadening in the region around 142-139 ppm can be seen in Figure 3D. (This change is more apparent in the LPL case in Figures 6D and 7A.) Intensity at 106 ppm was previously assigned to aromatic carbons ortho to methoxy functionalities (G2, S2, S5). Upon acetylation at the phenolic site in model compounds similar to syringyl and guiacyl units,the chemical shift of this carbon was shown to move to lower shielding by about 8 ppm (22). After acetylation, the sharp peak at 106 ppm in Figure 3A is significantly reduced and the lower-shielding shoulder at 116 ppm becomes more pronounced in Figure 3D. An increase in intensity at 123 ppm is also observed. Previous solution-state 13C NMR studies (25)have assigned this resonance to G5 of an acetylated guaiacyl unit, agreeing well with our analysis here. Other spectral changes that occur for ROL upon acetylation are elimination of intensity between 85 and 90 ppm, an increase in intensity at 75 ppm, and a slight shift in intensity around 64 ppm. These changes can be explained easily on the basis of acetylation of the aliphatic side chain. Intensity in the region of 85-90 ppm was previously assigned to eth-

erified Ca and Cj3 carbons and intensity at 63 ppm was assigned to C y O H carbons. Examination of most structural models for lignin (2-6) reveals that a very large fraction of nonaromatic OH groups are present at the Cy position and only rarely at the Ca and Cj3 positions. Thus, we would expect acetylation of the aliphatic side chain to occur primarily at the Cy site. After acetylation of the aliphatic side chain at the C r position, the chemical shifts of the Ca and C@carbons have been previously shown (25)in the solution state to move to higher shielding at 75 and 81 ppm, respectively, while the C r chemical shift moves to around 64 ppm. All of these changes are clearly observable in Figure 3A,D. Resonances that remain essentially constant upon acetylation give the intensity a t 137 ppm due to alkyl-bearing aromatic carbons (Gl, Sl), the intensity at 154 ppm due to aromatic carbons with methoxy groups attached (G3, S3, S5), and the peak at 56 ppm, which was assigned to methoxy groups attached to aromatic rings. Once again, all of the chemical shift assignments made above are verified by the interrupted decoupling results shown in Figure 5A,D. The difference spectrum obtained by subtracting the spectrum of unmodified ROL (Figure 3A) from the spectrum of acetylated ROL (Figure 3D) is shown in Figure 4A. This figure shows far more fine structure than the difference spectra obtained for the methylation reaction on ROL or LPL. Chemical shift changes, for both the aromatic ring and the side chain, are more diverse upon acetylation than methylation (22,25),thereby adding to the complexity of the difference spectrum given in Figure 4A (and Figure 7A, for the LPL, case). Nevertheless, decreases in intensity upon acetylation at 148, 106,85-90, and 63 ppm and increases at 171, 75, and 21 ppm can all clearly be observed, illustrating the structural changes discussed above. The acetylation treatment was also applied to lodgepole pine lignin (LPL) and the results are shown in Figure 6D, with the interrupted decoupling result shown in Figure 8D for comparison. As was the case in the methylation experiment, changes in the relative intensities for LPL are smaller upon acetylation than for ROL. However, all of the applicable spectral changes noted above for ROL are also observed for LPL, most notably the decreases in intensity at 148,106, and from 85 to 90 ppm. The distinct shoulder at 143 ppm due to the acetylated aromatic hydroxyl groups (G4, S4) is more clearly observable for LPL than for ROL. Intensity due to alkyl-bearing aromatic carbons (137 ppm; G1, Sl), aromatic carbons with methoxy groups attached (154 ppm; G3, S3, S5), and methoxy carbons on the aromatic ring (56 ppm) remain constant with acetylation, as expected. The intensity changes can all be seen in the difference spectrum shown in Figure 7A. Once again, each of the assignments is further supported by the interrupted decoupling result shown in Figure 8A,D. Quantitation in Lignin Spectra. An issue that is often raised, especially in the chemical analysis of complex organic solids like wood, is the ability of solid-state 13CCP/MAS NMR to yield quantitative results. A detailed set of relevant relaxation experiments was carried out on each of the lignin samples discussed above in order to address this concern. Given in Table I are the results of these experiments. Values are given for the following three relaxation parameters: (1) TIH,the proton spin-lattice relaxation time; (2) TcH,the cross polarization relaxation time; (3) TlpH,the proton spin-lattice relaxation time in the rotating time. Values for each of these are given for every clearly discernible peak in each of the lignin spectra. These three parameters are pivotal in determining the quantitative significance of the relative intensities of a CP/MAS spectrum, and the effects of each are discussed below. Note that for every sample there exists a large variation in the values obtained for all of the relevant relaxation pa-


177

Table I. Relaxation Parameters for Specific Peaks in I3C Spectra of Lignin Samples

sample

154

148

137

105

89

84

75

67

63

56

TlH, 5 TCH, ms TipH,ms

0.86 0.77 9.1

0.90 0.81 8.6

0.90 0.65 8.7

0.98 0.21 5.4

1.0 0.18 4.9

1.0 0.18 4.6

1.0 0.19 4.3

1.2 0.18 4.6

0.95 0.20 6.2

0.90 0.30 8.9

RbL

ROL methylated

T 1 ~s , TCH~ ms TlpH,

ms

154

148

135

105

89

84

75

67’

61

56

0.55 1.0 11.4

0.54 0.96 11.4

0.55 0.87 12.5

0.67 0.25 8.8

0.68 0.26 7.6

0.66 0.22 8.8

0.70 0.23 7.9

0.70 0.21 7.6

0.65 0.26 10.6

0.57 0.35 12.6

ROL acetylated

T ~ Hs, TCH,ms TloH?

optimum exptl parameters

relaxation parameters at following chemical shifts (ppm)

relaxation parameter

sample


relaxation parameters at following chemical shifts (ppm)


sample

sample

optimum exptl Darameters

relaxation parameters at following chemical shifts (pprn)


ms

171

154

135

103

75

63

56

21

0.94 0.55 19.0

0.86 0.55 17.0

0.81 0.48 18.9

0.96 0.15 20.5

0.92 0.18 20.3

0.92 0.13 20.9

0.87 0.20 21.1

0.93 0.22 21.9

rrepr 5

4


154

148

133

123

113

105

89

84

75

65

63

56

TiH, s TCH,ms Tlp~ ms,

0.97 0.46 9.9

0.84 0.46 8.4

0.80 0.34 8.2

0.81 0.16 8.9

0.82 0.12 9.6

0.81 0.09 6.5

1.06 0.10 7.3

0.98 0.09 6.5

0.91 0.10 5.5

0.86 0.08 7.5

0.82 0.09 7.7

0.81 0.16 9.7

LPL

sample LPL methylated

sample LPL acetylated

relaxation parameters at following chemical shifts (pprn)


relaxation parameters at following chemical shifts (ppm) 154

148

133

123

113

105

89

84

75

65

63

56

T l ~s, TCH,ms TipH,ms

0.63 0.58 10.4

0.56 0.54 10.9

0.50 0.39 10.5

0.51 0.11 13.1

0.52 0.11 13.4

0.65 0.09 9.3

0.53 0.08 11.0

0.55 0.08 10.2

0.59 0.09 8.6

0.61 0.08 10.6

0.58 0.09 10.3

0.62 0.16 12.5

Tcontaet,

ms

1.4

optimum exptl parameters T,,~,s T ~ ms 4

1.4

optimum exptl parameters T , , ~ ,s rcontact, ms 4

1.19



relaxation parameters at following chemical shifts (pprn) 170

152

148

138

133

123

113

102

83

72

64

56

20

T , , ~ ,s

T , , , ~ ~ ~ ms ,

T l ~s, TCH,ms Tlp~ ms,

1.04 0.56 18.0

0.96 0.55 17.3

0.87 0.31 19.7

0.70 0.40 24.6

0.85 0.35 20.2

0.91 0.15 19.1

0.82 0.14 18.6

0.94 0.15 20.8

0.85 0.17 20.6

1.08 0.13 18.8

1.07 0.16 20.3

1.06 0.22 17.9

1.07 0.22 20.0

4

1.4

rameters. Unfortunately, this complicates the significance of the intensities shown in the spectra presented in this paper. To ensure that proton spin-lattice relaxation was essentially complete, and that therefore T1H effects would not distort the observed spectral intensities, a repetition time of 4 s was chosen for all samples. This is a t least four times the largest T i p H value displayed in Table I and should therefore be adequate for this aspect of the issue of quantitativeness. However, the effects of TcH and T i p H variations are not as straightforward to handle. lH-13C polarization transfer, represented by the rate constant, TCH-l, and rotating-frame proton spin-lattice relaxation, represented by the rate constant, TlpH-l, are competing effects which determine for each peak the percent of the theoretical limit of CP-enhanced 13C magnetization that is observed for a given set of experimental conditions. This relationship is given by eq 1(26)) where M(T)

is the observed magnetization for a given CP contact time, r, and Mc.. is the carbon magnetization obtained under conditions of maximum CP-enhancement and infinitely long TlpH, i.e., the equilibrium l3C magnetization multiplied by Y1H/Y13C

(where the y’s represent magnetogyric ratios). The large variations in TCH and TipHshown in Table I and the fact that the larger TcH values are often close to the smaller TlpH values for a particular sample eliminate the possibility of choosing an optimal set of experimental conditions that would result in the observation of 100% theoretical magnetization (intensity) for each peak in each spectrum. It therefore seemed advisable to choose for each sample a “compromise”contact time that would result in “reasonablyquantitative” intensities for all peaks. These values were calculated from the TCH and T i p H values by choosing a contact time that resulted in equal losses in magnetization due to the effects of the largest T C H value and the smallest TlpH value for each sample. The contact times that were chosen in this way for each sample and used to obtain the spectra shown are also given in Table I. Of course, knowing the TCHand T l p H values, one can “correct” the intensities observed for a given contact time to obtain the corresponding theoretical CP-enhanced magnetizations. Analysis of the data given in Table I shows that for all of the lignin samples of this study the relative intensities displayed in Figures 3 and 6 reflect the actual carbon contents to within 30% at worst and to within roughly 10% in most cases. For both woods, the acetylated lignin samples yield

~

~

~

178


Table 11. Chemical Shift Assignments for Solid Lignins and Modified Lignins sp2 carbons chemical shift, ppm assignment

171- 70 154 148

-O*C(=O)CH3

142- 39 138- 35 130 125- 23

G4,0 S4" G1, S1 Ca G6,G5,"C@

116- 13

G5, G2" S2." S6" G2, S2, S6

106-102

G3, S3, S5 G4, S4

sp3 carbons

chemical shift. ppm

assignment

90-84 81 75-72

CB-OR, Ca-R CPb Cy-OR, Cab C4-OR

64 63

Cyb

61 56

S4-OC*HsC Ar-OC*H3, Cy-OC*H," -OC(=O)C*H,

21-20

Cy-OH, Cp-OAr

Chemical shift assignable to this carbon after acetylation at S4 or G4. Chemical shift assignable to this carbon after acetylation at the Cy position. Chemical shift assignable to this carbon only after methylation of lignin. intensities that are overall the most accurate, with an 8% deviation being the worst case. The other LPL samples show intensities that may deviate by as much as 15%. The methylated and untreated ROL samples, on the other hand, yield spectral intensities that may deviate by up to 30%. For each of the samples, intensity due to methoxy carbons (at 56 ppm) is the most accurate representation of carbon content in the spectrum. The observed relative intensities for substituted aromatic carbons tend to be lower than intensities reflecting actual carbon contents and are usually responsible for a large portion of the deviations noted above. For example, in acetylated LPL the observed intensities reflect carbon contents to within 1 %, except for peaks due to substituted aromatic carbons, which can be low in intensity by as much as 7 % . To extract purely quantitative information from specific spectral regions by application of eq 1 would require either well-resolved peaks or reliable deconvolution techniques. Problems of spectral overlap, which certainly exist in a macromolecular system as complex as lignin, render this task almost impossible at the present time. However, the uncertainties indicated above, e.g., an error range of less than roughly 20%, and often better, do not preclude the kinds of conclusions on structural changes discussed in this paper. And, as indicated above, if more accurate conclusions are required, one can improve the quantitativeness by making corrections based on eq 1. More Detailed Comparison of Lignins and Modified Lignins. A summary of the chemical shift assigments discussed above is given in Table 11. By use of these chemical shift/structure relationships, it is possible to draw more detailed conclusions concerning the nature of red oak and lodgepole pine lignins and their behaviors upon methylation and acetylation. Two of the most obvious differences between the 13C CP/MAS spectra of ROL and LPL in Figures 3A and 6A are the relative sizes of the peaks at 56 and 154 ppm. Softwoods are generally considered to contain lignin macromolecules that are constructed from guaiacylpropane units, whereas hardwood lignins have been shown to contain mixtures of both guaiacylpropane and syringylpropane units (27). Table I1 shows the peak a t 56 ppm to be due to methoxy groups attached to aromatic rings and the resonance a t 154 ppm to aromatic carbons with methoxy groups attached. The relative intensities of both of these peaks are higher for ROL than LPL. Red oak, a hardwood, contains syringyl as well as guaiacyl functionalities and, as such, is expected to have a

higher methoxy content than lodgepole pine, which contains predominantly guaiacyl units. Typically, hardwood methoxy content is estimated to be roughly between 12 and 15% of the total carbon content in lignin, while softwood methoxy content is thought to be roughly 7-11% (28). The peak a t 56 ppm is sharp and well-defined in the 13CCP/MAS spectra of both ROL and LPL, suggesting the possibility of using solid-state NMR to quantitate aromatic methoxy content. This would provide an excellent check on those analytical techniques that rely upon solubilizing the lignin fraction as a first step. It is first necessary, however, to accomplish a detailed line-shape analysis of this region in the lignin spectra. Deconvolutions (not shown) of the ROL and LPL spectra shown in Figures 1B and 2B were carried out by using the minimal number of Gaussian lines necessary to simulate accurately the entire spectrum of each lignin. The area of each peak determined in the simulation was corrected by using the relaxation data given in Table I and applying eq 1. In those cases for which accurate simulation necessitated the use of a peak for which good relaxation data could not be obtained (usually due to spectral overlap), the adjustment was determined by employing the appropriate relaxation parameters for the closest or overlapping peak. Although this procedure adds some error to the calculations, this level of error is both unavoidable and minimal in the present case. The results given in Table I show that the percent magnetization observed for each peak does not widely vary within a small chemical shift range. Thus, intensity adjustments implemented in this manner should be nearly as accurate as if all intensity in the spectrum was contained within clearly resolved peaks for which relaxation data were accessible. In addition, any error that this approximation would create will affect only the calculation of total percent carbon content, since the peaks in question are present only in areas not of critical interest. Calculation of total methoxy content was carried out in this way for both LPL and ROL. For the lignin samples, determination of methoxy content was based on only the peak at 56 ppm for reasons discussed above. Calculation of methoxy content for LPL gave a value of 5.8%. This value is slightly lower than the 7-11% determined by conventional methods, as discussed above. Methoxy content in ROL was determined to be 7.4%, also based on intensity at 56 ppm. This value is uncharacteristically low for typical hardwoods, which generally yield methoxy contents of 12-1570, as determined by conventional methods. Unfortunately, values obtained for this specific hardwood were unavailable from the literature for comparison. Another area of interest is the determination of the number of free phenolic groups in lignin. Estimating the percent of carbons which contain free phenolic groups based on intensity at 148 ppm is difficult due to the spectral overlap problems. The intensity distribution in the region around 154-148 ppm is unquestionably complex. Although we have identified intensity a t 154 ppm to arise from aromatic carbons with methoxy groups attached and intensity at 148 ppm to be due to phenolic carbons, spectral overlap in this region from other moieties, such as other Ar-0-R structures and ?-hydroxyphenyl units, undoubtedly exisb and complicates the meaning of any calculation based on intensities in this region. However, some valuable semiquantitative information is certainly accessible. Values obtained in this manner were 4.8% for LPL and 5.0% for ROL. Little work has been done by classical chemical methods to compare with these estimates (28); however, these values seem entirely reasonable in light of the methoxy contents determined in this work and described above. Another area of vital interest in lignin chemistry is the ratio of syringyl (S) and guaiacyl (G) units (28). Attempts to derive


this ratio based solely on aromatic intensity are precarious, a t best, due to the wide range of spectral overlap problems mentioned above. However, careful consideration of line shape and choice of reference peaks can yield valuable semiquantitative information. One possible way of estimating the S/G ratio is to consider the intensities at 148 and 56 ppm. Intensity due to phenolic C-OH carbons at 148 ppm can arise from one S site and one G site, while intensity due to methoxy groups attached to aromatic rings at 56 ppm can arise from two S sites and one G site. Thus, the intensity ratio, 156/I14a, should reflect the ratio (G + 2S)/(G + S). Determination of the S/G ratio in this manner suffers chiefly from possible contributions to the intensity at 148 ppm from p-hydroxyphenylfunctionalities. However, in most cases, one would expect this contribution to be small, if a t all significant. Generally, these moieties are fur less abundant in the lignin than are G or S. With the p-hydroxylphenyl contribution not considered, the S/G ratio determined in this manner for ROL is 0.5 and the value obtained for LPL is 0.2. These ratios are consistent with the concept of softwoods consisting of chiefly guaiacylpropane-type polymers and hardwoods containing a mixture of S and G moieties. The value of 0.5 obtained for ROL is consistent with typical hardwood S/G ratios (27),as determined by classical chemical methods. In softwoods, however, S/G ratios are often much lower than 0.2, as we found for LPL; but no general S/G ratio can be given for softwoods (27). Values obtained by classical methods for either ROL or LPL could not be found for exact comparison with our results. One intriguing possibility that resulted from this study is the potential use of methylation for the determination of S/G ratios. Methylation of guaiacyl sites yielded methoxy intensity at 56 ppm, while methylation of syringyl moieties gave rise to intensity at 61 ppm. When I3C-1abeledCH31is used, these two peaks are sharp and well-defined, which would allow accurate deconvolution. Calculation of S/G ratios in this manner would require a methylation method that is selective for only aromatic hydroxyl groups and one that is exhaustive for these sites. Neither of these criteria are satisfied by the procedure used in the current study. Nevertheless,the proper choice of a methylation treatment should provide an exceptionally promising method for the accurate determination of S/G ratios.

SUMMARY AND CONCLUSIONS A typical high-resolution 13C NMR spectrum of a liquid lignin solution contains nearly a hundred resolvable lines (2-5)) and the complexity of assigning each of these resonances to specific structural moieties is becoming more difficult as higher-field spectrometers and more advanced techniques for resolving more lines become available. This situation reflects the large number of different carbon environment types found in lignin, resulting in spectral overlap and the inability of model compounds to represent the lignin macromolecule completely. Solid-state 13C NMR spectra typically have broader lines, making the overlap problem more severe. Although this problem places certain limitations on the ability of CPIMAS 13CNMR to yield highly quantitative results on subtle structural details in lignin and wood, one can nevertheless achieve a useful level of quantitation, while avoiding the analytical uncertainties associated with limiting samples to liquid extracts. In this study we have clearly identified

179

many of the resonances in the solid-state 13CNMR spectra of HC1-hydrolysis lignins, summarized in Table I. While these results are certainly not all inclusive, largely due to spectral overlap, the identities of many peaks and regions of intensity are well-established. Use of these assignments has permitted the successful probing of some aspects of lignin chemistry, such as determining the ratio of syringyl to guaiacyl moieties and the behavior of these lignin samples upon methylation and acetylation. Thus, solid-state CP/MAS 13C NMR is a powerful technique for the study of lignin in whole wood, as well as in solid lignin preparations, and the potential for future work is clear. At the present level of capabilities of solid-state CP/MAS 13CNMR, quantitation of lignin spectra must be carried out within limitations of the types discussed above. As higher sample spinning speeds, better line-narrowing techniques, and higher fields become available, spectral overlap problems may decrease. However, even within the limits of current technology, valuable and reliable semiquantitative information is obtainable. Registry No. Cellulose,9004-34-6;hydrolytic lignin, 8072-93-3.

LITERATURE CITED Chem. Eng. News, 1984, Sept. 24, 19. Nimz, H. Angew. Chem., Int. Ed. Engl. 1074, 13, 313. Krlngstad, K. P.; Merck, R. Holzforschung 1983, 37,237. Ralph, J.; Obst, J. R. Holzforschung 1983, 37,297. Nimz, H. H.; Tochlrner, J.; Stahle, M.; Lehman, R.; Schlosser, M. J . wood chem. Technol. 1984, 4 , 265. Lapierre, C.; Monties, 6.; Gulttet, E.; Laliemand, J. Y. Holzforschung 1084, 38, 333. Bartuska, V. J.; Maclel, G. E.; Bolker, H. I.; Fleming, B. I. Holzforschung 1980, 34, 214. Maciel, G. E.; O'Donnnell, D. J.; Ackerman, J. J. H.; Hawkins, B. H.; Bartuska, V. J. Makromol. Chem. 1081, 182, 2297. Schaefer, J.; Sefcik, M. D.; Stejskal, E. 0.; McKay, R. A.; Hail, P. L. Macromolecules 1981, 1 4 , 557. Kolodzlejskl, W.; Frye, J. S.; Maciel, G. E. Anal. Chem. 1082, 5 4 , 1419. Pines, A.; Gibby, M. G.; Waugh, J. S. J . Chem. Phys. 1973, 59, 569. J . Am. Chem. SOC. 1078, 9 8 , 1031. Schaefer, J.; Stejskai. E. 0.; Schaefer, J.; Stelskal, E. 0.Topics in Carbon-I3 NMR Spectroscopy; Levy, G. C., Ed.; Wiley: New York, 1979; Vol. I. Pfeffer, P. E.; Hicks, K. 6.; Frey, M. H.; Opella, S. J.; Earl, W. L. J . Carbohydr. Chem. 1984, 3, 197. Schuk, T. P.; Preto, R. J.; Pittman, J. L.; Goldstein, 1. S. J . Wood Chem. Technol. 1082, 2 , 17. Liotta, R. Fuel 1970, 58, 724. Liotta. R.: Rose. K.: HiDDo. E. J . Ora. Chem. 1981, 46, 277. Wind, R. A.; Anthonlo,'F. E.; Duijve&jn, M. J.; Smidt, J.; Trommel, J.; devette, G. M. C. J . Magn. Reson. 1082, 48, 125. (19) Frye, J. S.; Maclel, 0. E. J . M g n . Reson. 1082, 48, 125. (20) Haw, J. F.; Maciel, G. E.; Biermann, C. J. Holzforschung 1984, 38, 327.

(21) Haw, J. F.; Maciel, G. E.; Linden, J. C.; Murphy, V. G. Holzforschung 1985, 39, 99. (22) Silverstien, R. M.; Bassler, G. C.; Morrili, T. C. Spectrometric Identification of Organic Compounds, 4th ed.; Wiley: New York, 1981; p 265. (23) Breltmaier, E.; Voelter, W. NMR Spectroscopy, 2nd ed.; Verlag Chemle: New York, 1978; pp 150, 157. (24) Opella, S. J.; Frey, M. H. J . Am. Chem. SOC. 1979, 101, 5854. (25) Nlmz, H.; Robert, D.; Falx, 0.;Nemr, M. Holzforschung. 1981, 35, 16. (26) Mehring, M. High Resolutlon NMR Spectroscopy in Solids, 2nd ed.; Springer-Verlag: Berlin, 1963; p 153. (27) Browning, B. L. The Chemistry of Wood; Krieger: New York, 1975; p 256. (28) Fengel, D.; Wegener, 0.; Woad: Chemistry, Ultrastructure, Reactions; Waiter de Gruyter: Berlin, 1964; p 149.

RECEIVED for review May 8, 1986. Accepted September 4, 1986. The authors gratefully acknowledge partial support of this research by the Colorado State University Experiment Station.

Qualitative and quantitative analysis of solid lignin samples by carbon

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