Quantitative characterization of a hardwood milled wood lignin by nuclear magnetic resonance spectroscopy

The structure of Eucalyptus grandis milled wood lignin (MWL) was investigated by 2D 1H-13C HSQC, HMQC, and 1H-1H TOCSY correlation NMR techniques and by quantitative 13C NMR as well as by the permanganate oxidation degradation technique. The combination of 2D NMR and quantitative 13C NMR spectroscopy of nonacetylated and acetylated lignin preparations allowed reliable identification and calculation of the amount of different lignin structures. About 85% of side-chain moieties were estimated on the structural level. This information was substantiated by data on the quantity of various functional groups and interunit linkages as a whole. A modified method for calculation of the h:g:s ratio has been suggested and compared with previously suggested approaches. E. grandis MWL has been determined to have an h:g:s ratio of 2:36:62. The amounts of various phenolic/etherified noncondensed/condensed guaiacyl and syringyl moieties were approximately estimated. E. grandis MWL contained approximately 0.60/Ar of beta-O-4 moieties along with small amounts of other structural units such as pino/syringyresinol (0.03/Ar), phenylcoumaran (0.03/Ar), and spirodienone (0.05/Ar). The degree of condensation was estimated at approximately 21%; the main condensed structures are 4-O-5 moieties (approximately 0.09/Ar). The structure of E. grandis MWL was compared with those of other lignin preparations isolated from various hardwoods.     

Structural characterization of lignin from triploid of Populus tomentosa Carr

To improve yields while minimizing the extent of mechanical action (just 2 h of planetary ball-milling), the residual wood meal obtained from extraction of milled wood lignin (MWL) was sequentially treated with cellulolytic enzyme and alkali, and the yields of MWL, cellulolytic enzyme lignin (CEL), and alkaline lignin (AL) were 5.4, 23.2, and 16.3%, respectively. The chemical structures of the lignin fractions obtained were characterized by carbohydrate analysis, gel permeation chromatography (GPC), Fourier transform infrared (FT-IR) spectroscopy, and various advanced NMR spectroscopic techniques. The results showed that the lignin isolated as MWL during the early part of ball milling may originate mainly from the middle lamella. This lignin fraction was less degradable and contained more linear hemicelluloses and more C═O in unconjugated groups as well as more phenolic OH groups. Both 1D and 2D NMR spectra analyses confirmed that the lignin in triploid of Populus tomentosa Carr. is GSH-type and partially acylated at the γ-carbon of the side chain. Two-dimensional heteronuclear single-quantum coherence (13C-1H) NMR of MWL, CEL, and AL showed a predominance of β-O-4' aryl ether linkages (81.1-84.5% of total side chains), followed by β-β' resinol-type linkages (12.2-16.4%), and lower amounts of β-5' phenylcoumaran (2.1-2.6%) and β-1' spirodienone-type (0.4-1.4%) linkages. The syringyl (S)/guaiacyl (G) ratios were estimated to be 1.43, 2.29, and 2.83 for MWL, CEL, and AL, respectively.     

Solution-state nuclear magnetic resonance study of the similarities between milled wood lignin and cellulolytic enzyme lignin

The structures of milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL) have been analyzed using traditional chemical methods and solution-state NMR techniques. Comparisons of the results obtained reveal that subtle differences exist between the two lignin preparations. Thioacidolysis produced higher monomer yields from CEL than MWL, suggesting MWL has a more condensed structure. Quantitative (13)C NMR determined the degree of condensation in MWL to be 0.43 unit per aromatic moiety as compared to 0.36 in CEL. The MWL also contained a lower amount of beta-O-4' substructures per aromatic ring than CEL, 0.41 versus 0.47, respectively. Carbohydrate analysis revealed that the MWL may contain a higher proportion of middle lamella material as compared to the CEL. Because the middle lamella is considered to have a more condensed lignin structure, on the basis of the bulk polymerization theory, these results could explain the differences in beta-O-4' and degree of condensation.     

Characterization of lignin structures and lignin-carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR spectroscopy

To characterize the lignin structures and lignin-carbohydrate complex (LCC) linkages, milled wood lignin (MWL) and mild acidolysis lignin (MAL) with a high content of associated carbohydrates were sequentially isolated from ball-milled poplar wood. Quantification of their structural features has been achieved by using a combination of quantitative (13)C and 2D HSQC NMR techniques. The results showed that acetylated 4-O-methylgluconoxylan is the main carbohydrate associated with lignins, and acetyl groups frequently acylate the C2 and C3 positions. MWL and MAL exhibited similar structural features. The main substructures were β-O-4' aryl ether, resinol, and phenylcoumaran, and their abundances per 100 Ar units changed from 41.5 to 43.3, from 14.6 to 12.7, and from 3.7 to 4.0, respectively. The S/G ratios were estimated to be 1.57 and 1.62 for MWL and MAL, respectively. Phenyl glycoside and benzyl ether LCC linkages were clearly quantified, whereas the amount of γ-ester LCC linkages was ambiguous for quantification.     

Elucidation of the structures of residual and dissolved pine kraft lignins using an HMQC NMR technique

Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft pulp and pulping liquor have been undertaken using the (1)H-(13)C HMQC NMR technique, GPC, and sugar analysis to elucidate the reaction mechanisms in kraft pulping and the lignin reactivity. A modified procedure for the isolation of enzymatic residual lignins has resulted in an appreciable decrease in protein contaminants in the residual lignin preparations (N content < 0.2%). The very high dispersion of HMQC spectra allows identification of different lignin moieties, which signals appear overlapped in 1D (13)C NMR spectra. Elucidation of the role of condensation reactions indicates that an increase in the degree of lignin condensation during pulping results from accumulation of original condensed lignin moieties rather than from the formation of new alkyl-aryl structures. Among aryl-vinyl type moieties, only stilbene structures are accumulated in lignin in appreciable amounts. Benzyl ether lignin-carbohydrate bonds involving primary hydroxyl groups of carbohydrates have been detected in residual and dissolved lignin preparations. Structures of the alpha-hydroxyacid type have been postulated to be among the important lignin degradation products in kraft pulping. The effect of the isolation method on the lignin structure and differences between the residual and dissolved lignins are discussed.     

Understanding the degree of condensation of phenolic and etherified C-9 units of in situ lignins

A novel approach for the quantification of the degree of condensation at the C(5) position of etherified and phenolic phenylpropane (C-9) units of in situ lignin is described. This is achieved by degrading unmethylated and methylated wood by thioacidolysis and analyzing the resultant product mixtures by quantitative (31)P NMR spectroscopy. Applying this new method to compression wood and normal wood from Pinus radiata showed that, whereas 41-47% of etherified guaiacyl C-9 units are condensed at the C(5) position, almost all phenolic guaiacyl C-9 units exist as uncondensed moieties. Analysis of milled wood lignin (MWL) isolated from the same wood by (31)P NMR spectroscopy before and after thioacidolysis showed that the phenolic guaiacyl C-9 units were more condensed than those in the in situ lignin. This is likely due to partial cleavage of the more condensed etherified linkages during the lignin isolation, leading to a relative increase in condensed phenolic guaiacyl C-9 units.     

Comparative evaluation of three lignin isolation protocols for various wood species

Milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and enzymatic mild acidolysis lignin (EMAL) were isolated from different wood species and characterized by various techniques. The EMAL protocol offered gravimetric lignin yields 2-5 times greater than those of the corresponding MWL and CEL. The purities of the EMALs were 3.75-10.6% higher than those of their corresponding CELs, depending upon the wood species from which they were isolated. Molecular weight analyses showed that the EMAL protocol isolates lignin fractions that are not accessed by the other procedures evaluated, while 31P NMR spectroscopy revealed that MWL is more condensed and bears more phenolic hydroxyl groups than EMAL and CEL. The yields and purities of EMAL, MWL, and CEL from hardwood were greater than those obtained for the examined softwoods. Structural details obtained by DFRC (derivatization followed by reductive cleavage)/31P NMR revealed different contents of condensed and uncondensed beta-O-aryl ether structures, dibenzodioxocins, and condensed and uncondensed phenolic hydroxyl and carboxylic acid groups within lignins isolated from different wood species.     

Analysis of technical lignins by two- and three-dimensional NMR spectroscopy

Modern multidimensional NMR spectroscopic methods were applied to investigate the effects of kraft pulping and oxygen delignification on lignin side-chain structures. In addition to the two-dimensional HSQC measurements, the three-dimensional HSQC-TOCSY technique was utilized to elucidate the (1)H-(1)H and (1)H-(13)C correlations of individual spin systems and thus indicate a certain lignin side-chain structure. Unlike earlier, nonlabeled samples were used for 3D measurements. According to 2D and 3D NMR spectra, most of the structures identified in milled wood lignin (MWL) are still present in technical lignins after kraft pulping and oxygen delignification. Although the main reaction during kraft pulping is the cleavage of beta-O-4 linkages, these structures are still left in spent liquor lignin as well as in residual lignin. The amount of coniferyl alcohol and dihydroconiferyl alcohol end groups, as well as some unidentified saturated end groups, is higher in technical lignins than in MWL. Contrary to our earlier observations, no diphenylmethane structures were observed in any technical lignins. Vinyl aryl ether structures could not be detected in technical lignins either.     

Lignin structural variation in hardwood species

A comprehensive lignin structure analysis of ten industrially relevant hardwood species is presented. Milled wood lignin (MWL) was isolated from each species using a modified protocol and all milled wood lignin preparations were analyzed through quantitative (13)C NMR spectroscopy, elemental analysis, methoxyl analysis, sugar analysis, and nitrobenzene oxidation. Nitrobenzene oxidation and ozonation were carried out on extractive-free wood, alkali-extracted wood, milled wood lignin, and alkali-extracted lignin. Milled wood lignin isolated by the modified protocol was found to be representative of the total lignin in alkali-extracted wood. Significant variations in lignin structures, such as syringylpropane/guaiacylpropane ratio (S/G ratio), arylglycerol-β-aryl ether (β-O-4), degree of condensation, and elemental and methoxyl contents, were found among the hardwood species studied. These structural variations among species appear to be correlated to a single factor, the syringyl/guaiacyl ratio. A new method to predict the S/G ratio of total lignin in wood was developed, using a calibration line established by the syringaldehyde/vanillin (S/V) ratio (nitrobenzene oxidation) and the S/G ratio ((13)C NMR) of milled wood lignin (MWL).     

CPMAS 13C NMR and IR spectra of spruce and pine litter and of the Klason lignin fraction at different stages of decomposition

Fresh and decomposed spruce and pine litter and the Klason lignin fraction of spruce needles at different stages of decomposition were studied by CPMAS ¹³C NMR and IR spectroscopy as well as by chemical methods. It was shown that decomposition of needles is accompanied by an increase in aliphatic substances and carboxyl group content; the amount of polysaccharides is reduced. It is assumed that stable aliphatic compounds like cutin and lipids of microbial origin will accumulate during litter decomposition and humification. Aromaticity is low and does not alter drastically. The NMR spectra of the Klason lignin fraction show pronounced peaks at 30, 55, 115, 130, 150 and 175 ppm. Obviously, this fraction contains appreciable amounts of aliphatic and carboxyl carbon besides the typical aromatic units of lignin. During decomposition aromaticity decreases whereas the relative amounts of aliphatic substances and carboxyl groups increase. This is probably due to splitting of aromatic ring structures and side chains. The findings agree with the results from chemical analyses.     

Elucidation of lignin structure through degradative methods: comparison of modified DFRC and thioacidolysis

Milled wood and milled wood lignin (MWL) samples were subjected to DFRC and thioacidolysis. Despite the fact that both methods selectively cleave aryl ether bonds, substantial differences in results were obtained. Lignin thioacidolysis gave total molar yields of degradation monomer products in the range of 3.5-7 mol % higher than DFRC. GPC analysis showed that the thioacidolysis-treated lignin was degraded to a lower average molecular weight than that treated by DFRC. Contrary to results reported for lignin model compounds, these results indicate that the DFRC method does not completely or efficiently degrade the lignin polymer. In fact, the DFRC-degraded lignin retained much of the characteristics of the original MWL. Elemental analysis revealed the presence of bromine in the DFRC-treated lignin, and two-dimensional (1)H-(13)C HMQC NMR spectroscopy showed the presence of beta-O-4 linkages in the DFRC-treated lignin. No beta-O-4 interunit linkages were detected in the thioacidolysis-treated lignin. These results are consistent with the lower monomer yields and the higher average molecular weight of the DFRC-treated lignin and indicate inefficiency in the chemistry of the method, probably due to steric constraints of the polymeric nature of lignin.     

Toward a better understanding of the lignin isolation process from wood

The recently developed protocol for isolating enzymatic mild acidolysis lignins (EMAL) coupled with the novel combination of derivatization followed by reductive cleavage (DFRC) and quantitative (31)P NMR spectroscopy were used to better understand the lignin isolation process from wood. The EMAL protocol is shown to offer access at lignin samples that are more representative of the overall lignin present in milled wood. The combination of DFRC/(31)P NMR provided a detailed picture on the effects of the isolation conditions on the lignin structure. More specifically, we have used vibratory and ball milling as the two methods of wood pulverization and have compared their effects on the lignin structures and molecular weights. Vibratory-milling conditions cause substantial lignin depolymerization. Lignin depolymerization occurs via the cleavage of uncondensed beta-aryl ether linkages, while condensed beta-aryl ethers and dibenzodioxocins were found to be resistant to such mechanical action. Condensation and side chain oxidations were induced mechanochemically under vibratory-milling conditions as evidenced by the increased amounts of condensed phenolic hydroxyl and carboxylic acid groups. Alternatively, the mild mechanical treatment offered by ball milling was found not to affect the isolated lignin macromolecular structure. However, the overall lignin yields were found to be compromised when the mechanical action was less intense, necessitating longer milling times under ball-milling conditions. As compared to other lignin preparations isolated from the same batch of milled wood, the yield of EMAL was about four times greater than the corresponding milled wood lignin (MWL) and about two times greater as compared to cellulolytic enzyme lignin (CEL). Molecular weight distribution analyses also pointed out that the EMAL protocol allows the isolation of lignin fractions that are not accessed by any other lignin isolation procedures.     

Structural characterization of the lignin from the nodes and internodes of Arundo donax reed

Milled wood lignin (MWL) and dioxane lignin (DL) from different morphological regions (nodes and internodes) of Arundo donax reed were subjected to a comprehensive structural characterization by (13)C, (1)H NMR, FTIR, and UV spectroscopies and functional analysis. The permanganate and nitrobenzene oxidation methods were also applied to the in situ lignins. Both node and internode lignins are HGS-type lignins, with a significant amount of H units (including p-coumaric acid type structures). The S/G ratio (1.13-1.32), the weight-average molecular weight (20,400-24,500), the methoxyl group content (0.90-0.98), the phenolic hydroxyl group content (0.23-0.27), and the aliphatic hydroxyl group content (1.00-1.09) are not very different in the lignins from nodes and internodes. However, some structural differences between node and internode lignins were observed. The former has much more phenolic acids (p-coumaric and ferulic), 8.8% in node versus 1.2% in internode and less beta-O-4 (0. 32 and 0.49 per aromatic unit in node and internode, respectively). In situ node lignin is more condensed than internode lignin.     

IR-, 1H-NMR- und 13C-NMR-spektroskopische Untersuchungen dioxanlöslicher Substanzen in Coniferenstreu

IR, ¹H and ¹³C NMR spectroscopic studies of dioxane soluble substances in coniferous litter Litter of mixed spruce and larch stands was extracted with dioxane. The extracts were studied by IR, ¹H NMR and ¹³C NMR spectroscopy. The NMR spectra revealed the dominance of aliphatic hydrocarbons. NMR peaks attributed to aromatic structures intensified after pressure extraction of the litter with dioxane. There is no conformity of the spectra of our dioxane soluble substances with spectra of lignin. Results of FTIR spectroscopic studies are in agreement with the NMR results.     

Accurate and reproducible determination of lignin molar mass by acetobromination

The accurate and reproducible determination of lignin molar mass by using size exclusion chromatography (SEC) is challenging. The lignin association effects, known to dominate underivatized lignins, have been thoroughly addressed by reaction with acetyl bromide in an excess of glacial acetic acid. The combination of a concerted acetylation with the introduction of bromine within the lignin alkyl side chains is thought to be responsible for the observed excellent solubilization characteristics acetobromination imparts to a variety of lignin samples. The proposed methodology was compared and contrasted to traditional lignin derivatization methods. In addition, side reactions that could possibly be induced under the acetobromination conditions were explored with native softwood (milled wood lignin, MWL) and technical (kraft) lignin. These efforts lend support toward the use of room temperature acetobromination being a facile, effective, and universal lignin derivatization medium proposed to be employed prior to SEC measurements.     

Studies on the effect of ball milling on lignin structure using a modified DFRC method.

The structures of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and residual lignin (REL) from a loblolly pine were analyzed using a modified derivatization followed by reductive cleavage (DFRC) method developed to allow the quantitative determination of three different structural monomeric products originating in lignin: phenolic beta-O-4, alpha-O-4, and etherified beta-O-4 structures. Results show that MWL and CEL are structurally identical, with an increased phenolic beta-O-4 content compared to that of the original Wiley milled wood. These results indicate that the portion of lignin linked to carbohydrates and that not linked to carbohydrates are structurally the same. Modified DFRC analysis of the effect of ball milling on the structure of lignin in wood, MWL, CEL, and REL indicate that vibratory ball milling does not change the lignin structure provided certain precautions are taken. Specifically, dry vibratory ball milling under a nitrogen atmosphere causes substantial structural changes including condensation, whereas vibratory ball milling in toluene had little effect on the lignin structure. This indicates that the structural differences observed in MWL and CEL arise because of the extraction procedure, which preferentially extracts phenolic lignin structures. MWL and CEL are representative of the total lignin in wood; however, due primarily to the solvent extraction process, higher phenolic hydroxyl contents are observed. Nitrobenzene oxidation showed structural results similar to those from the modified DFRC method.     

Structural characterization of guaiacyl-rich lignins in flax (Linum usitatissimum) fibers and shives

The structural characteristics of the lignins from flax (Linum usitatissimum) fibers and shives were studied. Significant differences in the content and composition of the lignin from both parts were observed. The lignin contents were 3.8% in the fibers and 29.0% in the shives. Analysis by Py-GC/MS indicated a H:G:S molar ratio of 13:72:15 in the milled wood lignin (MWL) isolated from flax fibers and a molar ratio of 5:87:8 in the MWL isolated from flax shives. In addition, 2D-NMR showed a predominance of β-O-4' aryl ether linkages, followed by β-5' phenylcoumaran and β-β' resinol-type linkages in both MWLs, with a higher content of condensed linkages in flax shives. Thioacidolysis (followed by Raney nickel desulfurization) gave further information on the lignin units involved in the different linkages and confirmed the enrichment of G units. The thioacidolysis dimers released were similar from both lignins, with a predominance of the β-5' followed by β-1' and 5-5' structures.     

Characterization of milled wood lignin (MWL) in Loblolly pine stem wood, residue, and bark

Milled wood lignin samples from Loblolly pine stem wood, forest residue, and bark were isolated and characterized by quantitative (13)C and (31)P nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC) for molecular weight determination. Results from (13)C NMR show the stem wood and forest residue samples have similar functional group contents. However, the bark has fewer methoxyl groups, β-O-4 structures, dibenzodioxocin, and side chains than the other two lignins. The bark lignin has the highest amounts of p-hydroxyphenyl (h) and C-5 condensed lignin, stem wood has the lowest, and the residue lies between. (31)P NMR analysis indicates that bark lignin contains more C-5 substituted phenolics and fewer aliphatic hydroxyl groups than the lignin isolated from stem wood or residue. The molecular weight distribution analysis indicates the bark lignin has higher weight-average molecular weight (M(w)) and polydispersity index than the lignin recovered from stem wood or residue.     

Determination of arylglycerol-beta-aryl ethers and other linkages in lignins using DFRC/(31)P NMR

An analytical method for lignins has been developed that involves derivatization followed by reductive cleavage (DFRC), depolymerization, and quantitative (31)P NMR spectroscopy. This technique detects and quantifies the various ether linkages present in softwood residual kraft lignins (RKL) and milled wood lignins (MWL). In addition, the technique supplies new quantitative information about beta-aryl ethers linked to condensed and noncondensed aromatic moieties, including dibenzodioxocins. Within RKL, beta-aryl ether bonds connected to condensed phenolic moieties predominated over those connected to noncondensed phenolic moieties. In addition, the amount of DFRC monomers determined by gas chromatography was minute in the RKL but large in the MWL. This indicates that almost all noncondensed beta-aryl ether linkages were cleaved during kraft pulping. The method offers new avenues for the detailed investigation of the bonding patterns of native and technical lignins.     

Lignin changes after steam explosion and laccase-mediator treatment of eucalyptus wood chips

Eucalyptus globulus chips were steam exploded followed by treatment with a laccase-mediator system (LMS) under different experimental conditions. Removal of hemicelluloses and, to a lesser extent, lignin was observed. Thermogravimetic analyses of whole meal obtained from chips before and after steam explosion indicated an increase in lignin degradation temperature due to lignin condensation. In contrast, application of LMS treatment caused a reduction in lignin and polysaccharide degradation temperatures. Lignins were isolated from wood samples before and after each treatment and analyzed by 2D NMR and (13)C NMR. An increase in carboxyl and phenolic hydroxyl groups and a significant decrease in β-O-4 structures were found in steam-exploded samples. The most relevant changes observed after laccase treatment were increased secondary OH and degree of condensation.