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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.

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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.

The fate of lignin from wheat straw, Miscanthus, and Loblolly pine after pretreatment by a non-toxic and recyclable ionic liquid (IL), [C2mim][OAc], followed by enzymatic hydrolysis was investigated.

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.

Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy was applied to intact and isolated loblolly pine wood samples to identify potential structural changes induced by tree age, milling, lignin extraction, or naturally occurring mutations. Special attention was paid to ketone and aldehyde as well as nonpolar alkyl groups, which could be observed at low concentrations (<2 in 1000 C) using improved spinning-sideband suppression with gated decoupling. Carbonyl structures were present in intact wood, and there are more keto groups than aldehydes. Their concentrations increased from juvenile to mature wood and with milling time, whereas extraction did not alter the C=O fraction. Significant amounts of aldehyde and dihydroconiferyl alcohol residues were present in coniferyl aldehyde dehydrogenase-deficient wood, confirming solution-state NMR spectra of the corresponding lignin. These results demonstrate the utility of solid-state NMR as an assay for changes in the lignin structure of genetically modified plants.

Advanced solid-state NMR was employed to investigate differences in chemical structure and heterogeneity between milled wood lignin (MWL) and residual enzyme lignin (REL). Wiley and conventional milled woods were also studied. The advanced NMR techniques included 13C quantitative direct polarization, various spectral-editing techniques, and two-dimensional 1H-13C heteronuclear correlation NMR with 1H spin diffusion. The 13C chemical shift regions between 110 and 160 ppm of two lignins were quite similar to those of two milled woods. REL contained much more residual carbohydrates than MWL, showing that MWL extraction more successfully separated lignin from cellulose and hemicelluloses than REL extraction; REL was also of higher COO, aromatic C-C, and condensed aromatics but of lower aromatic C-H. At a spin diffusion time of 0.55 ms, the magnetization was equilibrated through the whole structure of MWL lignin, but not through that of REL, indicating that REL is more heterogeneous than MWL.