The increasing research attention onto the utilization of biomass as feedstock for the production of sustainable materials and chemicals has been directed toward an in-depth understanding of plant cell wall natural structures and their constituents, which are consisted mainly with cellulose, hemicellulose, and lignin. A better understanding of these issues, on one hand, can guide the development of new efficient pretreatment technologies and robust catalysts for the catalytic separation and conversion of biopolymers; on the other hand, can provide new avenues to rationally designing of bio-energy crops with improved processing properties by either reducing the amounts of lignin present or providing a lignin that is easier to degrade. Traditionally, the elucidation of wood structure (lignin) usually follows the destruction-analysis process (e. g. Klason method) due to the insolubility of lignocellulose in conventional organic solvents, and the information obtained does not represent the natural structure of lignin.

Lignin is a complex aromatic chemical polymer present most commonly in wood. As an integral part of the secondary cell walls of plants, it is one of the most abundant organic polymers on the earth, exceeded only by cellulose. In 2007, Jiang et al. investigated the solubility of lignin in different ionic liquids, and the results showed that the order of lignin solubility for varying anions was: [MeSO4]- > Cl — > Br- ^ [PF6]-. This result indicated that the solubility of lignin is principally influenced by the anions of ILs [36]. Further 13C NMR analysis of lignin and lignin model compounds presented that 13C signals using ILs as the solvent is shifted up-field by d 0.1-1.9 ppm in comparison to 13C NMR data acquired using dimethyl sulfoxide (DMSO) as the solvent.

The full dissolution of lignocellulosic materials in ILs provided a new homo­geneous media without degradation of their components for the structural analysis of plant cell wall and lignin. Kilpelainen et al. demonstrated that the fully acet — ylated Norway spruce in ionic liquids was soluble in CDCl3, which allowed the first recording of the solution state 1H NMR spectra of intact acetylated wood. The careful integration of P-O-4 signals for lignin in the 1H NMR spectrum yielded a value of 7.3%, which was in good agreement with the anticipated value of 8% [21]. Further in situ quantitative 31P NMR analysis of spruce dissolved in ionic liquids showed the presence of 13.3 mmol/g hydroxyl groups. This value was close to the theoretically calculated value of 15.7 mmol/g based on traditional methods [37]. Analysis of different pulverization degrees provided semi-empirical data to chart the solubility of Norway spruce in IL [amim] Cl, and further method refinement afforded an optimized method of analysis of the lignin phenolic functionalities, without prior isolation of the lignin from the wood fiber [38, 39].

ILs not only can be used as solvents for catalysis and biomass dissolution, but also can be used as solvents for nuclear magnetic resonance analysis directly. Ragauskas et al. synthesized a series of perdeuterated pyridinium ILs for the direct dissolution and NMR analysis of plant cell walls. Due to the high melting point of pyridinium salts, a co-solvent DMSO-d6 was used to reduce the viscosity of the resulting mixtures, for example, a mixture of 1:2 [Hpyr] Cl-d6/DMSO-d6 was able to dissolve Poplar up to 8 wt% at 80°C in 6 h. Further in situ 1H NMR and 13C NMR analysis showed the full structural map of signals from cellulose, hemi- cellulose, and lignin. For example, the signals at d 61.5, 74.1, 75.8, 76.9, 80.1, and

103.0 ppm were in part attributed to cellulose. Whereas, the lignin methoxyl group corresponding to the signals at d 57 ppm and d 58-88 ppm could be attributed, in part, to Cp in b-O-4, Cc/Ca in P-O-4, P-5, and b-p. The signal at d 106 ppm was attributed to C2/6 resonance of syringyl-like lignin structures, and between 110 and 120 ppm to C2, C5, and C6 resonance of guaiacyl-like lignin structures. The properties and easy preparation of perdeuterated pyridium molten salt [Hpyr]Cl-d6 offer significant benefits over imidazolium molten salts for NMR analysis of plant cell walls; furthermore, the use of non-ball-milled samples in this study can pro­vide a more efficient and accurate characterization of lignin in the plant cell walls compared with the results from traditional methods [40]. Although lignin can provide a renewable source of phenolic polymers, a high lignin content has proved to be a major obstacle not only in the processing of plant biomass to biofuels, but also in other processes such as chemical pulping and forage digestibility. There­fore, precise analytic techniques for efficient lignin content assessment of a large number of samples are in high demand. Further study from Ragauskas’s group reported a linear extrapolation method for the measurement of lignin content by the addition of a specific amount of isolated switchgrass lignin to the biomass solution, and the integration ratio changes could be measured in the quantitative 1H NMR spectra with non-deuterated DMSO as the internal standard. The results showed comparable lignin contents as the traditional Klason lignin contents. They demonstrated that this direct dissolution and NMR analysis of biomass provided a new venue for rapidly assessing the lignin contents in large numbers of ‘‘new’’ plants in biofuel research [41].