4.3.1 Analytical Techniques

Advances in a variety of analytical techniques have provided valuable insight into the mechanisms involved in delignification and cellulose dissolution. Optical and fluorescence microscopy enabled the study of wood expansion (Fig. 4.2) [70, 71] and switchgrass dissolution [42] in ionic liquids at the micron scale. Distinct autofluorescence from cellulose and lignin signatures distinguish the cellulose-rich cell walls and the lignin-rich cell corners and middle lamellae in poplar and switchgrass [42, 70, 71]. Optical and scanning electron microscopies have been particularly useful in visualizing IL interacting with heterogeneous native biomass. They revealed structural changes after dissolution, regeneration, and chemical functionalization [22, 30, 36, 38, 41, 42, 44, 72].

X-ray diffraction provided an insight into structural changes occurring at the atomic scale in cellulose during its dissolution and regeneration [7, 36, 38, 71, 73]. It was used to monitor in situ the loss of cellulose crystallinity in poplar, ramie fibers [71], switchgrass, eucalyptus, and pine wood [73]. After the regeneration of dissolved pine and spruce sawdust, X-ray diffraction revealed a change of crystal structure from the native cellulose I to the cellulose II structure [7, 36]. Neutron scattering was used to estimate the surface roughness of switchgrass, eucalyptus, and pine after their IL pretreatment [73].

These structural changes were supplemented by analyses of the biomass chemical composition. The distinct Raman signatures of cellulose and lignin have made hyperspectral Raman imaging a powerful tool to map the chemical com­position of native biomass [74] and its evolution during pretreatments [70, 71]. IR spectroscopy was commonly used to assess purity [4, 39, 42], loss of hemicel — luloses/lignin after the dissolution [36,42], chemical functionalization [6, 7, 30,48], cleavage of b-O-4 bonds in lignin models [75,76]. It can also probe the interactions between the anion and cation in the IL and the hydrogen bonding network [77]. FTIR spectroscopy combined with principal component analysis was used to distinguish lignins from bagasse, softwoods, and hardwoods [78]. Efforts were made to use IR spectroscopy as a method to quantify glucose and cellobiose in [EMIM] [OAc]. The IR absorption of multiple bands in glucose and cellobiose was found to vary with concentration and empirical nonlinear relations between the absorbance and the concentration were derived [79].

Optical absorption spectroscopy offers a quick way to quantify the saccharification of purified substrates, such as Avicel, and native biomass.

The 2,4-dinitrosalicyclic reagent acid assay has been widely used to quantify reducing sugars, including glucose [26,41,44,45,80]. However, on native substrates (municipal solid waste, paper mill wastes, or agricultural wastes), the method suffers from the interference from other chemicals and impurities [78,81]. Due to the variety and heterogeneity of native biomass, it has also been difficult to find adequate standards to establish Beer-Lambert relations between the absorbance and the sugar concentration, particularly for lignin which has different ratio of syringyl and gua — iacyl units [78]. ILs, such as [BMIM][Cl], also absorb strongly in the UV range. Optical absorption analyses are also complicated by chemical alterations of the biomass during pretreatments [78].

Analytical techniques, such as mass spectrometry [44, 69], HPLC [26, 43, 44, 46], high-performance anion-exchange chromatography (HPAEC) [41, 45, 50], have been used to identify hydrolysis products. HPLC and HPAEC can quantitate the amount of reducing sugars produced during cellulose hydrolysis. Size exclu­sion chromatography was used to determine the molecular weight distribution of milled woods and their dissolution products in ILs [33].

Another widely used analytical technique is NMR. The variety of isotopes available ( H, C, P, Cl) has made NMR spectroscopy a versatile method to characterize chemical functionalization [7, 30, 82], assess purity of products [32], identify/quantify hydrolysis/dissolution products [3, 4, 31, 32, 36], study the structure of milled native biomass (poplar, switchgrass) [29] and lignin [83], and study hydrogen bonding in cellulose dissolution [84, 85].

The combination of all these techniques has provided a wealth of information at multiple length scales about the chemical composition and structure of the pre­treated biomass. Quantitation of reaction products allowed for the optimization of reaction conditions, such as temperature and IL composition, and revealed the critical factors affecting the delignification and hydrolysis of cellulose.