It is generally accepted that various types of chemical linkages between lignin and carbohydrate are present in LCC structures, and the most commonly proposed types are benzyl ether, benzyl esters, γ-esters and phenyl glycosides. The work of Balakshin et al. (2011) is a representative publication that observed various linkages by high-sensitivity NMR analysis. For the LCCs prepared in this study, the very high molecular masses of the structures were preserved. This made it difficult to detect and assign the NMR signals for the lignin–carbohydrate bonding because there was substantial influence from the high molecular mass, which caused a severe decrease in the signal intensity, especially when ordinary-sensitivity NMR analysis was performed (see below). However, some indirect evidence was collected, demonstrating the presence of lignin–carbohydrate linkages in the polymeric LCC structures.
First, the high molecular masses of the LCCs were revealed by alkaline SEC using UV detection at 280 nm (Figure 2), implying that lignin, which is responsible for the 280 nm adsorptions, is attached to various high-molecular-mass polysaccharides.
Second, LCCs demonstrate a very different solubility to free lignin. In a previous report, a dioxane/water solution was used at a ratio of 96:4 to dissolve the free lignin during milled wood lignin (MWL) preparation (Björkman, 1956), and used at a ratio of 82:18 to isolate lignin via acid hydrolysis from wood or pulp (Gellerstedt et al., 1994). When the solubility of the obtained LCC fractions was investigated in a dioxane/water solution (90:10 v/v), the GL and GML fractions were not soluble, and only a trace amount of XL was soluble (Table 1), possibly because the xylan portion that was attached by the lignin was itself partially dissolved in this solution. For spruce wood itself, only extractives were directly soluble (Table 1).
Third, as mentioned above, although the lignin aromatic signals were clearly observed for the XL fraction using the 2D HSQC NMR technique, with a 400 MHz NMR instrument (Figure 3), no aromatic signals were observed using the same NMR equipment as for GL or GML. NMR assignments of the main polysaccharides in XL were based on previous data (Teleman et al., 2000, 2002; Evtuguin et al., 2003). The cross-peaks at δ 4.76/108.3 (H1/C1), 3.83/82.0 (H2/C2), 3.65/77.7 (H3/C3), 3.97/86.5 (H4/C4) and (3.45; 3.61)/62.0 (H5/C5) are from α-l-(14) arabinosyl units, and those at δ 4.25/102.0 (H1/C1), 3.03/72.9 (H2/C2), 3.23/74.2 (H3/C3), 3.50/75.5 (H4/C4) and (3.16; 3.87)/63.4 (H5/C5) are from β-d-(14) xylosyl units. In the analysis, the XL was directly dissolved in solution as it had the smallest molecular mass of the three LCCs, whereas the GL and GML fractions had larger molecular masses and required acetylation to facilitate the required dissolution. A representative 2D HSQC NMR spectrum for GL is shown in Figure 4, after polysaccharide assignment as described by Lu and Ralph (2003). In this case, the lignin aromatic signals in the 1H NMR spectrum (Figure 5) were already very weak due to the short spin–spin relaxation time (T2) caused by the large size of the polymers (Zhang and Gellerstedt, 2007). Solid-state 13C cross polarization-magic angle spinning (CP-MAS) NMR was performed for GML and lignin-free glucomannan (GM) (Figure 6) in order to overcome the solubility difficulty encountered when performing solution-state NMR, and the assignments for GM are based on previous data (Rakhimov et al., 2004). Here, lignin signals (105–160 ppm for aromatic carbons, approximately 87.5 ppm for lignin side-chain carbons and 55.3 for methoxy carbons) were easily observed by comparison of the 13C CP-MAS NMR spectra for GML with those for GM. However, the resolution of the 13C CP-MAS NMR spectra is too low to reveal any more structural details. Using an NMR instrument with a cryo-platform and cryo-probe at 750 MHz, i.e. high-sensitivity solution-state NMR, greatly enhanced 2D NMR spectra were obtained for both original GML and acetylated GML; here, the analysis of GML was performed after acetylation of GML in order to achieve a high enough solubility in CDCl3, and thus the lignin aromatic signals from the G units were more clearly observed (Figure 7). Carbohydrate signals, assigned on the basis of previous data (Lu and Ralph, 2003; Qu et al., 2011), were also clearly observed in the spectrum. The cross-peaks at δ 4.41/100.4 (H1/C1), 4.79/71.6 (H2/C2), 5.06/72.3 (H3/C3), 3.71/75.9 (H4/C4), 3.55/72.6 (H5/C5) and (4.05; 4.36)/61.9 (H6/C6) are from β-d-(14) glucosyl units, and those at δ 4.64/97.8 (H1/C1), 5.32/68.5 (H2/C2), 5.03/70.6 (H3/C3), 3.87/72.6 (H4/C4), 3.50/72.6 (H5/C5) and (4.17; 4.28)/62.5 (H6/C6) are from β-d-(14) mannosyl units. The ‘disappearance’ of the lignin signals in the ‘ordinary’-sensitivity NMR (400 MHz) analysis confirmed the high-molecular-mass characteristics of the LCCs, which are the result of chemical linkages between the lignin and the polysaccharides.