NMR Analysis of LCC and Lignin Preparations

Complete structural information (sub-units, lignin linkages, and lignin-carbohydrate linkages) of both LCC and lignin preparations was determined using a Bruker AVANCE 600 MHz spectrometer, in which the quantitative ¹³C NMR and 2D-HSQC NMR spectra were obtained using dimethyl sulfoxide-d₆ (DMSO-d₆) as the solvent. The sample preparation steps for NMR analysis were described previously (Dong et al., 2020). For ¹³C NMR analysis, the acquisition parameters were 25°C, 90° pulse width, 1.7 s relaxation, and 1.2 s of acquisition time. For 2D-HSQC NMR analysis, 160 transients (scans per block) were acquired using 1024 data points in F2 (¹H) dimension with an acquisition time of 53 ms and 256 data points and F1 (¹³C) dimension with an acquisition time of 5.14 ms. The amounts (per 100 Ar (C₉₀₀)) of lignin linkages and lignin-carbohydrate linkages in both LCC samples were calculated from a combination of quantitative ¹³C and 2D HSQC NMR according to Zhang and Gellerstedt (2007) using the following formulas:

In the formulas, I_x is the integral value of a position signal in lignin linkages (β-O-4, β-β, and β-5) in the 2D spectra region. BE, PhGlc, and γ-Est are the amounts of benzyl ether, phenyl glycoside, and γ-ester LCC linkages. During the integration process, all the integration was conducted at the same contour level.

The contents of aliphatic hydroxyl and phenolic hydroxyl in LCC and lignin preparations were determined by quantitative ³¹P NMR using a Bruker AVANCE 600 MHz spectrometer, in which endo-N-hydroxy-5-norbornene-2, 3-dicarboximide (e-NHI) was used as the internal standard. The samples [40 mg of solid was dissolved in 0.5 mL of anhydrous pyridine/CDCl₃ mixture (1.6:1, v/v)] were prepared according to our previous report (Huang et al., 2017). The acquisition parameters with a total number of 256 scans were used to obtain the spectra in ³¹P NMR analysis.