2.6. Analysis of Lignin Characteristics

FT-IR (VERTEX70, Bruker, Karlsruhe, Germany) was used to analyze lignin characteristics. The dried samples were embedded in KBr pellets at concentrations of 1 mg/100 mg KBr. All spectra were recorded in absorption band mode over the range of 4000 to 500 cm⁻¹.

For the acetylation of lignin samples, 50-mg lignin sample was dissolved in 2 mL pyridine in a 20-mL glass vial, followed by the addition of acetic anhydride (2 mL). The mixture was sealed and allowed to react at room temperature for 24 h. The acetylated lignin was precipitated in ice DI-water with constant stirring and collected by filtration. The collected acetylated lignin was washed with DI-water and vacuum-dried.

The molecular weight distribution of lignin samples was determined by GPC (WATERS Alliance HPLC e2695, Waters, MA, USA). Approximately 2 mg of acetylated lignin sample was dissolved in tetrahydrofuran and then filtered through a 0.45-μm filter. An Agilent 1200 series high performance liquid chromatography (HPLC) equipped with an ultraviolet detector (UV) at 254 nm was used to conduct GPC analysis of lignin molecular weight distribution. Polystyrene standards with molecular weights ranging from 139 to 16,000 g/mol were used to calibrate the molecular weight based on retention time.

NMR (AVANCE III 500 MHz, Bruker, Karlsruhe, Germany) were recorded according to literature method [7], by using a spectrometer equipped with a DCH cryoprobe. HSQC spectra was recorded at 25 °C using the Q-CAHSQC pulse program. Matrices of 2048 data points for the ¹H-dimension (13 to −1 ppm) and 1024 data for the ¹³C-dimension (160 to 0 ppm) were collected, with the relaxation delay set at 6 s [7]. The lignin samples were dissolved in 0.5 mL of dimethylsulfoxide-d6 (DMSO-d₆) and chemical shifts were referenced to the solvent signal (2.50/40.21 ppm).