2.4. Lignin Characterization

The molecular weight distributions of lignin were analyzed by size-exclusion chromatography (SEC) based on the methods reported by Guerra et al. [47]. The lignin fractions were acetobrominated before chromatographic analysis [48]. Briefly, a 5 mg lignin sample was firstly mixed with 900 μL glacial acetic acid and 100 μL of acetyl bromide. Then, the mixture was subjected to 2 h stirring. After acetobrominatation and the subsequent removal of excess glacial acetic acid and acetyl bromide, the acetobrominated lignin was dissolved in 1 mL tetrahydrofuran (THF, HPLC grade) and the resultant solution after filtering with a syringe filter (0.45 μm) was injected to SEC systems for molecular weight distribution analysis.

The functional groups of lignin were quantified by running ³¹P NMR [49]. A ~30 mg lignin fraction was dissolved in 100 µL dimethylformamide (DMF) and 100 µL of pyridine. The internal standard for quantification was 40 mg/mL of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide (e-HNDI) (Sigma Aldrich) while the relaxation reagent was 5 mg/mL of chromium (III) acetylacetonate (Sigma Aldrich). Phosphorylation of lignin was achieved by adding 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane, and then the lignin derivative was dissolved in CDCl₃ before performing ³¹P NMR analysis.

A MicroDouble-shot Pyrolyser Py-2020iD (Frontier Lab, Fukushima, Japan) was connected with a Shimadzu GC/MS-QP 2010 apparatus (Kyoto, Japan) to conduct the Py-GC/MS/FID analysis. The separation was achieved by a capillary column RTX-1701 (Restec, Bellefonte, PA, USA) with dimensions of 60 m length, 0.25 mm width and 0.25 μm internal diameter. The pyrolysis was performed at 500 °C with a heating rate of 600 °C s⁻¹. The other operation conditions and data analysis simply followed the ones reported previously [50].