Lignin Characterization by Nitrobenzene Oxidation

Lignin was characterised on 3 or 4 biological replicates using the nitrobenzene oxidation method [64 (link)]. 10 mg of CWR were digested with 2 mL of 2 M NaOH and 30 μL nitrobenzene at 165 °C for one hour (Hach LT200 system). After centrifugation, ca. 1500 μL of supernatant was collected and 10 μl of vanillin-D3 (Sigma-Aldrich) at 10 mg/mL in 1,4-dioxan were added as a surrogate standard. Nitrobenzene was removed by four washing steps with ethyl acetate (1 mL, vortexing/centrifugation cycle). The pH of the solution was adjusted to 2–3 by adding approximately 200 μL of 6 N HCl solution. The oxidation products were recovered by two successive extractions with 1 mL ethyl acetate (vortexing/centrifugation cycle) followed by cleaning with 500 μl of saturated NaCl solution and drying with Na₂SO₄. The GC-MS analysis was performed after trimethylsilylation, realized by addition of 50 μl of Bis(trimethylsilyl)trifluoroacetamide (BSTFA) to 50 μL of dried extract and derivatization at 60 °C for 30 min. Quantitative analyses were performed using a HP-5MS column (30 m × 0.25 mm, 0.25 μm, Agilent) installed in a 7890B-5977A GC-MS system (Agilent). Injection was done at 250 °C in splitless mode. The oven program started at 40 °C for 5 min, increased to 230 °C at 10 °C/min, then to 320 °C at 40 °C/min and was kept at 320 °C for 10 min. Salicylic acid-D4 was used as internal standard.