Standard chemicals: p-Hydroxybenzaldehyde(H), vanillin(G) and syringaldehyde (S) were purchased from Sinopharm Chemical Reagent Co., Ltd. The sample was extracted with benzene-ethanol (2:1, v/v) in a Soxhlet for 4 h, and the remaining pellet was collected as cell wall residue (CWR). The procedure of nitrobenzene oxidation of lignin was conducted as follows; 0.05 g CWR was added with 5 mL 2 M NaOH and 0.5 mL nitrobenzene, and a stir bar was put into a 25 mL Teflon gasket in a stainless steel bomb. The bomb was sealed tightly and heated at 170°C (oil bath) for 3.5 h and stirred at 20 rpm. Then, the bomb was cooled with cold water. The chromatographic internal standard (ethyl vanillin) was added to the oxidation mixture. This alkaline oxidation mixture was washed 3 times with 30 mL CH2C12/ethyl acetate mixture (1/1, v/v) to remove nitrobenzene and its reduction by-products. The alkaline solution was acidified to pH 3.0-4.0 with 6 M HCl, and then extracted with CH2CI2/ethyl acetate (3 × 30 mL) to obtain the lignin oxidation products which were in the organic phase. The organic extracts were evaporated to dryness under reduced pressure 40°C. The oxidation products were dissolved in 10 mL chromatographic pure methanol.
HPLC analysis: The solution was filtered with membrane filter (0.22 μm). 20 μL Solution was injected into HPLC (Waters 1525 HPLC) column Kromat Universil C18 (4.6 mm × 250 mm, 5 μm) operating at 28°C with CH3OH:H2O:HAc (25:74:1, v/v/v) carrier liquid (flow rate: 1.1 mL/min). Calibration curves of all analytes routinely yielded correlation coefficients 0.999 or better, and the detection of the compounds was carried out with a UV-detector at 280 nm.
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