2.5. Analytical methods
The moisture content of solids was measured by drying the sample at 105 ± 1 C in an oven until constant weight. The ash
content was determined by the standard procedure TAPPI T211. Structural carbohydrates and acid insoluble lignin composition were characterised by the method of Puls [25]. Cellulose and hemicelluloses were determined on the basis of monomer contents measured after a two-step acid hydrolysis. In a first step, 300 mg of free-extractive dry material was hydrolysed with 3 cm3 of 72% (v/v) H2SO4 at 30 C for 60 min. In a second step, the reaction mixture was diluted to 4% H2SO4 and autoclaved at 121 C for 1 h. The system was then filtered on a Gosh filter No. 4. The solid fraction, acid insoluble lignin (AIL), was dried and weighed. Sugars (Dglucose, D-xylose, L-arabinose and D-galactose) in hydrolysates were determined by normal phase HPLC (Merck Hitachi) with refraction index detector by using an Aminex HPX-87H column, at 45 C. The mobile phase was composed of 5 mM H2SO4 with a flow rate of 0.6 cm3 min1. Acid-soluble lignin (ASL) was determined using the protocol described by Sluiter et al. [26]. Extractives were determined gravimetrically after soxhlet extraction. It was used a twostep extraction process to remove ethanol/acetone (1/2, v/v) soluble materials and ethanol soluble materials. All the analyses were carried out in triplicate.
2.5. Analytical methods
The moisture content of solids was measured by drying the sample at 105 ± 1 C in an oven until constant weight. The ash
content was determined by the standard procedure TAPPI T211. Structural carbohydrates and acid insoluble lignin composition were characterised by the method of Puls [25]. Cellulose and hemicelluloses were determined on the basis of monomer contents measured after a two-step acid hydrolysis. In a first step, 300 mg of free-extractive dry material was hydrolysed with 3 cm3 of 72% (v/v) H2SO4 at 30 C for 60 min. In a second step, the reaction mixture was diluted to 4% H2SO4 and autoclaved at 121 C for 1 h. The system was then filtered on a Gosh filter No. 4. The solid fraction, acid insoluble lignin (AIL), was dried and weighed. Sugars (Dglucose, D-xylose, L-arabinose and D-galactose) in hydrolysates were determined by normal phase HPLC (Merck Hitachi) with refraction index detector by using an Aminex HPX-87H column, at 45 C. The mobile phase was composed of 5 mM H2SO4 with a flow rate of 0.6 cm3 min1. Acid-soluble lignin (ASL) was determined using the protocol described by Sluiter et al. [26]. Extractives were determined gravimetrically after soxhlet extraction. It was used a twostep extraction process to remove ethanol/acetone (1/2, v/v) soluble materials and ethanol soluble materials. All the analyses were carried out in triplicate.
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