2.7. Anion-exchange chromatographic method
The oxalate soluble fraction (OSP) was purified by anion-exchange chromatography as described (Mazumder et al., 2004 and Ray and Lahaye, 1995b). The crude polysaccharides were applied to a DEAE–Sepharose FF (acetate form) column (21 × 4 cm) equilibrated previously with 0.05 M sodium acetate buffer, pH 5.5. The column was washed with the same buffer (400 ml: fraction OF1) and then eluted successively with 400 ml of 0.2 M (fraction OF2), 0.7 M (fraction OF3) and 1.5 M NaOAc buffer pH 5.5 (fraction OF4) in a stepwise manner. Fractions (40 ml) were collected and analyzed for their total sugar and uronic acid contents. Appropriate fractions were concentrated, dialyzed against water, and finally lyophilized.