2.7. Purification of crude metabolites by column chromatography

An aliquot of three grams of concentrated extract of SEB32 was further separated by column chromatography. Activated silica gel (230–400 mesh, MERCK) was packed on to a glass column (450 mm × 40 mm) with the maximum height of 30 cm using methanol:ethyl acetate (30: 70) solvent and 3 g of extract was loaded on the top of silica gel. Fractions eluted successively with 50 ml of methanol and ethyl acetate (30: 70). Purity of the active metabolites was confirmed by analytical TLC as well as by gas chromatography analysis using the method described by Wilkins [45].