Preparative separation of isoflavone components in soybeans using high-speed counter-current chromatography

Four isoflavone components were purified from soybean extract by high-speed counter-current chromatography (HSCCC). Two types of multilayer coil separation columns were used: a small column made of standard 2.6-mm I.D. PTFE (polytetrafluoroethylene) tubing with a 260-ml capacity and a large column of convoluted PTFE tubing of 5.7-mm average I.D. with a 1200-ml capacity. Separation was performed with a two-phase solvent system composed of hexane–ethyl acetate–1-butanol–methanol–acetic acid–water (1:2:1:1:5:l, v/v) by eluting the lower aqueous phase at 2 ml/min (small column) and 5 ml/min (large column) at a revolution speed of 700 rpm. From 500 mg of crude sample the small column yielded 33 mg of daidzin, 41 mg of genistin, 27 mg of 6″-O-malonyldaidzin and 24 mg of 6″-O-malonylgenistin. The large convoluted column separated, from 3 g of crude sample, 203 mg of daidzin, 241 mg of genistin, 158 mg of 6″-O-malonyldaidzin and 135 mg of 6″-O-malonylgenistin all at over 90% purity. The convoluted tubing facilitated preparation of a large multilayer coil due to its high flexibility.