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 separ ation 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 o f convoluted PTFE tubing of 5.7-mm average I.D. with a 1200-ml capacity. Se paration was performed with a two-phase solvent system composed of hexane-e thyl acetate-1-butanol-methanol-acetic acid-water (1:2:1:1:5:1, v/v) by elu ting 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-m alonyldaidzin and 24 mg of 6 " -O-malonylgenistin. The large convoluted col umn separated, from 3 g of crude sample, 203 mg of daidzin., 241 mg of geni stin, 158 mg of 6 "O-malonyldaidzin and 135 mg of 6 " -O-malonylgenistin al l at over 90% purity. The convoluted tubing facilitated preparation of a la rge multilayer coil due to its high flexibility. (C) 2001 Elsevier Science B.V. All rights reserved.