Separation and purification of isoflavones from a crude soybean extract by high-speed counter-current chromatography

A set of isoflavones with a broad range of polarity including daidzin, glycitin, genistin, acetyldaidzin, glycitein, acetylgenistin and daidzein was separated from a crude soybean extract by high-speed counter-current chromatography using a two-step operation. Three solvent systems were used: chloroform–methanol–water (4:3:2, v/v); chloroform–methanol–n-butanol–water (4:3:0.5:2, v/v); and methyl tert.-butyl ether–tetrahydrofuran–0.5% aqueous trifluoroacetic acid (2:2:0.15:4, v/v). The first solvent system was used for separating less polar isoflavones and the second for more polar isoflavones by eluting the lower organic phase. Genistin and glycitin, which were only partially resolved in the chloroform system, were separated by the third solvent system. Each isolated component showed 98–99% purity as determined by high-performance liquid chromatography analysis. Their structures were identified by LC–MS.