Isolation and purification of isoflavones from soy flour by high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) was applied to the isolation of isoflavones from soy on a preparative scale. Two different protocols were developed for the isolation of monoglucosylated/acetylated isoflavones as well as malonylated derivatives. Analysis of malonylated derivatives requires a quick and gentle extraction/separation procedure and the use of acid-free solvents for HSCCC. An appropriate solvent system for the isolation of 6"-O-malonylgenistin is the biphasic mixture of tert.-butyl-methyl ether/n-butanol/acetonitrile/water (1/3/1/5). In the case of monoglucosylated/acetylated isoflavones an enrichment and clean-up stop (i.e. adsorption on XAD-7 resin) is preferably carried out prior to the HSCCC separation. The separation is then achieved by the application of tert.-butylmethyl ether/acetonitrile/water (2/2/3) and tert.-butylmethyl ether/acetonitrile/water (6/3/8) as solvent systems. Peak purity of the isolated compounds was checked by HPLC-MS and H-1-NMR spectroscopy. The isolated compounds can be used for studies on metabolism and bioavailability of isoflavone derivatives.