Purification of a flavonoid sample by countercurrent chromatography

A quercitrin rich vegetal flavonoid sample was fractionated using a new hydrodynamic CCC apparatus based on the Ito scheme IV design. The Kromaton CCC apparatus has two spools with a tube coiling allowing work with two different apparatus volumes. The small "analytical" volume was 94 mt and the large "preparative" volume was 1.07 L. The fractionation of the flavonoid sample was done using the two machine volumes. The bi phasic liquid system was a non chlorinated solvent system, butanone/water. UV detection was possible at 330 nm. In one run, 330 mg of the flavonoid sample could be fractionated by the CCC machine in six peaks that were collected in nine fractions. The HPLC and MS analysis of the fractions allowed identification of the main constituents of the sample. Quercitrin, a rhamno-flavonoid, was the major compound making up more than 88% w/w of the studied sample. The three minor identified flavonoids were quercetin, kaempferol, and kaempferol-3-rhamnoglucoside. Six very minor impurities were detected by the HPLC analysis of the fractions, but they were not unambiguously identified.