High Performance Countercurrent Chromatography for Quantitative Analysis of Five Flavonoids

Abstract

The stability of stationary phase in the rotating coil of modern countercurrent chromatography, and the repeatability of chromatographic process of target compounds were the focal points in this report. A PTR CCC-1000 chromatograph mounted with either column set for semipreparative (1.6mm id, total capacity of 338mL) or 3-layer analytical for rapid screen (0.85mm id, total capacity of 51 mL) was used to perform quantitative analysis of five flavonoids. The same solvent system, CHCl₃/MeOH/H₂0 (4:3:2), reported for separa tion of quercetin (1) apigenin and leuteolin (2) was adopted in this study. The chromatographic parameters such as k' (capacity factor), K (partition coefficient), N (number of the theoretical plate), and Ps (peak resolution) were determined from both sets of chromatograms, and compared thereafter. The repeatability of chromatographic process was evaluated through repeated injections of respective compounds, and presented as c.v. (coefficient of variation) values. Time for analysis of first four flavones was within one and half hour, while the last isoflavone took additional one hour, when the semipreparative column set was used. The 3-layer analyt ical column set reduced the assay time to within one hour, but produced heavy bleeding after a large amount of flavonoids mixture was introduced. Other solvent systems such as Hexane/EtOAc/MeOH/H₂O (1:1:1:1) and 1-ButOH/H₂O were also included to monitor stationary phase retention between two column sets.