EFFECTIVE ON-LINE COMBINATION OF HIGH SHEAR TECHNIQUE AND CONTINUOUS SAMPLE INJECTION IN HIGH-PERFORMANCE COUNTER-CURRENT CHROMATOGRAPHY FOR ISOLATION AND PURIFICATION OF POLYPHENOLS IN GREEN TEA

Abstract

A new sample preparation method of high shear dispersing emulsifier (HSDE) coupled with continuous sample injection in high-performance counter-current chromatography (HPCCC) was successfully used for the extraction and online isolation of caffeine and two polyphenolic compounds from green tea. The lower phase of hexane-ethyl acetate–methanol–water (1:8:1:8, v/v/v/v) was used as both the HSDE solvent and HPCCC mobile phase. In one time sample loading, 1.5 mg of (-)-epicatechin-3-gallate, 5.0 mg of (-)-epigallocatechin-3-gallate, and 3.1 mg of caffeine with the purity over 95.0% determined by high performance liquid chromatography (HPLC), could be obtained from about 0.1 g of raw material of green tea. Ten time sample injections were realized in HSDE extraction−HPCCC separation process, and the quantity could be magnified about decuple, 13.8 mg of (-)-epicatechin-3-gallate, 46.3 mg of (-)-epigallocatechin-3-gallate, and 28.9 mg of caffeine could obtain from 1.0 g of raw material of green tea. The compounds were analyzed by HPLC and identified by their chromatograms and their retention time compared with that of authentic reference compounds. It was the first time combining HSDE with continuous sample injection in HPCCC for effective isolation and purification of active compounds from natural products. The mechanism of HSDE extraction was illustrated.