Enhanced Separation and Elution of Catechins in HPLC Using Mixed-Solvents of Water, Acetonitrile and Ethyl Acetate as the Mobile Phase

Abstract

We conducted a study for the separation of four catechins ((–)-epicatechin (EC), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG) and (–)-epigallocatechin gallate (EGCG)) in tea by HPLC with an electrochemical detector at different temperatures (25–50°C). Various solvent compositions of mixed solvents of water, acetonitrile (AN) and ethyl acetate (EA) were used as eluting solvents. The compositions of the solvents were as follows: water, 90–70% (v/v); AN, 10–30% (v/v); EA, 0–5% (v/v). The binary mixtures of water (90–70% (v/v)) and AN (10–30% (v/v)) were not able to separate the peaks of EC and EGCG under all of the different solvent compositions. However, ternary mixed-solvents containing a small amount of EA (3–5% (v/v)) with water (85–83% (v/v)) and AN (12% (v/v)) separated four catechins sufficiently, especially the peaks of EC and EGCG, within a 10 min elution time at 40°C. The effect of the solvent composition is discussed in terms of the E_T^N and D_II,I values, solvent structure and extraction constants of catechins into EA. The formation of a pseudo stationary phase in which ODS is partly covered by EA is proposed for the enhanced separation of EC and EGCG in the presence of EA. Catechins in three kinds of tea were determined under an optimum condition.