The hexane-ethyl acetate-methanol-water system for the separation of theaflavins from black tea (Camellia sinensis) using high-speed counter-current chromatography

Theaflavins (TFs) from black tea leaves were separated using high-speed countercurrent chromatography (HSCCC). Isobutyl methyl ketone (4-methyl-2-pentanone) was used to separate the TFs from a hot water extract of black tea leaves. Three different compositions of the biphasic solvent system hexane–ethyl acetate-methanol-water (2:5:2:5 solvent system A) 1:4:1:4 (solvent system B) and 1:5:1:5 (solvent system C) were studied. The aqueous phase was used as the mobile phase in the head to tail elution mode. Flow rates of 2.0 – 2.8 mL min^-1, revolution speeds of 800-1000 rpm and a 200 mg sample of TF in 5 mL of the aqueous phase with settling times 17-18 secs (solvent systems B and C) gave the best separations. Three peaks were separated in the HSCCC traces obtained using solvent systems B and C, and each peak was monitored by high performance liquid chromatography (HPLC).Separation of theaflavin monogallates was not achieved, but solvent system Cresulted in a complete separation of theaflavin and theaflavin digallate.