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

N Savitri Kumar,P A Nimal Punyasiri,W A M B Wijekoon

doi:10.4038/cjs.v45i2.7391

Abstract

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.

Highlights

Tea made from the tender leaves of Camellia sinensis, is one of the most widely consumed beverages in the world
Distilled solvents were used for high-speed countercurrent chromatography (HSCCC), while high performance liquid chromatography (HPLC) grade solvents (Merck, Mumbai, India) and water purified by a Milli-Q system (Millipore, Bedford, MA, USA) were used for HPLC
TFs are completely extracted from a tea extract by iso-butyl methyl ketone (IBMK) and part of the TR fraction which is acidic. IBMK has a lower solubility in water and is more effective than EtOAc, in liquid–liquid extraction

Summary

Introduction

Tea made from the tender leaves of Camellia sinensis, is one of the most widely consumed beverages in the world. Three principal types of tea - green tea, oolong tea and black tea are manufactured from the young green shoots of the tea plant Camellia sinensis. Oxidative polymerisation of polyphenols takes place during oolong and black tea production, in the presence of the enzyme polyphenol oxidase.Oxidised or ‘fermented’ teas contain the more complex flavanoids theaflavins (TFs) and thearubigins (TRs) formed by enzymic oxidation of the flavan-3-ols in the green tea shoots (Haslam, 2003).TFs are bright orange coloured pigments with acidic properties, and contribute to the colour and strength in the brew, andalso determine the quality of black tea(Smith and White, 1965). A combination of HSCCC and Sephadex LH-20 chromatography was used by Yang et al to separate TFs from a sample of black tea (Yang et al, 2008)

Methods

Results

Conclusion