ISOLATION AND PURIFICATION OF FOUR INDIVIDUAL THEAFLAVINS USING SEMI-PREPARATIVE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3,3'-digallate (TF3) are the major theaflavins, which mostly contribute to the quality and bioactivity of black tea. In this study, a rapid isolation and purification of the four major individual theaflavins from crude theaflavins mixture was established. The crude theaflavins mixture was prepared by enzymatic oxidation of tea polyphenols using immobilized polyphenol oxidase and then fractionated using a Mitsubishi SP-207 resin chromatography with an elution gradient of 20%, 30%, 40%, 50%, and 70% aqueous ethanol to obtain a mixture of theaflavins with 80% purity (TF80). The TF80 was further purified using a semi-preparative high performance liquid chromatography (HPLC) equipped with a C18 column using isocratic elution with water/acetonitrile/glacial acetic acid (73.5:26:0.5, v/v/v) at a flow rate of 5mL/min as optimized operating conditions. The purity of the isolated individual theaflavins were 92.48% for TF2A, 90.05% for TF2B, 92.40% for TF3, and 73.02% for TF1, respectively.