Preparative Isolation and Purification of L‐Theanine by HPLC

Abstract

A preparative extraction HPLC method has been developed to purify theanine from green tea. The green tea residua, after extracting catechin with ethyl acetate (EtOAc), was dissolved with 50°C hot water and purified through a 732 cation resin column to obtain a green tea extract containing 50% theanine. Then, the extract was isolated and purified to theanine with a purity higher than 98% using preparative HPLC. The preparative separation of theanine was performed by injecting 1.5 mL of filtered 50% theanine extract solution onto a Water Prep Nova‐pak HR C18 reversed‐phase HPLC column (19 mm × 300 mm, 60 Å, 6 µm). A UV‐VIS detector, set at 210 nm, was used to monitor the eluate profile. The yield of theanine in the preparative HPLC method was 70.4%. Peak identification of theanine was confirmed using RP‐HPLC, photo‐diode‐array (PDA) detector and electrospray mass spectrometer.