Coupling Chromatographic Techniques For Preparative Isolation Of Tea Ingredients And Its Detection On Antioxidant Activity

Rapid separation and detection of complex active ingredients were difficult problems in the natural products research. In this work, three on-line liquid chromatography tandems were developed for one-step preparative separation of components and screening of individual antioxidants from the tea water extractFirstly, according to the different separation mechanism between the molecular exclusion chromatography and reversed-phase chromatography, a new two-dimensional medium pressure liquid×preparative HPLC (2D-MPLC x prep-HPLC) was constructed. The Sephadex LH-20medium-pressure column was applied as the first dimension and the C18preparative column as the second one. One-step preparative separation of18compounds,16of which were identified by MS,1H-NMR and even by less-sensitive13C-NMR, could be achieved using this system, exhibiting great advantages in analytical efficiency.Secondly, on the basis of a switching interface with a switching valve as well as two equivalent holding columns, an on-line two-dimensional chromatography coupling of counter-current chromatography and high-performance liquid chromatography (2D CCC×prep-HPLC) was developed. The CCC acted as the first dimensional isolation column with an aqueous two-phase solvent system of isopropanol and16%sodium chloride aqueous solution(1:1.2, v/v), and a preparative column packed with C18had been employed as the second dimensional separation for effluents pre-fractionated. Under the optimum separation conditions, the present integrated system was successfully applied in a one-step preparative separation of11pure compounds from the water extract of tea.9compounds were identified by NMR and MS. The results demonstrated that the on-line2D CCC×LC method was very efficient for the preparative separation of the ingredients from the tea water extract.Finally, a fast and efficient online HPLC-FRAP equipment system for screening of antioxidants from the six kinds tea water extract was developed. Based on the known compounds which were achieved by the above two-dimensional chromatography,8antioxidants were identified from tea the water extract of Tie Guan Yin, of which catechins (EC) and epigallocatechin gallate (EGCG) were the main antioxidants.