Preparative separation of a challenging anthocyanin from Lycium ruthenicum Murr. by two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

The preparative separation of anthocyanins by HPLC often suffers from insufficient separation selectivity. In this work, a two-dimensional liquid (LC-LC) method was established to efficiently purify a challenging anthocyanin in Lycium ruthenicum Murray. Reversed phase liquid chromatography (RPLC) was used in the first-dimension preparation to fractionate the sample for its high separation efficiency. After the optimization of second-dimension methods, hydrophilic interaction chromatography (HILIC) was applied to further isolate the anthocyanin for the good orthogonality to RPLC. To improve HILIC separation for anthocyanins, stationary phases and mobile phases were investigated systematically. A satisfactory result was obtained on a zwitterionic Click XIon column with 1% phosphoric acid as an acidic additive. Using the above method, the anthocyanin and three new alkaloids were isolated from L. ruthenicum for the first time. This RPLC/HILIC method solved the coelution problem of anthocyanin and basic non-anthocyanins in one-dimensional HPLC, benefiting from the significantly improved separation resolution.