Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography-diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring.

Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC-DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate-ethanol-water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).