ABSTRACT
In this work, a new online preparative high-performance liquid chromatography was developed for the fast and efficient separation of complex chemical mixtures from natural products. This system integrates two chromatographic systems into an online automatic separation system using the technique of multiple trap columns with valve switching. The sample was first separated into 18 subfractions in the online preparative high-performance liquid chromatography, and the sample eluents were then diluted and captured online on 18 trap columns by the multiple trap columns technique, respectively. Each subfraction retained on the trap column was transferred online to the separation column for the second separation. Finally, the target compounds were purified by appropriate separation conditions and multiple heart-cutting strategies. Importantly, the system was successfully used to separate 18 high-purity flavonoids from the crude extract of Epimedium koreanum Nakai online in one step. The entire separation time was approximately 20 h, and the structures were characterized by the high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and nuclear magnetic resonance. This online preparative high-performance liquid chromatography system represents an efficient and rapid separation system that has the potential for a wide array of applications in the separation of complex chemical components from natural products.