A new consecutive high-performance counter-current chromatography approach based on co-current and overlap mode for...

Conventional consecutive separation methods based on overlapping injection modes produce interactions between samples as the separation time increases. This will lead to a reduction in the purity of the target due to the gradual saturation of the stationary phase distribution. To enhance the purity of the sample preparation and separation efficiency, we devised a consecutive separation method under high-performance counter-current chromatography (HPCCC) utilizing co-current elution and overlapping injection. The method was used for the separation and preparation of olive leaves for their main bioactive polyphenolic constituents. Six HPCCC separations were conducted in reversed-phase mode using a two-phase solvent system consisting of petroleum ether/ethyl acetate/water (1:40:40, v/v). Finally, 302.7 mg of luteolin-4'-O-β-D-glucoside and oleuropein were isolated from 720 mg of crude sample with purities of 93.3% and 96.2%, respectively. The optimization strategy outlined in this study can be used to enhance the extraction of bioactive components from olive leaves.