pH-gradient counter-current chromatography isolation of natural antioxidant chlorogenic acid from Lonicera japonica Thumb. using an upright coil planet centrifuge with three multi-layer coils connected in series

A new pH-gradient counter-current chromatography method for the isolation of chlorogenic acid from flowers and buds of Lonicera japonica Thumb. has been successfully established using a novel upright coil planet centrifuge with three multi-layer coils connected in series with 600 mL capacity. The crude extracts were first prepared by direct extraction with hot water and following concentration to remove the solution. Then the two-phase solvent system composed of ethyl acetate–n-butanol–water (2:1:3, v/v) was applied to the separation. Its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and base lower phase with 10 mM NH3 were employed as mobile phase with gradient elution in the head to tail mode. As a result, 330 mg quantity of crude extract was purified in one-step separation for 180 min, yielding 20.5 mg chlorogenic acid with over 98% purity. Structure of the compound is further identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR).