An efficient method for the preparative separation and isolation of ginkgolic acids from the sarcotesta of Ginkgo biloba L by pH-zone-refining counter-current chromatography coupled with inner-recycling mode

An efficient method for the preparative separation and isolation of ginkgolic acids (GAs) from the sarcotesta of Ginkgo biloba L was developed by combining pH-zone-refining counter-current chromatography (PZRCCC) with inner-recycling mode. Two grams of GAs extract were separated by PZRCCC using a two-phase solvent system of n-heptane–ethyl acetate–methanol–water (2:1:3:1, v/v) with 10 mM HCl added to the upper phase as the retention acid and 10 mM triethylamine (TEA) in the lower phase as the elution base. In a one-step PZRCCC separation, two GA compounds, C15:1 and C17:2, were obtained with purities over 98% along with two mixtures. The mixtures were further separated by inner-recycling CCC mode with n-heptane–ethyl acetate–methanol–acetic acid (5:4:1:1, v/v) as the solvent system. Five main GAs were successfully separated with purities over 98%. The structures of the separated GAs were determined based on standard samples coupled with Electrospray Ionization Mass Spectrometry (ESI-MS) results. This separation method of coupling PZRCCC with inner-recycling CCC is effective for the preparation of GAs from the sarcotesta of G. biloba and could be further applied to separate organic acids of low polarity in other natural products.