Geranium wilfordii Maxim (Chinese name “laoguancao”), is a popular traditional Chinese medicinal plant used in the therapy of rheumatism, osteoporosis, and diarrhoea. Corilagin and Geraniin, though regarded as important polyphenols components in traditional Chinese medicine (TCM), are often difficult to separate from crude extracts. Recently adsorption chromatography using cross-linked 12% agarose gel, tentatively considered to be based on hydrogen bond retardation, was introduced for the separation and puri？cation of compounds from natural products.

Researchers with Institute of Process Engineering (IPE) established high-speed counter-current chromatography using a new liquid–liquid/solid three-phase system for the separation of the polyphenols corilagin and geraniin from a crude extract of Geranium wilfordii Maxim in one step.

In the experiments, the first step was to prepare standard and crude samples. Two milligram of each ？avonol aglycons and dihydroxybenzoic acid standards were dissolved in 5 mL mobile phase by sonication, respectively. Two mixtures were centrifuged before column injection. The crude extract powder (800 mg) was dissolved in 20mL 40% methanol. The HSCCC sample was prepared by diluting 2 mL of the crude extract solution with 3 mL of an equilibrated mixture of the aqueous and organic phases of the solvent system.

The next important step was the HSCCC separation. The selected solvent system was prepared by thoroughly mixing organic solvents, water, and the cross-linked 12% agarose microspheres. The two liquid phases were divided just before use and degassed by ultrasonication. The lower phase was used as stationary phase and was mixed by magnetic stirring. After establishing a hydrodynamic equilibrium in the column, the retention of the stationary phase of each solvent systemwas calculated followed by sample injection. Appropriate fractions were collected, vacuumevaporated, and analyzed by HPLC.

Experimental results showed that the optimized three-phase system was composed of n-hexane/ethyl acetate/methanol/acetic acid/water and to which was added 10-m average diameter microspheres of cross-linked 12% agarose at the ratio of 0.2:10:2:1:5 and 0.1 g/mL, respectively. The purities of geraniin and corilagin were 82 and 90%, which were determined by HPLC at 280 nm. A 14.5 and 7 mg of geraniin and corilagin were puri？ed from 160 mg crude extract with the yields of 70 and 78%, respectively.

A new liquid–liquid/solid three-phase system was established and successfully used to separation polyphenols by high-speed counter-current chromatography with acceptable purity and recovery, which indicates the potential of LLS three-phase system for separation of polyphenols with similar structures and adds the selection range of alternative solvent systems for HSCCC separation of natural products.

The paper was published in Journal of Separation Science.