Separation and purification of polyphenols from the extract of hawthorn (Crataegus laevigata) was investigated with high-speed counter-current chromatography (HSCCC). Solvent systems were examined according to partition coefficients of target components and n-butanol-ethyl acetate-water (1:1:2, v/v/v) system was chosen. The critical experimental parameters of HSCCC were optimized and determined with response surface methodology (RSM) as following: Revolution speed was 850 rpm, flow-rate was 1.5 mL/min and temperature was 25 degrees C. Under these conditions, 500 mg of crude extract was introduced into the HSCCC system and a product of 9.7 mg with purity over 94.8% was obtained in 260 min, which was identified as chlorogenic acid with UV, LC-MS and H-1 NMR. The result proved that RSM is an efficient method to optimize HSCCC conditions, and HSCCC could be a very effective approach to separate and purify chlorogenic acid from natural source. (C) 2010 Elsevier B.V. All rights reserved.