High-speed counter-current chromatography (HSCCC) is a versatile technique in preparative separation and purification.of pure compounds from complex matrices. As a preparative chromatography, there is a need to maximize the column production. Based on the plate theory of Van Deernter, the effect of the sample load on the separation was investigated in a preparative HSCCC with a 1000 ml column capacity. The test samples of hydroquinone, pyrocatechol and phenol were separated using a two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (1: 1: 1: 1, v/v/v/v) at different sample loads. The results showed that for the case of HSCCC, the agreement of the effect of sample load on peak height and peak width between the Van Deermer's theory and the experiments is excellent. Furthermore, the factors limiting the mass load, including.the resolution between the peaks, the partition isotherm and the solute solubility were also discussed. (c) 2007 Elsevier B.V. All rights reserved.