High speed countercurrent chromatography (HSCCC) was successfully used for the separation and preparation of isomeric compounds. The influence of various two-phase solvent systems on the resolution of the natural catechin isomers of (-) -epigallocatechin gallate (EGCG) and (-) -gallocatechin gallate (GCG) from green tea polyphenols and the synthetic isomeric compounds of para-, ortho-, meta-bromoanilline was investigated. The results indicated that the catechin isomers of (-) -EGCG and (-) -GCG can be isolated through two runs of preparative HSCCC with two-phase solvent system composed of n-hexane-ethyl acetate-water (1: 10: 10 in volume ratio). The upper organic phase was used as stationary phase and the lower aqueous phase as mobile phase, while the mobile phase was operated at a flow rate of 3.5 mL/min, and the apparatus rotated at 800 r/min. High performance liquid chromatographic analysis of (-) -EGCG and (-) -GCG revealed that the purity was all over 98%. The isomeric compounds of para-, ortho-, meta-bromoaniline can also be effectively isolated through a single run of preparative HSCCC with the mobile phase flow rate of 3.0 mL/min and the apparatus rotation speed of 800 r/min by using tetrachloromethane-chloroform-methanol-water (7: 3: 7: 3 in volume ratio) as two-phase solvent system, while the lower phase was selected as stationary phase and the upper phase as mobile phase.