High-performance centrifugal partition chromatography (HPCPC) has been successfully applied to the separation of four protoberberine quaternary alkaloids, namely palmatine, jatrorrhizine, columbamine and pseudocolumbamine, from a methanolic extract (M1, 1.47 g) of Enantia chlorantha Oliver stem bark. For their isolation, two successive biphasic solvent systems composed of dichloromethane-methanol-water (48:16:36, v/v) were selected. The aqueous-rich phase was the stationary phase and the organic-rich phase was the mobile phase. The first system, containing potassium perchlorate, allowed to isolate 600 mg of palmatine, and to obtain 146 mg of a mixture (M2) containing only jatrorrhizine, columbamine and pseudocolumbamine. The second biphasic system, prepared with water alkalinized with sodium hydroxide, was employed to isolate the M2 components. This system applied to the purification of 70 mg of M2 allowed to obtain 16 mg ofjatrorrhizine and 13 mg of columbamine. To obtain pseudocolumbamine (16 mg), the elution mode was reversed, the aqueous-rich phase becoming the mobile phase, and the organic-rich phase becoming the stationary one. Analytical reversed-phase high-performance liquid chromatography, NMR, high-resolution mass spectrometry and UV spectrometry were used to verify the identity and the purity of the isolated compounds.