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pH-Zone-refining counter-current chromatography (PZRCCC) was successfully used for preparative separation isoquinoline alkaloids from Phellodendri Cortex. Two target compounds of berberine and palmatine were completely resolved by a two-phase solvent system chloroform/methanol/water (4:2:2, v/v), with 10 mmol L-1 HCl in upper stationary phase and 10 mmol L-1 TEA in lower organic phase. From 2.0 g of the crude alkaloids, palmatine (448 mg, 95.1%) and berberine (159 mg, 95.4%) were obtained in one-step PZRCCC separation. The chemical structures were identified by ESI-MS and NMR data. The results indicated that PZRCCC separation is an excellent preparative separation method for isoquinoline alkaloids from Phellodendri Cortex.