AbstractThree novel pyridine derivatives were successfully separated from their partially purified synthetic mixtures by pH‐zone‐refining counter‐current chromatography using a multilayer coil planet centrifuge designed in our laboratory. After equilibration of a two‐phase solvent system consisting of methyl tert‐butyl ether–tetrahydrofuran–water at an optimized volume ratio of 4:6:7, triethylamine (10 mM) was added to the organic phase as a retainer and hydrochloric acid (10 mM) to the aqueous phase as an eluter. Separation runs of 1.20, 1.16, and 1.15 g of three samples yielded three pyridine derivatives in weights of 306, 255, and 314 mg at a high purity of over 98.5, 99.0, and 98.2% (determined by HPLC), respectively. The structures were identified by electron impact mass spectrometry and 1H NMR.