Pyridoxine, pyridoxamine and pyridoxal were separated quantitatively from each other by gel filtration chromatography on Sephadex LH-20 (1.35×195 cm) using 5% propanol in 10 mM potassium phosphate buffer (pH 6.6). Their phosphates were also separated quantitatively by Sephadex G-15 column (1.35×195 cm) and 0.1M potassium phosphate buffer (pH 6.6). Each column and fraction collector was shielded from light by covering them with black paper. The procedures were proposed as methods for the quantitative determination of various types of vitamin B_6 compounds.