Fundamental studies on the separation of pyridoxal-5-phosphate by thin layer chromatography and by cellulose ion exchanger column chromatography.

Abstract

As the fundamental studies on the quantitative determination of pyridoxal phosphate, its separation from other B6 analogues was investigated by using thin layer chromatography and cellulose ion exchanger column chromatography and the following findings were obtained.1. The fluorescence and the Gibbs coloration of pyridoxal phosphate, pyridoxine, pyridoxal, pyridoxamine, pyridoxamine phosphate and pyridoxine phosphate on the thin layer were individually different according to the kinds of thin layer adsorbents.2. The limit concentration for detection of pyridoxal phosphate and other B6 analogues on the thin layer was about 10-4M. In the case of real development, the limit concentration of pyridoxal phosphate may be greater than 10-3M.3. Dioxane-water (7:3) was found to be the suitable solvent for separating pyridoxal phosphate from other B6 analogues on the cellulose thin layer. But if the reproducibility of Rf value is exactly requested, the rigid control of the experimental condition is needed.4. The recovery of the pyridoxal phosphate spotted on cellulose thin layer was 96.9%. Thin lager chromatography is thought to be the method to be tried once for separation of pyridoxal phosphate from other B6 analogues. But when its concentration is less than 10-4M, concentrating operation of the sample seems to be necessary.5. DEAF-, TEAS-, SM- and SE-cellulose ion exchanger were preliminaly investigated for the separation of pyridoxal phosphate from other B6 analogues. It was found that 0.01 N HCI was suitable for separation in the case of DEAE cellulose column, 0.01M acetate buffer (pH 4.4) in the case of TEAE-cellulose column, 0.01N acetic acid and 0.01M acetate buffer (pH 4.7) in the case of SM-cellulose column and 0.001M acetate buffer (pH 4.7) in the case of SE-cellulose column.6. The recovery of pyridoxal phosphate was 90 to 98% in the case of TEAE-cellulose column and 97 to 99% in the case of DEAF-cellulose column. Subsequently, ion exchanger column chromatography is considered to be able to separate pyridoxal phosphate from other B6 analogues.