Thin-layer chromatography of vitamin B12 and its analogues

Abstract

The separation of vitamin B12 from its analogues is difficult because they are chemically closely related to cyanocobalamin. On the grounds of our experiments we suggested a new method for the separation of corphyrins on dry alumina (basic, second degree of activity according toBrockmann). As solvent system the mixture of isobutanol, isopropanol and water (1 : 1 : 1) was used. The separation with good compact spots was obtained of the following corphyrins: cyanocobalamin (vitamin B12), factor B12 III, pseudovitamin B12, factor A, factor B and factor V (nB). A new simple and rapid method for separation of hydroxocobalamin and cyanocobalamin on thin layer of dry alumina (neutral, second degree of activity according toBrockmann) was reported by us. Advantage was taken of the property of hydroxocobalamin to form with NH4 OH a compound of the cobalichrome type. Under these conditions cyanocobalamin does not change so that both compounds can be easily separated. The best separation was obtained by mixture of isobutanol, isopropanol and water (1.5 : 1 : 1.25) as solvent system (with addition of NH4 OH until the ph was 8.5). On the grounds of the results from attempts on the separation of cyanocobalamin from its analogues we reported a method for quantitative determination of vitamin B12 in microbial fermentations, commercial preparations, and concentrates. The method involved the precipitation of vitamin B12 and other analogues (accompanying it) as coper-cyanide complexes, the decomposition of complexes with addition of NaCN-solution to obtain the dicyanocomplexes, the separation of cyanocobalamin on thin layer of dry alumina and its colorimetric determination to 361 nm.