[57] Chromatography of folates on sephadex G-10

This chapter discusses the chromatography of folates on sephadex G-10. Several methods of column chromatography have been developed for the separation of folates. Different kinds of ion exchangers, gel-chromatographic materials, as well as carriers for affinity chromatography have been applied for separation purposes. After chromatographic separation, the folates in effluent can be detected in various ways. The spectrophotometric detection is limited by its relatively low sensitivity and can be disturbed by many naturally occurring substances. In chromatography of folates on sephadex G-10, the folates are separated on a sephadex G-10 column and detected spectrophotometrically or by microbiological assay with Lactobacillus casei. Alternative detection with some other testing strains, for example, Streptococcus faecalis or Pediococcus cerevisiae, is also possible for the differentiation of various forms of folates. The method is especially suitable for the separation of folic acid from other folates, and of dihydrofolic acid derivatives from their tetrahydrofolic forms. Comparison of the chromatographic patterns of samples, treated with and without conjugase, enables to distinguish free and bound forms of folates. The method described can also be used for the purification of the synthetic folates that usually contain other folate derivatives.