Abstract
This chapter describes the separation of pteroyl-oligo-γ-L-glutamates by high-performance liquid chromatography (HPLC). The introduction of suitable microparticulate columns, having high efficiency, has made rapid analysis of folate coenzymes by HPLC possible. HPLC offers excellent resolution, relatively high sensitivity, and quantitative recovery of components in addition to the speed of analysis, thus offering many advantages over the conventional methods of column chromatography and microbiological assay. Two analytical methods are described in this chapter for the separation of pteroyl-oligo-γ-L-glutamates. Both require a suitable liquid chromatograph equipped with gradient elution capability and appropriate detectors. In both cases, commercially available columns, packed with microparticulate bonded phases on siliceous support, are employed. The first method uses a strong anion-exchange column and gradient elution, with aqueous phosphate buffer, in order to separate oligoglutamates in the order of increasing number of glutamyl residues. In the second method, a C-18 reversed-phase column is used with gradient elution at increasing concentration of acetonitrile in the aqueous eluent. Under these conditions the elution order is reversed. Concomitant use of the two analytical methods facilitates the identification of the individual peaks on the basis of different quantitative structure-retention relationships.