Retention behavior of pteroyl-oligo-γ-l-glutamates in reversed-phase chromatography

The effect of eluent pH on the retention of pteroyl-oligo-γ-l-glutamates containing up to eight glutamyl residues is investigated in reversed-phase chromatography with octadecyl-silica column. When the carboxylic groups of the solutes are largely undissociated, at pH 2, the retention of oligoglutamates increases with the number of glutamyl residues and the elution order parallels that in anion-exchange chromatography. At sufficiently high eluent pH the carboxylic groups are dissociated and the elution order is reversed so that solute molecules having smaller number of charges. i.e. less glutamyl residues, are retained stronger. The logarithm of capacity factor, with the exception of folic acid, is linearly dependent on the number of glutamyl residues over a wide range of eluent pH. The dependence of the capacity factor on pH for oligoglutamates is quantitatively interpreted considering the different dissociation constants for the α- and γ-carboxyls. The results suggest that in reversed-phase chromatography the selectivity of separation for polyionogenic compounds can be drastically modulated by changing the pH of the eluent.