Separation of Folk Acid Compounds by Gel Chromatography on Sephadex

The separation of folate compounds has been achieved by gel chromatography and the effect of buffer concentration on the elution pattern of folate derivatives was investigated by Sephadex G-15 and G-25 column chromatography. Highly conjugated polyglutamates appear in the effluent considerably sooner when eluted with water than would be expected from their molecular weights. Monoglutamates, except IO-formyltetrahydropteroylglutamic acid, are retarded on G-15 and G-25 in phosphate buffer (pH 7.0) solvent system; the situation becomes more apparent as the buffer concentration is increased. Chromatography on Sephadex G-25 with 0.1 M phosphate buffer, pH 7.0, containing 0.2 M E-mercaptoethanol gives a satisfactory separation of pteroylpolyglutamates containing 2 to 7 glutamic acid residues.