Separation of Folic Acid Compounds by Gel Chromatography on Sephadex G-15 and G-25

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 10-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 2-mercaptoethanol gives a satisfactory separation of pteroylpolyglutamates containing 2 to 7 glutamic acid residues.