Thin-layer chromatographic analysis of hydrophilic vitamins in standards and from Helisoma trivolvis snails

Separations of the hydrophilic vitamins thiamine (B1), riboflavin (B2), niacin (B3), pyridoxine (B6), cobalamin (B12), ascorbic acid (C), and folic acid have been compared using 14 mobile phases and commercially available, precoated silica gel and chemically bonded silica gel thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) plates. The best separations of individual and mixed vitamin standards was achieved on silica gel plates with 1-butanol–chloroform– acetic acid–ammonia–water, 7:4:5:1:1, benzene–methanol–acetone–acetic acid, 70:20:5:5, and chloroform–ethanol–acetone–ammonia, 2:2:2:1, as mobile phases. Previously reported detection reagents selective for the hydrophilic vitamins were also tested and compared. To determine the usefulness of these chromatographic systems for analysis of biological samples, hydrophilic vitamins in Helisoma trivolvis snails (Pennsylvania strain) were identified and B2 was quantified by videodensitometry. Because of the amount of non-vitamin, water-soluble substances that gave fluorescence-quenched zones in the snail extracts analyzed, it was necessary to use natural color or fluorescence or a selective vitamin spray-reagent, in combination with RF values, to identify vitamins in biological samples.