Thin-Layer Chromatographic Separation of Isomers of Ascorbic Acid and Dehydroascorbic Acid as Sodium Borate Complexes on Silica Gel and Cellulose Plates

Abstract

A thin-layer chromatography (TLC) method is described for isomers of ascorbic acid (AA) and their oxidation product, dehydroascorbic acid (DHAA), on sodium borate impregnated (B) silica gel and cellulose plates. Borate complexes of AA and DHAA could be separated from each other, with AA migrating further. For comparative purposes, TLC was also attempted using direct (D), reversed-phase (RP), and reversed-phase sodium borate (RP-B) TLC plates with and without metaphosphoric acid (MPA). A reasonable separation of the three AA isomers is achieved on D and RP cellulose plates. Using an RP−silica−MPA plate the three AA isomers as well as the three DHAA isomers are fairly separated from their own group members. The separation of six stereoisomers, three AA isomers, and three DHAA is achieved using a D-cellulose−MPA plate. This procedure has been adopted to separate and identify AA and its oxidation product DHAA in food products, pharmaceutical preparations, and biological tissues and fluids. Keywords: Ascorbic acid; borate complexes; metaphosphoric acid

Production of o-diphenols by immobilized mushroom tyrosinase

“…Concerning ascorbic acid. AH 2 in the presence of borate forms a complex (step 12) (Obi et al, 1998;Roomi and Tsao, 1998;Tsao and Young, 1996) and only a small free fraction remains in the medium to act as reductant. Therefore, borate permits a reserve of AH 2 throughout all the process, while its free concentration is insufficient for it to act as substrate of the enzyme.…”

Hydrophilic Vitamins

“…Thin-layer chromatography has been widely used to determine ascorbic acid concentrations in foodstuffs (41)(42)(43), pharmaceutical preparations (43)(44)(45), and biological materials (43,46,47). Mushrooms contain reducing substances with chemical properties similar to those of ascorbic acid, and osazones were formed from the reducing substances in 19 kinds of edible mushrooms (42).…”

“…A TLC method has been described for isomers of ascorbic acid and their oxidation product, dehydroascorbic acid, on sodium borate-impregnated silica gel and cellulose plates (Table 10) (43). This procedure has been adopted to separate and identify ascorbic acid and dehydroascorbic acid in fresh orange and lime juices, pharmaceutical preparations (ascorbic acid), and guinea pig tissues (liver, kidney, and eye lens) and fluids (plasma and urine).…”