Paper chromatographic estimation of L-ascorbic and D-araboascorbic acids.

Abstract

1. Paper chromatographic separation (ascending method) of the mixture of L-ascorbic and D-araboascorbic acids was carried out by using the papers impregnated with 3% metaphosphoric acid and water-saturated methyl ethyl ketone as a solvent system. 6 to 40 μg per spot of each isomer could be clearly distinguished by spraying 0.013% indophenol solution, the difference in RF values for both acids being close to 0.1. Developing temperature of 20 to 25° and developing time of 5 to 5.5 hours were recommended for the separation.2. Effects of moisture and partition liquids in the stationary phase were also studied. By increasing the concentration of glycerol in 3% metaphosphoric acid, the locations of both the isomers were varied without change of separating ratio. By using this procedure, the stability of the RF values for the acids was increased. This technique may be effective for separating the substances from natural products which contain some impurities having the same RF values as these acids.3. Influences of some carbohydrates and their derivatives on the separation of the isomers were studied at various temperatures. L-Ascorbic and D-araboascorbic acids were shown to be completely separated from carbohydrates even if the isomers existed in the dehydro-forms.4. After separation, the isomers were estimated by 2, 4-dinitrophenyl hydrazine method. Experimental results were treated by analysis of linear regression and 95% fiducial limits of regression lines were determined. 95% fiducial limits of regression lines were ±3 to 4μg for both compounds at a range of 10 to 50μg and ±2 to 3μg for 4 to 10μg.