HPLC Separation of Ascorbic and Dehydroascorbic Acids

Ascorbic acid is an essential vitamin for humans and also functions as a potent antioxidant. Its oxidized form, dehydroascorbic acid, is similarly important. Due to their high polarity, both ascorbic acid and dehydroascorbic acid are not effectively retained by traditional reverse-phase HPLC methods.

A robust and generic HPLC method has been developed for the separation of these two compounds using a Zodiac HST SB mixed-mode column. This approach provides excellent resolution and peak shape for both analytes. The method is well-suited for analyzing ascorbic acid and dehydroascorbic acid in biological fluids.

Proteins present in such samples are not retained on the column due to the repulsive interactions with the stationary phase, allowing for cleaner analysis. Detection can be performed using standard techniques such as UV, ELSD, CAD, or LC/MS.