Abstract
This chapter focuses on the analysis of ascorbic acid, dehydroascorbic acid, and transformation products using ion-pairing high-performance liquid chromatography with multiwavelength ultraviolet and electrochemical detection. Preparative separation of transformation products is carried out by DEAE-Sepharose column chromatography. Elution is carried out with 10 m M sodium phosphate buffer, pH 6.0. The eluates are examined by absorption spectrum and electrochemical detection–high-performance liquid chromatography (ECD–HPLC) analysis. L -ascorbic acid (AsA), erythro- L -ascorbic acid (EAsA), and the reductant R-345, possess an absorption maximum at 345 nm. Analysis by ECD–HPLC reveals three main ECD-active spots—AsA, EAsA, and a reductant possessing an absorption maximum at 290 nm. The latter reductant has a retention time a little less than that of AsA, and it is eluted in the void volume on a DEAE-Sepharose column, showing a less negative charge. Its absorption maximum at 290 nm in neutral aqueous solution shifts to 335 nm above pH 9.0.
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