Assay of L( + )-Ascorbic Acid in Buttermilk by Densitometric Transmittance Measurement of the Dehydroascorbic Acid Osazone

Abstract

Abstract A study is presented for the quantitative analysis of ascorbic acid in buttermilk samples, using densitometric transmittance measurements. The procedure is based on oxidation of ascorbic acid to dehydroascorbic acid, followed by reaction with 2,4-dinitrophenylhydrazine to form the dinitrosazone. The osazone is separated from interfering substances in the sample by thin layer chromatography (TLC) on silica gel G plates developed with chloroform-ethyl acetate-acetic acid (60+35+5) . The TLC plates are scanned with a flying-spot densitometer. The relationship between integrated signal and concentration is linear for 0.08—1.00 fig ascorbic acid (coefficient of variation 3—4%). Deviations from Beer's law start to appear at levels higher than 1.00 fig ascorbic acid. A mean coefficient of variation of 3.5±1.1% (P = 95%) was established for standard spot measurements (3 spots averaged) on 15 chromatoplates. Recovery of ascorbic acid added to buttermik was 98% (coefficient of variation 7.2%). Results of this study are compared with those reported for spectrophotometric, titrimetric, and potentiometric procedures. The proposed method is less accurate because of interferences and the subsequent variables which arise as a result of taking transmittance measurements through an opaque medium.