Spectrophotometric measurements on ascorbic acid and their use for the estimation of ascorbic acid and dehydroascorbic acid in plant tissues

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Abstract
L-Ascorbic acid at pH 6.8 or over has emax. (265 m[mu]) of 1.65-1.655 x 104. This value agrees with that of Lawendel (1956) but an effect of sorbitol reported by him could not be confirmed. At pH between 1.0 and 2.0, in metaphosphoric acid or in hydrochloric acid, [epsilon] max (244 m[mu]) is 1.05 x 104. This value is about 15% lower than that reported by Lawendel. An isosbestic point which is independent of pH occurs at 250 m[mu] where [epsilon] is 8.85 x 103. Light-extinctions obey Beer''s law at 265 and 245 m[mu] for concentrations of ascorbic acid up to 35 [mu]g/ml in buffered solutions of pH 6.8 or above and pH 1-2 respectively, and at 250 m[mu] regardless of these pH limits. A spectrophotometric method for the estimation of small amounts of ascorbic acid and dehydroascorbic acid in extracts of leal tissues is described. Satisfactory agreement was obtained between (a) indophenol titrations and (b) spectrophotometric changes resulting from oxidation by cucumber oxidase, and subsequent reduction of dehydroascorbic acid by either homocysteine or hydrogen sulphide. Good recoveries of ascorbic acid and dehydroascorbic acid were obtained when small amounts were added to extracts after grinding. When such additions were made before extraction, losses of ascorbic acid up to 35% occurred and these were recovered as dehydroascorbic acid. The conversion of ascorbic acid into dehydroascorbic acid during extraction is greater for added ascorbic acid present in an extracellular location than for that present within tne cells.