Determination of Ascorbic Acid in Presence of Interfering Substances by the ‘ Continuous-Flow’ Method

been concerned in a study of the nutritional value of dehydrated foods.These, it was found, under exceptional conditions of preparation, namely, if exposed to unduly high temperatures during either processing or storage, were liable to become caramelized with the consequent production of the reductone-like substances.'I'hus a method for differentiating between the ascorbic acid and the interfering substances in them became necessary.The only quantitative test hitherto available was one depending on the different rates of condensation with formaldehyde (Lugg, 1942; Mapson, 1943 b).This method, however, had the disadvantage of being time-consuming and relatively laborious.It was felt, moreover, that the results in the formaldehyde test would gain in conviction if they could be shown to agree with those obtained by a second method based on an entirely different principle.The other author had previously been interested in the possibility of applying to this problem of differentiation between ascorbic acid and interfering substances the principle of the 'continuous-flow ' apparatus.This apparatus had been first devised by Hartridge & Koughton (1923) for their physico-chemical investigations on haemoglobin, and provides a means whereby the rate of very rapid chemical changes may be accurately measured.It will be recalled that it was used over I o years ago by Millikan (1935) for obtaining presumptive evidence of the identity with ascorbic acid of the reducing substances present in the wall of the gut (Hopkins & Slater, 1935); he did not, however, apply it to the actual estimation of ascorbic acid or its differentiation from other substances, which is the problem with which we have been concerned.We undertook, therefore, a somewhat detailed study of the rates of reaction of ascorbic acid and other reducing substances, as influenced by various changes in the experimental conditions (such as pH, concentration of reactants, solvent, temperature, etc.), using the continuous-flow apparatus for the purpose.As a result we feel able to recommend a reliable and convenient technique which may be used, first, to detect whether or not the interfering substances are present, and secondly, if so, to make a quantitative estimation both of them and of the ascorbic acid.It will be shown that biological tests confirm the accuracy of this new method, and that the results agree also with those obtained by the formaldehyde method. I. GENERAL ACCOUNT OF PROCEDURE AND FINDINGSFor simplification of presentation, a brief outline will first be given of the analytical procedure which was ultimately adopted, together with a short statement of some of the more important conclusions reached; the more detailed descriptions of the construction of the apparatus, of the technique of its use, and of the various theoretical studies, control tests and checks, can then follow more conveniently in later sections.The continuous-jbw apparatus.The equipment is shown in Fig. I.Of two exactly similar bottles, one ( A ) contains a solution of indophenol dye of standard strength (I ml.= 0.04 mg. of ascorbic acid), and the other (B) an extract of the ' unknown' made up at such a dilution as to be precisely equivalent in its total reducing capacity to the strength of the dye.The degree of dilution necessary is ascertained by means of a preliminary measurement of the total reducing titre, determined photo-electrically