The Micro Determination of Citric Acid by the Thunberg Methylene Blue Method.

Abstract

This method 1 is based on the capacity of an enzyme found in cucumber seed to liberate hydrogen from citric acid for the reduction of methylene blue to the leuco compound. The speed of decolorization varies with the amount of citrate up to a certain concentration. Beyond this “critical” concentration there is no further decrease in decolorization time.

The least amount of citrate necessary for maximum speed of decolorization is influenced by the nature of the enzyme extract; this depends on the source of the cucumber seed, the amount of grinding and centrifuging in its preparation, the H ion concentration (during extraction and the reaction proper) and on the time the extract stands before use. Enzyme extracts less than 40 minutes old rapidly decolorize Me-blue even in the absence of citrate; with increasing age of the extract the decolorization time is greatly prolonged unless citrate is present. In practice enzyme extracts about 2 1/2 hours old have proved most satisfactory in revealing the “critical” citrate concentration. Of 20 determinations made on standard freshly prepared citrate solutions 15 were within 6% of the average. The other 5 varied more widely perhaps because of slight irregularities in the preparation of the enzyme extract. Adams and Boothby 2 suggested extraction of the seed with water instead of with 0.87% K₂HPO₄ at pH 8.2 because water extracts contain less extraneous substances which have an accelerating effect like that of citrate. The advantage gained by this procedure is somewhat offset by the higher “critical” concentration of citrate necessary, and by the absence of buffer, other than that supplied by the seed.

The sensitivity of the method is directly proportional to the amount of methylene blue present in the reaction tube. Large amounts of methylene blue (0.3 cc. of 1:30,000 solution) permit accurate reading; small amounts (1:120,000) are difficult to read but allow the estimation of as small amounts of citrate as 0.2 mg. per 100 cc.