AN IMPROVED METHOD FOR VITAMIN C ASSAY(MODIFICATIONS OF THE ROE-KUETHER'S DINITROPHENYL-HYDRAZINE COLORIMETRIC METHOD)
- VernacularTitle:抗坏血酸的定量法(Roe-Kuether二硝基苯肼比色法的改良法)
- Author:
Chengchih SU
;
Yuansi KU
- Publication Type:Journal Article
- From:
Acta Nutrimenta Sinica
1956;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Instead of using active carbon as oxidizing agent as described in the briginal Roe-Kuether method, the present authors adapted O2 and ascorbic acid 6xida.sc to oxidize ascorbic acid. The interfering substances such as reductone and sugars are thus eliminated and the specificity of the Roe-Kuether method for ascorbic acid is, therefore, highly increased, and the time for the determination is shortened. The details of the working conditions have been worked out. The modified procedure may be briefly outlined as follows:Extraction: To x gm of sample add 4x ml of 5% metaphosphoric acid. Grind with sand, add 5x ml of water, stir and centrifuge. Take the supernatant clear layer for analysis, its pH is about 1.7 and the concentration of metaphosphoric acid is about 2%. In case of a liquid sample, both the metaphosphoric acid and water may be added directly to the [sample solution before centrifugation.Oxidation: Delivery 2.5 ml aliquot of the extract to a test tube graduated at 10 ml. Place the tube in a water bath at 37℃, and pass O2 through a capillary tube into the test solution for 15 minutes (about 9000 ml O2 for 15 minutes). Remove the tube from the warm water and adjust the pH of the extract to 6 with 2N NaOH (0.13ml). Add 0.1 ml of ascorbic acid oxidase solution and again pass O2 for another 5 minutes. Readjust the pH of the test solution to its original value (about 1.7) by adding 0.13 ml of IN HCl and finally add 6.% trichloroacetic acid to the 10 ml mark. Through this treatment, the ascorbic acid is quantitatively converted into dehydroascorbic acid and the interfering substances are completely removed.Color Comparison: To each of 2 colorimetric tubes add 4 ml aliquot of the oxidized extract and 2 drops of 10% thiourea. After thorough mixing, set one tube to serve as a blank, and to the other add 1 ml of 2% 2,4-dinitrophenylhy-drazine solution. Mix and incubate the latter tube at 37℃ for exactly 3 hours. Place both tubes in ice water and to each add slowly 5 ml of 85% H7SO4 (the addition should take 1-2 minutes). The ascorbic acid content of the unknown solution (mg/100 ml) is determined by interpolation of the optical density values on a standard curve prepared by a series of gradient ascorbic acid solutions.
