Colorimetric Determination of Ascorbic Acid - ACS Publications

LITERATURE. CITED. (1; Anderson, D. H., personal communication. (2) Anderson, D. H., and Woodall, N. B., Anal. Chem., 25,1906. (1953). (3) Di Stefano,...
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V O L U M E 26, NO. 9, S E P T E M B E R 1 9 5 4

(2) Anderson, D. H., and Woodall, N. B., ANAL.CHEM.,25, 1906 (1953). (3) Di Stefano, V., Neuman, W. F., and Rouser, G., A r c h . Biochem. and B i o p h y s . , 47, No. 1, 218 (1953). (4) Schiedt, U. (reported by H. Hausdorff), A p p l . Spectroscopy, 7, NO.2, 75-84 (1953). (5) Schiedt, U., 2. Naturforsch., 8b, 66-70 (1953). (6) Schiedt, U., and Reinwein, H., Ibid., 7b, 270-7 (1952). (7) Stimson, AI. M., and O'Donnell, M. J., J . Am. Chem. Soc., 74, 1805 (1952).

comparison of the unknown with either galactoseamine phosphate or galactoseamine has not yet been possible, as neither of these substances was available in pure form. ACKNOWLEDGMENT

The authors Lvish to acknowledge valuable technical assistance from R. G. Smith and D. H. Anderson of the Spectroscopy Laboratory, Eastman Kodak Co.

RECEIVED for review April 2, 1954. .4ccepted June 3, 1954. Based on work performed under contract with the United States Atomic Energy Commission a t the University of Rochester Atomic Energy Project, Rochester, N.Y,

LITERATURE CITED (1

.-lnderson, D. H., personal communication.

Colorimetric Determination of Ascorbic Acid New Developments Concerning the Reaction with Diazotized 4-Methoxy-2-Nitroaniline MORTON SCHMALL, C. W. PIFER,

E. G. WOLLISH, ROBERT DUSCHINSKY, and HAROLD GAINER

Nutey, N. 1.

Hoffmann-La Roche Inc,,

A

(IV). With n-araboascorbic acid (isoascorbic acid) the corresponding L-erythronic acid lactone (111) is formed in an analogous manner, which upon alkaline hydrolysis yields the identical sodium salt (IV). When the absorbancies of the pure isolated dye and of the color produced by an equivalent of ascorbic acid subjected to the method described ( 4 ) were compared, it was found that the reaction proceeded with a yield of 90%. Considering the rather complicated mechanism, this yield is surprisingly high. The appearance of a negative charge a t the a-nitrogen atom is in accord with the known a-sodium salt of phenylhydrazine (3). IDEYTIFICATION O F BLUE COMPOUYD AND P R E P 4 R i T I O I Resonance forms such as ( V ) undoubtedly contribute to the OF 4YALOGS formation of an intense color. This form (V) also includes Two of the authors (Duechinsky and Gainer) identified the enolization in the group -NHCO- of (IV) which increases the final reaction product as the deep blue disodium salt of oxalic number of double bonds in conjugation. Compounds of analacid 4-methoxy-2-nitrophenylhydrazide (IV). The mechanism ogous type, such as l-(p-nitrophenyl)-2-acetylhydrazine are of formation of the oxalyl compound is still not completely knoa-n to produce a deep red color with alkali ( 2 ) . -4number of clarified. Whatever the mechanism may be, it appears that related acylated 0-, m-, and p-nitrophenylhydrazines which were ssvorbic acid (I) and the 4-methoxy-2-nitrobenzenediazonium prepared are given in Table I. For the production of an intensely cation (11) undergo an oxidation-reduction reaction, probably colored sodium salt, @-acylationas well as ortho- or para- nitraafter forming an adduct, while the substituted benzene diazonium tion, is essential. moiety forms a hydrazide. The ascorbic acid part suffers, in Compounds 1, 2, 4, 6, 10, and 11 were obtained from ascorbic addition to oxidation, an opening of the furan ring. The resultant acid and the requisite diazonium salt as indicated above. The product is the 4-methosy-2-nitrophenylhydrazideof the aformyl compounds 3, 9, and 12 were prepared either by formylaoxalate of D-threonic acid lactone (111). Further action of tion of the hydrazine or by refluxing of the corresponding oxalic alkali on (111)yields finally the blue disodium oxalate derivative acid monohydrazide in acetic or propionic acid. A mixture of compounds 5 and 7 was obtained when 2nitro-4-methoxy-phen~-lhydrazine was heated 0 0 !I I \ with ethyl oxalate. The diacetyl compound ('~--COH SO?