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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
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.

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

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

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

Colorimetric Determination of Ascorbic Acid

New Developments Concerning the Reaction with Diazotized 4-Methoxy-2-Nitroaniline
Hoffmann-La Roche Inc,,


Nutey, N. 1.

COLORIMETRIC method for the determination of ascorbic acid has been previously reported by three of the authors ( 4 :. This involved reaction of diazotized Pmethoxj--2-nitroaniline with ascorbic acid in acid medium followed by addition of nlkali to yield an intenselj- blue color. By comparison, only colorless or light yellow reaction products had been described by Erlbach ( 1 ) and Weidenhagen and Wegner ( 5 ) after studying the rmction between ascorbic acid and benzenediazonium salts. The blue compound from ascorbic acid with a nitroeubstituted benzenediazonium salt n-as therefore investigated.

(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 number of knoa-n to produce a deep red color with alkali ( 2 ) . -4 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? <'--?~---XH S H D - O C B Xo. 8 was prepared by refluxing the hydrazine with acetic anhydride. I n contrast to the ci +K&>>-OC'H3 -+ 0" monoacylated derivatives, i t developed the '\ maximum blue color only upon treatment C"-COH CH---('=O with excess alkali, which seems t o indicate that HOC" (I1 IIOCH i hydrolysis of the acetyl group in position R2 has I to precede the formation of color. CHy-0


(111) (Compound 2 , Table I )


Jc2Sa (IV) Compound 4a. Table I )



I n a previous paper (4) a number of modifications of the basic method were described in order to eliminate certain interferences.

1522 Table I. Related Nitrophenylhydrazines





1 2

R 2-NO2 2-NO2 2-NO2 2-NO2

H H 4-CHa0 4-CHaO H H H H


Ra COCO-L-threonyl COH

Formula CsHiOsNs CizHiiOsNa

4 4a 5


: E: C,

Analyses, 3 Calcd. Found C, 42.67 c , 43.01 H, 3.13 H, 3.45 C, 44.31 C, 44.40 H, 3 . 1 3 1; ; : N, 1 2 . 8 1 C, 45.50 C, 4 6 , l 5
1 ;; :

M.P., C.
152-153 176-177 171-172 177 116-116 75

Color in Alkali Purple Purple Blue Blue Blue Blue Blue

Disodium salt of cmpd. 4 2-iYOz 4-CHa0 H 2-NO8 4-CHaO H

COCOzCzHr COCO-L-threonyl

Cd3706NaNaz CiiHisOeNa CiaHnOsNa

H, Na, C, H, N, C, H,

39.56 4.06 15.37 46.64 4.63 14.84 43.95 3.69 45.77 3.84 49.43 4.90


:c, :
H, H,

240::: 39.96 H, 4.37 Na, 1 4 . 9 3 C, 46.52


43.43 3.65 45.79 3.54



c, c,




4-CHaO 2-CHsO 2-CHsO 2-CHaO 2-CH30



CiiHiaOsNa CaH90rNa CaHoOeNa CisHraOsNs CsHsOrNa

9 10
11 12

5-NO2 &NO2



N: 1 5 . 7 3



4:: ; H , 3.56 N , 11.83 c. 4 5 , 5 0

H, 4.70 N , 15.89 C, 45.55 H, 4.10 C, 42.28 H, 3.64 N, 11.24 C, 45.21 H, 4.46 N, 20.26

C, 49.86

101-102 214-215 170-171 93-94 199.5-201

Blue Light yellow Light yellow

Light yellow Deep orange

An additional procedure has been developed which offers good possibilities for the determination of ascorbic acid in media where the previously reported techniques could not be successfully applied. In this procedure n-butyl alcohol is substitut,ed for ethyl alcohol or isopropyl alcohol since this alcohol is miscible with the aqueous sample solution in the ratios used. Moreover, a t the point of extraction a separation of the n-butyl alcohol from the aqueous phase can be accomplished by the addition of ethyl ether.


When samples of milk or powdered milk, and fortified animal feed were tested, either no color or only rapidly fading colors could be produced with the original procedure or its modifications. However, when the above described technique was applied to these samples (Table 11),relatively stable colors could be obtained.

Table 11. Determination of Ascorbic Acid

Sample Results Powdered milk (enriched with 0.4 mg. of ascorbic A B acid per gram) C Fortified animal feed (enriched with 0.1 mg. of D ascorbic acid per gram) Found, ?Ig. Ascorbic Aoid/Gram 0.38 0.39 0.41 0.095

Procedure. Two milliliters of amino reagent ( 4 ) are pipetted into a 250-ml. glass-stoppered Erlenmeyer flask, followed by addition of 2 ml. of nitrite reagent. The solution i s swirled to the disappearance of the orange color of the amino reagent, 75 ml. of n-butyl alcohol are added, and the contents of the flask are mixed, A sample in solution or finely divided form, adjusted to contain approximately 0.5 t o 2 mg. of ascorbic acid, is added. Samples in solution should be dissolved in a 0.5% solution of aqueous oxalic acid while in the case of a solid, 5 ml. of 0.5% oxalic acid solution are added to the mixture, which should then be thoroughly shaken for several minutes. The mixture is transferred (filtering, if necessary) to a 500-ml. separator. Twenty-five milliliters of 10% sodium hydroxide are added, followed b y about 150 ml. of ethyl ether. The contents are well shaken and the layers are allowed to separate. The bottom layer is drawn off into a 200-ml. volumetric flask. The solvent in the separator is Fashed three times with 15-ml. ortions of 10% sodium hydroxide, and the washings are a d d e i to the volumetric flask which is brought to volume with water. TWO standards and a blank are prepared as described in the previous publication ( 4 ) , and treated in the same manner as described above for the sample. The colors are compared a t a wave length of 570 mF, setting the spectrophotometer or colorimeter a t zero absorbancy with the blank.

The authors have not thoroughly investigated the application of this method for the determination of ascorbic acid in biological fluids. The following preliminary experiments suggest that a more sensitive procedure, based on this method, might be developed for biological fluids, where ascorbic acid levels of a very small order can be expected. Urine. The method as originally described was applied to a sample of urine, where 1 mg. of ascorbic acid was added to 5 ml. of urine and quantitative recovery was obtained, using the iodine blank technique ( 4 ) . Blood Serum. One milligram of ascorbic acid was added to 2 ml. of blood serum and quantitatively recovered, utilizing the above described butyl alcohol extraction procedure.

The authors are indebted to 8 1 Steyermark and his staff of this organization for performing the elementary microanalyses, and to E. G. E. Shafer for helpful suggestions.

(1) Erlbaoh, H., Ber., 68, 534 (1935). Hyde, E., Ibid., 32, 1811 (1899). hlichaelis, h., Ann., 252,266 (1889). (4) Schmall, &I., Pifer, C. W., and Wollish, E. G., ANAL.CHEM.,25, 1486 (1953). (5) Weidenhagen, R., and Wegner, H., Ber., 72, 2010 (1939).
(2) (3)
RECEIVED review March 9 , 1954. Accepted M a y 28, 1954. for