207
V O L U M E 19, NO. 3, M A R C H 1 9 4 7 surface film, approximately 10 ml. of alcohol are mixed with the suspension by running it in rapidly from a coarse buret tip. The tubes are centrifuged for 10 minutes a t 2000 r.p.m., after which the fluid can be decanted without loss of precipitate. Two water washings are performed by twice adding 10 to 15 ml. of water, centrifuging, and decanting. To each washed precipitate is then added 1 ml. of chromogenic reagent. The end of each tube is flipped with the finger to stir up the precipitate and the mixture is heated on the steam bath for 15 minutes. Without waiting for the tubes to cool, 4 ml. of saturated sodium carbonate solution are added and the blue color which results is measured photoelectrically. No effort is made to eliminate residual diatomaceous earth, since it was not found to interfere significantly with the colorimetry. There is no appreciable variation in color density for a t least 10 minutes and no appearance of turbidity when the mercurous chloride precipitate has been adequately washed. A color-concentration curve obtained using a Cenco Photelometer with its red filter is given in Figure 1. Unknown samples to which the mercuric chloride method can be directly applied are treated in the same manner as the standards and the formic acid concentration is determined by comparison of the color density measurement with the standard values. Formic acid in biological samples such as blood is preliminarily separated from interfering substances by protein precipitation and low-temperature vacuum distillation in the presence of a strong acid. I n the case of blood it was found satisfactory to centrifuge a mixture of 1 ml. of blood with 1 ml. of water and 1 ml. of 10% sulfosalicylic acid, then t o add approximately 0.02 ml. of sulfuric acid to the separated supernatant and to vacuum-distill this t o dryness a t room temperature. This distillation can be conveniently carried out in a n appropriate evacuated and sealed tube (6) with the condenser portion cooled in a dry ice bath. When blood samples were treated in this way, recoveries of added formic acid in amounts of 30 to lo00 micrograms per ml. averaged 98% in nine single analyses, with an average absolute deviation from the mean of 5.1 yo. DISCUSSION
The curve of Figure 1reveals a deviation from Beer’s law which is most apparent at the lowest concentrations and a separation of duplicate points from the line equivalent to approximately 1
microgram. The disproportionately low color intensity in the 0to 5-microgram range is probably due to loss of some mercurous chloride by solution in the wash waters, while the scattering may be accounted for by mechanical losses. Keither of the factors was improved through reduction of the scale of the procedure and employment of centrifuge tubes of 3-ml. volume, but slight benefit is obtainable through use, of wash water saturated with mercurous chloride. The specificity of the colorimetric procedure for formic acid is no better than that of other methods based on reduction of mercuric chloride, and samples t o be analyzed must be free of other reducing substances such as ascorbic acid and formaldehyde. However, the increased sensitivity of the colorimetric method does eliminate the need for the commonly employed concentrating procedures involving lengthy distillations or evaporation of considerable volumes (1, 2, 3, 6, 7 , IS). Such procedures constitute a recognized source of error due either to decomposition of the acid or to its formation from carbohydrates (3, 4,8). ACKNOWLEDGMENT
The author wishes to acknowledge the valuable technical assist ance of Helen E. Pentz. LITERATURE CITED
(1) Benedict, E. M., and Harrop, G. A., J . Btol. Chem., 54, 443
(1922). (2) Claren, 0.B., 2. physiol. Chem., 276,97-107 (1942). (3) Dakin, H. D.,Janney, N. W., and Wakeman, A. J., J . B i d . Chem., 14,341 (1913). (4) Droller, H., Z. physiol. Chem., 211,57(1932). (5) Grant, W. M., IND. ENG.CHEM.,ANAL.ED.,18,729 (1946). (6) Grossfeld, J., and Payfer, R., Z . Untersuch. L e b m i t t . , 78, 1-30 (1939). (7) Krause, A. C., and Weekers, R., Arch. ophtalmol. (Paris),3,226-9 (1939). (8) &?senheher, J.. Ber., 41,1009 (1918). (9) Pirie, N.,Biochm. J., 40,100 (1946). (10) Porter and Ruyssen, Compt. rend., 82,1504 (1876). (11) Riesser, O.,2. physiol. Chem., 96,355 (1915-16). (12) Stepp, W., and Zumbusch, H., Deut. Arch. klin. Med., 134.112 (1920).
Oxidative Bromination in the Determination of Malic Acid and Aspartic Acid Micromethod f o r Determination of Beta-Alanine HEIKKI SUOAIALALNEN AND EVI ARHIMO Department of Biochemistry, Alcohol Research Laboratory, Helsinki, Finlend
F
OR the determination of malic acid in biochemical xork
Pucher, Vickery, and Wakeman (21; cf. 22) have developed a very specific micromethod which is based upon permanganate oxidation in acid solution containing potassium bromide. The reaction product, when distilled with steam in an acid 2,4dinitrophenylhydraxine solution, yields an insoluble dinitrophenylhydrazine derivative. This compound is filtered off, dried, and then dissolved in pyridine. On addition of sodium hydroxide to the pyridine solution and dilution with water, a measurable blue color is developed. This method has been used as such-i.e., without previous deamination with nitrous acid (cf. 2l)-by Arhimo (1) for the determination of aspartic acid, and it has proved to be exceptionally specific for this purpose. Of the other amino acids
tested by him, only tyrosine and dioxyphenylalanine were found to interfere’ whereas glutamic acid did not. The method can be successfully employed in the determination of aspartic acid, since aspartic acid, together with glutamic acid, may be separated from the other amino acids, according to Foreman’s ( 7 ) principle, and the organic mono- and dicarboxylic acids, including malic acid, are extractable with ether from the amino acids in solution. Subsequently Laine (IO) calculated the extinction values for tyrosine, although the method in this case seems less appropriate because of the comparatively low extinction values. Although considerable study has been given t o the nature of the oxidation product of malic acid, a detailed explanation of the chemical reactions that occur has not yet been obtained (21). Arhimo (1) has suggested dihromoxalacetic acid as an interme-
ANALYTICAL CHEMISTRY
208
The 2,4-dinitrophenylhydrazinederivative, formed from malic acid and obviously
. also from aspartic acid, B-alanine, and tyrosine, by brominating permanganate oxidation, was identified as glyoxal-2,4-dinitrophenylosazane. The latter is obtained from dibromoacetaldehyde,but not from bromal, showing that dibrornoacetaldehyde, in all probability, functions as intermediate in the reaction. There is, according to Dakin's assumption, an analogy in the behavior of dichloroacetaldehyde, when aspartic acid is oxidized by chloramine-T. The micromethod for determination of malic acid developed by Pucher, Vickery, and Wakeman is based upon this reaction. The method has been applied by Arhimo to the determination of aspartic acid and may be used to determine &alanine.
diate. Recently Greenberg (8)has claimed that malic acid, being first brominated to dibromomalic acid, ought to be oxidized to dibromoxalacetic acid. The dibromoxalacetic acid could then be separated by steam distillation and combined with dinitrophenylhydrazine in acid solution to yield a hydrazone which is insoluble in water. The behavior of citric acid on being converted into pentabromoacetone (19-12) suggests an analogous reaction mechanism in the case of malic acid-Le., an oxidative bromination. The mechanism of this brominating and simultaneously decarboxylating oxidation in the citric acid determination, the so-called Stahre's reaction, has been elucidated by Wirhlk ( 2 7 ) and described by Kometiani (9). However, such a behavior of malic acid has never been mentioned. Since the elucidation of the reaction mechanism seems to be of importance for the development and application of the excellent method of Pucher, Vickery, and Wakeman, the authors have att.empted t o solve this question. The blue color, developed from the 2,4-dinitrophenylhydrazine derivative by addition of alkali, indicates that a dinitrophenylosazone is formed (cf. 2 ) . The authors have isolated and crystallized this compound and found it to be glyoxal-2,4-dinitrophenylosazone. This is in favor of dibromoacetaldehyde as the reaction product formed by permanganate oxidation from malic acid in the presence of bromine. Dibromoacetaldehyde reacts with hydroxylamine hydrochloride to form glyoxime, as proposed by Wittorf ( 2 6 ) . Since the permanganate oxidation of tyrosine yields acetic acid ( 5 ) ,the formation of dibromoacetaldehyde from tyrosine is also conceivable. The hydration of dibromoacetaldehyde in aqueous solution (18) retains it in the water phase on extraction with petroleum ether to remove the water-insoluble pentabromoacetone. This is in agreement with the appearance of dibromoacetaldehyde as an intermediate in the malic acid determination of Pucher, Vickery, and Wakeman. Dibromopyruvic acid reacts with phenylhydrazine, yielding the phenylosazone of glyoxalcarbonic acid (14). At room temperature treatment of hydroxypyruvic acid wit,h phenylhydrazine solution yields this compound via tartronic acid semialdehyde but a t a higher temperature, 50" to 60" C., the reaction leads to the formation of glyoxalphenylosazone (6,Zs). CHBrl
CH : S . NH . C6H,
co .--f C: N . XH. C COOH COOH Dibromopyruvic acid
CHO
CHOH
CHzOH
~ fH ~ CHOH f- COH f- co COOH COOH COOH Tartronic acid Hydroxysemialdehyde pyruvic acid
If dibromopyruvic acid functions as an intermediate in malic acid determination, glyoxaldinitrophenylosazone would be formed when the steam-volatile oxidation product reacts with dinitrophenylhydrazine. It is also possible that heating the oxidation solution preceding the distillation decarboxylates the primary oxidation product. Considering these alternatives, the
authors have prepared the dinitrophenylhydrazine derivative by adding dinitrophenylhydrazine on the one hand to the oxidation solution directly, and on the other hand to the cold distillate, obtained by distillation through a spiral condenser. In either rase they have established by titration that the dinitrophenylhydrazine derivative did not contain a carboxyl group. The decomposition points of the p-nitrophenylosazones of glyoxal and of glyoxalcarbonic acid differ very little from each other, the decomposition point of glyoxal-p-nitrophenylosazone being 310-11" C. (25, 28) and that of glyoxalcarbonic acid pnitrophenylosazone 310" C. (4). Since the same similarity may exist with regard to the 2,4-dinitrophenylosazones,the authors have solved the problem by using phenylhydrazine. According to the latest report of Sprinson and Chargaff ($3; cf. 6, 12) the difference between the decomposition points of the two phenylosazones is considerable, glyoxalphenylosazone being decomposed a t 169" C., and glyoxalcarbonic acid phenylosazone a t 222223' C. The phenylosazone isolated by the authors from the oxidation solution and from the distillate proved to be the derivative of glyoxal. Although asparagine reacts very poorly in the determination according to Pucher, Vickerg, and Kakeman a dinitrophenylhydrazine derivative can be separated from the oxidation sohtion with good yields. However, the authors have not succeeded in crystallizing this compound. The fact that the oxidation product of asparagine is not volatile with steam, and that its dinitrophenylhydrazine derivative is an osazone, giving a blue color when alkali is added, suggests the possibility that it may be the 2,4-dinitrophenylosazone of glyoxalcarbonic acid amide : CH: N.N H . CsH,. (SOz)? C : N . X H . CGHa. (XOJz CO. YH, On oxidation of asparagine with sodium hypochlorite, Larigheld (11) obtained the corresponding phenylhydrazine derivative, and Dakin (S), using the sodium salt of p-toluenesulfonamide (chloramine-?') as the oxidizing agent, obtained both the phenylhydrazine and the p-nitrophenylhydrazine derivatives. Dakiii describes the reaction phases as follom: COOH CH,KH* CHZ
CHO
CH: iX.N H . C B H ~
+ CCl, + C:?\T.PITH.C6H6
CO. SHZ
CO. SHC
CO, KHZ
Especially remarkable in this connection is the finding that in oxidizing aspartic acid with chloramine-?', derivatives of glyoxal are separated in large amounts from the oxidation solution, when phenyihydrazine or aromatic diamines are added. According to Dakin, this Oxidation of aspartic acid with chloramine-T leads t o the formation of dichloroacetaldehyde, the CHn-group being chlorinated supposedly in the following way:
V O L U M E 19, NO. 3, M A R C H 1 9 4 7 COOH CH.SH2 --+ CHO +CHO
+CHO
CH,
CH:
cc1,
COOH
COOH
COOH
CHCl,
Khen methyl aspartic acid was used, the corresponding derivatives of methyl glyoxal were obtained. As a summary of the foregoing the following reaction mechanism for the method of Pucher, Vickery, and Wakeman is proposed : COOH CH2 HCSH?
\ -
COOH
CHI
\
CHO 1 KO2
COOH CH? HCOH COOH
-.
CH:N S H a S 0 2
CHBr2 CHO
The Phenylhydrazine Derivatives. Attempts to prepare a phenylhydrazine derivative from malic acid according to the method of Pucher, Vickery, and Wakeman, but using hydrochloric phenylhydrazine solution in the receiver, led to the formation of deeply colored products. This was induced supposedly by liberating hydrogen bromide and was avoided by using sufficient amounts of sodium acetate. The phenylhydrazine derivatives were, therefore, prepared by adding sodium acetate to the phenylhydrazine solution containing acetic acid. They were crystallized by the addition of sufficient water to their hot alcoholic solutions to produce a slight turbidity. The phenylhydrazine derivative so obtained from malic acid decomposedat 170-172" C.; the decomposition point of the phenylosazone, derived directly from the oxidation solution, was 169171' C. When a spiral condenser was used in the distillation and the compound was then separated by addition of phenylhydrazine to the cold distillate, the result was the same. Thus, as regards the decomposition points, all the phenylhydrazine derivatives were identical with glyoxalphenylosaxone. The decomposition points were determined with the meltingpoint apparatus of Dr. Linstrom, using rapid heating a t the rate of 1" C. every 2 or 3 seconds, according to Emil Fischer. SPECIFICITY OF METHOD AND EMPLOYMENT FOR DETERMINATION O F 8-ALANINE
h'
/
/
/'
CH:N.NHC>SOr
s0 2
HCKH,
COOH I n order to ascertain the validity of tLis scheme the authors have prepared the 2,4-dinitrophenylhydrazinederivatives on the one hand from malic acid according to Pucher, Vickery, and Wakeman, and on the other hand directly from glyoxal and from dibromoacetaldehyde. If the scheme is valid, the same derivative, glyoxal-2,4-dinitrophenylosazone,ought to be obtained from all these different components.
On the basis of the above reaction scheme the authors made some determinations of the specificity of the method. In addition to those acids which, according to Pucher, Vickery, and Wakeman, failed to give any precipitate they have found that admixtures of 25 mg. of t h e following substances did not interfere: ethanol, dioxytartaric acid, and dioxymaleic acid (prepared according to Neuberg and Schwenk, 15). Acetaldehyde, on the other hand, interferes slightly and so does ascorbic acid, although the latter behaves like carbohydrates, yielding a precipitate even if no potassium bromide is added. Of the amino acids, e-alanine does not interfere and asparagine only slightly. 8-Alanine, however, reacts in a manner analogous to malic acid. Since only small quantities of p-alanine were available, the authors were compelled to perform the determinations with commercial preparations which evidently were not completely pure. The results, on a malic acid basis, suggested a purity of 9570 with Schuchardt's p-alanine and 9370 with Scbering-Kahlbaum's preparation.
EXPERIiMENTAL
The 2,4-Dinitrophenylhydrazine Derivatives. The 2,4-dinitrophenylhydrazine derivative formed in the malic acid determination according to Pucher, Vickery, and Wakeman was prepared from malic acid (Schuchardt, pro analysi). After repeated recrystallization from glacial acetic acid its decomposition point was 322" C. Glyoxal-2,4-dinitrophenylosazonewas prepared from glyoxal sodium bisulfite (Schuchardt) in hydrochloric solution with a calculated excess of 2,4-dinitrophenylhydrazine. Various preparations, repeatedly recrystallized from glacial acetic acid, decomposed a t 322-324' C. 2,4-Dinitrophenylosazone from Dibromoacetaldehyde. Dibromoacetaldehyde was prepared according to Mylo ( I S ) and the fraction boiling a t 135-141 O C.was used for the preparation of the osazone. This, recrystallized from glacial acetic acid, decomposed a t 323' C. The osazones were all alike in appearance and crystallized from glacial acetic acid in beautiful red needles. I n pyridine solution diluted with water they all yield the blue color, characteristic of the dinitrophenylosazones when alkali is added. Keuberg and Simon (16) report the decomposition point of the 2,4-dinitrophenylosazoneof glyoxal to be 318", Lucas and Stewart (12) 323",and Strain (24) 326-328O.C. 2,4-Dinitrophenylhydrazine Derivative of Bromal. Bromal was prepared according to Mylo (13; cf. I ? ) , the fraction boiling between 174" and 180" C. being collected; bromal hydrate, puriss. (Schering-Kahlbaum), was also used. The dinitrophenylhydrazone of bromal crystallized from glacial acetic acid in reddishyellow needles. Recrystallized, it decomposed at 261-262' C. In a pyridine solution diluted with water the addition of alkali precipitated it partly, the solution remaining reddish. In contrast with the glyoxalosazone, it dissolved in an alcoholic potassium hydrouidp solution with a violet color.
LITERATURE CITED
(1) (2) (3) (4) (5)
Arhimo, A. A., S u m e n Kemistilehti, B12, 6 (1939). Bamberger, Ber., 32, 1803 (1899). Dakin, Biochem. J . , 11, 79 (1917). Ibid., 13, 398 (1919). Denis, J . Biol. Chem., 10, 74 (1911). (6) Fischer, Baer, and Xidecker, H e h . C h i m . A c t a , 20, 1226 (1937). (7) Foreman, Biochem. J . , 9, 463 (1914). (8) Greenberg, in Sahyun, "Outline of the Amino Acids and Proteins", p. 115, New York, Reinhold Publishing Corp., 1944. (9) Kometiani, 2. anal. Chem., 86, 359 (1931). (10) Laine, S u o m e n Kemistilehti, B13, 18 (1940). (11) Langheld, Ber., 42, 2360 (1909). (12) Lucas and Stewart, J. Am. Chem. S O C . ,62, 1792 (1940). (13) Mylo, Ber., 45, 645 (1912). (14) Nastvogel, Liebigs Ann. Chem., 248, 85 (1888). (15) Keuberg and Schnrenk, Biochem. Z., 71, 104 (1915). (16) Xeuberg and Simon, Ibid., 256, 485 (1932). (17) Organic Syntheses, Vol. XVII, p. 18, New York, John Wiley & Sons Co., 1937. (18) Pinner, Liebigs Ann. Chem., 179, 67 (1875). (19) Pucher, Sherman, and Vickery, J. Biol. Chem., 113, 236 (1936). (20) Pucher, Vickery, and Leavenworth, IND.ENG.CHEM.,ANAL ED.,6, 190 (1934). (21) Pucher, Vickery, and Wakeman, I b i d . , 6, 288 (1934). (22) Pucher, Wakeman, and Vickery, Ibid., 13, 244 (1941). (23) Sprinson and Chargaff, J . Biol. Chem., 164, 417 (1946). (24) Strain, J . Am. Chem. S O C . ,57, 758 (1935). (25) Summerbell and Rochen, Ibid., 63, 3241 (1941). (26) Wittorf, J . russ. phys.-chem. Ges., 32, 88 (1900). (27) Wohlk, 2. anal. Chem., 41, 77 (1902). (28) Wohl and Neuberg, Ber , 33, 3096 (1900).
