ANALYTICAL CHEMISTRY
206 Reliable vapor pressure data can be obtained with the method described above and a number of results are presented in Table I. I n each case the observations are made on a sample of approximately 5 cu. mm. Studies on the application of the method were restricted to conditions under which the highest vapor pressure measured was less than 100 mm., principally because the vapor pressure measurements were subsequently used to define the optimum conditions for the purification and separation of certain organic liquids by vacuum distillation. There is no reason why the method cannot be applied over greater pressure and temperature ranges, although a t higher pressures distillation from $he liquid column may limit the number of observations that can be made from any one sample. As observations made on a sample of approximately 5 cu. mm. permit the construction of a vapor pressure versus temperature curve, it is clear that the heat of vaporization of an organic liquid can also be determined on a milligram scale.
ACKNOWLEDGMENT
The authors wish to express their indebtedness to P. A. Shafler, Jr., and to Edward Bennett for their contributions in the course of this investigation. LITERATURE CITED
Emich, F., Monatsh., 38, 219 (1917). Hershberg, E. B., IND. Exo. CREM.,ANAL.ED.,8, 312 (1936). (3) International Critical Tables, Vol. 3, New York, McGraw-Hill
(1) (2)
Book Co., 1928. (4)
Sartori, M., “The War Gases”, New York, D. Van Nostrand Co.,
1940. ( 5 ) Schneider, F., “Qualitative Organic Microanalysis”, New York, John Wiley, 1946. CONTRIBUTION 1074 from the Gates a n d Crellin Laboratories of Chemistry, California Institute of Technology. This paper is based on work done for t h e O 5 c e of Scientific Research and Development under Contract OEMsr325 with the California Institute of Technology.
Colorimetric Micromethod for Determination of Formic Acid W. MORTON GRANT Howe Laboratory of Ophthalmology, Harvard University Medical School, Boston, Mass. The sensitivity of the mercurous chloride method for formic acid determination can be increased approximately one hundredfold by means of a colorimetric procedure based on the reduction of a .mixture of phosphomolybdic and phosphotungstic acids. This method may be used to determine formic acid in blood.
M
ETHODS frequently employed for the determination of formic acid consist of the measurement of the amount of mercurous chloride or of carbon dioxide formed in the reaction of formic acid with mercuric chloride. Both gravimetric ( 1 , 3, 10) and oxidative titrimetric (6, 7, 1 1 ) procedures have been used to measure the mercurous chloride, but neither procedure is successfully applicable to the determination of less than milligram quantities of formic acid (4, IS). Quantities in the range of 0.1 to 1 mg. can be determined by the carbon dioxide method (9). To increase the sensitivity of the mercurous chloride method, a colorimetric procedure has been developed which permits the determination of quantities of formic acid as small as 5 to 30 micrograms in 1 ml. with a n accuracy of * 1 microgram. The present method is based on the chromogenic reduction of a mixture of phosphomolybdic and phosphotungstic acids by the washed mercurous chloride precipitate from the formic acid-mercuric chloride reaction. Formic acid itself does not give a color with the reagent.
PROCEDURE
For determining the relationship of colorimetric reading to formic acid concentration a series of dilutions of standard sodium formate solution is prepared containing the equivalent of 0 to 30 micrograms of formic acid per ml. One milliliter of each solution is placed in a 15-ml. conical centrifuge tube with 0.5 ml. of mercuric chloride reagent and the mixture is heated on a steam bath for 3 hours. The upper two thirds of the tubes should be protected from excessive heating in order to prevent evaporation. ( A satisfactory steam bath cover and tube support for this purpose consist of a sandwich of sponge rubber between aluminum plates with perforations into which the tubes fit snugly.) The tubes are next allowed to cool completely and 2 ml. of diatomaceous earth suspension are added to increase the bulk of the precipitate. To overcome the tendency of some of the particles to stay in a
REAGENTS
Mercuric Chloride Reagent. An aqueous solution is employed containing 20 grams of mercuric chloride, 30 grams of sodium acetate, and 8 grams of sodium chloride in 100 ml., as generally used by others. Suspension of Diatomaceous Earth. A mixture of 20 mg. of Dicalite laboratory Filteraid and 100 ml. of water is shaken up each time before use. Alcohol. U.S.P. 95% ethyl alcohol is mixed with activated charcoal and filtered to remove anv imwrities which - suspended may interfere with the analysis. Chromogenic Reagent. Ten grams of phosphotungstic acid and 130 grams of phosphomolybdic acid are dissolved in sufficient water a t room temperature to give a volume of 400 ml., and then 50 ml. of concentrated hydrochloric acid and 50 ml. of 85y0 phosphoric acid are added. Although a small sediment forms, the supernatant light yellow solution is stable for several weeks in a stoppered Pyrex bottle. The gradual appearance of a green tinge is attended by a slight increase in the color of the blank. Sodium Formate Standard. A freshly prepared solution is used containing 44.4mg. of sodium formate per liter, which is equivalent to 30 micrograms of formic acid per milliliter. sodium Carbonate, saturated aqueous solution.
0
5
10
15,
20
25
30
Formic Acid, Microgram#
Figure 1.
Color-Concentration Curve
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-
