A N A L Y T I C A L EDITION
August, 1944
hydroxide. They have identified this pigment as the neo-8carotene U of PolgAr and Zechmeister (If). They report that this carotenoid has no vitamin A activity. It may, therefore, become necessary to calculate the carotene composition on the basis of more than two components. This may be possible by the use of spectrophotometric data obtained a t other critical points on the absorption curve of the extract. It is hoped that a continuation of spectral studies will furnish data that will be useful in this connection. LlTERATURE CITED
(1) Beadle, B. W., and Zscheile, F. P., J . Biol. Chem., 144, 21 (1942). (2) Cary. H.H., and Beckman, A. O., J. O p t i d SOC.Am., 31, 692 (1941). ( 3 ) Fraps, G. S., and Kemmerer, A. R., J . Assoc. OLEcial Agr. Chem., 22, 190 (1939). (4) Kernmerer, A. R., and Fraps, G. S.,IND. ENG.CHEM.,. ~ N A L . ED.. 15,714 (1943).
Determination of Citric
515
(5) Kemrnerer, A. R . , and Fraps, G. S., J. Am. Chem. SOC.,66, 305 (1944). (6) Kernohan. Geo., Science, 90, 623 (1939). (7) Moore, L. A., IND.ENQ.CHEM.,ANAL.ED., 12, 726 (1940). (8) Moore. L.A., and Ely, R., Zbid., 13, 600 (1941). (9) Pepkowitz. Leonard P.. J . Biol. Chem., 149,465 (1943). (10) Peterson, W. J., Hughes, J. S., and Freeman, H. F., IND.ENG. CHEM.,ANAL.E D . ,9,71 (1937). (11) Polgfu, A., and Zechmeiater. L.. J . Am. Chem. SOC.,64, 1856 (1942). (12) Silker, Ralph E.,Schrenk, W. G., and King, H. H., Abstracts of papere, 107th Meeting AM.CHEM.SOC., 1944. (13) Wall, M. Er and Kelley, E. C., IND. ENG.CHEM.,A N ~ LED., . 15, 18 (1943). (14) Wiseman, I-I. C., Kane, E. A., Shinn, L. h.,and Cary, C. A . , J . Agr. Research, 57,635 (1938). PEEBENTHD before the Division of Biological Chemistry at the 107th Meeting of the AMERICANCHEIIICAL SOCIETY, Cleveland, Ohio. Contribution 285, Department of Chemistry. This work is supported by the Kansas Industrial Development Commission.
Acid
in Fermentation Media
and Biological Materials DAVID PERLMAN, HENRY A. LARDY,
AND
MARVIN J. JOHNSON
Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison,
Several modifications have been made in the colorimetric penbbromoacetone method for the determination of citric acid as proposed b y Pucher, Vickery, and associates. The time required per sample
C
ERTMN strains of the mold, Aspergillus niger, convert a
large proportion of carbohydrate substrates to citric acid. With most strains of this organism some oxalic acid is also produced. I n studying the factors affecting the production of citric acid by this mold (3),it was necessary to have rapid and reasonably accurate methods for the determination of these two acids in the presence of each other. Since both acids have rather insoluble calcium salts, these salts have been used for their determinations in fermentation media by Doelger and Prescott (Z), Wells, Moyer, and May (8), and others. Calcium oxalate is much less soluble than calcium citrate in cold, slightly acid solution, and the two salts may be separated in this manner. Calcium citrate is insoluble in hot neutral solutions. These two acids are usually the main acidic products of this fermentation, and for routine work the difference in adidity between the total acid and that due to oxalic acid may be considered to be due to citric acid. This is a somewhat uncertain procedure, as the mold produces many other acids, and an independent method for the determination of citric acid is desirable. The wlumetric method of Wilkinson, Siphard, Fulmer, and Christensen (9) involves preliminary precipitation of the lead salts of the oxalic and citric acids. The acids are then regenerated and titrated with alkali and ceric sulfate. This method is not very specific for citric acid, as-many fermentation acids form rather insoluble lead salts. A more specific method for the determination of citric acid depends upon its conversion to pen tabromoacetone, which may be estimated gravimetrically or colorimetrically. This conversion is based on the fact that when citric acid is oxidized with potassium permanganate in the presence of bromine, under controlled conditions, the acid is converted quantitatively to penta bromoacetone. Deysher and Holm (1) have discussed the diffi-
Wfs.
is reduced, while accuracy has not been diminished. This method has been applied to the determination of citric acid in fermented media and tissue extracts with satisfactory resulb.
culties in the gravimetric determination of this derivative of citric acid. Pucher, Vickery, and associates (4, 6, 6) have estimated the pentabromoacetone colorimetrically by a method based on the yellow color formed by the addition of pentabromoacetone to a sodium sulfide solution. This colorimetric method is somewhat involved and requires quantitative extraction of the pentabromoacetone from the reaction solution. Purinton and Schuck (7) have modified the original method slightly but still require quantitative extraction of the pentabromoacetone and several other rather involved procedures. I n the modification described, no attempt has been made to extract the pentabromoacetone quantitatively from the reaction solution. Instead, advantage is taken of the distribution coefficient and a single extraction is made. A single extraction has been found to remove R constant amouct of the total pent,abromoacetone i n any series of samples of uniform volume. This modification has made possible thc development of a .more convenient procedure. EXPERIMENTAL
REAGENTS.Sulfuric acid, equal volumes of 95% sulfuric acid and water. 1 M potassium bromide, bromine water (iaturated), 3% hydrogen peroxide, petroleum ether (acid-washed Skellysolve B). Dioxane-water mixture, equal volume of dioxane anti Ivater. Sodium sulfide eolution, 4 grams of sodium sulfide nonahydrate per 100 cc. of solution. 1.5 N and 0.1 N potassium permanganate. PROCEDURE. If the samples are known to contain reducing material, aliquots preferably containing less than 23 n ~ g .of citric acid are placed in 2.5 X 20 cm. (1 X S inch) Pgrcs test tubes and 2 cc. of the sulfuric acid solution are added. Tile total volume is adjusted to about 20 cc. and the samples are boiled for a few minutes. The solutions are then cooled and 3 to 5 cc. of bromine water are added. After 10 minutes, any precipitate
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Table 1. Recovery of A d d e d Citric A c i d from Solutions Citric Solution -4a Solution Ba Solution CC Acid Added Citric Recovery Citric Recovery Citric Recovery per acid of added acid of added acid of added found acid found acid acid Sample found % MQ. % % Mo Mg. Mg. 0
... . .
0
.
0.35 0.37 Av. 0 . 3 6
.....
0.24 0.25
.. ...
Av. 0 . 2 4
0.20
0.19
...
95.0 100.0
0.57 0.56
105.0 100.0
0.44 0.43
100.0 95.0
0.40
0.39 0.41
97.5 102.5
0.75 0.74
97.5 95.0
0.63 n.65
97.5 102.5
0.80
0.78 0.80
97.5 100.0
1.18 1.16
102.5 100.0
1.04 1.07
100.0 103.8
1.00
1.01 1.02
101.0 102.0
1.36 1.37
100.0 101.0
1.25 1.24
101.0 100.0
1.20
1.23 1.19
102.5 99.2
1.56 1.58
100.0 101.7
1.44 1.47
100.0 102.5
1.60
1.58 1.89
98.8 99.4
1.95 1.99
100.0 102.5
1.85 1.88
100.6 102.5
1.79
99.4 98.3
2.10 2.17
96.3 100.6
2.00 2.06
97.8 101.1
1.80
b c
1.77
Citric acid added to distilled water. Citric acid added to fermentedsynthetic medium. Citric acid added to fermented molasaes medium.
that has formed is removed by centrifugation. The supernatant liquids are decanted off, and adjusted to known volumes. If the samples do not contain appreciable amounts of reducing material they may be adjusted to known volumes without this preliminwy bromine treatment. Aliquots of these solutions, preferably containing 0.2 to 1.8 mg. of citric acid, are placed in test tubes (the 18 X 150 mm. size is convenient) and 0.3 cc. of the sulfuric acid, 0.2 cc. of tlie potassium bromide, and 1 cc. of the strong potassium permanganate solutions are added. The total volumes are adjusted to about 5 cc. and the tubes are allowed to stand for 5 minutes at room temperature. At the end of this period they are chilled in an ice bath, and the excess Permanganate is decolorized with the hydrogen peroxide solution. Care must be taken to keep the reaction mixtures below 5 ” C. during this step. Any excess peroxide is removed with the weak permanganate. The total volumes are then adjusted to 10 cc. (the test tubes should have a 10-cc. calibration mark for this purpose) and 13 cc. of the petroleum ether are added. The tubes are stoppered, shaken vigorously, and centrifuged (to break any emulsion that might, ,be formed). Colorimeter test tubes are prepared containing 5 cc. of the water-dioxane mixture and 5 cc. of the sodium sulfide solution, and IO-cc. portions of the petroleum ether extract containing the pentabromoacetone are added. The colorimeter tubes are then stoppered, shaken vigorously, and centrifuged. The color produced should be a light yellow and will be fully developed in 5 minutes. It is stable for several hours. The absorption is determined in a photoelectric colorimeter a t 450 mfi, Light absorption by the solution has been found to be reasonably con.stant, from 400 to 450 mH. A tube containing no citric acid, but which has gone through the same procedure, is used as a 100yo transmission standard. At least two known samples of citric acid should be run with each set of analyses. The color follows Beer’s law, as is shown in Table I, column 3, and the standards are used to calculate k in the equation log T = kc. If too large a sample of citric acid has been used, a second smaller aliquot of the petroleum ether extractpay be taken, thus avoiding another complete analysis. As is shown in Table I, the method gives satisfactory results when between 0.2 and 1.8 mg. of citric acid is present, in the sample. Larger samples of citric acid may be used. I n such cases larger volumes of petroleum ether for extraction of the pentahromoacetone, or smaller aliquots of t,he petroleum ether extract containing the pentabromoacetone, may be used. However, several known solutions must always be determined in exactly the same manner as the unknown samples. Recoveries of added citric acid from two types of fermentation media are also shown in Table I. Solution B contained citric acid, oxalic acid, and unfermented sucrose as the major organic constituents. The solution had been prepared for analysis by
Vol. 16, No. 8
removing the mycelium, and adjusting the fermented liquor to a known volume. Various amounts cJf citric acid were added, as shown in Table I, to an aliquot o f this solution corresponding to 0.02 cc. of the original fermented liquor. The eame procedure was repeated with solution C, a partia!Iy fermented beet molasses medium. This method has also been applied to the determination of citric acid in tissue extracts. Quantitative recoveries were obtained when citric acid was added to muscle extracts and human Berninal fluid. Isocitric acid, cis-aco:iltic acid, tmns-aconitic acid, and oxalacetic acid do not interfere with this method (Table 11) when present in biologicai samples. Gluconic acid, which is often found in media fermented by A . niyer, does not interfere. Pucher et al. (6) list several other acids often found in fermentation media which are not converted to pentabromoacetone under t,he conditions of this method and thus do not interfere with the determination of the citric acid. The following critical points have been noticed: .4n excess of hydrogen peroxide in the solution before the petroleum ether extraction leads to low recoveries. The p r m ence of excess potassium permanganate leads to high recoveries. The solution must be thoroughly ctilled before excess permanganate is removed; otherwise, recoveries are erratic. Some stabilizing agcnt, must be present to stabilize t,he colored reaction product of the pentabromoacteorie and the sodium sulfide. Both 50% dioxane-wzter and 50% pyridine-water eolutions have proved satisfactory. Interfering materials may be removed from the petroleum ether by acid washing. The pentabromoacetorie should not be allowed to remain in the petroleum ether for more than 15 minutes.
Table Subatance Tested
II. Specificity of Method Maximum Sample IJscd
MQ. Isocitric acid
cis-4conitic acid Irana-Aconitic acid Oxslacetic acid Gluconic acid
18.8 1.74 1.74 6.0 5.0
Equivalent d Citric Acid Found MR.
