V O L U M E 2 6 , NO. 2, F E B R U A R Y 1 9 5 4 behavior waq obtained using hydroxylamine sulfate in 0.1X sulfuric acid. On the other hand a linear relationship from 1 to 50 X lO-b-Tf, the maximum concentration studied, was obtained when a platinized electrode was substituted for the smooth platinum. In addition, constancy of the current &-as reached much faster and the sensitivity n’as much greater. ACKXOWLEDG,M ENT
One of the authors (L. B. Rogers) wishes to thank the Atomic Energy Commission for partial support during this study. LITERATURE CITED
Conant, J. B., Chem. Revs., 3, 1 (1926). Ephraim, F., “Inorganic Chemistry,” Third English Ed., (translated by Thorne, P. C. L., and Ward, A. AI.), London, Gurney and Jackson, p. 650, 1939. Eyring, H., Marker, L., and Kwoh, T. C., J . P h y s . &. Colloid Chem., 53, 1453 (1949). Fichter, Fr., “Organische Elektrochemie,” pp. 119-20, Ann Arbor, Rlich., Edwards Bras., 1946. Fieser, L. F., J . Am. Chem. Soc., 52, 5204 (1930). Gaylor, V. F., Conrad, A. L., and Elving, P. J., A N a L . CHEM., 25, 1078 (1953). Goon, E., “Polarography of Hydrazine,” B.S.thesis, Massachusetts Institute of Technology, RIay 1950. Griess, J. C., Jr., and Rogers, L. B., unpublished studies, 1947. Hendenburg, J. F., and Freiser, H., ASAL. CHEW, 25, 1355 (1953).
295 (10) Julian, D. B., and Ruby, W. R., J . Am. Chem. Soc., 72, 4719 (1950). (11) Kolthoff, I. M.,and Jordan, Joseph, Ibid., 75, 4869 (1953). (12) Kolthoff, I. XI., and Lingane, J. J., “Polarography,” New York, Interscience Publishers, 1946. (13) Kolthoff,I. &I., and Rosenblum, C., “Acid-Base Indicators,” p. 244, New York, Macmillan Co., 1937. (14) Laitinen, H. A., and Kolthoff, I. M., J . P h y s . Chem., 45, 1079 (1941). (16) MacNevin, W. AI., and Sweet, T. R., Quart. J . Studies Alc., 13, (16) 46 (1951). (17) Merritt, C., Jr., and Rogers, L. B., unpublished studies, 1952. (18) Miller, H. H., and Rogers, L. B., Science, 109, 61 (1949). MUler, R. H.. J . Am. Chem. Soc.. 69, 2992 (1947). (19) Nernst, W.,and Merriam, E. S., Z. p h y s i k . Chem., 53, 235 (1905). (20) Petrocelli, J. V., and Paolucci, A. A., J . Electrochem. Soc., 98, (21) 291 (1951). Popoff and Kunz, J . Am. Chem. Soc., 51, 352 (1929). (22) Rogers, L. B., Miller, H. H., Goodrich, R. B., and Stehney, (23) 4.F., -4N.4L. CHEM.,21, 777 (1949). Schmidt, A., Z . Elektrochem., 44, 699-708 (1935). (24) Turk, E. H., Hyman, H., and Rogers, L. B., unpublished studies, 1949. (25) T%’ik,on, R. E., and Youtz, M. -4.,I n d . Eng. Chem., 15, 603 (1923). (26) Zlotowski, I., Roczniki Chem., 14, 640-90 (1934). RECEIVED for review April 29, 1953. Accepted November 12, 1953. Presented in part a t the Pittsburgh conference on Analytical Chemistry, March 1952. This paper was abstraoted from a thesis submitted by 5. 9. Lord, Jr., in partial fulfillment of the requirements for the Ph.D. degree, Massachusetts Institute of Technology, February 1952.
Polarographic Determination of Citric Acid Polarography of Pentabromoacetone PHILIP J. ELVING’
and
ROBERT E. VAN ATTA*
The Pennsylvania State University, State College, Pa.
The determination of citric acid is important in agricultural, biochemical, and medical studies. A n analytical method has been developed which utilizes the conversion of citric acid to pentabromoacetone recommended in earlier methods, but substitutes a direct polarographic measurement for the subsequent petroleum ether extraction, oxidation-reduction treatment, and final titration procedure. The citric acid is essentially quantitatively converted to pentabromoacetone by the procedure of Hargreaves, Abrahams, and Vicliery. D-Isocitric acid, one of the components in the Krebs tricarboxylic acid cycle,
T
HE determination of n-isocitric acid and of citric acid, into which D-isocitric acid can be converted, is of considerable
importance in the study of organic acid metabolism in plant tissues. Several analytical methods have been described for the determination of citric acid, of which that of Krebs and Eggleston (1, 6, 7), as modified by Pucher, Vickery, and Leavenworth (8) with further modification by Hargreaves, Abrahams, and Vickery (2), has apparently proved most successful. The method is based on the oxidation of citric acid by permanganate in the presence of bromide to pentabromoacetone. The latter compound is extracted with petroleum ether and decomposed with sodium sulfide. The Sendroy silver iodate procedure (9) is then used to determine the bromide liberated by the sodium sulfide treatment.
*
Present address, University of Michigan, Ann Arbor, Mich. Ada, Ohio.
* Present address, Ohio Northern University,
can be similarly determined after its conversionto citric acid. The precision of the polarographic method is comparable to that of the older method with a considerable saving in the time required. A graphical method for the precise evaluation of diffusion currents is presented which corrects for the geometrical effect of the polarographic wave resulting from the presence of manganous ion in the test solutions. The three polarographic waves observed for pentabromoacetone are apparently due to dibromoacetic acid and bromoform, into which the pentabromoacetone is quantitatively hydrolyzed.
Although the accuracy and precision of the modified Krebs and Eggleston method are satisfactory, the procedure is rather tedious and time-consuming because of the petroleum ether extraction and its attendant difficulties and the care required in the subsequent iodate procedure. It was evident that a polarographic method for the direct determination of pentabromoacetone would increase the convenience of the procedure, providing that the polarographic behavior of the ketone proved suitable for such an application. Accordingly, the polarographic behavior of pentabromoacetone has been briefly investigated in order to determine the optimum conditions for adaptation of the results to the Krebs and Eggleston method for the determination of citric acid. The preliminary stages in the polarographic modification of the method are identical with those of Hargreaves, Abrahams, and Vickery ( 2 ) with respect to the conversion of citric acid to pentabromoacetone. The resulting reaction mixture is then
296
ANALYTICAL CHEMISTRY
treated so a~ to produce a stock ketone solution suitable for the preparation of a polarographic test solution. Thus, the extraction and sodium sulfide, iodate, and thiosulfate titration procedure phases of the older method are eliminated.
Table I. Range 0 to 2 4 to 6 8 to 9 . 5 10.5 to 12.5
EXPERIMENTAL
Materials. All chemicals used were C.P. or analytical reagent grade. Pentabromoacetone was prepared from citric acid by the method described by Hargreaves, Abrahams, and Vickery ( 2 ) for the conversion of this acid to the ketone. One hundredmilligram quantities of citric acid were taken, in order that the pentabromoacetone would be formed in sufficient quantity to be separable from the reaction mixture. The precipitated ketone was filtered, washed well with cold water, and recrystallized from petroleum ether. The resulting crystalline compound melted a t 73" to 74" C. [literature (S), 73" '2.1. The compound was analyzed for bromine by the Stepanov method and was found to contain 88.4y0bromine (calculated, 88.3%).
a
Buffer Solutionsa Components KC1 with added HCI ?iaOAc a.ith added HOAc "IC1 with added NHa Na2HPOI with added NaOH
All buffers were prepared with an ionic strength of 0.531
water-jacketed. Water a t 25" =k 0.1' C. was circulated throughout the jacket. All potential measurements were made and are reported versus the saturated calomel electrode a t 25" C. X t r o gen used for deoxygenation was purified by bubbling through concentrated sulfuric acid and alkaline pyrogallol, and was then conditioned by passage through distilled water and a portion of the solution being examined. Polarographic Pentabromoacetone Procedure. Solutions used to determine the optimum conditions for the polarographic determination of pentabromoacetone m-ere prepared by pipetting 5 ml. of 3.0 mM pentabromoacetone stock solution (50% ethyl alcohol) into a 50-ml. volumetric flask and diluting to the mark with the desired buffer; the buffer solutions used, prepared by the usual methods, are listed in Table I. The electrolysis cell was rinsed with a portion of the test solution and another portion was added to the final bubbler in the nitrogen purification train. Nitrogen was then passed through the solution for 10 minutes, after which the tube was withdrawn and a nitrogen atmosphere maintained over the test solution during electrolysis. The polarographic values and constants were calculated in the usual manner from the rerorded polarograms; in the presence of manganese the geometrical technique described below was used. Citric Acid Conversion Procedure. The following procedure was used in preparing for polarographic examination solutions of pentabromoacetone from known quantities of citric acid.
*drt
I V.
4 6V.
Figure 1. Typical Polarograms Obtained in Determination of Citric Acid Curves are numbered with respect to the number of milligrams of citric acid present in original sample The measured diffusion current ie the vertical distance between the extended base line (dashed line) and the encircled point of intersection
Transfer an accurately measured volume of a solution containing between 0.5 and 10.0 mg. of citric acid to a 125-1111, Erlenmeyer flask and dilute to approximately 10 ml. Add 2 ml. of 1 8 s sulfuric acid and boil for 5 minutes on a hot plate; after cooling to room temperature, add 1 ml. of 20% metaphosphoric acid, 2 ml. of 1.V potassium bromide, and 5 ml. of 1.5AV potassium permanganate. (The temperature should not exceed 22" C. during the oxidation and bromination of the citric acid.) Allow the mixture to stand without stirring for 10 minutes and then add ice-cold 3% hydrogen peroxide with stirring until the solution is decolorized. Thus far, the procedure is essentially the same as that of Hargreaves, Abrahams, and Vickery ( 2 ) . The rractions may be represented as follows: CI&-C'OOH HO-&-COOH
I
anf H-2
I
+ ('0+ H20
b-0 I
__c
C"2-C'OOH
CH2COOH Br
C'H2C'OOH Stock solutions of pentabromoacetone (3.0 mM) were prepared by transferring the required amount of the com ound to a 50ml. volumetric flask, dissolving in 26 ml. of 9 5 8 e t h y l alcohol, and diluting to the mark with distilled water. A stock solution of citric acid containing 1 mg. of the acid per ml. was prepared for the analytical investigation; a 10 mM solution of manganous chloride was also prepared for experiments on the effect of excess manganese. Apparatus. A Leeds and Xorthrup Electro-Chemograph, T y e E, wm used to record olarograms. Beckman Model G pH meters were use8for p H measurement: a Type E electrode was used for all measurements above pH 10. The capillary used for the dropping mercury electrode was prepared from Corning marine barometer tubing and had an m value of 1.28 nig. per second (open circuit in distilled water) a t a mercury head of 50 cm. The m213t1/6 value for a t (drop-life) on the limiting current portion of the third wave of pentabromoacetone (- 1.35 volts versus saturated calomel electrode) was 1.413 n1g.2/3 second-' 12. The H-type polarographic cell used ( 5 ) contained a saturated calomel electrode and a potassium chloride-agar-fritted glass diak salt bridge; the entire H-cell was
C"&OOH
H2S04
I
C=O I
I
C"2COOH
Br-A-Br Kbfn04 C=O I
-KBr
' i H-C-Br
+ CO +
('02
4- Hz0
I
Br The conversion to pentabromoacetone is 98% of theory ( 2 ) . To the resultant reaction mixture, add 1.5N potassium permanganate dropwise until a faint pink coloration indicates the presence of an excess of this reagent; a slight excess of permanganate is necessary in order to eliminate the polarographic wave caused by the reduction of hydrogen peroxide which masks the pentabromoacetone wave. Add one drop of 0.1% methyl orange indicator, followed by the dropwise addition of 5AVsodium hydroxide until the color of the mixture changes from deep pink to chocolate brown. At this point, the solution is slightly acidic. Add 13 ml. of 95% ethyl alcohol, shake for a few seconds, and transfer the mixture to a 50-ml. volumetric flask. ilfter dilution to the mark with distilled water, allow the misture to
297
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4 stand for 10 minutes, so that the flocculent precipitate of manganese dioxide and manganous hydroxide may settle. The supernatant liquid is suitable for polarographic examination. Transfer 10 ml. of this solution to a 50-ml. volumetric flask and dilute to the mark with the desired buffer solution. The polarographic procedure previously described (polarographic pentabromoacetone procedure) can now be applied to this test solution.
The occurrence of such a hydrolysis was substantiated by experiments in which approximately equimolar mixtures of dibromoacetic acid and bromoform (about 0.3 mM in each species) were electrolyzed. The polarograms obtained were essentially identical with those recorded for 0.3 mJf pentabromoacetone under similar polarographic conditions. Selection of Buffer Medium. As the principal objective in the development of a polarographic method for the determination Measurement of Diffusion Currents. In order to minimize the of citric acid was the elimination of the extraction step, the error introduced into current measurements as a result of the medium selected for the electrolysis had to be one in which the geometrical effect of the manganous wave on the pentabromoinorganic ions present in the test solutions would not produce acetone wave, the following graphical construction method was interfering polarographic waves. Of the inorganic ions present most satisfactory for obtaining reproducible results (Figure 1). in the reaction mixture, only permanganate and manganous ions are polarographically reTable 11. Relation between Quantity of Citric Acid Taken and Diffusion Current ducible in potential range apMeasured for Third Wave of Pentabromoacetone plicable with the buffers under Citric Measured Diffusion Currents for Individual Series of Determinations. hficroamperes consideration ( 4 ) . The relaAcid Taken, 8td. tively small amount of perMg. A B C D" E F Mean der. Range, i d / C manganate present does not 10.00 0.493 0.505 0.510 0.484 0.493 0.501 0.498 0.0085 0.050 f 0.001 7.50 0.400 0.415 0,408 0.419 0.411 0.0070 0.055 =t0.001 interfere, since the perman5.00 0.320 0.320 0.312 o:ios 0.309 0:3i7 0.314 0.0054 0.063 =to.ooi 3.00 0,197 0.194 0.182 0.185 0.190 0.187 0.189 0.0031 0.063 f 0.002 ganate reduction wave occurs 0.123 0.130 0.126 0.120 0.125 0.126 0.0036 0.063 f 0.002 2.00 0.130 a t zero applied potential versua 0.072 0.070 0.066 0.076 0.067 0.069 0,0038 0.069 f 0.004 1.00 0.065 0.50 0,030 0.035 0.036 0.036 0.037 0.035 0.035 0.0022 0.070f0.004 the saturated calomel electrode. The large amount of c Polarograma shown in Figure 1. manganous ion present in the reaction nuxture is largely precipitated as manganous hyConstruct lines ABC, BD,EF, and G H , representing the averdroxide on neutralization of the mixture and on subsequent mixage currents for the various portions of the polarogram. Conture with the alkaline buffer solution. The manganous wave resultstruct a line parallel to line ABC through point M , the miding from the small amount of the ion remaining in solution occurs point of line EF. The vertical distance between the encircled a t - 1.45 volts. Because of the presence of this manganous ware, point of intersection and line ARC' is i d for the pentabromoacetone wave. the phosphate buffers were eliminated from consideration, since the third wave for pentabromoacetone (the wave most suitable This method gave reproducible currents even when sufficient for analytical purposes) coalesces with the manganous ion ware. manganous ion was added to the test solution prepared from 5 mg. of citric acid to produce a second wave five times the height of the one shown in Figure 1. ~
DEVELOPMENT OF T H E ANALYTICAL PROCEDURE
Polarographic Behavior of Pentabromoacetone. The applicability of the polarographic method for the determination of pentabromoacetone was tested by examining solutions of the ketone in each of the buffer systems listed in Table I. The final test solution contained 5% ethyl alcohol which came from the stock ketone solution. At such ethanol concentrations up to about 0.3 niM ketone solutions can be studied. The polarographic waves obtained for pentabromoacetone in the acidic region were completely unsuitable for analytical application, being long and drawn out, complicated by maxima effects and, in general, poorly defined. The situation in the alkaline region, however, was the reverse. Three waves were found in each of the ammonia buffers used (pH 8.2, 8.8, and 9.5); the first of these was masked somewhat by the mercury oxidation wave in each case, the second was well defined but geometrically unsymmetrical, while the third was a sharp, well-defined wave. The three waves appeared to be pH-independent; E112values were -0.12, -0.48, and - 1.22 volts, respectively. Three waves were also observed in phosphate buffers a t p H 10.5, 11.5, and 12.5. In the latter buffers, all three waves were fairly well defined; i d for the third wave was approximately twice that for each of the first two waves; Eli2 values were -0.15, -0.54, and - 1.37 volts, respectively. Further investigation of the basic polarographic behavior of pentabromoacetone is planned since, in the alkaline background solution used, a stoichiometric hydrolysis of pentabromoacetone apparently occurs to form dibromoacetic acid and bromoform.
I
I
!
1
I
3
I
,
5
,
I
7
!
I 9
C!TRlC ACID, MG
Figure 2.
Calibration Curve for Polarographic Determination of Citric dcid
The ammonia buffer a t pH 8 8 was selected as the most ideal medium from the analytical point of view for several reasons. The third wave for pentabromoacetone in this buffer is well defined and of relatively large current height per unit of concentration; E1/zfor this wave is sufficiently far removed from that for the manganous ion wave that interference would be a t a minimum; the medium is alkaline enough to ensure that a minimum of manganous ion would be left in solution. Although the ammonia buffer a t pH 9.5 meets these requirements, it has the undesirable feature that ammonia is lost through prolonged degassing of the solutions. Calibration in Terms of Citric Acid. -4 series of replicate determinations was made, starting with known amounts of citric acid ranging from 0.5 tQ 10.0 nig. The citric acid was converted to pentabromoacetone as previously described in the citric acid
ANALYTICAL CHEMISTRY
298
conversion procedure, polarographic stock solutions were prepared, and the polarograms were recorded. The measured currents for the third wave of pentabromoacetone are shown in Table 11. Typical polarograms for a set of citric acid experiments are shown in Figure 1; the curves are designated in terms of the number of milligrams of citric acid originally taken. A calibration curve for citric acid in terms of measured wave height for the pentabromoacetone third wave is shown in Figure 2; all the Table I1 data have been plotted, so that the scatter of the points on the curve represent a measure of the precision of the method. B better indication of precision is shown in the standard deviation values (Table 11). The precision is of the order expected for organic polarographic methods, ranging from about 1 part in 20 for the 0.5-mg. samples to 1 part in 50 for the 10-mg. samples. Effect of Ethyl Alcohol Concentration. The leveling off of the calibration curve (Figure 2) is apparently due to formation of large enough amounts of pentabromoacetone so that it is incompletely dissolved, with the resultant decrease in id per unit concentration. Such a decrease suggested that increasing the ethanol concentration might increase the solubility of the ketone. Experiments with 10 and 15% ethanol test solutions of 0.3 mM pentabromoacetone produced i d values approximately 5 and 10% smaller, respectively, than those obtained for 5 % ethyl alcohol test solutions. The effect of the ethyl alcohol concentration on id is apparently greater than the expected increase in solubility of the pentabromoacetone. Consequently, it is recommended that the procedure described with its dilution technique be applied to samples containing not more than 5 mg. of citric acid; large amounts of citric acid will require more dilution. Effect of Manganous Ion. To determine whether the manganous wave (the second wave in Figure 1) had any appreciable effect on the diffusion current measured for the pentabromoacetone wave, a 5-mg. sample of citric acid was converted to pentabromoacetone by the prescribed procedure and the polarogram for the resulting test solution was recorded. .4dditional manganous ion in the form of a 10 mM manganous chloride solution y a s added so that the manganous wave was increased to five times its initial size. No difference in i d for the pentabromoacetone wave was obtained as long as the graphical construction described for determining wave heights mas used. I n another experiment, several drops of 5 N sodium hydroxide were added in excess of the amount required to neutralize a 3-mg. citric acid reaction mixture in order to precipitate completely the manganese as manganous hydroxide. The resulting polarogram, after proper preparation of the test solution, is shown in Figure 1 (curve labeled 3); the calculated results are given in Tab!e 11, series D for 3 mg. The values obtained agree well with those for solutions in which the manganous wave was present, indicating that identical results are obtained for the i d of the ketone wave whether the manganous m-ave is present or not, providing the proper graphical interpretation of the polarograms is applied.
wave of pentabromoacetone as measured in this investigation was 2.18 for concentrations representing 5 mg. of citric acid or less. However, where i d / C is a constant or reproducible value, such a comparison is not necessarily required to obtain satisfactory results; by calibrating the polarographic procedure with known citric acid samples, no correction factor for the conversion of citric acid to pentabromoacetone is necessary and it is unnecessary to calculate the concentration of pentabromoacetone found. Determination of n-Isocitric Acid. D-Isocitric acid present in plant and animal tissue-for example, in organic acid metabolism study-can be determined by the polarographic citric acid procedure after enzymatic conversion of the D-isocitric acid to citric acid as described by Hargreaves, Abrahamp, and Vickery (2) and the references cited by them. EVALU4TION OF T H E POLAROGRAPHIC METHOD
The polarographic modification of the Krebs and Eggleston (1, 6, 7 ) method for the determination of citric acid offers two
major advantages over the modification of Hargreaves, Abrahams, and Vickery (3). The tedious and time-consuming extraction and subsequent iodate procedure phases of the latter modification are eliminated. The polarographic determination, starting with a sample containing citric acid or an aliquot part thereof, can be completed in about 1 hour, of which time three 10-minute periods (standing during oxidation, standing after stock solution preparation, and degassing period) do not require the attention of the analyst. The second advantage is that the results of the determination may be obtained quickly and easily by reference to a calibration curve, eliminating the necessity for corrections involving the percentage conversion of citric acid to pentabromoacetone (the calibration automatically accounts for this correction) and the blank determinations required in the iodate procedure (9). The precision of the polarographic method is of the order of 2 to 375, as compared with the 1 to 2% of the earlier method. The error in the polarographic determination may be minimized by taking suitable aliquots (representing 1 to 5 mg. of citric acid, the range in which the calibration curve is linear) and by running duplicate determinations; it is standard practice to titrate three aliquots in titration measurement ( 3 ) . There is little likelihood of interference with the polarographic method due to the presence of other reducible organic compounds, since any compound surviving the sulfuric acid treatment and subsequent oxidation procedure would have to give a polarographic wave in the range of - 1.0 to - 1.4 volts versus saturated calomel electrode in order to produce interference. ACKNOWLEDGMENT
The authors wish to express their gratitude to the U. S. Btomic Energy Commission for a grant which supported the research described. LITERATURE CITED
AKALYTICAL PROCEDURE FOR CITRIC ACID
(1)
Eggleston, L. V., and Krebs, H. V., Biochem. J . (London),45, 578
The recommended procedure for the polarographic determination of citric acid is as follows:
(2)
Hargreaves, C. A., 11, .4brahams, M. D., and Vickery, H. B.,
(3)
Jackson, C. L., and Adams, R., J . A m . C h e m
Prepare a polarographic test solution, starting with a solution containing from 1 t o 5 mg. (optimum range) of citric acid, according to the procedure prescribed under citric acid conversion procedure. Record the polarogram from -0.9 t o - 1.6 volts versus the saturated calomel electrode and determine the height of the first (pentabromoacetone) wave obtained, applying the graphical construction described. From the wave height obtained, determine the amount of citric acid in the original aliquot by reference to a calibration curve previously prepared for known amounts of citric acid.
It is standard polarographic practice to compare results obtained for different capillaries on the basis of diffusion current constant ( I )values ( I = id/Cmz/3t”6); the I value for the third
(1949). ANAL.
CHEM.,23, 467 (1951).
Soc., 37, 2533
(1915).
(4) Kolthoff, I. M., and Lingane, J. J., “Polarography,” Sew York, (5)
Interscience Publishers, 1946. Komyathy, J. C., Malloy, F., and Elving. P. J., A h - a ~ .CHEU.,
(6)
Krebs, H. A., and Eggleston, H. V.,Biochern. J . (London), 37,
24, 431 (1952). 334 (1943). (7) I b i d . , 38, 426 (1944).
(8) Pucher, G. W., Vickery, H. B., and Leavenworth, C. S., IND. ENG.CHEM., ANAL. ED.,6, 190 (1934). (9) Sendroy. J., J . Bid. Chem., 120, 335, 405 (1937). RECEIVED for review July 23, 1953 Accepted November 9, 1953. 4bstracted from a thesis submitted by Robert E. Van Btta as part of the requirements for the Ph.D. degree, The Pennsylvania State College, 1952.
