V O L U M E 2 3 , NO. 3, M A R C H 1 9 5 1 (2) Clift, G. D., and Fedoroff, B. T., “Manual for Explosives Laboratories,” Vol. 1, p. 19, Philadelphia, Lefax Society, Inc., 1942. (3) Dennis, L. hl., and Nichols, M. L., “Gas Analysis,” revised ed., p. 222, A-ew York, Macmillan Co., 1929. (4) Fischer, R. A., “Statistical Methods for Research Workers,” 10th ed., Edinburgh, Oliver and Boyd, 1946. (5) Giauque, W. F., and Kemp, J. D., J . Chem. Phys., 6, 40-52 (1938). (6) Guye, J . -4111.Chem. Soc.. 30, 155 (1908). (7) Johnston and Giauque, I b i d . , 51, 3194 (1929). (8) Kieselbach, Richard, IXD. ENG.CHEM.,S N ~ED., L . 16, 766-71 (1944). (9) Klemenec, A., and h’euman, K., Monatsh., 70, 273-5 (1937).
467 (10) Lunge and Bed, Z. angew. Chern., 19, 809, 858 (1906); 20, 1714 (1907). (11) Milligan, L. H., J. Phys. Chem., 28, 544-78 (1924-1925). (12) Scott, “Standard Methods of Chemical Analysis,” 5th ed., Vol. 1, pp. 653-5, New York, D. Van Nostrand Co., 1939. (13) Ibid., Vol. 2 , pp. 2418-19. (14) Tower, Z. anorg. Chem., 5 0 , 3 8 2 (1906). (15) Whitnack, G. C.,and Holford, C. J., AXAL.CHEM.,21, 801 (1949). (16) Whittaker, Sprague, and Skolnik, to be published.
RECEIVED July 25, 1950. Presented before the Pittsburgh Conference o n Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., February 15 t o 17. 1950.
Determination of Citric and d-Isocitric Acids CHESTER A. H-ARGREAVES, 11, MARJORIE D. ABRAHAMS, AND HUBERT BR.4DFORD VICKERY Connecticut Agricultural Experiment Station, New Haven, Conn. d-Isocitric acid is one of the components of the series of enzymatic reactions generally referred to as the tricarboxylic acid cycle of Krebs, a mechanism that is frequently advanced as the explanation of respiration in living cells. The substance is thus probably widely, if not uniTersally, distributed, and it is known to occur in substantial quantities in the leaves of certain plants. The Krebs and Eggleston method of determining isocitric acid depends upon the use of aconitase, which converts a definite proportion of the isocitric acid to citric acid, which is in turn oxidized and brominated to pentabromo-
S
EVER.41, fundamentally different methods for the determina-
tion of d-isocitric acid have been described (6, 8, 10). Of these, the method of Krebs and Eggleston, which depends upon t h e conversion of d-isocitric acid t o citric acid by the enzyme aconitase, has advantages t h a t commend it for use in the study of organic acid metabolism in plant tissues. Under the action of this enzyme, an equilibrium is established such that the ratio of citriccis-aconitic-d-isocitric acids a t 38’ C. is 89.5-3.9-6.6y0 ( 3 , 5 ) . Accordingly, it suffices to determine citric acid before and after .treatment of samples n i t h the enzyme; from the increase in citric acid, the sum of isocitric and &-aconitic acids can be calculated. Because cis-aconitic acid does not appear to be present in significant quantities in the plant tissues with which the authors have been concerned, the method may be used for the determination of isocitric acid in them. I n the course of a study of the Krebs and Eggleston (6) method for isocitric acid, a number of observations have been made t h a t throw light upon certain of the hitherto imperfectly understood details of the pentabromoacetone procedure for the determination of citric acid. -4s a result, modifications have been made of the method of Pucher, Vickery, and Leavenworth ( 1 5 )of determining citric acid which increase both its convenience and accuracy. T h a t the earlier titrimetric (12, 15) and colorimetric (14) methods leave something to be desired is obvious from a number of papers that describe modifications of one or another of the procedures ( 4 , 6, 9, 17, 19-61). The critical points appear t o be the acidity at which the oxidation is conducted, the rate of addition of the permanganate, the temperature, the time allowed for the oxidation, and the precise nature of the oxidizing agent. Under the conditions described by Pucher, Vickery, and Leavenworth, the yield of pentabromoacetone was close t o 90% of theory. Goldberg and Bernheim ( 4 ) showed t h a t the yield is a function of the acidity a t which the oxidation is carried out, ranging from about 90% in 1 N sulfuric acid to about 105%, owing to the formation of some hexabromoacetone, in 9 N acid, but diminish-
acetone. It has now been found that if metaphosphoric acid is present during the oxidation of citric acid, the conversion can be made essentially quantitative and that considerable latitude is then permissible in the conditions, such as acidity, time, temperature, etc., which it has hitherto been necessary to control with care. A marked improvement in the pentabromoacetone method of determining citric acid and, accordingly, of isocitric acid has thus been effected, the precision being now of the order of 1 to 2%. The way is thus opened for the study of the metabolism of isocitric acid in plants. ing a t still higher acidities. Approximately quantitative results were obtained in 4 t o 5 N acid. Similar observations were made by Taussky and Shorr (19) as well as in the present work. It was noted, in the course of oxidations of solutions of citric acid carried out after treatment of mixtures of citric and isocitric acids with aconitase and subsequent deproteinization with metaphosphoric acid, that the recoveries of citric acid as pentabromoacetone were invariably close t o 98y0 of theory. This vvas true even when the acidity was 1 or somewhat less, conditions that led to only S9% recovery with the usual oxidation procedure before treatment with aconitase. Furthermore, the appearance of these solutions during Oxidation differed, inasmuch as there was no development of turbidity nor formation of a brownish color owing to the separation of manganese dioxide; the color of the clear solution remained that of permanganate. hlore careful observation of the oxidation a t high acidities showed that the solutions also remained clear a t or above an acidity of about 4 -V. The difference in behavior between solutions before and after the treatment x i t h aconitase was traced to the presence of metaphosphoric acid. This reagent appears t o form complex compounds such that, if it is present, no precipitation of manganese dioxide takes place. The system is therefore homogeneous during the oxidation reaction and the end result is the formation of pentabromoacetone in essentially quantitative yield. The desirability of a homogeneous system is also illustrated by the behavior of solutions oxidized a t high acidity, which likewise give high yields. Accordingly, the conditions under which the oxidation and bromination are carried out were modified by adding a sufficient amount of metaphosphoric acid to prevent the separation of manganese dioxide. It was then found that a system had been developed that was no longer sensitive t o a number of the conditions, the rigid control of which had been regarded as essential by some workers with this method. As is clear from Table I, there was very little effect if the rate of addition of the permanganate were varied over a wide range; on the contrary, it seemed desirable t o
ANALYTICAL CHEMISTRY
468 add an excess all at once from a rapid pipet, and stirring was not necessary. Constant recowries of 98% were obtained at acidities from 1 N to 2 N , so that only approximate adjustment of the a(-idity is essential; a t higher acidities, the formation of hexabromoacetone appears to be increasingly stimulated. The time required for the reaction may be varied from 3 to 15 minutes, the cluantity of citric acid present is immaterial within reasonable limits, and the temperature may be a t any convenient point below 20" C., although it should not be allowed to exceed this more than a degree or so. On the other hand, in the absence of metaphosphoric acid, the extent of the reaction is greatly affected b j the rate of addition of the permanganate.
Table I.
Yield of Pentabromoacetone from Citric .4cid
(After oxidation with permanganate in presence of bromide, expressed as percentage of theorya) KO. of Yield, Variable Studied Detns. % 5 Time of addition of 5 ml. of KMnOd, seconds 18 88.8 (solution stirred, metaphosphoric acid absent) 45 25 94.1 99.5 90 4 Volume of 20% metaphosphoric acid, ml. (stir0 18 88.8 ring not necessary) 2 96.2 0.4 97.4 0.6 2 4 98.2 1.0 2.0 2 98.4 Time of addition of 5 ml. KhlnOl. seconds ( 1 . 0 7 42 98.0 nil. of metaphosphoric acid added) 2 30 97.4 45 2 97.8 2 60 97.6 2 90 97.6 600 2 98.2 Time allowed for oxidation t o proceed, minutes 1 2 94.8 (metaphosphoric acid added) 3 1 98.8 2 5 98.7 10 98.3 3 15 2 98.3 30 2 44.5 60 2 90.2 Concentration of HtSOn during oxidation, nor0.5 2 96.4 mality (met,aphosphoric acid added) 1.0 2 98.0 1.5 2 98.0 2.0 2 98.2 100.6 3.0 1 5.0 2 102,2 2 8.0 106.8 2 10.0 103.2 2 14.0 64.0 0.5 4 Quantity of citric acid taken, mg. (metaphos98.2 phoric acid added) 1 10 97.9 2 24 98.0 4 3 98.3 Temperature during oxidation, C . 3 -4 1 97.6 18-19 2 97.8 33-35 2 89.5 45-50 2 i5.a One variable was examined a t a time in each group of tests. Except in first group, Oxidation mixture was not Rtirred after addition of permanganate.
The other important modification of the original titrimetric method is the use of the Sendroy silver iodate procedure (18) for the determination of the bromide liberated by the sodium sulfide. (For the suggestion of this technique, the authors are indebted to the late G. W. Pucher, who, a t the time of his death, was engaged in preliminary tests of the applicability of the Sendroy method.) This change contributes greatly to the convenience and precision of the titration and makes it possible to extend the scale of the method to the microgram range if desired. REAGENTS
Sulfuric acid, 18 N (15); potassium permanganate, 1.5 .V ( 1 5 ) , potassium bromide, 1 M ( 1 5 ) ; phosphoric acid, 0.85 and 0.085 M (18). Phosphoric acid, 2.0 M . 136 ml. of concentrated phosphoric acid (specific gravity 1.7) made t o 1000 ml. with water. Nitric acid, 1.0 N . 32 ml. of concentrated nitric acid (specific gravity 1.42) made to 500 ml.; 0.05 h' obtained by twentyfold dilution. Sodium thiosulfate, 0.1 N stock solution. 24.82 grams of pentahydrate made to 1000 ml. with water and 5 ml. of chloro-
form added; 0.01 N reagent obtained by tenfold dilution of stock solution and standardized against 0.01 N potassium iodate daily. Potassium iodate, 0.1 N . 3.5569 grams of dried salt made to 1000 ml. with water; 0.01 N obtained by tenfold dilution. Potassium chloride, 12.5 millimoles per liter. 0.9319 gram of dried salt made to 1000 ml. with 0.085 M phosphoric acid. Sodium zulfide, 4% solution of the nonahydrate made in 250ml. lots ani1 kept refrigerated Then not in use; stable for a few weks. Metaphosphoric acid, 20%. 20 grams of analvtical reagent grade in 100 ml. of water -prepared a t room temperature and kept refrigerated. Phosphate buffer, pH i . 4 , 0.1 .M,prepared according to Clark (, 2,) . Hydrogen peroxide, 3%. Commercial solution kept refrigerated. Petroleum ether, boiling_ point 30" to 60" C. Analvtical . reagent grade. Sodium or potassium iodide, 10%. Prepared in 150-ml. quantities as needed and kept refrigerated; discarded when iodine can be detected by a test with starch indicator. Silver iodate. Prepared as dry powder according to directions of Sendroy (18) and stored in amber glass-stoppered bottle in desiccator. Starch indicator. Prepared according to Peters and Van dlyke (11).
Filter paper, chloride-free. 9-cm. Whatman No. 3 washed completely free from chloride with boiling water, dried, and stored in a closed container. Frozen beef heart. A fresh beef heart is trimmed of fat, cut into three or four pieces, and stored in a deep freeze unit. The necessary quantity of tissue is obtained by shredding the frozen m u d e on a household grater; the aconitase remains active for several months. PROCEDURE
Preparation of Solution. Extracts suitable for the deterniination of citric acid in plant tissues are prepared as directed by Pucher, Wakeman, and Vickery (16). To suitable ali uots, 2 ml. of 18 A' sulfuric acid are added and the solution, diquted to about 20 ml., is boiled for 5 minutes. After being cooled and filtered (most conveniently through a Gooch crucible into a beaker placed in a vacuum apparatus), 1 ml. of 20% metaphosphoric acid is added and the solution is diluted to 35 ml. and oxidized. Animal tissue extracts are deproteinized with 20% metaphosphoric acid according to the procedure of Krebs and Eggleston (6) and treated similarly, save that additional metaphosphoric acid is not needed. Oxidation and Bromination. To the solution at a temperatulc not exceeding 22" C. are added 2 ml. of potassium bromide and 5 ml. of potassium permanganate; after about 10 minutes without &ring, the temperature is brought to about 10" C. by means of an ice bath, and the color is discharged by the rapid dropwise addition of ice-cold hydrogen peroxide with stirring. The solu-' tion is transferred to a 125-ml. pear-shaped separatory funnel and the heaker is rinsed with several small portions of petroleum ether totaling approximately 25 ml. Great difficulty has been experienced in obtaining 125-ml. separatorv funnels suitable for this o-Deration. Funnels with outlef tubes bf 8-mm. inside diameter a i d about 70 mm. long are required so that the aqueous phase will drain freely. I t is usually essential to grind the stoppers to a perfect fit with the finest abrasive, if errors from the loss of petroleum ether are to he avoided. A trace of high grease is used for the stopcock but - grade not for the stopper. Isolation and Dehaloaenation of Pentabromoacetone. The separatory funnel is shaxen vigorously for a t least 30 seconds. Stubborn emulsions occasionally formed with animal tissue extracts can be broken by the addition of a few drops of 27& aqueous solution of turkey red oil. The aqueous phase is drawn off quantitatively (a second extraction is unnecessary) and the ether is washed four times with 3-ml. portions of water to remove all halide ion. The pentabromoacetone is then decomposed by shaking the ether briefly with two successive 3-ml. portions of sodium sulfide, the colored solution being drained each time into a 25-ml. volumetric flask. The ether is washed with 2-ml. portions of water until no further color is removed, two washings being usually sufficient. The washings are drained into the same flask and 2 ml. of 2.0 M phosphoric acid are added. The solution is then boiled gently on a hot plate for 5 t'o 6 minutes. A few tiny chips of quartz previously extracted with acid and thoroughly washed are added just before heating the solution (not before adding the acid!) in order to facilitate smooth boiling. The flask is theu cooled, exactly 5.00 ml. of 12.5 millimoles per liter of potassium chloride are added, and the solution is made to volume.
V O L U M E 23, NO. 3, M A R C H 1951
469
Titration of Halide. The contents of the flask are poured without rinsing into a 50-ml. conical flask which contains 0.25 gram of dry silver iodate previously measured into it with a glass spoon made for the purpose. The flask is stoppered and shaken on a platform shaking machine for 5 minutes and the suspension is poured through a dry chloride-free filter paper in a funnel. Suitable aliquots of the clear filtrate (usually 2 or 5 ml.) are at once pipetted into 100-ml. test tubes and 2 drops of 0.085 M phosphoric acid and 1 ml. of 10% sodium or potassium iodide are added. After approximately 5 minutes, the liberated iodine is titrated with 0.01 N thiosulfate by the customary technique, using itarch ae indicator. I t is standard practice to titrate three aliquots.
Table 11.
Recovery of Bromide from I'entabromoacetone
and of Citric Acid" r'itric Acid Taken Alf
Q
BromidP Found
Y o . of
Detns.
.
3.213 2.154 2,106 2.093 2.056 1.077 1.063 1 047 0 ,5265
%
.MQ.
98.30 98.13 98.01 98.03 97.83 97.48 97.93 97.88 98.24
2.157 2.104 2,094 2,052 1.071 1,052 1.045 0.527
A v . 98.036
42
Citric Acid Found
* 0 . 3 1R
3.242
Rerover\
"0
DETERMINATION OF ISOCITRIC ACID
100.32 100.51 99.91 100.05 99.81 99.44 99.91 99.86 100.15 O9.99 81
*o
.Average value 98.0'% of theoretical yield of llpntabromoacetone was riped Standard deviation is calculatrd for 1 2 drter-
t o compute citric acid found.
.l tjlank determination on each lot of sodium sulfide solutioii must be run. Six milliliters of sodium sulfide, 5 ml. of wat,cr, 2 nil. of 2 M phosphoric acid, and a few tiny quartz chips in a 25nil. volumetric flask are boiled on the hot plate to expel hydrogen sultide and carried through the subsequent procedure as dcscrilierd. The blank titration obtained includes the value for tht. :~tltledpotassium chloride as well as any halide in the reagent, iind lour or more individual titrations are made and averaged. Calculation. The citric acid equivalent to the titration value i i otitained from the following equations:
'I'ituttion . . value X normality factor X 1000 6 X ml. of aliquot = millimoles of iod:itc (or bromide) per liter ~lilliniolesfound
-
1)
niillimoles i n blank = net, millimoles of iodate (or bromide) per litcr ( 2 )
Set millimoles of iodate per - . ___.______
1itc.r
5 X 40 X 0.980
millimoles of citric acid per 25 nil.
(3)
niillimoles of iodate pl;ter_-x O.O6O(i 0.980 = ing. of citric acid per 25 nil.
(4)
=
St.1
ments of Pucher showed that the solubility c*orrec:tion could Iic neglected under these conditions. Accorclingly, 2.5 millimolex per liter of potassium chloride are added to thr solution after the hydrogen sulfide is expelled. The titration of this chloride along with any halide in the reagents furnishes the blank subtracted from all determinations. Table I1 shows a summary of a s ( 4 w of rxperiments 011 the recovery of citric acid over the range 0.5 to 3.2 mg. Howcver, even smaller quantities can be determinrd if the potassium chloride is omitted and corrections for the solubility of silver iodate as estimated by Sendroy are included in the calculations. The meaii recovery of bromide from the pentabromoacetone \-vas 98.04% of theory with a standard deviation of +0.315 or 0.32%. If this figure is employed in the calculation as shown in the equations, the mean recovery of citric acid is qunntitative.
The factor 0.980 represents the average percentage recovery of pcmtahronioacetone and is supported hy the data in Table 11. I t i.i~st.qupon 42 determinations which had a coefficient of variation o f 0.32%. The factor 0.9606 is one fifth of the molecular weight iif citric acid (as pentabromoacetone gives 5 equivalents of I J l Y J I l l i ( k ) divided hy 40, the ratio of 25 ml. to a liter. Ordinarily. r - i t i i r a acid will be computed in milligrams by Equation 4; howi * v i > rEquation , 3 is required to give the data used for the coniputntioti of isocitric acid described in a later paragraph. The final result is expressed in terms of the tissue by using the factor for the :Iliquot of the tissue extract originally taken. DISCUSSION
Sciidroy has shown that if the iodate ion concentration is less than 3 millimoles per liter, the application of a correction for the solubility of silver iodate is desirable. T o avoid the necessity of employing solubility curves, the conditions in the present prowdure have been adjusted so that t,he solution never contains less than 2.5 millimoles of iodate per liter. The preliminitry esperi-
Procedure. Extracts of plant tissues prepared as described by Pucher, Wakeman, and Vickery (16) contain an excess of alkali and acid is required in addition to the phosphate buffer of p H 7 . 4 to adjust the reaction correctly. A suitable aliquot of the extract is transferred to a 30-mi. beaker together with 5 ml. of p H i . 4 phosphate buffer solution. Roughly 1 gram of grated frozen heart muscle tissue is added and stirred into suspension with a rod, arid t,he mixture is titrated to pH 7.4 with 0.05 N nitric acid using a glass electrode. The volume of nitric acid required is noted. A similar aliquot of the extract is transferred to a 40-1Ill. graduated centrifuge tube, and 5 ml. of phosphate buffer and the amount of 0.05 -V nitric acid determined m above are added; 1 gram of heart muscle tissue is stirred in and the volume is made to 15 ml. with water. After the suspension has been thoroughly mixed, t,he tube is stoppered nit,h a plug of cotton and is incrihated a t 38" t,o 40" C. for 1 hour. To the warm suspension, 8 to 10 ml. of 207, metaphosphoric acid arc added, and the solution is cooled to room temperature, adjusted to a volume of exactly 25 ml. with water, and centrifuged. A tissue blank is treated likewise, water being substituted for t>heestract, and 10-ml. aliqiiots are taken for oxidation. The clear deproteinized solution is decanted arid a suitable aliquot (10 or 5 ml.) is taken for the determination of citric acid by the procedure described, save t,hat it is not necessary to add further metaphosphoric acid nor to boil and filter the solution after the addition of the 18 S sulfuric acid. The tissue blank needs to be detrrmined only occasionally; i t ifi small and remains constant during the period that the heart is usahle. The true blank arising from the muscle tissue is the difference between the tissue blank and the blank on the sodium sulfide solution. I n practice, this difference may be employed in finding tho tissue blank when a new solution of sodium sulfide is p r p pared.
Table 111. Recovery of Bromide from Pentahromoacetone Obtained by Oxidation of Citric 4cid in Presence of Bromide and Heart Muscle Extractives Citrir Acid Taken
N o . of
Detns
%
lfg
2.056 1.977 1.139
Bromide Found
5 4 8
98.03 97.65 98.45
17
Av. 98. 14 +0.43
Citric .kcid Found Mg. 2.0.56 1.970 1.447
Reco>err
9c 100.00 99.62 100 3 . )
ioo.nH
That the recovery of pentabromoacetone is not affected b j iu11dances present in the extract of the heart muscle employed as thtb source of aconitase is evident from the data in Table 111. Thew tests were carried out on the supernatant fluid obtained after incubating samples of heart muscle in the course of the determination of tissue blanks. KOadditional metaphosphoric acid was addrd. The recovery of pentabromoacetone was 98,14% of theory with a standard deviation for 17 experiments of *0.43, n result indistinguishable from that in Table 11. The recovery of ritric acid was also quantitative
ANALYTICAL CHEMISTRY
470 Calculation. The net millimoles per liter of iodate (or bromide) are computed by deducting the tissue blank or, otherwise, the sum of the blank on the sodium sulfide and the true blank arising from the heart tissue, from the millimoles per liter found by titration. Due allowance is made if the tissue blank represents an aliquot different from that used for the determination. The number of millimoles of citric acid per 25 ml. is then obtained from Equation 3. As has been pointed out by Krebs and Eggleston, if this quantity is denoted by X and if Y is the quantity of citric acid in an identical aliquot of the extract before treatment with aconitase and, further, if Z is the combined quantity of disocitric and cis-aconitic acid present originally in the aliquot, one may set up the equation
Y
0.11 + z = x’ + a 9 x
(5)
crystallization of the anhydride from moist ethyl acetate and chloroform as described by Krebs and Eggleston. Analysis of Mixtures of Citric and Isocitric Acid. To illustrate the accuracy of the methods, Table 1’ shows the results of determinations made upon three mixtures of citric and isocitric acid. These %ere single determinations, but were calculated from the results of triplicate titration of aliquote.
Table V. Citric -4cid Taken
.
Mo 0.6455 0.4303 0.8606
Recovery of Citric and d-Isocitric Acid from Mixtures Isocitric Citric Acid Isocitric Acid dcid Found Found Taken
.
Mo 1.013 1.351 0.6753
MQ.
0,646 0,432 0.863
% 100.1 100.4 100.3
Mo. 1.01 1.35 0.670
% 99.7 99.9 99.2
or V
z = l - y 0.89
This equation depends upon the equilibrium relationship among citric, cis-aconitic, and d-isocitric acid in the presence of aconitase. Table I V summarizes a number of observations of the proportion of citric acid present when citric, cis-aconitic, or disocitric acids were individually allowed to reach equilibrium in the presence of the enzyme. The average is 88.9 * 0.55%. Thus the total acidity from these three acids, after equilibration, is made up of citric acid and a quantity of d-isocitric and cisaconitic acid together equal to 11/89ths of the citric acid found.
Table IV. Proportion of Citric Acid Present after Equilibration of Citric, cis-Aconitic, and d-Isocitric Acids with Heart Muscle Aconitase No. of Equilibrium Standard Substrate Detns. Concentration Deviation %
Citric acid &Isocitric acid cis-Aconitic acid
32 24 4 60
89.27 88.71 88.64 85.93
10.59 10.42 *0.32 10.55
If cis-aconitic acid is neglected in the calculation, the values of X and Y in Equation 5 may be expressed in milligrams-that is, the citric acid is calculated from Equation 4 rather than from Equation 3. Determination of Citric Acid a t Equilibrium in Presence of Aconitase. I n order to obtain support for the results of Krebs and Eggleston (3,6) on the position of the equilibrium in the presence of aconitase, carefully purified specimens of each of the acids were prepared and samples were treated with heart muscle as described; citric acid was then determined. The results are summarized in Table IV. The citric acid employed was prepared by- dehydrating analytical reagent material. Titration indicated a purity of 100%. &Isocitric acid was purified by recrystallization ‘of the lactone ( I S ) from chloroform. The lactone was prepared from the dimethyl ester isolated from Bryophyllum leaves and melted a t 156” to 157” C. Hydrolysis of specimens of lactone on the steam bath in the presence of a small excess of 0.1 Y sodium hydroxide for 15 minutes opened the lactone ring and back-titration indicated a purity of 100%. I t was observed, however, that satisfactory standard solutions of isocitrate could not be obtained by alkaline hydrolysis of specimens of the dimethyl ester. Although titration data indicated high or complete purity, such solutions gave low yields of citric acid after incubation with aconitase. This anomaly has not been explained. cis-Aconitic acid anhydride was prepared according to Anschutz and Bertram (1)and obtained in large crystals of melting point 82” C., several degrees higher than was recorded by Malachowski and Maslowski ( 7 ) . Nevertheless, in spite of many recrystallizations from benzene, the anhydride still retained an impurity. A satisfactory specimen of the free acid was secured by
Analysis of Plant Tissues. The results of duplicate determinations of citric acid in 11 samples of tobacco leaf and 10 samples of Bryophyllum leaf were subjected to statistical analysis to obtain an estimate of the precision of the method. The citric acid content ranged from 6 to 111 me. per 100 grams of dry tissue. The standard deviation of a single determination was computed from the 21 differences between duplicates as 0.60 me. per 100 grams. Because of the wide range of the citric acid content of the samples, the expression of the error of a single determination as a percentage of the mean has no great significance; however, the value was *1.2’%. d similar analysis of a group of 15 pairs of duplicate determinations of isocitric acid in samples of Bryophyllum leaf gave the standard deviation of a single determination as 2.8 me. per 100 grams of dry weight. These samples were relatively constant in isocitric acid content, the range being from 131 to 176 me. per 100 grams. The error of a single determination \vas *1.7% of the mean. ACKNOWLEDGMENT
A post doctoral fellowship of the Kational Research Council for the academic year 1948-49 to one of the authors (C. A. H.) is gratefully acknowledged. LITERATURE CITED (1) Anschutr, R., and Bertram, IT., Ber., 37, 3967 (1904). (2) Clark, W. M., “Determination of Hydrogen Ions,’ ‘p. 212, Baltimore, Williams & Wilkins Co., 1928. (3) Eggleston, L. V., and Krebs, H. A, Biochem. J . , 45, 578 (1949). (4) Goldberg, A. S., and Bernheim, A. R., J . B i d . Chem., 156, 33 i19441. (5) Krebs, H. A., and Eggleston, L. V., Biochem. J . , 37, 334 (1943). (6) Ibid., 38,426 (1944). (7) Malachowski, R . , and Maslowski, &I., Ber., 61, 2531 (1928). (8) hlartius, C., and Leonhardt, H., Z . physiol. Chem., 278, 208 (1943). (9) Satelson, S., Lugovoy, J. I
