Determination of Malic, Tartaric, and Citric Acids in Fruit by Ion

Determination of Panthenol and Pantothenates in Multivitamin Preparations. Morton. Schmall and E. G. Wollish. Analytical Chemistry 1957 29 (10), 1509-...
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V O L U M E 25, N O . 11, N O V E M B E R

1953

with bitartrate, of ferric ion by phosphate and phosphoric acid, and of titanium by metaphosphoric acid. The influence of c o p per ixi the iodine titration was eliminated by the addition of citrate. Tribalat (6) precipitated perrhenate from alkaline solution to separate it from molybdate. The use of fluoride as a complexing agent is restricted because of the l o v solubility of the arsonium, phosphonium, and stibonium salts. Although the borofluoride ion, B R - , is quantitatively precipitated, an attempt to use this as a method for fluoride failed because of the rapid hydrolysis of this ion, SUhIMARY

Tetraphenylphosphonium chloride precipitates quantitatively, in the presence of sodium chloride, complex chloride salts of tin, cadmium, and mercury such as [(C6H&PI2SnC16. These cannot be weighed as such, because sodium chloride must be used in the wash solution. The excess of reagent may be titrated potentiometrically with standard iodine to form (CnHJdPI?. Tetraphenylphosphonium permanganate, perrhenate, and perchlorate are insoluble i n cold water and therefore both gravimetric and titrimetric methods are possible. For periodate the titrimetric method only can be used. Tetraphenylstibonium chloride may be used for the gravi-

1637 metric determination of permanganate and perchlorate, but in the latter case the low solubility of the reagent makes the method impractical. The salts are soluble in chloroform. The average error under recommended conditions is about zkO.06 mg. LITERATURE CITED

Affsprung, H. E., Barney, S . A., and Potrate, H. b.,AXAL. CHEM.,23, 1680 (1951). Bode, H., 2.anal. Chem., 133, 95 (1951). Ibid., 134, 100 (1951). Potratz, H. A., and Rosen, J. M., ANAL.CHEM.,21, 1276 (1949). Smith, W. T., Jr., Ibid., 20, 937 (1948). Tribalat, S., Ann. Chim., 4, 289 (1949); Anal. Chim. Acta, 3, 113 (1981). Tribalat, S., and Beydon, J., Ibid., 6, 96 (1952). Willard, H. H., Perkins, L. R., and Blicke, F. F., J . Am. Chem. SOC.,70, 737 (1948). Willard, H. H., and Smith, T. LI., IXD.EXG.CHEM..A N ~ LED., . 11, 186, 269, 305 (1939). RECEIVED for review May 15, 1953. Accepted August 10, 1953. From a thesis submitted by Lowell R. Perkins t o t h e Graduate School of t h e Unirersity of Michigan in partial fulfillment of the requirements for t h e degree of doctor of philosophy.

Determination of Malic, Tartaric, and Citric Acids in Fruit by Ion Exchange Chromatography HENRY H. SCHENKER' AND WILLIAM RIEMA.\: I11 School of C h e m i s t r y , Rutgers University, New Brunswick, N. J . The methods for the determination of malic, tartaric, and citric acids in fruit are very lengthy. This paper describes a procedure in which the acids are separated by ion exchange chromatography and then determined by oxidation with permanganate. All three acids can be determined in a fruit in 8 hours by the proposed method, and the error in each determination is usually ahout *0.1 mg. of acid. The sample may contain a maximum of 24 mg. of any one acid. The method is applicable to fruit, fruit juices, and fruit prodncts.

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111: methods gene] ally used for the determination of malic, taitaric, and citric acids in fruit are those described by the .lssoc*iation of Official Agricultural Chemists (1). These proceclures are very time-consuming. TT'ieland and Feld ( 5 ) suggested the use of paper chromatography for the separation and determination of the fruit acids; relative errors of about *lo% nre frequently incurred by this method. Isherwood ( 3 ) applied pnrtition chromatography to this prohleni by a procedure that involves a tedious extraction and then concentration of the fruit : i d s . These steps alone take 48 hours and fail to evtract the ncids qunntitatively. RECORIMENDEI) PROCEDURES

Reagents. ,411 reagents were of reagent grade. Eluant A was 0.0800 J f with sodium nitrate, 0.0013 .If with sodium t,etraborat,e, and 0.30 -21 with boric acid. Eluant B was 0.1G00 .lf with sodium nit,rate and had the same concentrations of sodium tetraborate and boric acid as eluant A. Both eluants viere prepared by weighing the required amounts of solutes (on an analytical balance i n the cases of sodium nitrate and tetraborate) and diluting ill a volumetric flask. The pH lyas then checked with a Beckman pH meter and, if necessary. was adjucted to 6.15 by the dropwise addition of 1 -1fsodium hydroxide. 1 Present address, Carothers Research Laboratory, Experiment Station, E . I , d u P o n t de Semours & C o , , Kilmington. Del.

Potassium permanganate, about 0 1 .\; was prepaied and standardized by the usual method. Standard oxalic acid of exactly the same normality as the permanganate was prepared. The normality was checked by titration with the permanganate. Sulfuric acid, 9 M , was prepared. Apparatus. A glass tube 3.8 sq. em. in cross-sectional area, provided with a sintered-glass filter disk of medium porosity, was filled t o a depth of 25 em. with Dowex-1, a strong-base anion exchange resin. The flow rate through this column waa controlled by a Hoffman clamp attached to a piece of rubber tubing fitted to the lower end of the filter tube. Excessively fine particles of resin had previous1 been removed by decantation, As the resin was received in the cxloride form, it was converted to the nitrate form by passing 0.5 M sodium nitrate through the column until the effluent gave a negative test for chloride. Then 1 liter of eluant -4was passed through the column. -1constant-temperature bath was maintained a t 30" zk 1'. Preparation of Juice. The juice was obtained by pressing or squeezing the fruit and was centrifuged and filtered through Whatman KO.41 paper. One milliliter of the juice was then titrated with 0.1 S sodium hydroxide to find the total concentration of fruit acid as a guide to the selection of the proper size of sample for the elution. Suffirient solid boric acid was added to the juice to give a 0.30 solution. The juice was stored in a refrigerator. Procedure for Elution. The column \vas prepared for use by the passage of 500 ml. of eluant A. A sample of 5 ml. or less, containing not over 24 mg. of any one acid, was pipetted into the column, and the inside w A l was rinsed with a small amount of eluant A. The column was drained until the meniscus just reached the resin. Then 627 ml. of eluant A were passed

ANALYTICAL CHEMISTRY

1638

DISCUSSION

Table I. sample SO.

1

2 3

.? 6 7 8 9 10 3Iean

Analyses of Known Mixtures of Malic, Tartaric, and Citric Acids

Malic Acid, N g . Taken Found Error 17.4 17.4 0 0 -0 5 16.8 16.3 16.2 16.0 -0 2 16.1 15,s -0 3 2.4 2.4 0 0 -0 1 15.4 15.3 -0 2 16.0 15.8 0.5 0.5 0 0 14.9 14.8 -0 1 18 4 18.4 0 0 -0 1

Tartaric Acid, h l g . Taken ~ ~ u n E~~~~ d 20.0 20.3 +0.3 22.5 22.8 t0.3 20.6 +O. 3 20.9 23.0 0.0 23 0 21.3 21.3 0.0 2.4 2.3 +o. 1 21.8 21.8 0.0 21.8 21.8 0.0 0.7 0 8 +o. 1 22.8 22.7 +o. 1 +o 1

Citric bcid, M g . Taken Found Error 18, o, 18.5 17.1 17.0 -0.1 17.3 -0.2 17.5 17.8 -0.1 17.4 19.0 19.8 2.6 2.4 -0.2 19.9 19.8 -0.1 18.7 -0.1 18.8 0.7 o,6 -0.1

through the column a t a flow rate of 0.8 em. or 3 ml. per minute. Then, without permitting the column to drain, 500 ml. of eluant B were passed through a t the same rate. The first 377 ml. of eluate were collected in a graduated cylinder and discarded. The next three fractions of 250 ml. each were collected in volumetric flasks. Flask 1 contained all the malic acid; flask 2 contained all the tartaric acid; and flask 3 contained all the citric acid. Thus each acid was separated from the others and collected in a solution readv for the determination with permanganate. Determination of Malic, Tartaric, and Citric Acids. Twentyfive milliliters of solution were aiDetted from each flask into separate 150-ml. beakers and laced in the constant-temperature bath a t 30" for 10 minutes. $hen 25 ml. of standard permanganate, also at 30 ', were pipetted into each beaker, and 10 ml. of 9 Jf sulfuric acid were added. Each beaker remained in the constant-temperature bath until oxidation was complete, 90 minutes for malic acid, 105 minutes for tartaric acid, and 60 minutes for citric acid. Thus each acid was ovidized completely to carbon dioxide and water. Then 25 ml. of standard oxalic acid were pipetted into each beaker. The solutions were heated to 60" and titrated with standard permanganate from a 5-ml. buret. Thus the amount of permanganate used in the final titration was equal to the amount used to oxidize the fruit acid. Three aliquots from each elution fraction xere generally analyzed in this manner. RESULTS

Preliminary Elutions. Over 100 elutious viere performed before the recommended conditions

were selected. These preliminary elutiorls were done by analyzing small fractions (11 ml.) of the eluate either qualitatively or quantitatively to find the total fraction tvithin which any one acid was contained (4). Dowes-1 (100- to 200mesh) was found to be superior to Dowes-2, .iinberlite IRA-100, rlmberlite IRA-410, and hmberlite IR-4B for this separation. Sodium nitrate was more satisfactory than the chloride, ____ but neither eluant alone was able to separate the three fruit acids. Attempts to achieve the separation by control of p H met with partial success. Akta pH below 3, malic acid could be separated from tartaric. and citric acids, whereas a pH of 9.00 gave a separation of citric acid from malic and tartaric. However, there is no pH at which a noncomplesing eluant citn separare all three acids. S e s t , the effect of metals in the eluant as complesing agents was tried. Cadmium, aluminum, and nickel failed in t,his role. Finally, boric acid was tried as a complexing agent and led to the recommended method. The addition of sodium borate enabled the boric acid to serve as both complesing agent and buffer. pH of 6.15 proved to be most satisfactory for the separation. A concentration of 0.0800 -11 sodium nitrate (eluant -4) was selected when it was found that 0.1000 Jf or more concentrated sodium nitrate failed to separate malic from tartaric acid. However, if only eluant A is used, removal of citric acid from the column would require 3050 ml. of this eluant, and the elution mould last 17 hours. A considerable saving of reagents and time is gained by switching to eluant B as soon as the malic acid is removed from the column, as in the recommended procedure. Flon- rat,es less than the recommended value have little effect on the elution esrept to make it more time-consuming. Incomplete separation of malic and tartaric acids results from greater flow rates. If the sample contains more than 21 mg. of any one arid, especially malic or tartaric acid, the separation is likely to be incomplete. Figure 1 shows a typical elution graph obtained under the recommended conditions. The proposed procedure determines, of c'ourse, the t'otal quantity of any given acid, both free acid and salt. For this reason, the sum of the normalities of the three acids is always slightly greater than the normality of acid determined by titration with sodium hydroxide.

ii::

-:,;

Table I shows the results of the analyses of ten knoir n mixtures of the fruit acids. In each case, the weighed acids were dissolved in 5 ml. of eluant .4, transferred to the column. and treated as described above. Table I1 gives the results of three analyses of one specimen of lemon juice. Analysis 1 revealed the presence of only citric acid, as reported by other investigators (6). Known aeights of malic and tartaric acids %ere added to the second and third samples. The results indicate excellent recoveries of the added acids as well as high precision in the determinations of citric acid. Table I11 shows the results of tKo analyses of the juice of Concord grapes. Good precision and accurate recovery of added citric acid are seen. Table 11. Analyses of 0,300-311. Samples of Lemon Juice Two juices from t n o different hatches of freestone peaches Tartaric Acid, h k . citric Acid .inalysip Malic Acid, XIg. Found, Rlg. Added Found Added Found NO. were analyzed (Table IT-). The results indicate again good precision and accurate recovery of added acids. Furthermore, tartaric acid was found in both hatches of peaches, although this acid has not been previously found in peaches. This is not surprising, because the concentration of tartaric acid in peaches is too small to be determined or detected by the classical procedure Table 111. Analyses of 5.00-311. Saniples of Grape Juice ( 1 ) . I t is also possible that some other acid in the peach, such as .inalysis Malic Acid Tartaric . h i d Citric Acid. llg. S O . Found, M g . Found, L l g . .Idded Found I n juice galacturonir, polvgalacturonic, or gluconic, was eluted in the 1 10.6 19.6 0.0 0.8 0.8 third fraction and therefore mistaken for tartaric acid, a s the 2 10.8 19.4 1.2 1.9 0.7 authors' investigation did not include a study of these acids. Finally, 3.00 ml. of the grape juice and Table IV. Analyses of 3.00-Ml. Samples of Peach Juice 3.00 ml. of specimen 2 of peach juice were Tartaric Acid, hfg. Malic Acid. hIg. Citric Acid Specimen .Inalysis SO. No. .Idded Found In juice .Idded Found I n juice Found, Jfg. mixed and analyzed. Table V reveals that good agreement was obtained he1 1 0.0 7.9 7.9 0.0 2 3 2.3 20.4 1 2 7 8 155 7 7 133 157 2 4 20.5 tween the quantities of acids found and > 3 0 0 6 7 6.7 0.0 0.6 0.6 9 0 the quantities expected from the results 7 4 0 0 6.9 6.9 1 , 2.3 0 6 9.0 .~ ~ ~ _ _ ~ Tahles I11 and IV. ___-_____._~~ ~

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~

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V O L U M E 2 5 , NO. 11, N O V E M B E R 1 9 5 3 Determination of Fruit Acids by Oxidation. Under the iecommended conditions of oxidation, the fruit acids react according to the following half-equations.

+ 3H20 + 12H+ + 12e + 2H2O +.4COs + 10Hf + 10e + 5H20 6C02 + 18H+ + 18e

HzC4H405 H2C4HlOs H3C~Hb07

+4 2 0 2

1639 error in determination of comparable quantities by the proposed method is about 0.1 mg. Furthermore, the proposed method is much more rapid. The classical procedure for the determination of any one of the major fruit acids requires about 2 days; but all three acids can be determined by the proposed method in 7 . 5 hours, during only 3 of which the operator's attention ip required.

-+

Thus the equivalent weights of malic, tartaric, and citric acids of the molecular weights, respectively. are 1/12, l/10, and A large excess of permanganate is required to oxidize the fruit acids quantitatively under the recommended conditions. Therefore, it was necessary to take a one-tenth aliquot of each fraction. Thus the minimum ratio of permanganate to fruit acid in terms of equivalents was 11, and quantitative oxidation occurred in the specified times in the 30' bath. Longer periods are not desirable because of possible decomposition of the permanganate. The periods required for quantitative oxidation in a 25" bath are 120 minutes for malic acid, 165 for tartaric, and 60 for citric. The temperature of the eolution rises sonieivhat upon the addition of 9 JI sulfuric acid, but no steps are required to cool the mixture except to keep it in the bath.

3.0- ELUTION OF MALIC, TARTARIC,

Table V. Analysis of AIixture of Grape and Peach Juices Malic Acid, Mg. 6 4 6.8

I n 3.00 nil. of grape juice In 3.00 ml. of peach juice Total expected Found Difference

Table VI.

CITRIC ACID

13.2 13.3

Tartaric d c i d . Jlg. 11.7 0.6 13.3 12.4

+o. 1

TO.1

Citric .icid, JIg. 0 3 9 0 9.5 9.7 t o .2

Elution of Other Fruit Constitueiits Constituent

111. of Eluate Containing Constituent

Common s u ar, Succinic aci% .' Lactic acid Malic acid Tartaric acid Oxalic acid Citric acid

23- 91 91- 148 68- 240 383- 610 645- 793 690- 8 9 0 907-1100

F I G U R E I.

A minor disadvantage stems from the nonuniformity of commercial ion exchange resins. A different batch of Dowex-1 (100to 200-mesh) might have slightly different properties and hence require slight changes in the elution conditions. It is advisable, therefore, to test each column of Dowex-1 before using it for this determination by running separate elutions of known quantities of malic, tartaric, and citric acid. In each rase, small fractions of the eluate should be analyzed in order to determine the limits of eluate within which each acid is contained. ACKNOWLEDGMENT

VOLUME OF ELUATE, MI-. The oxidimetric method of determining the isolated fluit u i d s a a s tested by dissolving weighed quantities of a fruit acid in 250 ml. of the appropriate eluant and then applying the recommended procedure. The average error for each acid was thus found to be f 0 . 1 mg. Other Constituents of Fruit Juice. An error would be incurred in the analysis, of course, if any other reducing agent in the juice appeared in the second, third, or fourth fraction of the eluate along with the malic, taitaric, or citric acid. To investigate this point, small fractions of eluate were examined for the presence of several other possible fruit constituents, in order to find the total fraction of eluate within which the constituent was eluted under the recommended conditions. The results, summarized in Table VI, indicate that oxalic acid is the only constituent of fruit juice that is likely to interfere and that it would introduce an error in the determination of tartaric acid only. This is not serious, because oxalic acid is seldom present in fruit (6) and never in excess of 0.03% (equivalent to 0.1 mg. of tartaric acid per ml. of juice). Furthermore, oxalic acid can be distinguished from tartaric acid by the difference in the velocity of its reaction with permanganate. Advantages. The proposed method is much more accurate than the classical procedure-for example, a collaborative study ( 2 ) indicated an average error of 3 mg. in the determination of tartaric acid in apple jelly by the traditional method, whereas the

The authors gratefully express their indebtedness to the Rutgers Research Council for financial support during this investigntion and to R. C. Dell for assistance in the study of the effect of pH. LITERATURE CITED

(1) Assoc. Official dgr. Chemists, "Offic. Methods of Analysis,"

7th ed., Washington, D. C., 1950. (2) Hai.tman, B. G., J . Assoc. Ofic. Agr. Chemists, 14, 459 (1931). (3) Isherwood, F. A , , Biochem. J . , 40, 688 (1946). (4) Schenker, H. H., doctor's thesis, Rutgers University, 1952. (5) W e l a n d , T., and Feld, U., 2. angew. Chem., 63, 258 (1951). (6) Winton, A. L., and Winton, K. B., "Structure and Composition of Foods," T'ol. 11, Chap. 11, New York, John W'iley R- Sons,

1935. RECEIVED for reviem January

21, 1953

A c r e y t e d August 18, 1933

Colorimetric Microdetermination of Boron by the Curcumin-Acetone Solution Method-Correction I n the article on "Oolorimetric Microdetermination of Boron by the Curcumin-Acetone Solution Method" [Silverman. Louis, and Trego, Katherine, ANAL.CHEX, 25, 1264 (1953)l a t the bottom of the f i s t column on page 1265 the last sentence under "Stock Boron Solution" should read: One milliliter equals 0.1 mg. of boron.

LOWS SILVERMAN