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IDENTIFICATION OF SMALL AMOUNTS OF ORGANICCOMPOUNDS BY DISTRIBUTION STUDIES

VII. SEPA RATION AND ESTIMATION OF NORMAL FA TTY ACIDSB Y YOSHIO SATO , GUY T. BAR RY,* AND LYMAN C. CRAIG

(From the Labora tories of Th e Roc kefeller Institu te for Med ical Res earch, New York)(Received for publica tion, March 2S , 1947)

The estimation and identification of small amounts of the lower fattyacids have always been a troublesome problem, particularly to the fermen-tation chemist and to the organic chemist carrying out degradative studiesin which these acids are formed as fragments of a larger molecule. Severalways of approaching the problem are at hand, depending on whether ornot unequivocal identification is required. Perhaps the best knownpreliminary approaches have been through the use of the Duclaux con-stants (1) and the partition method of We&man and coworkers (2). Thepartition method is suitable for identifying other organic acids (3) as wellbut requires some supplementary procedure for demonstrating homo-geneity.

More recently the possibilities in the use of partition chromatographyfor the identification and estimation of the lower fatty acids have beeninvestigated (4, 5). The procedure permitted quantitative resolutionunder carefully standardized conditions and with a suitable silica gel.However, the position of the band for a given acid was not constant butvaried with the init ia l amounts of acids used. This observation wouldsuggest either interfering adsorption effects by the silica gel or a non-linear partition isotherm.By way of comparison it was of interest, in connection with the generalstudy under way in this laboratory, to learn whether the technique ofcounter-current distribution (6) could be satisfactorily applied to thequalitative and quantitative estimations of the lower fatty acids. Thiswould be only a preliminary step in the extension of the study to thehigher fatty acids. It was hoped that a system could be found whichwould not show a large shift of partition coefficient with change of con-centration. Our studies have shown this latter to be possible and havealso indicated that at least the lower members of the normal fatty acidscan be separated readily by the procedure.

The resolution obtainable in twenty-four transfers is plainly shown bythe representative distribution given in Fig. 1. This was made with amixture of 57.1 mg. of acetic acid (b.p. 11%119, 760 mm.; m.p. 15.5-

* Fellow of the National Institute of Health, Was hington, D. C.501

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502 DISTRIBUTION STUDIES. VII16; neutralization equivalent 60.5), 51.0 mg. of propionic acid (b.p.139-140; neutralization equivalent 73.3), 55 mg. of normal butyric acid(b.p. 162-163; neutralization equivalent 89), and 50.0 mg. of n-Valerieacid (b.p. 184-185; neutralization equivalent 103). The system used wasisopropyl ether-2.2 M phosphate buffer at a pH of 5.17 (21). The buffercontained 1.94 moles of KHzPOa and 0.26 mole of Na2HP04 per liter.

At the completion of the distributing part of the operation it was neces-.sary to devise some simple method for estimating quantitatively thetotal acid present in both phases of each tube. This was accomplished bymaking use of the quantitative aspects of simple successive extraction insuch a manner that the complete extraction of the water-soluble acid

2 4 6 8 10 12 14 16 18 20 22 24No. of tube

FIG. 1. Twenty-four transfer distrib ution of a mixture of ace tic, prop ionic, buty-.ric, and Valerie acids . X, Curve 1, 0, Curve 2; l indicate s calculate d curves forseparate bands; Curve 3, acetic acid, Curve 4, propionic acid, Curve 5, butyric acid,Curve 6, Valerie acid.from the aqueous phase was not required in order to calculate the totalamount present per tube. In plotting a curve, however, it is not necessaryto plot the total amount in each tube, since some amount proportional tothe total will be just as satisfactory. This proportional amount can berepresented by a single extraction or the sum of two or more extractions,as shown in Fig. 1. Two or more successive extractions permit calcula-tion of the partition coefficient or proportionality factor by extractionequation (2).

The following procedure was employed. Each of the tubes was acidi-fied with 1 cc. of 8 M phosphoric acid, and after equilibration and centrif-ugation the upper layers were removed. An aliquot (1 cc.) of each wastitrated against standard 0.0103 M NaOH. The amount of alkali required

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SA TO , BARRY, AND CRAIG 503for each tube is shown by Curve 1. Appreciable amounts of phosphoricacid were found not to be extracted by the ether. Identical volumes offresh isopropyl ether were then added to each of the lower layers and asecond extraction made. A 1 cc. aliquot of each was again titrated. Thesum of this and the previous titration then gave the values shown byCurve 2. These data were then sufficient for locating the position ofeach band, even though complete extraction from the lower layers hadnot yet been achieved.

From Curve 2 the partition coefficients of 0.09, 0.50, 2.24, and 10 can beestimated (7) and theoretical curves fitted to each, as shown by Curves 3to 6 for acetic, propionic, butyric, and Valerie acids respectively. In thisit is assumed that the maximum tubes of the central bands are homog-eneous and that 0 and 1 of the first band and 23 and 24 of the last bandare homogeneous. As can be seen from Fig. 1, the sum of these fourcurves approximates very closely Curve 2. The theoretical curves maytherefore be used for the quantitative deductions to follow. However,the data thus far are sufficient for identification and rough estimation.

Though it can be predicted from the theory of counter-current distri-bution that certain of the tubes are essentially pure, since a nearly linearisotherm is indicated from the shape of the curves, it appeared of con-siderable interest to demonstrate this fact experimentally. Accordingly,the butyric acid remaining in Tubes 15, 16, and 17 was combined, trans-ferred to a volume of 8 cc. of isopropyl ether, and subjected to a twenty-four transfer distribution in exactly the same way and in the same syst.emused for the original mixture. The result of this experiment is shown inFig. 2. A theoretical curve fitted to the experimentally derived curve isalso shown. The symmetry of this curve is at once evident and it willbe noted that the maximum agrees very well with the maximum of thebutyric acid band df the first distribution. That the acid in Tubes 15,16, and 17 of Fig. 1 was of high purity is now unquestionable. Thisexperiment further indicated that the deviation from a linear isot,herm issmall for butyric acid at the concentration range used.

The quantitative aspects of the distribution may now be developed.The leakage at the ground surface of the distribution apparatus is only amatter of a few drops with a well seated machine. Even if it should be acc. or more, the loss may be neglected in view of the total volume of 400cc. in both phases. Experimentally, any loss tends to be distributedevenly among al l the tubes. A loss approximating 1 to 2 per cent, holy-ever, did occur in removing the solutions from the machine. The solu-tions were individually removed by means of a hypodermic syringe wi-ith along needle and, in order to shorten the process, rinsing of each tube wasneglected. For a critical experiment requiring greater precision, thetubes could be rinsed.

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504 DISTRIBUTION STUDIES. VIIIn calculation of the percentage of each acid in the original mixture, the

first step is that of finding the total amount of acid in a given tube whichis shown by the curves to be of high purity. This is the sum of thatdetermined from the two successive extractions plus the fraction remainingunextracted, and may be calculated by formulae (1) and (2). Formula(1) permits the partition coefficient for the solvent and the acidified bufferto be calculated, where WI is the amount from the first extraction and WZis the amount from the second extraction. WI and Wz may be expressed(0 W2 Krl- .---=-WI Kr -I- 1in terms of the volumes of the standard alkali required. K is the partitioncoefficient. T is the ratio of the volumes of the upper and lower phases

0 2 4 6 8 10 12 14 16 18 20 22 24

Fro. 2. Redistribution of the acid in Tubes 15, 16, and 17 of Fig.mental; l , calculated. 1 0, experi-

used in the extraction. This formula gives a sufficiently accurate estimateof K for the purpose when the substance has a linear partition isothermand when WJW, is not greater than approximately 0.7. When Wz/W,is greater than 0.7, either the volume of the extract must be increased ora solvent used which has a more favorable partition coefficient.

From the extraction formula the fraction, X, of acid extracted on thefirst extraction is given by formula (2). By application of this formula(2) Krxc -Kr + 1to the data obtained with Tube 22 of Fig. 1 the value of 0.912 is obtainedfor X. The total amount of valeric acid in Tube 22 (Fig. 1) is thereforerepresented by 1.59 X 1.00/0.912 X 8 = 13.96 cc. of 0.0103N NaOH. When

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S.4T0 , BARRY, AND CRAIG 505a correction is made for the small amount of butyric acid shown to bepresent in this tube by Curve 5, the result of 13.00 cc. is obtained. Thisis the equivalent of 13.65 mg. of valeric acid.

In the binomial expansion a given term is always a fixed fraction ofthe total. The fraction of the total Valerie acid in Tube 22 can be calcu-lated (7) as a term of the expansion to be 0.281. The total valeric acidrepresented by Curve 6 is therefore 13.65 X 1.00/0.281 = 48.7 mg.

Calculation of the total acid represented by Curve 5 in the same waygave 56.8 mg. of butyric acid and for Curve 4, 50.3 mg. of propionic acid.A sufficient fraction of acetic acid was not extracted by the two successiveextractions to give the calculations in this case the desired accuracy. Theamount of acid in Tube 2 was accordingly derived by two further succes-sive extractions with 16 cc. volumes of diethyl ether. This permitted54 mg. of the acid to be demonstrated in the first band under Curve 3.This value was checked by complete extraction of Tube 2.

For routine work the number of extractions and titrations made inobtaining sufficient data for interpretation could be very much reduced.It would not be necessary to extract or titrate every tube but only enoughto show definitely the position of the peaks. Greater precision could bederived by extracting with 16 cc. volumes instead of 8.

In order to evaluate the quantitative aspects of this distribution thehomogeneity of the acid preparations used ini tia lly should be established.This is especially so in view of the fact that the method of counter-currentdistribution has had wide application for homogeneity studies in thewar time synthetic antimalarial program and more recently in the prepara-tion of penicil lin. It has frequently shown the presence of considerableamounts of impurity when other methods have failed.

Of the four acids used in this study it appeared that the n-valeric acidmight be most open to question. A twenty-four transfer distributionwas accordingly made on 48 mg. of this acid in exactly the same way asfor the mixture of acids. The result obtained is shown in Fig. 3 and isagain in agreement with the theory. The acid recovery calculated asabove was 97 per cent. It was thought in the beginning that the mostprobable impurity might be isovaleric acid. Isovaleric acid in the samesystem was found to have a partition coefficient of 5.5, as compared tothat of 7.7 for Valerie acid. Appreciable amounts of the former would beevident as a deviation from the theoretical at Tubes 16 to 18.

Isobutyric acid (b.p. 153- 154; neutralization equivalent 90), on the otherhand, proved to have a partition coefficient very similar to that of butyricacid and could not be satisfactorily separated in the system used. Itwould thus not be recognized if it were present as an impurity. It isinteresting to note that Elsden (5) also was not able to separate isobutyric

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506 DISTRIBUTION STUDIES. VIIacid from normal butyric acid by partition chromatography on silica gel.It was considered probable that the acetic and propionic acids used were

% 1.4.4,++ 1.2.2a.J?J? 1.0.0$44& 0.6.6

$jj 0.6.60.4.40.2.20 2 4 6 8 100 122 144 166 188 200 222

1.6

L

FIG. 3. Twenty-four transfer distribution of valeric acid. 0, experimental; 0,calculated.1600

0 2 4 6 8 10 12No. of tube

FIG. 4. Twenty-four transfer distribution of acetic. acid. Cl, firstfirst plus second extraction, 0, calculated curve.

extraction; 0,

of satisfactory purity for this particular study, in view of the physicalconstants and high recoveries obtained in Fig. 1. Nevertheless, a sample

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SA TO , BARRY, AND CRAIG 507of acetic acid was distributed in the same system used for the mixture ofacids, except that the volume of the upper layer was doubled. Theresult is shown in Fig. 4. Two successive extractions of the acidifiedlower layers were made. They are given in Curves 1 and 2. The absenceof an appreciable amount of propionic acid in the sample used is thusdemonstrated.

A point of interest can be derived by comparing the distribution ofvaleric acid alone and the corresponding band which emerged from theacid mixture in Fig. 1. The theoretical curve for the former which is inagreement with the experimental values is calculated on the basis of apartition coefficient of 7.7, and this value is in agreement with the exper-imentally determined partition coefficient. On the other hand, it is neces-sary to base the calculation of the theoretical curve for the acid on thevalue 10 for that obtained with the mixed acids. This is an appreciableshift of the position of the band and is a result which has been checked bya duplicate run of the four mixed acids made in a slightly different buffer.We have noted such a shift in other distributions when one of the com-ponents occupied a position either far to the left or to the right of thediagram (partition coefficient of 10 or O.l), but have not noted such ashift as yet when the component occurred near the center of the diagram(partition coefficient of 1). In the present case it afforded a slightlybetter separation of butyric and valeric acids than expected. The posi-tion of the butyric acid band was essentially the same either in the mixtureor alone, as the comparison of Figs. 1 and 2 will show.The agreement of the experimental curve in Fig. 3 with the theoreticalcurve would indicate a linear partition isotherm. This has been checkedexperimentally over the concentration range employed.

SUMMARYThe use of the method of counter-current distribution for the separation

and quantitative estimation of the volatile normal fatty acids (C&Z,) hasbeen investigated. A system and procedure satisfactory for the purposeof estimation to within 2 or 3 per cent have been described.

BIBLIOGRAPHY1. Duclaux, E., Ann. chim . et phys., 2 , 289 (1574) ; Trait6 de microb iologic, Paris, 3,

384 (1900).2. Werkman, C. H., Ind. an d Eng . Chem ., An al. Ed., 2, 302 (1930). Osburn, 0. L.,

Wo od, H. G., and Werkman, C. H., Ind. and Eng . Chem., An al. Ed,, 8,270 (1936).3. Dermer, 0. C., and Dermer, V. H., J. Am . Chem . Sot., 66, 1653 (1943).4. Ramsey, L. L., and Patterson, W. I., J. Ass n. Oficial Agr. Chem., 28, 644 (1945).5. Elsde n, S. R., Bioche m. J., 40, 252 (1.946).6. Craig, L. C., J. Bio l. Che m., 156,519 (1944). Craig, L. C., Golu mb ic, C., Mighton,

H., and Titus , E., J. Biol. Chem., 161,321 (1945).7. Willia mso n, B., and Craig, L. C., J. Bio l. Chem., 168, 687 (1947).

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