Liquid-Liquid Extraction Analysis CALVIN GOLUMBIC Office of Synthetic Liquid Fuels, V, S. Bureau of Mines, Bruceton, Pa.
The technique of liquid-liquid extraction, more particularly the partition of substances between pairs of immiscible solvents, is a very convenient tool for analysis of a great diversity of compounds. To review the present status of the subject, the basic theory of fractionation by partition between immiscible solvents, usually called countercurrent distribution, was developed. Fundamental data on the partition of pure substances were obtained and used in the qualitative and quantitative analysis of mixtures of organic compounds. The accuracy of the method compared favorably with spectroscopic methods. The most diverse types of mixtures—
phenols, heterocyclic bases, and polynuclear hydrocarbons—were successfully analyzed. Even very sensitive materials, such as the antibiotics, can be analyzed without undergoing change. The basic technique can be applied to diverse substances with only minor changes in procedure. The method does not require prior knowledge of the type of subin the mixture or calibration data on pure stances substances of known identity, which is a requisite for most methods. As it can be used for actual isolation of the components of a mixture, the technique is very useful to the organic chemist and the chemical engineer.
chemist is familiar with the technique of resolving
and are separated. In multiple contact extraction, one of these phases, defined as the raffinate, is successively extracted with fresh portions of the other phase, the solvent, as shown in Figure 1. The phase withdrawn at each stage is the extractant. The circles represent the mixing vessels. Each operation of once
mixtures by distributing the components between immisciEVERY ble solvents. The qualitative and quantitative analysis of solutions by this technique may be called liquid-liquid extraction analysis. For "ideal” or “perfect” solutions, the ratio of the concentration of a component in the two liquid phases (Ci and C2) is a constant at constant temperature, Ci/C2 k; k is called the distribution constant or partition coefficient. If more than one component is involved in an ideal solution, Nernst’s law of independent distribution applies—i.e., each component distributes itself independently of the others. Extraction techniques are fundamentally of two types: single or multiple contact (sometimes called co-current contact) and countercurrent. The former is a tool for removal or recovery of a constituent from a mixture; the latter may also be employed for this purpose, but it is primarily a fractionation procedure, suitable for separating the individual components of a mixture. Both techniques can be applied to almost any substance that is capable of distribution in an immiscible solvent system.
mixing and separation of layers constitutes
an
equilibrium stage.
=
s
i
Totol
s
s
*s
J
j
J
To illustrate the operation of the method, the case may be considered in wrhich the raffinate layer is denser than the extract layer, and the partition isotherm of solute is linear. After n stages, the fraction of the solute remaining in the raffinate phase is given by the
Jill
Figure
1.
E
E
S.
ra^I°s of the
•
—
E
Conceivably, this procedure could be applied to other homologous individual components are distributed in accordance with the distribution law and, in addition, have appreciably different partition coefficients. Multiple contact extraction finds important application in problems involved in the recovery of a valuable constituent from a mixture or in the removal of a troublesome impurity. In fact, large scale liquid-liquid extraction is seldom undertaken for purposes other than these. Solvent extraction and refining processes Recent uses of this are examples of multiple contact extraction. method, which are of possible technical importance, are the removal of fatty acids from aqueous solution by extraction with derivatives of furan {31) and the removal of mineral acids from acid hydrolyzates of proteins by extraction with long-chain aliphatic tertiary amines, such as methyldioctylamine {SI). In the laboratory, also, multiple contact extraction is a short-cut solution to many problems of recovery and purification. To cite one recent example {44)i preparations oi tetrahydro-2-naphthol, made by catalytic reduction of 2-naphthol and containing small but difficultly removable quantities of the parent compound, were readily and completely freed of the contaminant, 2-naphthol, by selective extraction with a buffer solution of appropriate pH. series in which the
Extract Raffinate Solvent
Both procedures can be arranged as discontinuous—i.e., batchwise—or continuous processes. For large scale applications, attention is, of course, focused on continuous operation. Recent improvements in the design of continuous liquid-liquid fractionation equipment {49) undoubtedly will increase the number of industrial applications, particularly in the antibiotic field, in which separation by extraction offers many advantages {6, 7). In the laboratory, however, the discontinuous techniques are preferred, because the equilibrium stages are more easily observed. For this reason only the discontinuous techniques of the co-current and countercurrent extraction procedures are discussed in this paper. SINGLE AND MULTIPLE
are
These relations constitute the basis for the application of mul tiple contact extraction in the quantitative analysis of the lower homologous fatty acids {63, 68). Thus, by repeated extraction of an aqueous solution of the fatty acids with an immiscible sol1 extractions), followed by titravent (n components require n tion of the aqueous phase initially and after each extraction, sufficient information is obtained for setting up simultaneous equations, which are solved to determine the quantity of each acid.
Scheme of Multiple Contact Extraction E. R.
^
concentrations and volumes, respectively, of the two phases (3$). At each stage a fraction kR{l/kR + l)n of solute is removed in the extractant. When the raffinate is the lighter phase, {kR/ kR + 1)" of solute will remain in the raffinate and each nth extract will contain {1/kR + 1) {kR/kR + l)"_l of solute.
Solute--O—R~~0—R~~0~R—~0 E
>w^el-e ^ an 1. This has been found to be the case in many instances—e.g., the results given in Table IV show that the 0 values for 2,4- and 3,5-xylenol in the alkaline region were three times that at neutral pH. The situation was also improved for a mixture of 3,5- and 2,4-xylenols, and strikingly so for a mixture of 2-methyl and 8-methvlquinolines.
In the discussion so far, association effects have been ignored. In actual practice, however, these effects would be encountered frequently, and their influence on separability should be considered. It can easily be shown that if dimerization occurs in the organic phase' (28), the observed partition coefficient will be as follows: k'
=
2.12[H.4] K
k 1
+
iH +]
(15)
where A is the association constant and [HA ] the total concentration of acid in the organic phase.
7
pH
11
1.9
2.7
1.6
4.9
2.5
4,7
pH
4
m-Ethylphenol
—
Wi
pH
w-Cresola p-Cresol 2.4- Xylenoi" 3.5- Xylenol
(13)
The observed partition coefficient is thus a simple function of pH; the slope of the curve, log k' vs. pH, should be a straight line with a slope of 1; for bases, the slope would be +1. This relation has been found to be valid for a large number of weak acids and bases (27, 28). Equation 13 may also be used for an approximate determination of ionization constant of a weak acid (or base) merely by making an additional measurement of the partition coefficient of the un-ionized acid (or base). This was done in a number of instances, and it was found that the approximate ionization constants wen' usually within 0.1 to 0.2 pK unit of exact values (27, 28). It follows from Equation 12 that the 0 value of any two weak acids is given by the expression: k
1
In addition to the constants, k and K. which were involved before, the association constant now plays a role. Obviously, if kc/kd, Kd/Kc, and Ac/Aa are all >1, the effect of association would be to increase the 0 value. In other words, it is possible that, at least in some instances, increasing the concentration of solutes to promote association may have a beneficial effect upon
logarithmically: log k'
...... K
(12)
K or, expressed
The 0 values for two acids which dimerize will be:
close to 1,
by use of alkaline buffer salts. This adjustment causes extensive ionization in the aqueous phase. The question then arises as to the influence of the ionization constants of these acids on their separability. Under these circumstances, the observed partition coefficient is the resultant of two equilibria: (1) distribution of the un-ionized acid between the two phases and (2) ionization of the acid in the aqueous phase. The observed partition coefficient, k', is then related to the dissociation constant of the acid, K, and the distribution constant of the un-ionized acid, k, by the following expression (28):
1.4 Quinolineb Isoquinoline 1.5 d-Methylquinoline^ 8-Methylquinoline 3.7 2-Methylquinoline 8-Methylquinoline Solvent pair. Cyclohexane-0.5 M phosphate buffer. Solvent pair. Cyclohexane-phosphate, citrate buffer. b
11
b
3.7 4.2
28.0
Nature of Results. Under ideal conditions, it is possible to make the following observations by countercurrent distribution: (1) determine the minimum number of components in a system; (2) isolate these on a small scale or obtain enough data to develop a large scale isolation method; (3) test the purity of any isolated component; (4) obtain physical constants of the components, such as their partition coefficients and approximate ionization constants, which may be a valuable aid in the qualitative identi fieation of the components; and (5) calculate the concentration of the various components. As an incidental detail in obtaining the distribution pattern, the ultraviolet and infrared absorption spectra of the components can also be obtained even without isolating them in the conventional manner. A recent investigation in which all these points of information were obtained by countercurrent distribution was concerned with the composition of the phenolic fraction of coal hydrogenation oil (29). Close-boiling distillate fractions of the phenolic material were subjected to 53-plate distributions. The distribution patterns, determined by ultraviolet spectroscopy, were then used to estimate the number of components and their approximate amounts and to calculate the partition coefficients and ionization constants of the components. From these data, together with infrared absorption spectra of selected tubes, tentative conclusions could be drawn as to the structure of some of the components before any isolation work was done. Major constituents were isolated by multiple contact extraction procedures also based on the partition data. The extinction coefficients of the pure substances were determined, and these values permitted a precise determination of the amounts of the constituents in the
ANALYTICAL
1216
original fraction. Whenever there was doubt about the purity of an isolated compound, a homogeneity test (25) wascarriedout, as described by Craig et al. (18). SCOPE, ADVANTAGES, AND LIMITATIONS COUNTERCURRENT DISTRIBUTION
OF
Countercurrent distribution is a fractionation technique that applied with equal facility to the most diverse substances. Its wide scope is evident from the list of compounds for which it has yielded valuable qualitative information (Table V). Conspicuously absent from this list are aliphatic hydrocarbons, aldehydes, ketones, and esters. Whether countercurrent distribution will prove useful for these classes of compounds is a question which cannot be answered until appropriate investigations have been made. Published reports on the countercurrent separation of mercaptans also are lacking; however, data in the literature on the partition coefficients of alkyl mercaptans (72) leave no doubt that homologous members of this class of compounds can can be
be separated and analyzed.
Unfortunately, the quantitative aspects of countercurrent distribution have been utilized in comparatively few instances. This technique is eminently suitable for quantitative as well as qualitative analysis of mixtures. For routine quantitative work, it is necessary to obtain only one complete distribution curve for a given series of analyses; thereafter the analyses can be confined to a few tubes. From w'ork done at the Bureau of Mines, it appears that the distribution method is particularly applicable to the quantitative analysis of isomers and homologs when used in conjunction w'ith ultraviolet and infrared spectral measurements (71).
Table V. Fatty acids
Types of Compounds Studied by Countercurrent Distribution (.3,
48)
Amino acids (16) Aromatic acids (34, 4?) Polypeptides (16, 70) Phenols (27, 28, 64)
Antibiotics
Penicillins (5, 6, 17)
Streptomycins (42, 46, 56, 60, 61) Xanthomycins (65)
Aromatic amines (26)
Heterocyclic bases (27, 58) Antimalarial bases (14, 18, 59) Veratrine alkaloids (23) Polynuclear hydrocarbons (15, 24) Azulene hydrocarbons (45) Nucleotides (33) Heparin (43) Vitamins Biotin (8) Hormones Pituitary oxytocic factor (S8)
The many points of information, already mentioned, that are available from a distribution pattern may be cited among the advantages of countercurrent distribution. In addition to these, the countercurrent distribution technique wastes no material; all material put into a distribution machine can be recovered quantitatively. The distribution treatment is a mild one and can be employed at low temperatures, so that the most sensitive types of material can be handled. The distribution technique requires, as a rule, little development work; once a suitable solvent system is know'n, countercurrent distribution can be carried out immediately and the results interpreted as discussed above— for example, all that was really needed in the recent application of distribution to phenolic compounds was to consult a handbook to select the appropriate buffers. With neutral compounds, the search for a suitable solvent system may be prolonged. This may be construed as a drawback to the use of the method; however, an amazing variety of immiscible solvent systems exists. Any work on critical-solution temperature phenomena, for instance, will divulge a number of unusual systems (22). The necessity for working at low concentration in order to approach ideal conditions is a disadvantage of the distribution method. Various means of circumventing this difficulty have been employed (13). If a distribution machine with a large
CHEMISTRY
number of units is available, the sample can be divided and placed among several tubes at the start with no loss in resolution or significant interference W'ith interpretation of the distribution pattern. Samples of 20 to 30 grams are used regularly in the 220-tube Craig machine. Preliminary fractionation in large separatory funnels has also been used for increasing the quantity of material treated. The possibilities for separation in regions of high concentrations should be explored. Certainly, ideal conditions that result in symmetrical distribution curves and precise mathematical analysis can be sacrificed for the sake of utility'. If distribution bands are skewed instead of symmetrical, the separation is no less satisfactory, provided that the bands are well separated. The customary countercurrent distribution procedure is carried out in equilibrium stages. Increased resolution may be possible by operating under conditions of disequilibrium. This aspect is being explored by Craig (4). The complexity of the mixtures that can be treated by the countercurrent distribution method is limited by the number of extracting units in the distribution instrument. For example, with the 54-tube instrument, a maximum of about four or five components can be segregated into definite bands. However, if the partition coefficients of the components are very close to each other, even fewer components can be distinguished because of severe overlapping of the distribution bands. The shape of the distribution pattern will thus signal the need for greater resolution, which can be accomplished by a number of alternative procedures already discussed. Recent Applications. The first extensive application of countercurrent distribution dealt with such complex molecules as the antimalarials and penicillins rather than with simple substances (14, 17, 59). In the past year or two successful efforts have been made to extend its use from the analysis of chemicals of purely biological interest to simple organic compounds. Thus, it has been found a valuable adjunct to precise fractional distillation in unraveling the exceedingly complex mixtures obtained upon hy'drogenation of coal (29). It should prove equally useful in the examination of coal tar and petroleum chemicals. Recent work reported by two laboratories (48, 50) indicates its applicability to the chemistry of fats and oils. Marvel and Richards (40) have recently used the countercurrent distribution technique to advantage in separating oxidative cleavage products of polymers. Its possible application to the fractionation of polymers may be foreshadowed in a recent investigation of high-molecularweight phenols in a coal-hydrogenation asphalt (36). It is not too much to expect that eventually proteins will also be examined by this method; the analysis of high-molecular-weight polypeptides has already proved feasible (16). Polypeptides of this type cannot be handled even by the most refined chromatographic techniques (53). The use of “carriers” for adjustment of unfavorable partition coefficients was initiated only recently (42). By this means, the distribution technique was extended to such hydrophilic substances as streptomycin (42, 46, 56) and heparin (43). The possibility of employing countercurrent distribution for the study of tautomerism is a new development. From evidence based on countercurrent distribution studies, Titus and Fried (61) demonstrated that streptomycin exists in several tautomeric modifications, the relative proportions of w'hich depend on the pH. No work has been reported on the countercurrent distribution of inorganic compounds. Very likely, some of the recent striking separations in this field (61, 62) that w'ere achieved by partition chromatography- on paper and by' ion exchange resins could be duplicated in liquid-liquid extraction. Whether any practical advantage would be gained by the extension of countercurrent distribution to this field is another matter. A chromatographic tube can be set up in any laboratory, but distribution separations of great sensitivity require special equipment. On the other
VOLUME
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1951
hand, the latter procedure is simpler in theory and interpretation' and is not subject to as many variables. In the selection of the preferred fractionation technique in any given instance, it seems best to base the decision on the properties of the compounds to be treated and on the particular type of information desired. ACKNOWLEDGMENT
The author wishes to thank L. C. Craig for many helpful discussions.
(35) Hunter, T. G., and Nash, A. W., Ind. Eng. Chem., 27, 836 (1935). (36) Husack, R., and Golumbic, C,,J. Am. Chem. Soc., 73,1567 (1951). (37) Jantzen, E., “Dasfractionierte Distillieren und das fractionierte Verteilen,” Dechema Monographie, Vol. V, No. 48, p. 81, Berlin, Verlag Chemie, 1932. (38) Livermore, A. H., and du Vigneaud, V., J. Biol. Chem., 180, 365 (1949). (39) Martin, A. J. P., and Synge, R. L. M., Biochem. J., 35, 1358 (1941). (40) Marvel, C. S., and Richards, J. C., Anal. Chem., 21, 1480 (1949) (41) Nichols, P. L., Ibid., 22, 915 (1950). (42) O’Keefe, A. E., Dolliver, M. A., and Stiller, E. T., J. Am. Chem. Soc., 71,2452 (1949). (43) O’Keefe, A, E., Russo-Alesi, F. M., Dolliver, M. A., and Stiller, E. T..Ibid., 71, 1517 (1949). (44) Orchin, M., and Golumbic, C., Ibid., 71, 4151 (1949). (45) Plattner, A., Heilbronner, E., and Weber, S., Helv. Chim. Acta, 32, 574 (1949). (46) Plaut, G. W., and McCormack, D. R., J. Am. Chem. Soc., 71, 2264 (1949). (47) Rudkin, G. O., and Nelson, J. M„ Ibid., 69, 1470 (1947). (48) Sato, Y., Barry, G. T., and Craig, L. C., J. Biol. Chem., 170, 501 (1947) (49) Scheibel, E. G., Chem. Eng. Progress, 44, 681 (1948). (50) Scholfield, C. R., Dutton, H. J., Tanner, F. W., Jr., and Cowan, J. C., Am. Oil Chemists’ Soc., 25, 368 (1948). (51) Smith, E. L., and Page, J. E., J. Soc. Chem. Ind. (London), 67, 48 (1948). (52) Spedding, F. H., Voight, A. F., Gladrow, E. M., and Sleight, N. R„ J. Am. Chem. Soc., 69, 2777 (1947). (53) Stein, W. H., and Moore, S., Cold Spring Harbor Symp., 14, 179 (1950) (54) Stein, W. H., and Moore, S., J. Biol. Chem., 176, 337 (1948). (55) Stene, S., Ark. Kem. Mineral. Oeol., 18H, No. 18 (1944). (56) Swart, E. A., J. Am. Chem. Soc., 71, 2942 (1949). (57) Thorne, C. B., and Peterson, W. H., J. Biol. Chem., 176, 413 (1948) (58) Tinker, J. F., and Brown, G. B„ Ibid., 173, 585 (1948). (59) Titus, E. O., Craig, L. C., Golumbic, C., Mighton, H. R., Wempen, I. M., and Elderfield, R. C., J. Org. Chem., 13, 39 (1948). (60) Titus, E. O., and Fried, J., J, Biol. Chem., 168, 393 (1947). (61) Ibid., 174, 57 (1948). (62) Tompkins, E. R., Khym, J. X., and Cohn, W. E., J. Am. Chem. Soc., 69, 2769 (1947). (63) Tsai, K. R., and Pu, Y., Anal. Chem., 21, 818 (1949). (64) Warshowsky, B., and Schantz, E. J., Ibid., 20, 951 (1948). (65) Watanabe, S., and Morikawa, K., J. Soc. Chem. Ind., Japan, 36, 585B (1933). (66) Weizmann, C., Brit. Patent Application 4346/43. (67) Weizmann, C., Bergmann, E., Chandler, E. F., Steiner, H,, Sulzbacher, M., and Zimkin, E., J. Soc. Chem. Ind. (London), 67, 203 (1948). (68) Werkman, C. H., Ind. Eng. Chem., Anal. Ed., 2, 302 (1930). (69) Williamson, B., and Craig, L. C., J. Biol. Chem., 168, 687 (1947). (70) Wooley, D. W..Ibid., 179, 593 (1949). (71) Woolfolk, E. O., Golumbic, C., Friedel, R. A., Orchin, M., and Storch, H. H., Bur. Mines Bull. 487 (in press). (72) Yabroff, D. L., Ind. Eng. Chem., 32, 950 (1940). (73) Yabroff, D. L., and White, E. R., Ibid., 32, 950 (1940). .
BIBLIOGRAPHY
(1) (2) (3) (4) (5) (6)
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Archibald, R. C„ J. Am. Chem. Soc., 54, 3178 (1932). Arnold, R. T., and Richter, J., Ibid.. 70, 3505 (1948). Atchley, W. A., J. Biol. Chem., 176, 123 (1948). Barry, G. T., Sato, Y., and Craig, L. C., Ibid., 174, 209 (1948). Ibid., p. 221. Bartels, C. R., and Dolliver, M. A., J. Am. Chem. Soc., 72, 11
(1950). (7) Bartels, C. R., and Kleiman, G., Chem. Eng. Progress, 45, 589 (1949). (8) Bowden, J. P., and Peterson, W. H., J. Biol. Chem., 178, 533 (1948). (9) Bush, M. T., and Densen, P. M., Anal. Chem., 20, 121 (1948). (10) Bush, M. T., Goth, A., and Dickison, H. L., J. Pharmacol. Exptl. Therap., 84, 262 (1945 .
(11) Cornish, R. E., Archibald, R. C., Murphy, E. A., and Evans, H. M., Ind. Eng. Chem., 26, 397 (1934). (12) Craig, L. C., J. Biol. Chem., 155, 519 (1944). (13) Craig, L. C., and Craig, D., in Weissberger, ed., “Technique of Organic Chemistry,” Vol. Ill, Chap. IV, New York, Interscience Publishers, 1950. (14) Craig, L. C., Golumbic, C., Mighton, H., and Titus, E., J. Biol, Chem., 161, 321 (1950). (15) Craig, L. C., Golumbic, C., Mighton, H., and Titus, E., Science, 103,587 (1946). (16) Craig, L. C., Gregory, J. D., and Barry, G. T., Cold Spring Harbor Symp., 14, 24 (1950). (17) Craig, L. C., Hogeboom, G. H., Carpenter, F. H., and du Vigneaud, V., J. Biol. Chem., 168, 665 (1947). (18) Craig, L. C., Mighton, H., Titus, E., and Golumbic, C., Anal. Chem., 20, 134 (1948). (19) Craig, L. C., and Post, H. O., Ibid., 21, 500 (1949). (20) Dijck, W. J. D. van, and Schaafsma, A., U. S. Patent 2,245,945 (1941). (21) Fieser, L. F., Ettlinger, M. G., and Fawaz, G., J. Am. Chem. Soc., 70, 3228 (1948). (22) Francis, A. W., Ind. Eng. Chem., 36, 764 (1944). (23) Fried, J., White, H. L., and Wintersteiner, 0., J. Am. Chem. Soc., 71, 3260 (1949). (24) Golumbic, C., Anal. Chem., 22, 578 (1950). (25) Golumbic, C., J. Am. Chem. Soc., 71, 2627 (1949). (26) Golumbic, C., and Goldbach, G., Ibid., 73, 3966 (1951). (27) Golumbic, C., and Orchin, M., Ibid., 72, 4145 (1950). (28) Golumbic, C., Orchin, M., and Weller S., Ibid., 71, 2624 (1949). (29) Golumbic, C., Woolfolk, E. O., Friedel, R. A., and Orchin, M., Ibid., 72, 1939 (1950). (30) Gross, P. M., Chem. Rev., 13, 91 (1933). (31) Guinot, H. M„ and Chassaing, P., U. S. Patent 2,437,519 (1948). (32) Hill, in Taylor, ed., “Treatise on Physical Chemistry,” 2nd ed., p. 467, New York, D. Van Nostrand Co., 1930. (33) Hogeboom, G. H., and Barry, G. T., J. Biol. Chem., 176, 935 (1948). (34) Hogeboom, G. H., and Craig, L. C., Ibid., 162, 363 (1946).
.
.
.
.
Received September 2, 1950. Presented before the Division of Physical and Inorganic Chemistry, Symposium on Analytical Methods Based on Heterogeneous Equilibria, at the 118th Meeting of the American Chemical Society, Chicago, 111.
