190
I'A~UPATI MUKERJEE AXD A ~ H O KRAY A
Vol. 67
THE EFFECT OF UREA ON AMICELLE FORMATION AKD HYDROPHOBIC BONDING BYPASUPATI MUKERJEE AND ASHOICA RAY Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 36, India Received M a y 1,2961 Dissolved urea is suggested as a probe for studying water structure contributions to micelle formation and hydrophobic bonding. Critical micelle concentrations (c.m.c.) of dodecylpyridinium iodide in water and urea solutions at 25 and 45" determined by ultraviolet spectrophotometry are reported. The nature of the spectra and their intensities indicate that the nature of the micelles does not change drastically on the addition of urea. The c.m.c., however, is raised t o a moderate extent, the effect being the same a t 25 and 45'; urea thus weakens hydrophobic bonding and the effect seems t o be independent of temperature. Arguments suggesting the importance of contributions other than those due t o water structure are presented. The relation of these findings to the stability of proteins is briefly indicated.
Introduction Recent investigations1-&have indicated that micelle formation in aqueous solution is primarily an entropydirected process in which enthalpy changes play a minor role, although considerations based on the loss of interfacial energy, taken for granted until re~ently,"~ predict large enthalpy changes. The currently accepted explanation3 invokes the iceberg picture of Frank and Evans.lo A wide variety of phenomena, including hydrophobic bonding in genera1,ll can be explained qualitatively on this basis. The possibilities remain, however, that interfacial effects are not entirely negligible, that enthalpies from several sources cancel each other in part, that a proper evaluation of pre-micellar association12may alter the thermodynamic picture, and that a substantial entropy contribution, in the case of aliphatic systems, arises from the orientations and bendings of the chain, which may be restricted in water and freer in a non-aqueous environment.lJ3 The present paper deals with the use of dissolved urea as a probe for investigating the water structure contribution to micelle formation and hydrophobic bonding. The choice of urea rests on its two outstanding properties in aqueous solutions, namely, its great ability to undergo hydrogen bonding with water, due to the presence of three potential bonding centers on each molecule, and its small effect on the polarity of water. Urea actually increases the dielectric constant of water appreciably and surface tension slightly. At high concentrations, therefore, it should markedly reduce the cooperative structure of water itself, which is primarily due to hydrogen bonding and responsible for the solvent structure effects,10814 without unduly af(1) G. Stainsby and A. E. Alexander, Trans. Faraday Soc., 46, 587 (1950). (2) E . D. Goddard and G. C. Benson, Can. J . Chem., 35, 986 (1957). (3) E. D. Goddard, C. A. J. Hoeve, and G. C. Benson, J . Phys. Chem., 61, 593 (1957). (4) P.White and G. C. Benson, Trans. Paradav Soc., 56, 1025 (1959). (5) B. D. Flockhart, J. Colloid Sei., 16, 484 (1961). (6) P.Debye, A n n . N. 1.' h a d . Sei.,61, 575 (1949). (7) 34. Nakagaki, J. Chem. SOC.Japan, 72, 113 (1951). (8) Y. Ooshika, J . ColZoid Sei., 9, 254 (1954). (9) I. Reich, J . Phys. Chem., 60, 257 (1956). (10) H. S.Frank and M.W.Evans, J. Chem. Phgs., 13, 507 (1945). (11) 'VV. Kauzmann, Adnan. Protein Chem., 14, 1 (1959). (12) P. Mukerjee, K. J. Mysels, and C. I. D u b , J . Phvs. Chem., 62, 1390 (1958); P. Mukerjee, ibid., 62, 1397 (1958); P.Mukerjee and K. J. Mysels, ibid., 62, 1400 (1958); P. Mukerjee, ibid., 62, 1404 (1958); F. Van Voorst Vader, Tvans. Faradag Soe., 67, 110 (1961); D. Eagland and F. Franks, Nature, 191, 1003 (1961); K. J. Mysels and P. Kapauan, J . CoZEoid Sei., 16, 481 (1961); G. D. Parfitt and A. L. Smith, J . Phys. Chem., 66, 942 (1962). (13) R. €1. Aranow and L. Witten, ibid., 64, 1643 (1960). (14) H. S.Frank and A. S.Quist, J . Chem. Phgs., 34, 604 (1961).
fecting the interfacial effects and the concomitant restrictions on the freedom of movement of organic solutes. I n view of the possibly great significance of hydrophobic bonding in the chemistry of proteins in aqueous solution,ll the effect of urea on micelle formation may contribute to the understanding of the denaturing action of urea. From this point of view, a short study similar to ours has been published recently,16 after our work was completed. The object of our study is mainly to understand the detailed nature of hydrophobic bonding. The results are in fair agreement with the recent work15but in strong disagreement with some older rneasurements.l6
Experimental Materials.-Dodecylpyridinium iodide was prepared from dodecylpyridinium chloride (purity better than 95%), obtained from Milton Industrial Chemicals, England, by washing with ether, recrystallization from dioxane, precipitation from concentrated potassium iodide solution in water by cooling, repeated twice, followed by three recrystallizations from water. Analytical reagent variety urea was used. C.m.c. Determination.-On micelle formation, the spectra of long chain pyridinium iodides change.17J8 The c.m.c. values were determined by following this change in absorption, as shown in Fig. 1. Measurements at several wave lengths a t 290 mp and above were made in a Hilger spectrophotometer using silica cella of 1-cm. path lengths. The cell chambers were thermostated at 25.0 f 0.2' and 45.0 f 0.2". Measurements at different wave lengths gave c.m.c. values in agreement to within 1%. The reproducibility of the c.m.c. waa within 1-2%. The measurements in pure water tended to be somewhat uncertain, presumably because of triiodide formation in small amounts. Some results, therefore, were obtained in dilute sodium thiosulfate solutions. The effect of changing the thiosulfate concentration was also studied.
Results and Discussion The c.m.c. data are summarized in Table I. The effect of thiosulfate is small and negligible compared to the effect of the urea addition. Our value in water a t 25' is about 5% higher than that reported by Harkins, et al.lg It is desirable to investigate if urea causes any change in the nature of micelles. The present spectrophotometric method can be adapted to give some information on this question. The investigations of Kosower and co-workerslg on short chain analogs such (15) W. Bruning and A. Holtzer, J . Am. Chem. Soc., 83, 4868 (1961). (16) M. L. Corrin and W. D. Harkins, ibid.,69, 683 (1947). (17) G. S.Hartley, KoEloid-Z., 88, 22 (1939). (18) W. D. Harkins, H. Krizek, and M. L. Corrin, J . CoZZozd ELL, 6, 676 (1951). (IS) E. M. Kosower and P E. Klmedinst, Jr., J . Am. Chem. Soc., 78, 3493 (1956): E. M, Kosower and J. C. Burbaoh, zbzd., 73, 5838 (1956).
191
EFFECTOF UREAON MICELLEFORMATION AND HYDROPHOBIC BONDING
Jan., 1963
0.5-
2.0
0.2-
d Ei 1 , 5 0 m E.l
d
z
42
$
0
2
%
2
8
0.1-
1.0
%
0 05-
0.5
0.02.
0
0
0.004
0.008
0.012
0.016
260
Moles/l.
Fig. 1.-Absorbance of dodecylpyridinium iodide a t 290 n~ as a function of its concentration in 0.0001 M sodium thiosulfate r t t 25" and varying concentrations of urea denoted by: 0, 0 M; A, 3.4 M ; n, 5.0 M . The c.m.c. values are marked by arrows.
C.M.C.
DATAON
TABLE I DODECYLPYRIDINIUM IODIDE
7 -
Urea concentration, M 0.96 3.4 5.9
-.
Medium
0
Water, 25' Water, 45' 0.0001 M NazSs03, 25' 0.001 M NazSsOa, 25' 0.001 M NaJih03, 45"
0.00526 .00670
.....
,00515
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.00930
.0139
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.00475
.00575
.00910
.0133
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.00563
.00710
,0110
,0157
.. ..
,....
8.0
0.00934 0.0136 .... .0118 .0171 0.0213
as methyl- and ethylpyridinium iodides leave little doubt that the appearance of the new absorption 011 micelle formation is due to charge transfer interactions between the iodide ions and the quaternary pyridinium ions. These short range interactions must be confined to the bound layer of the counterions on the micelle surface. They are also dependent on the polarity of the environment,20 so that the micellar bands can be interpreted in terms of the effective polarity a t the micelle surface.21 If urea changes the nature of these charge interacbions then the nature of the spectrum should be affected. If it alters the degree of dissociation, which is of importance in any thermodynamic calculation,22 the intensity of the absorption should change. Figure 2 shows two different spectra in water and 5.9 M urea. These were obtained by measuring the absorption of a solution appreciably above the c.m.c. against, one slightly above it. They are, therefore, charac-. teristic of the micelles. The logarithms of the optical densities are plotted against the wave lengths such that ( 2 0 ) E. M. Kosower, J . Am. Chsm. floc., 80, 3253 (1958). (21) P. Mukerjee and A. Ray, unpublished work. (22) P. Mukerjee, J . Phys. Chem., 66, 1375 (1962).
280
300 320 340 Wave length in mp.
360
380
400
Fig. 2.-Difference spectra of the micelles of dodecylpyridinium iodide in 0.0001 M sodium thiosulfate. Lower curve in water and the upper curve in 5.9 M urea.
any variation in only the micellar concentrations shifts the whole curve equally in the vertical direction. The two different spectra in water and 8 M urea are superposable within experimental error on shifting only the vertical scale, showing that the spectra are the same and, therefore, the nature of the charge interactions in the bound layer at t,he micelle surface are similar. The intensity of absorptions a t any wave length increases linearly with concentration above the c.m.c. in the concentratioii ranges studied (Fig. 1). The slope of the lines above the c.m.c. decreases by about 25% on passing from water to 5.9 M urea. If the molecular extinction coefficients are assumed to be constant, an increase in the degree of dissociation of the micelles is indicated, which is in conformity with the increase of the dielectric constant of the medium on adding urea, and the resultant decrease in the strength of the long-range electrical interactions. The magnitude, however, is small for our system though it may be larger for others.23 Figure 3 shows the relative increase in the c.m.c. with the concentration of urea. The results of Bruning aiild Holtzer16 on dodecyltrimethylammonium bromide are also shown. The trend of their data a t low urea concentration is very similar to ours but their value for 6 M urea deviates appreciably. Some older measurements on sodium lauryl sulfate gave very different results,16 even a slight decrease in the c.m.c. in 3 M urea. We ascribe this discrepancy to the shortcomings of the dye-spectral change method employed for the c.m.c. determinations, which have been examined in detai1.24 (23) E. K. Mysels, of the University of Southern California, LOBAngelets, has studied the conductivity of sodium lauryl sulfate in 6 M urea. The increase in dissociation in this case seems to be considerably more than in the case of dodecylpyridinium iodide (private communication). (24) P. Mukerjee and K. J. Mysels, J . Am. Chem. Soc.. 77, 2937 (1955).
192
PASUPATI MUICERJEE AND ASHOKA RAY V
9
6
8
Q
& V
I
li
0
A
1,ool 0
I
2
4 Molarity of urea.
Fig. 3.-The ratio of the c.m.c. in urea to the c.m.c. in the absence of urea as a function of the concentration of urea: 4, dodecylpyridinium iodide in water at 2 5 " ; Q , in water st 45'; 0, in 0.0001 M sodium thiosulfate a t 25"; A, in 0.001 M sodium thiosulfate a t 25"; 0, 0.001 M sodium thiosulfate a t 45'; V , dodecyltrimethylssmmonium bromide a t 25".'6
The c.m.c. in our system increases almost linearly with urea concentration. Urea clearly has appreciable influence on micelle formation and hydrophobic bonding. The effect of urea, moreover, seems to be independent of temperature. Measurements could not be carried out in 8 M urea a t 25' because of the precipitation of a white solid, presumably an inclusion complex of dodecylpyridinium iodide in urea. Up to 5.9 M urea, however, while the c.m.c. increases by a maximum factor of 2.8, the ratio of the c.m.c.'s a t the same urea and thiosulfate concentration a t 25 and 45' remains constant to within 1.5% of the average value of about 20%. This variation is within experimental error. Although thermodynamic interpretations in terms of c.m.c.'s alone have been shown t o be uncertaiqZ2 since the change in the dissociation in our system is nearly the same at 25 and 4 5 O , our results strongly indicate that urea affects mainly the entropy change in
Vol. 67
micelle formation, and not the enthalpy change, in accordance with our expectation that it modifies primarily the icebergs. Quantitatively, however, the effect of urea seems to be small, considering that in 8 M urea there are only five water molecules per molecule of urea, and that a decrease in the chain length by only two methylene groups raises the c.m.c. by a factor of 4. This suggests that water-structure effects may not be the whole cause of micelle formation. I n support of the possible importance of interfacial effects and the chain-entropy, two other evidences may be cited. The effect of short-chain organic additives such as methanol, ethanol, or acetone,26a t low molar concentrations, is much less than that of urea. The c.m.c. may even be lowered. At higher concentrations, however, in the 6-8 molar range and above, the c.m.c. increases much more steeply than with urea and to much higher values. The most likely explanation seems to be lowering of the surface tension of the medium in the case of the organic additives, and the consequent reduction of any interfacial energy and the chainentropy contributions. The other evidence comes from the recent study of Whitney and Tanford,26who found a considerably more pronounced effect of urea on the solubility of amino acids with aromatic side chains than one with an aliphatic one, although the expected entropy change on hydrophobic bonding is larger for the latter.1' This greater vulnerability of the aromatic system, for which the chain-entropy should be negligible, to urea also argues for a substantial chain-entropy contribution to the hydrophobic bonding of aliphatic systems. Finally, it may be remarked that the effects of urea and organic additives on micelle formation are clearly consistent with their denaturing effects on proteins1' in terms of weakening of hydrophobic bonds, although different factors may be involved, as indicated above. If the effect of urea on hydrophobic bonding is independent of temperature, as is suggested by our work, a promising differentiating tool may be obtained for the study of the complicated features of protein stability and denaturation. (25) A. F. H. Ward, Proc. R o y . SOC.(London), A176, 412 (1940); A. W. Ralston and C. W. Hoerr, J. Am. Chem. SOC., 68, 2460 (1946); A. W. Ralston and D. N. Eggenberger, J. Phys. Colloid Chem., 62, 1492 (1948); E. C. Evers s n d C. A. Kraus, J . A m . Chem. SOC.,70, 3049 (1948); C. A. Kraus, zbid., 70, 3803 (1948); G. L. Brown, P. F. Grieger, and C. A. Kraus, ibid., 71, 95 (1949); H. S. Young, P. F. Grieger, and C. A. Kraus, ibid., 71, 309 (1949). (26) P. L. Whitney and C. Tanford, J . B i d . Chem., 287, PC 1736 (1962).
