J . Phys. Chem. 1990, 94, 641 1-6420
should not deviate from unity by more than 1-3%, especially at the low electrolyte conditions that we have used in most of our experiments (0.08-0.20 M Na+). Unfortunately, it is practically impossible to make any predictions of the local activity coefficients inside the water pools. A variety of factors could affect these activity coefficients: (i) The presence of a strong electric field. (ii) Volume exclusion owing to the high ionic concentration near the micellar wall. (iii) Local deviation from electroneutrality in the double layer formed inside the water pools. (iv) Dielectric saturation of the solvent near the interface. (v) Perturbation of solvent structure near the interface. Even the local salt effect cannot be assessed on the basis of existing activity coefficient correlations such as (A.2), because the water pools are not locally electroneutral. Consequently, our strongest argument against the importance of all these effects is the absence of any experimental observation that would point to their existence.
Appendix B Standard State for the Interface and the Free Energy of Transfer. The partition coefficient is related to the Gibbs free energy of transfer from water to the surfactant interface. At equilibrium &nl = p;q.f or poaint
+ kBT In ( 7 F t x F ' )
= p'2q.f
+k
BIn ~(7:q.fX:q.f)
or finally AG $w ri
=
poaint
-
aq.f
a
= -kBT In Kx - kBT In
(TF~/$~'~) (B. 1)
The most convenient standard states for the chemical potentials appearing in this equation are (i) an infinitely dilute amino acid solution in pure bulk water and (ii) an interface formed under the excess electrolyte conditions at infinite dilution of amino acid.
6411
The definition of the interfacial standard state is different than the one given by Defay et a!9l The standard chemical potential depends not only on the temperature and pressure but also on all the factors that affect the average conformation of surfactant molecules in the interface and on the size of the micelle. This definition for the standard state is necessary in view of the nature of the system under study and since the surface tension of the droplets (required in the definition of ref 49) is not available as a function of system parameters. Of the factors that affect the standard state chemical potential of the solute in the interface salt concentration and type should be of particular importance, since they determine interfacial curvature4I and interfacial rigi d i t y . & ~Solvent ~~ type also affects interfacial rigidity and surface pressure, through its penetrating ability,47-51*53,56 so it should have a significant effect on the standard state. Other potentially important factors are intermicellar interactions, cosurfactants, and temperature. The experimental results presented in section 7 demonstrate the validity of the previous arguments. The bulk water activity coefficient is a function of the amino acid concentration and the salt concentration and type, although its deviation from unity is probably smaller than 5% under the conditions used (see Appendix A for more details). Very little can be said about the interfacial activity coefficient. Because of the definition for the standard state of the interface, which (we believe) cannot be decoupled from the accompanying electrolyte responsible for its formation, we anticipate that all environmental effects will be absorbed by the standard-state chemical potential. We therefore assume that the interfacial activity coefficient is unity for most of our experiments, given the low interfacial mole fractions of amino acid (A = 0-0.02 in most cases). This assumption is corroborated by results such as those of Figures 7 and 8. Similar assumptions have been made in the past by authors investigating solubilization in liposomes and miceIles.9.lI J ~ 8I In conclusion, and given all the previous warnings about the difficulty of interpretation of solubilization data and the approximations that we made in deriving eq IO, we believe that the quantity G is a true measure of the free energy of transfer of amino acid molecules to the AOT reversed micellar interface.
Amino Acids in AOT Reversed Micelles. 2. The Hydrophobic Effect and Hydrogen Bonding as Driving Forces for Interfacial Solubilization Epaminondas B. Leodidis and T. Alan Hatton* Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 021 39 (Received: September 12, 1989; In Final Form: February 13, 1990)
The free energy of transfer of a large number of amino acids from water to the surfactant interfaces of AOT/isooctane and DTAC/heptane/hexanol W/O microemulsions is found to correlate well with existing empirical hydrophobicity scales,indicating the importance of the hydrophobic effect as a driving force for interfacial solubilization. The importance of solutewater and solute-interface hydrogen-bondinginteractions and the possibility of specific chemical interactions with the AOT interface have also been assessed. The connection between reversed micelles and biological membranes and other surfactant assemblies is illustrated. The obtained free energies of transfer from water to the AOT interface constitute a novel hydrophobicity scale, which may be useful in membrane studies.
1. Introduction In the preceding paper of this series,l referred to here as part I , we demonstrated that it is possible to calculate the partition coefficient of amino acids between water and the surfactant interface of AOT reversed micelles by using simple phase-equilib(1) Leodidis, E. B.; Hatton, T. A. J . P ~ Y SChem., . previous paper in this
issue.
rium experiments. The dependence of the partition coefficient on the main system parameters was investigated. We concluded that the Partition coefficient G-as expressed in eq 10 of part I-is a true measure of interfacial association, and, as such, it may provide valuable insights into the driving forces for interfacial solubilization. In part 1 we were able to infer the location of amino acids in reversed micellar solutions by evoking the simple "surfacemonolayer" picture. Using a number of simplifying assumptions,
0022-3654/90/2094-6411$02.50/0 0 1990 American Chemical Society
6412
The Journal of Physical Chemistry, Vol. 94, No. 16, 1990
we postulated the existence of two uniform solubilization environments over which the solutes are distributed, namely, the water pools and the surfactant interface. The relative solute populations in these two environments were obtained quantitatively by using the calculated interfacial partition coefficient K"*. In the present paper we elucidate the driving forces responsible for this solute distribution between water pools and interface. An important additional goal of this paper is to show that reversed micelles are excellent membrane-mimetic systems, at least with regard to partitioning, by showing that solute partitioning in reversed micellar interfaces follows the general rules that have been established in the past for partitioning in biological membranes. The guidelines for this study were provided by the excellent work of Katz, Diamond, and Wright.2.3 On the basis of their analyses we hypothesized that the free energy of transfer of amino acids from water to the AOT interface is determined to a great extent by the interaction between the amino acid molecules and water. The hydrophobic effect and hydrogen bonding between solute and water are the two most dominant factors in this picture. In the case of a more polar surfactant interface a third factor becomes significant, namely, hydrogen-bonding interactions between solute and interface. This paper is structured around this initial hypothesis. In the next section we present a brief review of the existing literature on the investigation of driving forces for solute partitioning in surfactant assemblies. We also discuss empirical methods for quantifying the most important of these driving forces, and relating them to solute molecular properties. Section 3 contains a description of our experimental procedure. In section 4 we tabulated the interfacial partition coefficients and free energies of transfer to the AOT and DTAC interfaces of all the amino acids that we have investigated, along with a number of important molecular properties that are necessary for the ensuing analysis. The importance of the hydrophobic effect is examined by plotting the free energy of transfer vs the most popular hydrophobicity scales that exist today, with special emphasis placed on the octanol/water partitioning scale, as well as on scales using size-related molecular properties, such as molecular area and volume. In section 5 we use the results of section 4 to discuss the importance of solutewater hydrogen-bonding effects and the possible existence of specific "chemical" interactions of amino acids with the AOT interface. Section 6 contains a comparison between the AOT/ isooctane and the DTAC/heptane/hexanol reversed micellar systems, with respect to their solubilizing capacities for amino acids. The importance of interface-solute hydrogen-bonding interactions is illustrated. A summary of the important points of this paper is presented in section 7 . 2. On Driving Forces for Interfacial Solubilization in Surfactant Assemblies 1. Qualitative Partitioning Rules. A number of important studies on partitioning of small molecules between water and biological membranes have established general qualitative rules that determine the partitioning behavior. Although we discussed these rules in section 2.1 of part I , we believe that it is useful to summarize them once more in the context of this paper. According to the excellent reviews by Katz, Diamond, and Wright:2,3 (i) The hydrophobic effect is the major driving force for solute partitioning in membranes. The magnitude of the interfacial partition coefficient depends on the ability of the solute to break the structure of water. (ii) Polar substituents on the solute, which increase the solute's affinity toward water (e.g., through hydrogen-bonding interactions) reduce the partition coefficient. If solute-interface hydrogenbonding is possible, the partition coefficient may increase. (iii) The partition coefficient depends on the fluidity of the membrane and on all the factors that influence it. (2) Diamond, J. M.; Wright, E. M. Proc. R. SOC.London, E 1969, 172, 273. (3) Katz, Y . ; Diamond. J. M. J . Membr. Eiol. 1974, 17. 101, 121.
Leodidis and Hatton (iv) Empirical scales, such as the octanol/water partitioning scale, are often found useful in correlating partitioning data. As stated in part 1, these rules of thumb constitute the basis of the present investigation. 2. Quantitative Correlations with Empirical Scales. Solution scales: Forty years ago Collander observed that there tended to be systematic relationships among the partition coefficients of a family of compounds measured between water and different nonpolar solvent^.^ This is a manifestation of the fact that the same driving forces are responsible for partitioning in all cases, their magnitude and individual significance determined by the nature of the solutes and the nonpolar solvents. Thus, we may write expressions of the form
or
In
= s , , ~ In @Iw - r,,p/RT
where AC$ is the free energy of transfer of solute s from water to solvent a , K,*/" is the corresponding concentration-based partition coefficient, and sa,@ and are characteristic coefficients relating the partitioning between phase cy and water to that between phase p and water for a given family of compounds. Diamond and Katz have called the quantity sol,pthe selectivity ~oefficient.~ s:,~ generally increases as solvent a becomes more nonpolar, and it is a measure of the solvent system's sensitivity to changes in solute hydroph~bicity.~ Since the introduction of the octanol/water partitioning scale as an empirical quantitative measure of solute hydrophobicity and since its evolution as a major tool in drug d e ~ i g n ,it~ has . ~ been customary to generate a partitioning selectivity scale by using octanol as the reference /Isolvent and comparing all other solvents to octanol. The intercept also provides a measure of the "lipophilicity" of the solvent according to H a n ~ c h .A~ positive intercept indicates that the solvent is more lipophilic than octanol and vice versa. It has been found that empirical relations similar to eq 1 and 2 also hold when phases cy and/or /3 are ordered surfactant phases and not isotropic solvents. The partition coefficients of solutes between biological membranes and water, considered to be major components of membrane permeability to the solute^,^^^*^ often correlate with the octanol/water although indications to the contrary do e ~ i s t . *For ~ ~ this reason the octanol/water scale is considered extremely important in the discussion of the anaesthetic action of various compounds and in the general problem of examining the biological activity of drugs6 The octanol/water scale thus plays two important roles: (i) it offers an empirical method for quantifying the hydrophobicity of a solute, and (ii) it helps to characterize the interface as more hydrophobic or more hydrophilic with respect to octanol. Similar conclusions can be drawn using alternative "solution scales", many of which have been developed for amino Molecular size-based scales: In I972 Hermann proposed an alternative empirical scale for correlating solubilities and free energies of transfer.I2 He observed that the solubilities of organic compounds in water and the partition coefficients of nonpolar solutes between bulk solvents and water exhibit a high degree of correlation with the molecular areas or volumes of the solutes. (4) Collander, R. (a) Acta Chem. Scand. 1949,3, 717; (b) Acta Physiol. Scand. 1947, 13, 363. (5) Hansch, C.; Leo,A. (a) Substituent Comtants for Correlation Analysis i n Chemistry and Biology; Wiley, New York, 1979, (b) Leo, A.; Hansch, C.; Elkins, D. Chem. Rec. 1971, 71, 525. ( 6 ) Nogrady, T. Medicinal Chemistry, 2nd ed.;Oxford University Press: New York, (1987). (7) Walter, A.; Gutknecht, J. J . Membr. Biol. 1986, 90, 207. (8) Davis, S. S.;James, M. J.; Anderson, N. H. Faraday Discuss. Chem. Soc. 1986, 81, 313. (9) Gobas, F. A. P. C.; Lahittete, J. M.; Garofalo, G.; Shiu, W. Y.; Mackay, D. J. Pharm. Sci. 1988, 77, 265. (IO) Guy, H . R. Eiophys. J . 1985, 47, 61. ( I 1) Eisenberg, D.; McLachlan, A. D. Nature 1986, 319, 199. (12) Hermann, R. B. (a) J . Phys. Chem. 1972, 76, 2754. (b) Ibid. 1975, 79, I63
Amino Acids in AOT Reversed Micelles. 2 This is a manifestation of the structure of condensed phases, which is dominated by short-range excluded-volume interaction^.'^ A number of short-range intermolecular forces, as mediated by the presence of a solvent, also appear to depend strongly on solute shape and size.14 Hermann,12 S i n a n ~ g l u , ’and ~ Ben-NaimIS among others have tried to explain the correlation between free energies of transfer and molecular size parameters using simple statistical mechanical frameworks. Most of these theories divide the free energy of transfer into two principal components, following an old idea initially presented by Butler:I6 (i) the cavity term, which is the difference in the work necessary to create cavities in the solvents to accommodate the solute, before any intermolecular interactions are “turned-on”; (ii) the interaction term, which is the difference in solute-solvent interaction energies in the two media. The cavity term is roughly proportional to the molecular area of the solute, in both the soluophobic theory of SinanogluI4 and the scaled-particle theory of Pierotti used by Ben-Naim.lS+17The interaction term can be proportional to the volume of the molecule for short-range forces and for a solute with a well-defined “surface” of uniform hydroph~bicity.’~,’~ This proportionality will break down when the molecule contains a variety of residues that interact with the solvent in fundamentally different ways. This would, for example, be the case for nonpolar molecules containing polar moieties that can form strong hydrogen bonds5 Molecular size parameters and free energies of transfer between water and bulk nonpolar solvents are highly correlated, as has been shown repeatedly in the This establishes molecular size scales as a second major empirical tool to quantify solute hydrophobicity or hydrophilicity and its effect on partitioning behavior. Mixed scales: Mixed linear empirical scales have been devised in recent years, with the following goals: (i) to achieve the highest possible degree of generality, by simultaneously treating polar, nonpolar, and amphiphilic compounds on the same basis; (ii) to identify the major driving forces behind partitioning processes, by examining the relative magnitude and statistical significance of the coefficients in the linear correlations that supposedly reflect the impact of these driving force^.^'-^^ All these mixed scales correlate free energies of transfer with at least three parameters, related to the hydrophobicity, the electronic properties (dipole moment), and the ability of the solutes to act as hydrogen-bond donors or acceptors. Many of these scales are presented and discussed in the seminal work of H a n s ~ h .The ~ mixed scale that is based on the solvatochromic parameters has attracted much attention recently.21,22We shall not spend more time on this topic, since solvatochromic parameters are, to date, not available for amino acids. We find it pertinent to remark at this point that mixed-scale-based correlations have revealed the fact that the leading factors influencing partitioning phenomena are the solute’s hydrophobicity and its ability to act as a hydrogen-bond acceptor, in direct agreement with the empirical rules put forward in section 2.1. (1 3) Andersen, H. C.; Chandler, D.; Weeks, J. D. Adu. Chem. Phys. 1976, 34, 105. (14) Sinanoglu, 0. In Ratajczak, H., Orville-Thomas, W. J., Eds. Molecular Interactions; Wiley: New York, 1982; Vol. 3. (1 5 ) (a) Ben-Naim, A,; Ting, K.-L.; Jernigan, R. L. Biopolymers 1989, 28, 1309. (b) Ben-Naim, A. Hydrophobic Interactions; Plenum: New York, 1980. (16) (a) Butler, J. A. V. Trans. Faraday Soc. 1937.33, 229. (b) Barklay, 1. M.; Butler, J. A. V. Ibid. 1938, 34, 1445. (17) Pierotti, R. A. Chem. Reu. 1976, 76, 717. (18) Pearlman, R. S. In Yalkowsky, S.H., Sinkula, A. A,, Valvani, S. C., Eds.; Physical Properties of Drugs; Marcel Dekker: New York, 1980. (19) Leo, A.; Hansch, C.; Jow, P. Y. C. J. Med. Chem. 1976, 19, 611. (20) Moriguchi, 1.; Kanada, Y.; Komatsu, K. Chem. Pharm. Bull. 1976, 24, 1799. (21) Kamlet, M. J.; Doherty, R. M.; Fiserova-Bergerova, V.; Carr, P. W.; Abraham, M. H.; Taft, R. W. J. Pharm. Sci. 1987, 76, 14. (22) Leahy, D. E.; Carr, P. W.; Pearlman, R. S.;Taft, R. W.; Kamlet, M. J. Chromatographia 1986, 21, 473. (23) Yang, G.-Z.; Lien, E. J.; Guo, Z.-R. Quanz. Struct.-Act. Relaf.1986, 5, 12. (24) Moriguchi, 1. Chem. Pharm. Bull. 1975, 23, 247.
The Journal of Physical Chemistry, Vol. 94, No. 16, 1990 6413
3. Experimental Section I , Materials. Aerosol-OT (bis-(2-ethylhexyl) sodium sulfosuccinate) of 99% purity was obtained from Pfalz and Bauer (Waterbury, CT) and from Sigma (St. Louis, MO) and used as received. DTAC (dodecyltrimethylammonium chloride) was purchased from American Tokyo Kasei (Portland, OR). Although its original purity is reported to be higher than 99%, we recrystallized it from ether and methanol. Spectrophotometric grade heptane, isooctane, and hexanol were obtained from Mallinckrodt (St. Louis, MO) and used without further purification. Monobasic and dibasic phosphate salts of sodium and potassium as well as sodium and potassium chloride were purchased from Mallinckrodt and were also used without further purification. All amino acids were obtained from Sigma with the exception of homophenylalanine and (p-hydroxyphenyl)glycine, which were obtained from Chemical Dynamics (South Plainfield, NJ). All amino acids were used as received. The water used for the preparation of electrolyte and amino acid solutions was doubly distilled and deionized. 2. Methods. The detailed description of our experimental procedure is given in part 1. The partitioning experiments were performed at an initial external concentration of sodium phosphate buffer of 0.08 M, at a pH of 6.2-6.5. The amino acid concentration in the initial aqueous phase was set to 5 mmol/L to avoid significant perturbations of the reversed micelles by the interfacially incorporated solutes. AOT concentration in the initial organic solution was varied between 0.2 and 0.5 M. Higher AOT concentrations were necessary to obtain the partition coefficients of the more polar/smaller/less surface-active amino acids with sufficient accuracy. The DTAC experiments were performed using 0.3 M sodium chloride (no buffer) as the external electrolyte. The use of a chloride salt was deemed necessary to avoid any ion-exchange effects similar to those that have been reported in the AOT system.2s The initial organic phase was a 0.25 M solution of DTAC in a 4:1 (by volume) heptane/hexanol solution. The experimental procedure for the DTAC work followed the protocol established in the AOT experiments. 4. Investigation of Solubilization Driving Forces Using Empirical Correlations I . Presentation of a Database. All discussions below are based on the database provided in Table I. This table contains the interfacial partition coefficients of a number of amino acids and their free energies of transfer from water to the AOT and the DTAC interfaces, together with a number of molecular properties that are necessary for correlations based on solution and size scales. The free energies of transfer are calculated from the partition coefficients by using the equation
AGi:IP = -RT In KZ,s
(3)
The assumptions inherent in this equation have been discussed in detail in Appendix A of part 1. The free energy of transfer will be abbreviated below as AG,,. As can be seen in Table I, the sensitivity of the phase-equilibrium method is sufficient for determination of values larger than unity. This is quite satisfactory in view of the simplicity of the method, since we can detect the partition coefficients of all amino acids that have a larger affinity toward the micellar interface than toward water. On the other hand, the method is not sensitive enough to provide the partition coefficients of very small and/or very hydrophilic amino acids. We have not been able to calculate values for many of the 20 protein amino acids, and this was a problem when attempts were made to correlate the free energies of transfer to existing hydrophobicity scales. Further inspection of Table I reveals the absence of information for amino acids with side chains that are completely or partially charged at close to neutral pH. Thus, no information could be obtained for lysine, arginine, histidine, glutamic acid, aspartic acid, and ornithine. We have not been able to measure for glutamine (25) Leodidis. E. B.; Hatton, T. A. Langmuir 1989, 5, 741.
6414
The Journal of Physical Chemistry, Vol. 94, No. 16, 1990
-
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Amino Acids in AOT Reversed Micelles. 2
The Journal of Physical Chemistry, Vol. 94, No. 16, 1990 6415 TABLE 11: Results of Correlations of AG,, with Empirical Solution Scales
empirical scale Nozaki-Tanford2’ BuIl-Breese2* WolfendenZ9 octan~l/water’~ o ~ t a n o l / w a t e r ’(isoleucine ~ excluded) octanol/water3’ free energy of solution30 (tyrosine, proline excluded)
no. of compounds corr selectivity coeff in corr coeff 7 8 7 8 7 12
6
0.785 0.581 0.374 0.982 0.998 0.911 0.943
1.48
1.52 1.24
and asparagine; presumably their partition coefficients are too for small to be detected. We have not been able to measure (3,4-dihydroxyphenyl)alanine (DOPA), which degrades quickly to melaninz6 under our experimental conditions and over the equilibration period that we have used. In spite of these limitations the database of Table I is sufficiently extensive to permit us to draw unambiguous conclusions with respect to the solubilization driving forces. At this point we shall profit by the greatest advantage that amino acids offer as test solutes: Since the part of the molecule that is anchored at the interface is always the same, any differences in behavior can be ascribed to the side chains. 2. Correlations with Solution Scales. We have attempted to correlate the free energies of transfer of amino acids from water to the AOT interface with a variety of existing solution scales. The results of the correlations are summarized in Table 11. Although the lack of information for nonprotein amino acids has forced us to use only seven or eight compounds for many of these correlations, we may draw the following conclusions: (i) The Nozaki-Tanford scalez7is based on the transfer of amino acid side chains from water to ethanol and dioxane. The correlation that we obtained with the Nozaki-Tanford scale is not satisfactory, because of the significant polarity of these solvents, which cannot mimic the hydrophobic AOT interface. The selectivity factor obtained is not statistically significant, because of the low correlation coefficient. (ii) The Bull-Breese hydrophobicity scale2*is even poorer than the Nozaki-Tanford scale for correlating interfacial partitioning data. Strictly speaking, this is not a solution scale, since it is based on surface tension increments of aqueous solutions of amino acids. It has been included in Table I1 because of its popularity and because it can be argued that it considers partitioning of amino acids between bulk water and the more structured water layer at the air/water interface. (iii) The Wolfenden et al. scale29is also inappropriate for our purposes, since it uses water vapor as the nonpolar phase. (iv) A satisfactory correlation is obtained with the free energy of solution of amino acids,30if proline and tyrosine are excluded. The discrepancy observed for proline can be explained by the fact that proline is an imino acid and as such should not be expected to follow the correlation. Tyrosine is extremely insoluble in water, because of dimer formation. Its free energy of solution probably contains additional contributions that render it unsuitable for correlations. The correlation between AG,r and the free energy of solution is shown in Figure 1. (v) The correlation with the octanol/water scale is superior to all the previously considered cases. This indicates once more the suitability of this scale for hydrophobicity studies. Octanol has been suggested as the best available membrane-mimetic ~ o l v e n t . ~ It is quite nonpolar because of its long hydrophobic tail. On the other hand it does offer the potential for significant hydrogen(26) Young, T. E.; Griswold. J. R.; Hulbert, M. H. J . Org. Chem. 1974, 39, 1980. (27) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211. (28) Bull, H. B.; Breese, K. Arch. Biochem. Biophys. 1974, 161, 665. (29) Wolfenden, R.; Anderson, 1.; Cullis, P. M.; Southgate, C. C. B. Biochemistry 1981, 20, 849. (30) Hutchens, J. 0. In Sober,H. A. Handbook of Biochemistry; Selected Data for Molecular Biology; CRC Press: Cleveland, OH, 1970 p 118.
6416
The Journal of Physical Chemistry, Vol. 94, No, 16. 1990 00
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bonding interactions with its hydroxyl group. The correlation of our Act, with the octanol/water scale can be improved further if isoleucine is removed. Since we have no reason to doubt the accuracy of the available data for isoleucine, we must conclude that the AOT interface differentiates between linear and branched solutes in a manner different from that bulk solvents (seediscussion in section 5). A similar conclusion was drawn by Diamond and
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(31) (a) Treiner, C. J . Colloid Inrerfuce Sci. 1982, 93, 33. (b) Treiner, C.; Chattopadhyay, A. K. /bid. 1986, 109, 101. (c) Treiner, C.; Mannebach, M.-H. Ibid. 1987, 118, 243. (32) Collett, J . H.; Koo, L. J . Pharm. Sci. 1975, 64, 1253. (33) Azaz, E.; Donbrow, M. J . Colloid Interface Sci. 1976, 57, 1 1 . (34) Magid, L. J.: Kon-No, K.; Martin, C. A. J . Phys. Chem. 1981, 85, 1434. (35) (a) Pramauro, E.; Saini, G.;Pelizzeti, E. Anal. Chim. Acta 1984, 166, 233. (b) Pramauro, E.; Minero, C.; Saini, G.; Graglia, R.; Pelizetti, E. /bid. 1988, 212, 171. (36) Yunger, L. M.; Cramer, R. D. Mol. Pharm. 1981, 20, 602. (37) Pliska, Fauchere, J.-L.In Gross, E., Meienhofer, J., Eds. Peptides: Structure and Biological Function: Pierce Chemical Co.: Rockford, IL, 1979
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The applicability of the octanol/water scale to the problem at hand should come as no surprise. Numerous studies with membrane2*3,6and micellar s y ~ t e m s ~have l - ~ ~pointed to the value of this scale as a hydrophobicity indicator, and although the octanol/water scale has reportedly failed to produce satisfactory correlations in certain it has worked admirably well for the AOT interface. In Figure 2, top, we plot the free energy of transfer from water to the AOT interface as a function of the free energy of transfer from water to octanol. The octanol/water data were obtained from ref 36. The straight line in Figure 2, top, has been obtained with isoleucine excluded from the correlation. The slope of this line (the selectivity coefficient) is equal to 1.52, indicating that the AOT interface is a more hydrophobic environment for amino acids than is octanol. A more extensive correlation, using more compounds, is shown in Figure 2, bottom. The octanol/water data were obtained from Pliska et al.37 These authors provide calculated rather than measured A values (see eq 4 below), which we transformed into free energies of transfer using the glycine/water partition coefficient reported by Yunger and Cramer.36 While this reduces the accuracy and validity of the correlation, and in spite of poor octanol/water data for tryptophan, it is still interesting to note that a good correlation is obtained with a selectivity constant of I .24. Additional information can be obtained by using the octanol/water scale; specifically, it is possible to infer whether specific chemical interactions exist between the AOT interface and various chemical groups on the solutes. This is an important piece of information, particularly in respect to factors affecting enzymatic reactions in reversed micelles, since the reactivity of a substrate molecule strongly depends on its partitioning characteristics in the reversed micellar solution. As an example we mention the recent work of Bommarius, who conducted enzymatic oxidations
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of benzaldehydes to benzoic acids in reversed micelles.3s He found that the interfacial partition coefficients of benzaldehydes and the second-order enzymatic rate constant correlated very well with the octanol/water scale. We have investigated the presence or absence of chemical specificity of the AOT interface using para-substituted phenylalanines. Unfortunately, the octanol/water partition coefficients for these compounds are not available in the literature. The only alternative was to use A values obtained for related aromatic compounds. A A value for a family of compounds and a particular solvent is defined as AX = log P,x - log PA" (4) where P is the concentration-based partition coefficient between the solvent under consideration and water, A H is the 'parent" or "reference" compound (e.g., glycine for the family of amino (38) Bommarius, A. S . Enzymatic Reactions and Transport in Reversed Micellar Systems. PhD Thesis, MIT, Cambridge, MA, 1989.
Amino Acids in AOT Reversed Micelles. 2
The Journal of Physical Chemistry, Vol. 94, No. 16, 1990 6417
X = Phenylacetic Acid
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acids), and AX is a derived compound, in which chemical group X has replaced the specific "target" hydrogen atom of the parent compound. A values for para-substituted phenyl rings have been measured for a number of solute families.39 The family that is closest in chemical structure to the phenylalanines is that of the parasubstituted phenylacetic acids. Figure 3 shows the correlation between the free energy of transfer to the micellar interface, and this "effective" T scale for phenylacetic acids. The A value for the p-amino compound was not available and was estimated from data in Table I of ref 39. The correlation is excellent, indicating the absence of unusual chemical interactions. The two exceptions in this plot are the pnitro compound and the o-hydroxy compound. These deviations can be explained without evoking any specific chemical interactions. The o-hydroxy group of o-tyrosine is probably not sampling the apolar interior of the AOT interface, according to our surface-monolayer solubilization model, presented in part I . The polar environment that the &hydroxy group samples is almost equivalent to bulk water, as may be deduced by the fact that the interfacial partition coefficient of this compound is practically equal to that of phenylalanine (see Table I). The unusual electronic properties of the nitro group may be responsible for its deviation. We are after all using an effective A scale, assuming it is applicable to phenylalanines. Results presented in Figure 5 suggest that no specific interactions with the p-nitro group occur. Similar excellent correlations have been observed in the past between the octanol-water scale and the free energy of transfer from water to normal micelles for para-substituted benzoic acids?* halo phenol^,^^ and para-substituted phenol^.^^^^^ 3. Correlations with Molecular Size Based Scales. As stated in section 2, the free energy of transfer may be proportional to the molecular area or volume for a family of solutes. Following the pioneering work of Hermann,'* a variety of different solute molecular areas have been proposed for the empirical correlations of solubility and partitioning data. The differences between them and their advantages and disadvantages were reviewed recently by Pearlman.'*~40 Many amino acid molecular areas have also (39) Fujita, T.; Iwasa, J.; Hansch, C. J . Am. Chem. Soc. 1964.86, 5175. (40) Pearlman, R. S.In Dunn, W. J., Block, J. H.. Pearlman, R. S.,Us.; Partition Coefficient; Determination and Estimation; Pergamon Press: New York. 1986.
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6418
The Journal of Physical Chemistry, Vol. 94, No. 16, 1990 0 5 ,
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DTAC/hexanol interface as a function of their corresponding free energy of transfer from water to the AOT interface. been proposed in the past 15 years, because of the connection between side-chain area and "buriedness" in a protein interior.4145 Unfortunately, these data all refer to the 20 protein amino acids only, so they are not useful for our investigation. We have chosen to calculate van der Waals "all-trans" molecular areas and volumes for a number of protein and nonprotein amino acids, using the molecular area calculation program of the molecular simulator CHARMm. The correlations between the free energy of transfer to the micellar interface and (i) the partial molar volume of amino acids at infinite d i l ~ t i o n , ~(ii) ~ . ~their ' van der Waals volume from cHARMm, and (iii) their molecular area, also from cHARMm, are presented in Figure 4. The good correlations in all three plots are not surprising; it has been shown that these three correlational parameters are strongly interc~rrelated.'~~~~*~~ The straight lines in all these plots are not best-fit curves. They are the "minimum free energy" lines that would have been obtained if the amino acid side chains were purely hydrophobic (composed of carbon and hydrogen atoms). These are the partitioning lines determined by the hydrophobic effect. The presence of any other strong interaction between solute and water that increases the solute's affinity for water leads to higher free energies of transfer. Three different classes of solutes are found above the "hydrophobic-partitioning" lines: (i) solutes such as tyrosine and homoserine that contain one or more groups that can form hydrogen bonds with water; (ii) proline, which is an imino acid with different head-group interactions; (iii) large solutes that possess significant conformational freedom, for which the "all-trans" area and volume may be unsuitable for this correlation. In fact the average conformations of solutes such as cyclohexylalanine and homophenylalanine may be different in the ordered micellar interface than in water.6 Figure 4 offers a first illustration of the importance of hydrogen-bonding interactions in the determination of partitioning behavior. We will concentrate on this point in the next section. In closing the current discussion we mention that Figure 4 is a strong indication of the hydrophobic nature of the AOT interface and of the importance of the hydrophobic effect as a driving force for interfacial solubilization.
5. Effects of Specific Molecular Groups on Amino Acid Interfacial Partitioning A change in the chemical structure of the solute affects the interfacial partition coefficient by altering interface-solute and (41) Richards, F. M . Ann. Rev. Biophys. Bioeng. 1977, 6. 1 5 1 . (42) Chothia, C. Nature 1975, 254, 304. (43) Gelles. J.: Klapper, M. H. Biochim. Biophys. Acta 1978, 82, 784. (44) Frommel, C. J . Theor. Bioi. 1984, 1 1 1 , 247. (45) Rose, G. D.; Geselowitz, A. R.; Lesser, G. J.; Lee,R.H.; Zehfus, M. H. Science 1985, 229, 834. (46) Millero. F. J.; Lo Surdo, A.; Shin, C. J . Phys. Chem. 1978.82.784. (47) Shahidi, F.; Farrell, P. G. J . Chem. Soc., Furuday Trans. 1 1978, 74, 858.
Leodidis and Hatton water-solute intermolecular forces (enthalpic part of the free energy of transfer) and also the "degree of order" in water and in the interface (entropic part of AG,,). Following the example set by Diamond, Wright, and Katz>j we have used the database of Table I to assess the effects of various molecular groups on the partitioning behavior of amino acids. ( i ) Methylene Group. It has been repeatedly reported in the literature that free energies of transfer are linearly related to the number of carbon atoms in the molecule for solutes belonging to a specific family or homologous series. Increments of 4OC-IO00 cal/mol per each CH2 group have been reported for the free energy of transfer of solutes to normal micelles48 and to liposome^.^.^ Although the data in Table I are relatively scant, we can draw some conclusions by considering the following families: butyrine-norvaline-norleucine-a-aminooctanoic acid, valine-leucine, and phenylglycine-phenylalanine-homophenylalanine. The data indicate that the free energy of transfer increases by 900 rf: 150 cal/mol per additional methylene group. This value is consistent with previously reported results on micelles and l i p o ~ o m e s . ~It* ~ ~ is also quite large, indicating the strength of the hydrophobic effect. (ii) Effect of Branching. Even bulk solvents discriminate between linear and branched i s o m e r ~ . ~The J ~ degree of discrimination is greater in membrane structures, the partition coefficients of branched solutes being lower, because of the increased incompatibility of the branched solutes with the ordered i n t e r f a ~ e . ~ , ~ Our data are certainly consistent with these observations, as may be seen by comparing AG,, for the following families: norleucine-leucine-isoleucine-cycloleucine, norvaline-valine, and homophenylalanine-3-phenyl-2-aminobutanoic acid. The branched solutes have consistently lower partition coefficients, and it appears that the position of the branch on the molecule plays an important role as well! This is an illustration of the importance of steric geometric considerations in solute partitioning toward ordered interfaces. (iii) Effect of Unsaturated Bonds. Unsaturated bonds are capable of participating in hydrogen bonds, since they are Lewis b a ~ e s . ~This , ~ increases the solute's affinity for water and decreases its interfacial partition coefficient. The phenomenon is enhanced for triple bonds. Our results for the family norvaline-allylglycine-propargylglycine support the previous hypotheses. (iu) Hydroxyl Group (-OH). It has been widely recognized that the hydroxyl group increases the affinity of any solute toward water, through its ability to form three hydrogen bonds with ~ a t e r . ~This , ~ , will ~ increase the solute's solubility in water and decrease its tendency to preferentially partition into less polar phases. Thus, hydrogen bonding becomes a major driving force opposing the hydrophobic effect, reducing amino acid tendency for solubilization in the hydrophobic AOT interface. We have had the opportunity to assess the effect of the hydroxyl group on amino acid partitioning behavior using a number of solutes. Threonine and homoserine have essentially zero interfacial partition coefficients, although their molecular weights and areas are comparable to those of valine and norvaline. The interfacial partition coefficient of 5-hydroxytryptophan is considerably smaller than that of tryptophan, although its molecular volume is larger. The (hydroxypheny1)glycines and (hydroxypheny1)alanines are particularly interesting, because they demonstrate the importance of the location of the OH group on the side chain. The environment of the polar group becomes progressively more apolar as we move from the ortho to the para position. This finding strongly supports our picture of the surface-monolayer solubilization, put forward in part 1. (0)Amino Group (-NH,, >NH). This is another group capable of forming very strong hydrogen bonds with water. Unfortunately, the amino group tends to be ionized at pHs below 8 or 9. This excludes a significant number of available molecules from this study. Our conclusions will be based on @-aminopheny1)alanine (whose pK, is smaller than 6), which was investigated at a pH of 7.5. Its AG,, value indicates a significant reduction of the (48) (a) Bunton. C. A.; Scpulveda. L. J . Phys. Chem. 1979,83,680. (b) Hirose, C.; Sepulveda, L. Ibid 1981, 85, 3689.
Amino Acids in AOT Reversed Micelles. 2 partition coefficient owing to the presence of the amino group. Tryptophan, whose > N H group does not ionize, has a lower partition coefficient than would be expected on the basis of its size, as can be seen in Figure 4. The effect is rather small, however, the >NH group being able to form fewer hydrogen bonds with water than the amino group -NH2. (oi) Ether Oxygen (-@). Although not as capable of hydrogen-bond formation as the hydroxyl group, the ether bond is still sufficiently polar to produce a significant decrease of the partition coefficient. 0-Methylserine, 0-benzylserine, and @-methoxyphenyl)alanine, if compared to compounds where the ether link is replaced by a methylene unit, indicate a decrease of the partition coefficient by a factor of 2-10! These compounds are significantly more hydrophobic than their hydroxyl-containing homologues (e& 0-methylserine vs homoserine). (oii) Sulfur (-S-) and Sulfoxide (O=S=O) Groups. These are weakly polar groups that do have an impact on partitioning behavior, increasing the solute's affinity for water. Replacement of a methylene group by a -S- atom reduces K", by a factor of 1.5-2 (compare methionine to norleucine, and S-methylcysteine to norvaline). This effect is certainly small in the cases that we have investigated. The sulfoxide group reduces & more strongly because of its sulfydryl oxygens, capable of partial hydrogen bonding.* (uiii) Effecf of Halogen Atoms. The only relevant information available is from the data for para-halogenated phenylalanines. These results (and Figure 4,top and middle) imply that halogens are essentially equivalent to a hydrocarbon residue of the same size in their effect on AG,,. Similar conclusions were drawn by Rogers and Davis.49 This may not be true when the inductive effect of the halogens modifies the hydrogen-bonding ability of an adjacent polar group,2 but we did not have the opportunity to study such cases. 6. Effect of the Hydrophobic/Hydrophilic Character of the Interface on Amino Acid Partitioning All the results presented thus far pertain to the AOT interface. The evidence in Tables I and I1 and Figures 1-4 indicates that the AOT interface is very hydrophobic. Its selectivity constant with respect to octanol is 1.52, indicating a quite nonpolar effective solubilization environment for amino acid side chains. The possibility of forming solute-interface hydrogen bonds is quite low. This fact explains the significantly reduced partition coefficients for amino acids containing polar moieties on their side chains. To illustrate that the hydrophobicity or hydrophilicity of the interface has a profound effect on solute partitioning, we extended our investigation to consider partitioning of amino acids in the polar interface of reversed micelles formed in a DTAC/heptane/hexanol system. The reason for choosing this particular system was that it has recently been characterized with respect to the interfacial composition of the droplets under a variety of experimental condition^.^^-^ It was also shown at the same time that we can achieve a two-phase equilibrium between a reversed micellar solution and an excess aqueous phase under well-defined condition^.^^ The alternative to using the DTAC microemulsion would be to 'dilute" the AOT interface with aliphatic alcohols or other polar molecules capable of participating in hydrogen bonds. In such a case we would need to determine the mole fraction of this additional polar component in the reversed micellar interface, which was considered impractical for our present purposes. The interfacial partition coefficient K", of selected amino acids has been calculated by using a modified form of eq 10 of part 1 :
(49) Rogers, J. A.; Davis, S. S. Eiochim. Eiophys. Acta 1980, 598, 392. (50) bmmarius, A.; Pijeni, M, p.; petit, c.; Hatton, T.A., manuscript
in preparation.
The Journal of Physical Chemistry, Vol. 94, No. 16. 1990 6419 where R, is the ratio of moles of alcohol to moles of surfactant in the reversed micellar interface. This equation was derived from a straightforward extension of the surface-monolayer model of part 1, assuming that alcohol may also adsorb onto the surfactant interface and, in fact, participate strongly in the formation of the interface. Equation 5 assumes that all the surfactant is located at the oil/water interfaces, that the organic solvent is insoluble in water and does not adsorb at the interface, and that the interfacial composition ratio R, remains constant over a wide range of experimental conditions. All of the preceding assumptions are quite good. Bommarius has recently reported a constant R, value of 2.88 for this particular system under the experimental conditions of interest.38 In section 3 we described the experimental procedure used with the DTAC microemulsion system. As mentioned, the values of K", reported in Table I for the DTAC interface were obtained at an external salinity of 0.3 M NaCl (wo= 30). In paper 3 of this series we show that K", in the DTAC system depends on external salinity. A 'squeezing-out" effect is observed, similar to that reported for AOT microemulsions in part 1 . We selected an external salinity of 0.3 M NaCl for the DTAC experiments, based on the following considerations: (i) It is impossible to achieve a 'clean" two-phase equilibrium between a reversed micellar solution of DTAC and an excess aqueous phase for external salinities lower than approximately 0.15 M NaCI. The system separates into three phases at lower salinities. (ii) The assumption of constant interfacial composition (constant R,) appears to hold with good accuracy for external salinities greater than 0.2-0.3 M NaC1, according to B o m m a r i ~ s .For ~~ lower salinities the situation (with respect to R,) becomes uncertain. Given the different squeezing-out behavior in the AOT and the DTAC systems, as well as the difference in the surface charge sign of the two interfaces, it becomes unclear under which conditions it is appropriate to compare values for the two systems. In Figure 5 we show the free energy of transfer from water to the DTAC interface as a function of the free energy of transfer from water to the AOT interface. We can draw the following conclusions from this plot: (i) The DTAC interface is more polar than the AOT interface, because of the presence of large amounts of hexanol. Thus, the hydrophobic driving force for interfacial solubilization is considerably weakened, leading to values in the DTAC system that are much lower than the corresponding values for the AOT interface. (ii) An excellent correlation is obtained between the two sets of AGtr. Similar excellent correlations between the AGtr in different interfaces have been observed with different normal micellar system^^',^^ or with different biological membranes.*' There are a few notable deviations, involving compounds with polar moieties that can participate in hydrogen bonds. Hansch has observed that "minus" deviants in such correlations are generally strong hydrogen-bond acceptor^.^ The polar DTAC/hexanol interface is a much better solubilizing environment than the AOT interface for these compounds, because of its considerable propensity for hydrogen-bond formation. Thus there is a significant increase of the partition coefficients of these solutes over their expected values if no specific solute-interface interactions existed. Furthermore, this enhancement of interfacial partitioning appears to depend on the nature of the polar moiety on the solute, on the location of the polar moiety on the side chain, and on the "distance of immersion" of the residue from the "Gibbs dividing surface" of the surface-monolayer model. Thus we see a more significant enhancement of interfacial solubilization for m-phenylglycine than we see for m-tyrosine, since the m-hydroxy group in the first case is closer to the water/surfactant-alcohol interface and is given more possibilities for hydrogen bonding with alcohol molecules. This s-imple explanation presupposes that hexanol is largely anchored at the Gibbs dividing surface. A similar but weaker partition coefficient enhancement due to soluteinterface hydrogen bonding is also observed in the case of tryptophan, while in the
6420
J . Phys. Chem. 1990, 94, 6420-6425
case of methionine (whose polar character is quite weak) no effect is detectable. 7. Overview and Conclusions The data presented in this paper, although they constitute a far from complete picture of interfacial solubilization, permit us to draw the following conclusions: The main driving force for solubilization of amino acids in the AOT interface appears to be the hydrophobic effect, because of the very nonpolar nature of that interface. The entropic gain upon removal of the hydrophobic side chains from water and their immersion into the nonpolar AOT interface appears to be sufficiently strong to drive the solubilization process. The existence of polar moieties on the amino acid side chains that can increase the solute's affinity for water through hydrogen-bonding interactions significantly reduces the interfacial partition coefficient. Hydrogen bonding between solute and water opposes interfacial solubilization and is a significant driving force acting in a direction opposite to that of the hydrophobic effect. The possibility of hydrogen bonding between the solute and the interface leads to a significant increase in interfacial partitioning. At the same time, as the interface becomes more polar, the hydrophobic driving force is reduced. AI1 the previous results, as well as the discussion in section 5 on the effects of chemical groups on K",,are in perfect agreement with partitioning results obtained in the past with liposome^^,^ and micelle^.^'-^**^^ The results also agree with the empirical rules for membrane partitioning put forward by Collander4and more recently presented in full detail by Diamond and We feel that we have at least partly achieved one of the goals of this work, namely, to identify the similarities between reversed micellar solubilization and solubilization in biological membranes. We have thus been able to illustrate that reversed micelles are indeed excellent membrane-mimetic systems. The AG,, scale that we have generated in this work can be used
as a novel hydrophobicity scale. There are significant differences between this scale and the scales that are currently available in the literature. We have not been able to calculate free energy of transfer values for many polar or charged protein amino acids, and hence this scale cannot be used for protein-folding studies. On the other hand, this is a membrane-based solution scale, and hence it should be very useful in membrane studies. Since a large number of hydrophobic amino acids have been examined, this scale could also be useful in protein-engineering applications. The present results obtained with amino acids are of a much more general significance. Amino acid solubilization behavior should be shared by a variety of other, more hydrophobic molecules. Consequently, these results may be of use in reversed micellar enzymology. We believe that a number of unusual results that have been observed for enzymatic reactions in reversed micelles can be partly or even totally explained using partitioning arguments. It appears that the partitioning of enzymatic substrates in reversed micellar solutions has not been paid sufficient attention by reversed micellar enzymologist^.^^ In conclusion, we believe that with the present results we close a gap in the literature on reversed micellar solubilization of small guest molecules and that we offer some additional insights into the significance of the hydrophobic effect and hydrogen bonding for membrane partitioning studies. Acknowledgment. We acknowledge the assistance of Ellen M. O'Connell in much of the experimental work involved in this investigation. We would also like to thank Chris Antonsen and Martin Yarmush for the permission to use the CHARMm molecular simulator and also for teaching us how to use the molecular area calculation program. Finally, we would like to acknowledge partial financial support from the National Science Foundation under the Engineering Research Center Initiative to the Biotechnology Process Engineering Center (Cooperative Agreement CDR-8803014).
Solute-Solvent Interaction in Nonpolar Supercritical Fluid: A Clustering Model and Size Distrlbution Akihiro Morita and Okitsugu Kajimoto* Department of Pure and Applied Sciences, College of Arts and Sciences, the University of Tokyo, Komaba, Meguroku, Tokyo 153, Japan (Received: September 22, 1989; In Final Form: March 7 , 1990)
Solubility and solvatochromic shift of 4-(dimethylamino)benzonitrile (DMABN) in supercritical ethane were measured. The bathochromic shifts observed at various densities were much larger than expected from Onsager's reaction field theory. The discrepancy can be attributed to the solvent aggregation around DMABN even in nonpolar ethane. At low densities, the solvatochromicshift was found to be directly proportional to the fugacity of the solvent fluid. This relation was well rationalized by considering the size distribution of the cluster on a statistical mechanical basis using a grand canonical ensemble. With the help of intermolecular potential between ethane and DMABN, the cluster size distribution in low-density supercritical ethane was derived and the bathochromic shift per one solvent molecule was estimated.
Introduction Recent development of experimental techniques in spectroscopy, light scattering, and ultrafast laser systemi-3 enables us to study 1iquid+hase chemistry in such a sophisticated manner that we than in can discuss the phenomena at a molecular level
terms of a continuous bulk fluid concept. Another important technique, use of a supercritical fluid," also helps us to connect the molecular picture with the continuous fluid viewpoint. This particular medium makes it possible to study various phenomena in a controlled environment ranging from gaslike to liquidlike densities.
( I ) Solvation. Faraday Discuss. Chem. SOC.1988, No. 85. (2) Castnef, Jr., E. W.; Bagchi, B.; Maroncelli, M.;Webb, S. P.; Ruggiero, A . J.; Flemming. G. R. Eer. Bunsen-Ges. Phys. Chem. 1988, 92, 363. (3) Harrie. A . L.; Brown, J. K.: Harris, C. B. Ann. Rev. Pkys. Chem. 1988, 39. 341.
(4) Supercritical fluid solvents. Ber. Bunsen-Ges. Pkys. Chem. 1984, 88(9). ( 5 ) Yonker, C. R.; Smith, R. D. J . Phys. Chem. 1988, 92, 235. (6) Kajimoto, 0.; Futakami, M.; Kobayashi, T.; Yamasaki, K. J . Phys. Cheni. 1988, 92, 1347.
0022-3654/90/2094-6420$02.50/0
0 1990 American Chemical Society
