Crystallization of Organic Compounds in Reversed Micelles. I

Dec 1, 2000 - Crystallization of Organic Compounds in Reversed Micelles. ..... A large part of this curve is approximately linear, with deviations at ...
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Langmuir 2000, 16, 9996-10004

Crystallization of Organic Compounds in Reversed Micelles. I. Solubilization of Amino Acids in Water-Isooctane-AOT Microemulsions Junko Yano,†,‡ Helga Fu¨redi-Milhofer,† Ellen Wachtel,§ and Nissim Garti*,† Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel, and Faculty of Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel Received March 16, 2000. In Final Form: September 12, 2000 As a basis for crystallization studies, the solubilization of amino acids (glycine, l-histidine, and l-phenylalanine) in water-in-isooctane microemulsions stabilized by AOT (sodium di-2-ethylhexyl sulfosuccinate) was investigated. The maximum amount of amino acid that could be solubilized was determined by the solid-liquid extraction method, and the effect of the guest molecules (amino acids) on the size and shape of the microemulsion droplets and their thermal properties were determined using SAXS and DSC measurements, respectively. The solubilization of glycine molecules, which primarily dissolve in the water pool, was slightly lower than their solubility in pure water, decreasing with increasing concentration of AOT and increasing with increasing water content in the microemulsion. In contrast, the solubilization of phenylalanine, which is primarily located at the water/oil interface, exceeded several times the solubility in water, the solubilized amount increasing with increasing AOT and/or water concentrations. Histidine had characteristics intermediate between these two extremes. Solubilization of those molecules effected an increase in droplet size. The thermal analysis showed that loading of the microemulsion droplets with glycine has a much stronger effect on the thermal behavior of the emulsified water than has loading with phenylalanine. The low solubilization of glycine as compared to its solubility in pure water can be explained by the state of water within the microemulsion droplets, i.e., part of it is present as free water and part as water bound to the AOT headgroups. The loading of phenylalanine changed the shape of the microemulsion droplets from spherical to ellipsoidal, and with increasing droplet sizes, the [phenylalanine]/[AOT] molar ratio at the interface increased.

1. Introduction Reversed micelles [water in oil (W/O) microemulsions] are homogeneous, thermodynamically stable systems of nanosized domains of water dispersed within an immiscible organic (oil) phase and stabilized by a surfactant shell or, more frequently, a shell consisting of a suitable surfactant and a cosurfactant (usually an alcohol). These systems can be formed only in specific ranges of temperature, pressure, and composition and are therefore best described with phase diagrams. Because of the presence of supramolecular aggregates, W/O microemulsions have the ability to solubilize normally slightly soluble substances and have thereby found numerous applications as “microreactors” or “nanoreactors” for specific reactions,1-4 such as host-catalytic and enzymatic reactions, preparation of nanosized particles, and bioseparations. There has been a great deal of interest in exploring the solubilization mechanisms of foreign molecules into W/O microemulsions, as this is indispensable information for most application processes. The solubilization studies have been carried out mainly by liquid-liquid-phase equilib* Corresponding author. Tel: +972-2-6586574. Fax: +972-26520262. E-mail: [email protected]. † The Hebrew University of Jerusalem. ‡ Present address: Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, 7398528, Japan. § The Weizmann Institute of Science. (1) Pileni, M. P., Ed. Structure and Reactivity in Reverse Micelles; Elsevier: Amsterdam, 1989. (2) Fendler, J. H. Membrane-Mimetic Approach to Advanced Materials. Advances in Polymer Science 113; Springer: Berlin, Heidelberg, 1994. (3) Pileni, M. P. J. Phys. Chem. 1993, 97, 6961. (4) Sager, W. F. C. Curr. Opin. Colloid Interface Sci. 1998, 3, 276.

rium or phase transfer experiments.5-11 This method studies the equilibrium distribution of a solute between a reversed micellar system and an aqueous solution, determining interfacial partition coefficients as a measure of the degree of interfacial association of the solute.7 Luisi et al.5 used this method to show that the solute transfer is influenced to a large extent by the electrostatic interactions between the surfactant and the solute. Later, Adachi et al.8 recognized the effects of charged states of amino acids on the localization of the molecules in microemulsions. Using water-in-oil microemulsions stabilized by AOT (sodium di-2-ethylhexyl sulfosuccinate), Leodidis and Hatton have utilized partition coefficients to investigate solubilization driving forces,6,9 emphasizing the importance of hydrophobicity as a major driving force. It was shown that hydrophilic amino acids are solubilized primarily in the water volume, whereas hydrophobic molecules migrate to both the water pools and the W/O interface. The authors also explored the effects of salinity and interfacial curvature on the partition coefficients and examined the possibility of hydrophobic amino acids acting as cosurfactant.10,11 An alternative method, which was used for the solubilization of proteins and/or amino acids into reverse micelles, is the solid-liquid-phase extraction method.3,12 (5) Leser, M. E.; Luisi, P. L. Chimia 1990, 44, 270. (6) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6400. (7) Fletcher, P. D. I. J. Chem. Soc., Faraday Trans. 1 1986, 82, 2651. (8) Adachi, M.; Harada, M.; Shioi, A.; Sato, Y. J. Phys. Chem. 1991, 95, 7925. (9) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6411. (10) Leodidis, E. B.; Bommarius, A. S.; Hatton, T. A. J. Phys. Chem. 1991, 95, 5943. (11) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1991, 95, 5957. (12) Grandi, C.; Smith, R. E.; Luisi, P. L. J. Biol. Chem. 1981, 256, 837.

10.1021/la0004101 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/01/2000

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Table 1. Solubility in Aqueous Solution and Hydrophobicity of Three Amino Acids

a The solubility obtained in the present study agrees quite well with the value given by Nozaki and Tanford (ref 15). b The hydrophobicity scale for transfer of amino acid side chain from 100% organic solvent to water at 25 °C. These values were listed from ref 15.

Figure 2. Solubilization of glycine (mmol) in 100 g of microemulsion: (a) constant wt % AOT and (b) constant wt % water. In part a, the solubilized amount of glycine in water, equivalent as the water volume in the corresponding microemulsion, is indicated by the dotted line. In part b, the dotted lines (a, b, c, and d) denote the solubilized amount of glycine in water equivalent to that in the microemulsions having 10, 15, 20, and 25 wt % water, respectively. In parts a and b, each point includes the error in measurements of (0.2-0.4 mmol.

Figure 1. Ternary phase diagram of isooctane-AOT-water.16 Solubilization of amino acids was measured for the microemulsion compositions indicated by the closed circles with alphabetical symbols.

In this method the amino acid or protein powder is suspended in a preprepared microemulsion and solubilized by stirring. The dependence of the maximum solubilization values on the experimental conditions (composition of the microemulsion, water pool sizes, temperature, etc.) can then be determined. The method is convenient for the solubilization of water insoluble proteins13 and has also been used for biotechnological treatment of farinaceous natural products that contain large mixtures of proteins.14 Our aim in the present series of reports is to investigate the potential of reversed micelles to control the crystallization of solubilized organic compounds. For this purpose solubilization studies using the solid-liquid extraction method provide essential information, since (1) the characteristics of the solubilizing medium (i.e. water content, surfactant concentration, droplet sizes) are (13) Delahodde, A.; Vacher, M.; Nicot, C.; Waks, M. FEBS Lett. 1984, 172, 343. (14) Leser, M. E.; Luisi, P. L.; Palmieri, S. Biotechnol. Bioeng. 1989, 34, 1140.

directly defined; (2) the combination of three factors, the maximum solubilization, structural characteristics, and thermal behavior of solute-loaded microemulsions, can be obtained in the same system; and (3) after determining the solubilization parameters, crystallization can be induced simply by cooling without any intermediate steps, such as phase separation, being necessary. However, little systematic work concerning the effect of experimental parameters on solid-liquid extraction of organic molecules has so far been published. As model systems, we have therefore chosen amino acids as solute molecules and water-isooctane-AOT microemulsions as media of solubilization and crystallization. Because it is a well-defined system,5-11 one can characterize the structural and thermodynamical characteristics of amino acid-loaded microemulsions prepared by the solid-liquid extraction method. Also, the polymorphism and crystal structures of the amino acids are comparatively well-known in the bulk system. Therefore, effects of particle sizes and interfacial structures will be emphasized from the comparative studies on crystallization in the bulk system and in microemulsions. Thus, amino acids, with their wide range of hydrophobicity arising from different side chains and their charged states controlled by pH, can provide important insight into the solubilization and crystallization mechanisms of organic molecules, including the biochemically more important peptides and proteins.

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Figure 3. Solubilization of l-histidine (mmol) in 100 g of microemulsion: (a) AOT constant and (b) water constant. In part a, the solubilized amount of histidine in water, equivalent to the water volume in the corresponding microemulsion, is indicated by the dotted line. In part b, the dotted lines (a, b, c, and d) denote the solubilized amount of histidine in water, equivalent to that in the microemulsions having 10, 15, 20, and 25 wt % water, respectively. In parts a and b, each point includes the error in measurements of (0.2-0.4 mmol.

In the first paper of this series we examine the solubilization of three amino acids with different hydrophobicity,15 i.e., l-phenylalanine, l-histidine, and glycine (Table 1), in water-isooctane-AOT microemulsions, by means of solid-liquid extraction. We discuss the relationship between the localization of those molecules within the microemulsions and the changes in sizes and shapes of the water pools and the effect of structured water on the solubilized molecules. 2. Experimental Section 2.1. Materials. AOT of 99% purity was obtained from Sigma (St. Louis, MO) and isooctane (2,2,4-trimethylpentane) of > 99% purity was from Aldrich (Milwaukee, WI). These samples were used as received. Amino acids, >99% purity, were purchased from Sigma and used without further purification. Doubledistilled water was used for the preparation of microemulsions. 2.2. Solubilization Measurements. The compositions of the microemulsions used for the solubilization experiments (in wt %) are indicated in the phase diagram in Figure 1.16 The microemulsions were prepared by mixing the required amounts of isooctane, AOT, and water and shaking the mixture for several minutes by vortex. The maximum amount of amino acids that could be solubilized in a particular microemulsion, while maintaining a homogeneous L2 (water-in-oil microemulsion) phase, was determined at 25 °C by the following procedure. Small (15) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211. (16) Sager, W. F. C. Langmuir 1998, 14, 6385.

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Figure 4. Solubilization of l-phenylalanine (mmol) in 100 g of microemulsion: (a) AOT constant and (b) water constant. In part a, the solubilized amount of phenylalanine in water equivalent to the water volume in the corresponding microemulsion is indicated by the dotted line. In part b, the dotted lines (a, b, c, and d) denote the solubilized amount of phenylalanine in water equivalent to that in the microemulsions having 10, 15, 20, and 25 wt % water, respectively. In parts a and b, each point includes the error in measurements of (0.2-0.4 mmol. amounts (1-5 mg) of powdered amino acid were successively added to 3 g of microemulsion. The microemulsion was then shaken by vortex and ultrasonicated until a clear microemulsion was restored. The procedure was repeated until no more solute could be solubilized or, in some cases, at points closest to the stabilization boundary of the L2 phase, until the L2 phase collapsed. The saturated microemulsions were kept at constant temperature (25 °C) for 2 weeks. Finally, we confirmed under the microscope that no crystallization occurred during storage. 2.3. Differential Scanning Calorimetry (DSC). Calorimetric measurements were carried out using a Mettler TA 4000 thermal analysis system, equipped with a DSC 30 low-temperature cell. Microemulsion samples (10-15 mg) were sealed in 40 µL aluminum crucibles at room temperature. Samples were cooled by liquid nitrogen from 25 to -75 °C at a rate of 30 °C/min and then heated at a rate of 3 °C/min to 25 °C. From the cooling process, crystallization temperatures of the water pools, Tc, were determined, while the heating process yielded melting temperatures, Tm, and the corresponding enthalpies. DSC temperatures obtained in the first cycle (cooling and heating) were reproducible to (0.7 °C in the second cycle. 2.4. Small-Angle X-ray Scattering (SAXS) Measurements. The size and shape of microemulsion droplets were evaluated by SAXS measurements (Weizmann Institute of Science). Microemulsions with and without amino acids were prepared as described above. Each solution was sealed in a 1.5 mm diameter glass capillary which was then inserted into a copper sample holder thermostated by an alcohol-water bath. An Elliott rotating-anode X-ray generator operating at approximately 1.2

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Figure 5. SAXS curves, scattering intensity vs q (a and b) and the pair distance distribution function, P(r), (c and d) of glycineloaded microemulsions (a and c) and phenylalanine-loaded ones (b and d) at W ([water]/[AOT]) ) 24.4. All experiments were carried out at 2 wt % AOT (0.04 m). 100% of amino acid corresponds to the microemulsions containing the maximum amount of solubilized amino acid. Table 2. Structural Characteristics of Microemulsion Droplets Obtained by SAXS microemulsionsa pure glycinee phenylalaninee

P(r)

concn of amino acid (%)

Guinier Rg (nm)b

Rg (nm)b

Dmax (nm)c

form factord (nm)

0 50 100 50

3.79 4.09 4.29 3.95

3.62 3.83 3.97 3.89

9.6 10.4 10.9 11.6

R ) 4.67 R ) 4.94 R ) 5.13 a ) 4.58

c ) 5.80

100

4.43

4.20

11.7

a ) 5.20

c ) 5.85

P(r) ) (1/2π2)∫I(q)qr sin(qr) dq

Microemulsions having W ) 24.4 (AOT ) 0.04 m). Each value includes the error of (0.1 nm. c Dmax, the maximum dimension of the reversed micelles as determined by P(r) (Figure 5c,d). d R, radius of spherical droplets; a and c, semiminor and semimajor axes of ellipsoidal droplets. e Amino acid-loaded microemulsions. a

procedure.19 Then Rg values were obtained from the slope of a ln I(q) vs q2 plot, where q and I(q) are the amplitude of the scattering vector and the scattering intensity, respectively. In the method of Glatter, the pair distance distribution function P(r) was calculated by

b

kW produced Cu X-radiation which was monochromated by a Ni filter and 20 cm Franks mirror and collimated by a series of slits and height limiters. The scattering profile was measured by a linear position sensitive detector of the delay line type and was stored in a Z-80 based microcomputer as a 256 channel histogram. The distance from the sample to the detector was approximately 48 cm. The duration of each experiment was 1 h. The scattering from isooctane was subtracted as background from that of the microemulsion. Data were analyzed in two ways: the method of Guinier17 and the indirect transformation procedure of Glatter.18 The radius of gyration, Rg, was obtained independently by these two methods. In the Guinier analysis, data were first deconvoluted by the Lake (17) Guinier, A.; Fournet, G. Small Angle Scattering of X-rays; Wiley: New York, 1955. (18) Glatter, O. Small angle scattering and light scattering. In Neutron, X-ray and light scattering; Lindner, P., Zemb, Th., Eds.; Elsevier Science: New York, 1991; pp 33-60.

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where q is the amplitude of the scattering vector and r is a distance in real space. The expression gives the frequency of occurrence of vectors of length r, weighted by the electron density at either end of the vector. The radius of gyration, Rg, was obtained from

Rg2 )

∫ P(r)r ∞

0

2

dr/2

∫

0

∞

P(r) dr

(2)

The shape of the P(r) curve directly reflects the form of the microemulsion droplets for a monodisperse population.

3. Results 3.1. Solubilization of Amino Acids. The maximum amount of amino acids (glycine, l-histidine, and l-phenylalanine) that could be solubilized in microemulsions, as denoted in Figure 1, is plotted in two ways, along the constant surfactant or constant water lines respectively (Figures 2-4). 3.1.1. Glycine. Figure 2a shows the changes in the solubilized amount of glycine with increasing amounts of water at constant AOT concentration, while Figure 2b shows changes in solubilization with increasing amounts of AOT at constant water volume. For comparison, in both diagrams the solubilized amount of glycine in a volume of water that is equivalent to the water volume in the (19) Lake, J. Acta Crystallogr. 1967, 23, 191.

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Table 3. Dependence of Microemulsion Droplet Sizes on W as Obtained by SAXSa P(r) form factorc (nm)

W, [H2O]/[AOT]

Rg (nm)b

Dmax (nm)

pure

3.5 4.9 12.2 19.8 24.4

1.30 1.45 2.28 3.04 3.62

3.9 4.2 6.4 8.2 9.6

a ) 1.59 a ) 1.75 a ) 2.81 R ) 3.92 R ) 4.67

glycined

3.5 4.9 12.2 19.8 24.4

1.23 1.47 2.32 3.26 3.97

3.3 4.1 6.5 8.9 10.9

R ) 1.59 R ) 1.90 R ) 3.00 R ) 4.21 R ) 5.13

phenylalanined

3.5 4.9 12.2 19.8 24.4

1.42 1.55 2.44 3.39 4.20

3.9 4.6 6.7 9.3 11.7

a ) 1.57 a ) 1.75 a ) 3.00 a ) 4.23 a ) 5.20

microemulsions

c ) 1.95 c ) 2.10 c ) 3.20

c/a ) 1.23 c/a ) 1.20 c/a ) 1.14

c ) 1.95 c ) 2.30 c ) 3.35 c ) 4.65 c ) 5.85

c/a ) 1.24 c/a ) 1.31 c/a ) 1.12 c/a ) 1.10 c/a ) 1.13

a Data were measured by the microemulsions containing 0.04 m of AOT. The R and D b g max values were obtained using P(r). Each value includes the error of (0.1 nm. c R, radius of spherical droplets; a and c, semiminor and semimajor axes of ellipsoidal droplets; c/a, the semimajor to semiminor axis ratio. d Amino acid-loaded microemulsions containing maximum amount of solubilized amino acid.

corresponding microemulsions is indicated by dotted lines. It is seen that the solubilized amount of glycine in microemulsions was always lower than that in the aqueous solution. It decreased with increasing concentration of water at constant AOT concentration (Figure 2a) and with increasing AOT concentration at constant water content in the microemulsion (Figure 2b), except for the points closest to the stability boundary of the L2 phase (W/O microemulsion) (Figure 1). To confirm the low solubilization in microemulsions, an alternative procedure was also employed, whereby the microemulsion was prepared by introducing a saturated aqueous solution of glycine instead of water at 25 °C. In that case the homogeneous L2 phase was not formed, indicating that the maximum amount of glycine that could be solubilized was smaller than its solubility in water under the same experimental conditions. The reduction of the solubilization at points closest to the single L2 phase boundary may be observed in Figure 2a. We conclude that in this region the homogeneous L2 phase collapsed upon addition of a relatively small quantity of glycine. 3.1.2. l-Histidine. The solubilized amount of l-histidine in water-isooctane microemulsions stabilized with AOT is slightly higher than the corresponding amount dissolved in water (Figure 3a), indicating that a small fraction of the solubilized molecules is located at the W/O interface, while the majority is located within the water pools. At constant concentration of water in the microemulsion, solubilization decreased with increasing AOT concentration (Figure 3b), which may be interpreted as above. (iii) l-Phenylalanine. Figure 4 shows that the amount of phenylalanine solubilized in microemulsions is significantly higher than its solubility in an equivalent amount of water and increases with increasing water content (Figure 4a) and AOT concentration (Figure 4b). The curves showing the dependence of the amount of solubilized phenylalanine on the water content are linear over a large part of the concentration regime investigated (Figure 4a). Deviations from linearity were observed at low and high water content, i.e., at low and high water/AOT molar ratios, W. To confirm the location of the phenylalanine molecules within the microemulsions, we also checked the solubility in pure isooctane with and without the presence of AOT and found it to be negligible in comparison to the solubility in water. Therefore, the results suggest that the phenylalanine molecules are solubilized both in the water pools and at the water/oil interface.

3.2. Influence of Amino Acids on the Properties of the Microemulsions. In the following, the influence of solubilized phenylalanine and glycine on the size and shape of the water droplets and their thermal behavior are described. 3.2.1. Droplet sizes and shapes were determined for microemulsions containing 2 wt % of AOT with different W ratios. In Figure 5a,b, SAXS spectra of pure microemulsions with W ) 24.4 are compared with those of microemulsions of the same composition, containing 50% and 100% of glycine and phenylalanine, respectively. (Note that 100% of amino acid represents saturation, while 50% represents half of the maximum solubilized amount). In both cases, in the small angle region where qRg < 1.5, the slope of the scattering curve, and therefore Rg, increased with increasing amino acid concentration, indicating increasing droplet sizes in the presence of the guest molecules. The pair distance distribution function, P(r), of each sample was calculated according to eq 1 and plotted in Figure 5c,d. From the P(r) plot, the micellar radius of gyration, Rg, was calculated by eq 2, independent of the Guinier analysis. Close agreement between the Rg values determined by both procedures is shown in Table 2. The shape of the P(r) function (Figure 5c,d) indicates the basic geometry of microemulsion droplets as well, assuming monodispersity.18,20 The symmetrical shape obtained from pure and glycine-loaded microemulsions (Figure 5c) is characteristic of spherical droplets, with the glycine-loaded ones shifted to larger dimensions. Assuming monodisperse spherical droplets, we calculated the sphere radii from the equation Rg2 ) (3/5)R2, as listed in Table 2. In the presence of phenylalanine (Figure 5d), the peak shifted toward larger dimensions and the symmetry was distorted, indicating a change in shape from spherical to cylindrical or ellipsoidal. Assuming a prolate ellipsoidal shape and a monodisperse population, the form parameters were calculated from the relationship Rg2 ) (2a2 + c2)/5, where a is the semiminor axis and c ) Dmax/2, the semimajor axis (Table 2). The dependence on W of the structural characteristics of pure microemulsions and those saturated with glycine or phenylalanine is listed in Table 3. It is seen that at W e 12.2 the droplets in pure microemulsions are not spherical, but their symmetry is restored in the presence (20) Strey, R,; Glatter, O.; Schubert, K. V.; Kaler, E. W. J. Chem. Phys. 1996, 105, 1175.

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Figure 6. The crystallization temperature (Tc, °C), melting temperature (Tm, °C), and the enthalpy of fusion (∆Hf, J/g) of water pools in microemulsions at (a) point N (W ) 9.8) and (b) point C (W ) 24.4) in Figure 1. Tc, Tm, and ∆Hf are plotted as a function of amino acid concentration. 100% of amino acid corresponds to the microemulsions containing the maximum amount of solubilized amino acid. The closed squares, closed triangles, and open circles indicate the pure, phenylalanine-loaded, and glycine-loaded microemulsions, respectively. At each point, measurements were repeated several times by using samples which were prepared independently.

of glycine. On the other hand, in the presence of phenylalanine, all microemulsions are of ellipsoidal shape. 3.2.2. Thermal Behavior. Changes in the thermal behavior of water volumes in microemulsions upon solubilization of amino acids can be an indicator of their solubilization behavior. We have therefore measured DSC curves of water in microemulsions, as a function of the concentration of solubilized amino acids. From the results we obtained the temperatures and enthalpies of fusion (Tm, in °C, and ∆Hf, in J/g of water) and the crystallization temperatures (Tc, °C). Figure 6 shows the results for microemulsions of compositions corresponding to points N (W ) 9.8, Figure 6a) and C (W ) 24.4, Figure 6b) in Figure 1. As the amount of water is the same in both microemulsions, the droplet sizes decrease with increasing concentration of AOT (Table 3 and ref 1), i.e., rN < rC. This is reflected in the differences of the respective values obtained at 0% amino acid concentration, which show an increase in the amount of free water (from Tm and ∆Hf) and a decrease in the degree of supercooling (from Tc) with increasing droplet sizes. The difference between the

effects of solubilizing phenylalanine and/or glycine is also obvious: whereas an increase in the glycine concentration in the microemulsion significantly depresses the values of all parameters, loading with phenylalanine has only a slight effect. This suggests that glycine molecules are distributed within the water pools and consequently inhibit the crystallization of water. On the other hand, most of the phenylalanine molecules are probably located at the water/oil interface and therefore do not significantly affect the thermal behavior of water. Figure 7 shows ∆Hf values of the water pools in pure microemulsions and glycine-loaded microemulsions as a function of the concentration of AOT (Figure 7a, 10 wt % of water) and water (Figure 7b, 25 wt % AOT), respectively. It is seen that ∆Hf in pure microemulsions (solid squares) decreases with increasing AOT concentration (Figure 7a) but increases with increasing water content (Figure 7b). This is indicative of changes in the relation between free and bound water in the microemulsion droplets. When the water pools were saturated by glycine molecules, the ∆Hf values decreased, as shown by the open circles in

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In the following section, therefore, we discuss the effect of the properties of the water in the microemulsion on the relatively low solubilization of hydrophilic molecules. According to a recent review,21 water in W/O microemulsions can be roughly classified into free water, interstitial water, physically or chemically adsorbed surface water, and chemically bound water. These kinds of water have different enthalpies and temperatures of melting, as well as anomalously low supercooling temperatures.22 However, as a first-order approximation, the water in microemulsions can be viewed as consisting of only two kinds of water, i.e., free and bound.23-25 Figure 7 shows that the enthalpy of fusion of water increases with increasing water/surfactant molar ratio, W. Assuming that glycine is solubilized only by free water in the water pools, we calculated the expected amount of water available for solubilization in a given amount of microemulsion, ffree (wt % in microemulsion), by the following equation

∆Hf ffree ) ftotal ∆Hf*

Figure 7. The changes of enthalpy of fusion (∆Hf, J/g) of water pools in microemulsions along (a) the 10 wt % water line and (b) the 25 wt % AOT line in Figure 1. The closed squares and open circles indicate the ∆Hf values of pure microemulsions and glycine-loaded microemulsions which contain the maximum amount of solubilized glycine.

Figure 7 (see also Figure 6, top). The observed reduction may be due to the hydrogen-bond network in water being disturbed by the solubilized glycine molecules. 4. Discussion The results presented above show that the solubilization of the amino acids in the microemulsions, as compared to their solubility in water, increases in the order: glycine < histidine < phenylalanine. This is understandable, since the hydrophilic glycine molecule is primarily located within the water pools, histidine is located both in the water pools and at the interface, and the hydrophobic phenylalanine is mostly situated at the water/oil interface.7,8 We will now analyze in more quantitative detail the two extreme cases: (1) when the solute is situated primarily within the water pool, as exemplified by glycine, and (2) when the solute is associated with the W/O interface, as exemplified by phenylalanine. 4.1. Effect of Structured Water on the Solubilization of Hydrophilic Amino Acids. In the case of glycine, the maximum solubilization is lower than the solubility in pure water and only changes slightly with AOT concentration (Figure 2). Since the glycine molecules are located primarily in the water pools,7 the extent of solubilization should depend on the size of the microemulsion droplets and the state of the water within them.

(3)

where ftotal (wt % in microemulsion) is the total amount of water in the system and ∆Hf*is the heat of fusion of bulk water (340 J/g for our experimental conditions).26 Figure 8 (bottom) shows changes in ffree along (a) the constant (10 wt %) water line and (b) the constant (25 wt %) AOT line. From these values, and using the solubility of glycine in aqueous solution (330 mmol/100 g), the expected amount of solubilized glycine in the microemulsions was calculated. The values obtained (open squares, Figure 8, top) are only slightly lower than the observed ones (closed rhombs), indicating that free water is primarily responsible for solubilization. A small number of amino acid molecules may be solubilized in the bound water fraction, fbound () ftotal - ffree), as well. Assuming that fbound consists primarily of water bonded to the AOT headgroups, we calculated the approximate number of water molecules per AOT headgroup. The value increased from 4 to 6 molecules when W increased from 8.2 to 24.4. In comparison, Maitra23 found six water molecules per AOT headgroup at W > 10, but much less at smaller values of W. 4.2. Hydrophobic Amino Acids at the Water/Oil Interface. 4.2.1. Phenylalanine to Surfactant Molar Ratio at the Interface. The relatively high solubilization of phenylalanine is due to the sum of solubilized molecules in the free water of the water pools and at the W/O interface. In the preceding section on glycine, we demonstrated that the contribution of the bound water to the solubilization of the amino acid is almost negligible. The same is expected in the case of phenylalanine. By taking account of the quantity of bound water, we calculate the amount of phenylalanine molecules at the interface, Sint, as follows. The solubilization of phenylalanine in 100 g of microemulsion, SME (mol/100 g of microemulsion), is expressed as (21) Schulz, P. C. J. Thermal Anal. 1998, 51, 135. (22) Casillas, N.; Puig, J. E.; Olayo, R.; Hart, T. J.; Franses, E. I. J. Colloid Interface Sci. 1989, 5, 384. (23) Maitra, A. J. Phys. Chem. 1984, 88, 5122. (24) D’Aprano, A.; Lizzio, A.; Liveri, V. T. J. Phys. Chem. 1987, 91, 4749. (25) Boned, C.; Peyrelasse, J.; Moha-Ouchane, M. J. Phys. Chem. 1986, 90, 634. (26) Garti, N.; Aserin, A.; Ezrahi, S.; Tiunova, I.; Berkovic, G. J. Colloid Interface Sci. 1996, 178, 60.

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Figure 8. The available free water (wt % in microemulsion) for solubilizing glycine (bottom) calculated from the ∆Hf values in Figure 7 and solubilization of glycine (top): (a) water constant (10 wt %) and (b) AOT constant (25 wt %). In the bottom figures, dotted lines indicate the total amount of water which exists in the system. In the top figures, the open squares and closed rhombs show the expected solubilization of glycine calculated from the available free water (bottom figures) and the observed solubilization, respectively. The dotted lines in the top figures indicate the solubilized amount of glycine in water equivalent to that in the microemulsions.

Figure 9. The calculated [phenylalanine]/[AOT] molar ratio at the droplet interface along lines of constant AOT (10-30 wt % AOT) as a function of W ([water]/[AOT] molar ratio).

SME ) Sfree + Sint

(4)

where Sint and Sfree are the molar quantities solubilized at the interface and in free water, respectively. Sfree is obtained from the solubility in aqueous solution (Saq, mol/ 100 g of water) and the weight of free water in the system, wfree (g) as

Sfree ) Saq(wfree/100)

(5)

For simplicity, here we assumed that the number of bound water molecules per AOT headgroup is six (the maximum hydration number per AOT headgroup)23 and calculated wfree by

wfree ) wtotal - 6nAOTMw

(6)

where wtotal is the total amount of water in the microemulsion, nAOT is the molar fraction of AOT in the system, and Mw is the molecular weight of water. From the value of Sint determined from eq 4, the molar ratio of phenylalanine to AOT at the interface, [Phe]/[AOT]int, was obtained at different concentrations of AOT and plotted as a function of W (Figure 9). 4.2.2. Solubilization of Phenylalanine. In Figure 9, the ratio [Phe]/[AOT]int is seen to increase both with increasing amounts of water (i.e. with increasing W) and increasing AOT concentration in the microemulsions in the range 10 < W < 20, i.e., no chemical bonding but rather a kind of physical interaction between the amino acid and the AOT headgroups seems to be involved. As previously observed by Leodidis and Hatton,27 the main driving force for interfacial association seems to be the removal of the hydrophobic side chains from the water and not some specific association with the interface. For an explanation of the first point, we consider the curve representing 20-25 wt % of AOT in Figure 9. A large part of this curve is approximately linear, with deviations at W < 5 and W g 25. In general, the reason that interfacial association increases with increasing W is the increase in the droplet size under these conditions (see Table 3). With increasing droplet sizes, the curvature of the droplets becomes smaller, and consequently, much more space will be available for phenylalanine molecules to be accommodated at the interface as a cosurfactant. In fact, a study by Maitra showed that the apparent headgroup area of AOT molecule increases from 0.36 to 0.52 nm2 as W increases from 4 to 25.23 The deviation from linearity at W < 5 is easily explained if one considers (27) Leodidis, E. B.; Hatton, T. A. in ref 1, pp 270-302.

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literature data22-24 which show that at W < 5 most of the water in the microemulsion is bound to the AOT headgroups. The reduction in the [Phe]/[AOT]int ratio at W g 25 may be explained by changes in droplet sizes and shapes. As the droplet sizes become larger and the interfaces become flatter due to the decreased curvature, the surfactant cores may no longer be able to maintain the water pools, and as a consequence, the microemulsions are destabilized upon addition of small amounts of phenylalanine. As for the second point, at W < 10, the ratios [Phe]/[AOT]int at different AOT concentrations lie on the same line, but at larger W values the lines diverge. The following explanation may be appropriate. When the system contains less than 15 wt % AOT (full squares and closed circles in Figure 9), the composition of the respective microemulsion is close to the stability boundary (Figure 1). Therefore, destabilization occurs in the presence of relatively small quantities of phenylalanine. As the AOT content increases to 30 wt % (open squares in Figure 9), much more phenylalanine may be accommodated at the interface. On the other hand, the deviation from linearity occurs at small W, i.e., W > 15. The latter effect is probably due to the increased number of microemulsion droplets, which leads to an increased frequency of droplet collisions and consequently to earlier destabilization. 5. Conclusions The present investigation has focused on the mechanisms of solubilization of amino acids in AOT-based W/O microemulsions, with a view to maximizing the amount

Yano et al.

of solubilized amino acid in systems having different particle sizes. Using the solid-liquid extraction method we could, in particular, describe the solubilization of a hydrophilic amino acid, such as glycine, which was difficult to understand via the phase-equilibrium approach. In addition, we have clarified some structural and thermal characteristics of amino acid-loaded microemulsions. Our results may be summarized as follows. (1) The solubilization of hydrophilic glycine in microemulsions is lower than expected on the basis of its solubility in aqueous solution, due to the presence of bound water near the AOT interface. Solute molecules are primarily solubilized in the free water phase, but they also partition weakly into the bound water layer. Due to this characteristic localization, the thermal behavior of water is strongly influenced both by the presence of solute molecules and by the sizes of microemulsion droplets as well. In turn, the crystallization of glycine would also be expected to be strongly affected by water structure. (2) Hydrophobic amino acids such as phenylalanine and histidine migrate into the AOT interface layer and do not significantly affect the thermal behavior of the water pool. With increasing droplet size and therefore decreasing interface curvature, many more solute molecules can be associated at the interface and the [solute]/[surfactant] molar ratio at the interface increases. On the basis of these results, we have tailored the amino acid crystallization systems which will be described in the following paper. LA0004101