Crystallization of Organic Compounds in Reversed Micelles. II

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Crystallization of Organic Compounds in Reversed Micelles. II. Crystallization of Glycine and l-Phenylalanine 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, Rohovot, Israel Received April 26, 2000. In Final Form: September 12, 2000 The crystallization of glycine and l-phenylalanine from water-isooctane microemulsions stabilized by AOT (sodium di-2-ethylhexyl sulfosuccinate) has been investigated. Crystallization phenomena were strongly affected by the localization of the solubilized molecules within the microemulsion droplets. In the case of glycine, which is solubilized within the water pools, a significant reduction in crystal size was observed in the temperature range investigated (Ti ) 35 °C, Tc ) 5 °C). While crystals formed in aqueous solution usually grow to millimeter sizes, glycine crystals grown from microemulsions were submicron to micron in size. In addition, the R-form was only observed from aqueous solution, while the γ-form was the predominant form from microemulsions. In the case of phenylalanine molecules, which are located primarily at the W/O interface, morphology and polymorphism were also affected in addition to crystal size. While phenylalanine crystallizes from aqueous solution in the form of two polymorphs, i.e., the needlelike R-form and platelike β-form, upon crystallization from microemulsions, only the β-form appeared. The different crystallization mechanisms of the two amino acids are discussed.

1. Introduction Crystallization within confined spaces delineated by artificial membranes has critical implications in both biological and materials science research. From the biological point of view, the complicated and wellcontrolled interfacial catalytic crystallization processes that are often observed in living organisms have been of great interest in fundamental research.1,2 In materials science, the emphasis has been on different applications, such as the synthesis of nanosized particles for catalysis, semiconductors, optoelectronics,3-5 and purification techniques.6 Recent studies focused on the use of such systems to modify crystallization in order to obtain favorable crystalline forms for the creation of new products in industries ranging from pharmaceuticals to ceramics. There are two approaches to the crystallization in emulsified droplets: crystallization of emulsified melt or crystallization of solute molecules from emulsified solutions. In both cases, nucleation in the droplets may occur either in the water pool or in the W/O interfacial region, depending on the interaction between the crystallizing phase and the interfacial surfactant molecules and/or the nature of the solutions in the latter case. * 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) Mann, S.; Archibald, D. D.; Dydimus, J. M.; Douglas, T.; Heywood, B. R.; Meldrum, F. C.; Reeves, N. J. Science 1993, 261, 1286. (2) Addadi, L.; Weiner, S. Angew Chem. Int. Ed. Engl. 1992, 31, 153. (3) Pileni, M. P., Ed. Structure and Reactivity in Reverse Micelles; Elsevier: Amsterdam, 1989. (4) Sager, W. F. C. Curr. Opin. Colloid Interface Sci. 1998, 3, 276. (5) Fendler, J. H. Membrane-Mimetic Approach to Advanced Materials. Advances in Polymer Sci. 113; Springer-Verlag: Berlin, Heidelberg, 1994. (6) Davey, R. J.; Garside, J.; Hilton, A. M.; McEwan, D.; Morrison, J. W. Nature 1995, 375, 664.

Crystallization which occurs in the water pool has been utilized to study homogeneous nucleation, because the effect of heterogeneous nuclei (impurities) is minimized by compartmentalization. It has been shown that the probability of a droplet containing a heterogeneous nucleation center decreases with decreasing droplet sizes. Hence, in melt crystallization, the number of crystallized droplets decreases under certain conditions and the supercooling becomes larger in smaller droplets.7 Therefore, crystallization parameters such as induction times, polymorphic occurrence, and morphology may be strongly affected by the droplet sizes. The importance of compartmentalization in controlling crystallization from aqueous solution was demonstrated by Hirai et al,8 who studied crystallization of calcium carbonate in water-in-oil-in-water double emulsion systems. It was shown that both the physical and polymorphic forms of the particles could be controlled by choosing the conditions in the internal aqueous phase and the method of demulsification. In interface-catalyzed crystallization, crystallization phenomena are even more strongly affected. A pioneering study by Skoda and Van den Temple on the crystallization of triglyceride molecules showed9 that the catalytic activity increased the more the molecular structure of the emulsifier resembled that of the crystallizing phase. A mechanism was assumed whereby emulsifier molecules, adsorbed at the surface of the oil droplets, orient triglyceride molecules close to the surface and thus catalyze nucleation. This structural approach has been corroborated by Mutaftshiev and co-workers, who showed that in stearic acid-water emulsions the nucleation temperature is also affected by emulsifiers,10 and more recently by Davey et al,11 who (7) Turnbull, D.; Cech, R. E. J. Appl. Phys. 1950, 21, 804. (8) Hirai, T.; Hariguchi, S.; Komasawa, I.; Davey, R. J. Langmuir 1997, 13, 6650. (9) Skoda, W.; Van den Temple, M. J. Colloid Sci. 1963, 18, 568. (10) Cordiez, J. P.; Crange, G.; Mutaftschiev, B. J. Colloid Interface Sci. 1982, 85, 431.

10.1021/la000615+ CCC: $19.00 © 2000 American Chemical Society Published on Web 12/02/2000

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studied the nature of nucleation catalysis at the oil/water interface of m-chloronitrobenzene (m-CNB)-in-water emulsions using a wide range of emulsifiers. The latter authors found significant correlation of the crystallization temperature, morphology, and crystal orientation with the molecular packing and structure of the amphiphile monolayer at the oil/water interface. Droplet sizes are also thought to have an effect, since the rigidity of the molecular packing of the surfactants depends on the curvature of the interfaces. Recently, the possibility to use water-in-oil microemulsions for the crystallization of new polymorphic forms of organic crystals was demonstrated by Fu¨redi-Milhofer et al.12 When the artificial sweetener, aspartame, was crystallized from water-in-oil microemulsion systems emulsified by AOT (sodium di-2-ethylhexyl sulfosuccinate) a new crystal form with a distinct X-ray powder diffraction pattern was obtained. Aspartame is a dipeptide, consisting of a hydrophilic aspartyl and a very hydrophobic, esterified phenylalanine end. To understand the observed crystallization phenomena, it was therefore of interest to study the crystallization of amino acids of different hydrophobicity in the same crystallization media, taking into account their localization within the microemulsion droplets. To see the influence of the crystallization medium on polymorphism, we choose to investigate amino acids which are known to crystallize in more than one polymorphic form. Using these criteria, the crystallization of glycine and l-phenylalanine in water-in-oil microemulsion stabilized by AOT has first been studied. Of these, the hydrophilic glycine has been known to crystallize in three forms, called R, β, and γ.13-16 The crystal structures of those forms have been solved by X-ray singlecrystal structural analysis. The R- and β-forms are monoclinic (R, space group P21/n, a ) 0.510 nm, b ) 1.197 nm, c ) 0.546 nm, β ) 111.42°, Z ) 4, V ) 7.7 × 10-2 nm3; β, space group P21, a ) 0.508 nm, b ) 0.627 nm, c ) 0.538 nm, β ) 113.12°, Z ) 2, V ) 7.9 × 10-2 nm3),14,15 while the γ-form has a hexagonal crystal structure (space group P31 or P32, a ) 0.704 nm, c ) 0.548 nm, Z ) 3, V ) 7.8 × 10-2 nm3)16; R is the thermodynamically most stable form. There are still controversial points in the literature about the number of polymorphs of phenylalanine and their crystal structures. The controversies might be due to the structural disordering around the benzene rings. Using X-ray powder diffraction, Khawas found that two polymorphs, R and β, crystallize from aqueous solutions.17 More recently, Weissbuch et al.18 solved the single-crystal structure of platelike phenylalanine crystals. These crystals have a layered structure in which the lamellar distance is 3.0 nm, which corresponds to the bilayer structure. In the preceding paper of this issue,19 we reported the solubilization characteristics of glycine, l-phenylalanine, and l-histidine in a water-in-oil microemulsion stabilized (11) Davey, R. J.; Hilton, A. M.; Garside, J.; de la Fuente, M.; Edmondson, M.; Rainford, P. J. Chem. Soc., Faraday Trans. 1996, 92, 1927. (12) Fu¨redi-Milhofer, H.; Garti, N.; Kamyshny, A. J. Cryst. Growth 1999, 198/199, 1365. (13) Albrecht, G.; Corey, R. B. J. Am. Chem. Soc. 1939, 61, 1087. (14) Marsh, R. E. Acta Crystallogr. 1958, 11, 654. (15) Iitaka, Y. Acta Crystallogr. 1960, 13, 35. (16) (a) Iitaka, Y. Acta Crystallogr. 1958, 11, 225, (b) Iitaka, Y. Acta Crystallogr. 1961, 14, 1. (17) Khawas, B. J. Ind. J. Phys. 1985, 59A, 219. (18) Weissbuch, I.; Frolow, F.; Addadi, L.; Lahav, M.; Leiserowitz, L. J. Am. Chem. Soc. 1990, 112, 7718. (19) Yano, J.; Fu¨redi-Milhofer, H.; Wachtel, E.; Garti, N. Langmuir 2000, 16, 9996 (preceding paper in this issue).

Yano et al.

Figure 1. Microemulsion compositions used as media of crystallization for glycine and phenylalanine, denoted by closed circles with alphabetical symbols in the ternary phase diagram of isooctane-AOT-water (points a, b, and c for glycine and points a-g for phenylalanine).

by AOT. The results showed that the hydrophobic phenylalanine molecules are located primarily at the interface, in agreement with the literature.3 With increasing droplet size and therefore decreasing interface curvature, many more molecules can be associated with the AOT interfacial layer, and the [solute]/[surfactant] molar ratio at the interface increases. On the other hand, the hydrophilic glycine is mainly solubilized within the free water pools and slightly in the bound water layer. Histidine has a character intermediate between these two. In the present study, we have focused on the relationship between the location of solubilized molecules of glycine and/or phenylalanine and their crystallization behavior, in the context of the crystalline sizes, morphologies, and preferred occurrence of a certain polymorph. 2. Experimental Section 2.1. Materials. AOT of 99% purity and amino acids, >99% purity, were purchased from Sigma (St. Louis, MO), while isooctane (2,2,4-trimethylpentane) of >99% purity was obtained from Aldrich (Milwaukee, WI). All chemicals were used without further purification. Double-distilled water was used for the preparation of solutions and microemulsions. 2.2. Crystallization Procedure. Crystallization experiments were carried out from both aqueous solutions and microemulsions under the same conditions. Microemulsions having different compositions, as pointed out in Figure 1, were prepared by the same method as described in the first paper of this series.19 Microemulsions, saturated with respect to glycine (at 35 °C) or phenylalanine (at 25 °C), respectively, were prepared by adding the maximum amount of the amino acid that could be solubilized. The saturated aqueous solutions and microemulsions were kept in a water bath controlled at the initial temperature (Ti, 25 °C for phenylalanine and 35 °C for glycine) for 1 h and then immediately cooled and kept at constant crystallization temperature, Tc ) 5 °C. In the case of phenylalanine, the microemulsions were periodically examined by an optical microscope to detect the onset of crystallization (and thus estimate induction times), as well as the sizes and shapes of the crystals. In the case of glycine, crystal sizes were much below the detection limit of the optical microscope, so crystals were observed by transmission electron microscopy. For the determination of polymorphic forms, X-ray diffraction and/or electron diffraction patterns were taken as follows. 2.3. X-ray Diffraction Measurements. X-ray diffraction patterns of amino acid crystals were measured on a Rigaku Rotaflex X-ray diffractometer (40 kV, 140 mV, Cu KR, with Ni filter). Phenylalanine crystals formed from microemulsions were filtered and washed with hexane several times. During this procedure, morphological changes and polymorphic transforma-

Crystallization of Amino Acids in Microemulsions tion of crystals did not occur, as was confirmed by checking the morphology and diffraction patterns of control specimens, prepared without this procedure. Diffraction patterns were first measured from crystals obtained from solutions without crushing. Then, to confirm the polymorphic forms, we also measured diffraction patterns of crushed samples. 2.4. Electron Microscopy. Samples were prepared by suspending small amounts of the microemulsion in hexane solution. Observations were carried out by two different methods, as follows: (i) Suspensions were deposited on a copper grid coated with Formvar/carbon. The grids were observed by a JEOL CX100 electron microscope (80 kV, camera length L ) 760 mm). The indexing of diffraction spots was carried out by a program provided by Ecole Polytechnique de Lausanne, Switzerland. (ii) For the negative staining method, samples were deposited on copper grids and negatively stained with 1 wt % uranyl acetate. The samples were then examined with a Philips CM 12 electron microscope at accelerating voltage of 100 kV. 2.5. Small-Angle X-ray Scattering (SAXS) Measurements. The droplet sizes and shapes of amino acid-loaded microemulsions at Ti and Tc were measured by SAXS. Samples containing 2 wt % of AOT with different [water]/[AOT] molar ratios, W, were prepared in sealed glass capillaries (1.5 mm φ) at room temperature using the procedure described in the preceding paper.19 Then the capillary was introduced into the sample holder, in which the temperature was controlled by an alcohol-water bath. Measurements of scattering profiles were started after 10 min of thermal equilibration. Measurement and analysis of the profiles were carried out as described in the preceding paper.19 2.6. Differential Scanning Calorimetry (DSC). Decomposition points of amino acid polymorphs were investigated by differential scanning calorimetry (DSC). A Mettler TA 4000 thermal analysis system, equipped with a DSC 30 low-temperature cell, was used for the measurements.

3. Results 3.1. Crystallization from Aqueous Solution. When aqueous solutions of glycine, saturated at 35 °C, were cooled to 5 °C, only the R-form crystallized, as confirmed by the X-ray diffraction pattern shown in Figure 2a. Crystals grew to several millimeters during 1 day of aging at 5 °C (Figure 2b). When aqueous solutions of phenylalanine, saturated at 25 °C, were cooled to 5 °C, two crystal forms with different X-ray diffraction patterns (Figures 3a,b) and different morphologies, needle-shaped and platelike, were obtained. Crystallization started on the walls of the vessels, where needle-shaped crystals tended to grow in advance of the platelike ones. The decomposition points of the needle-shaped and platelike crystals, as measured by DSC, were ∼240 and ∼260 °C, respectively. The needlelike crystals were designated the R-form, because their X-ray powder pattern (Figure 3a) is similar to the pattern of the R-form of phenylalanine, reported by Khawas.17 The X-ray powder diffraction pattern of R suggests a nonlayered molecular structure. The X-ray powder diffraction pattern of the platelike crystals (Figure 3b) resembles the diffraction pattern calculated by Weissbuch (Figure 3c)20 from the crystal structure of platelike phenylalanine crystals prepared and characterized by Weissbuch et al,18 except for the strong (00l) reflections (l ) 2n - 1, where n is an integer), which are missing from our pattern (Figure 3b). Therefore, we concluded that the platelike crystals obtained in the present study have almost the same lateral packing, but the lamellar distance, d(00l), is half the size of Weissbuch’s,20 as illustrated in Figure 3d, i.e., d(00l) of our platelike crystals corresponds to 1.5 nm. In the present study, we have called this platelike crystal β-form. (20) Weissbuch, I. private communication.

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Figure 2. (a) The X-ray diffraction pattern and (b) the optical micrograph of glycine R. The scale bar shows 1 mm length.

To check the effect of AOT on the crystallization, we also crystallized phenylalanine and glycine from aqueous solution in the presence of 1 wt % of AOT, but did not observe detectable changes in crystal morphology or the crystallizing polymorph. 3.2. Characterization of Microemulsions Used for Crystallization. As a first approach we have determined the solubilization of glycine and phenylalanine in microemulsions, used as crystallization media, as well as the influence of the respective solute on the characteristics of the microemulsions. Most microemulsions had W < 20, since those having W > 20 were unstable at the crystallization temperature employed (5 °C, see also ref 21). However, the stability of phenylalanine-loaded microemulsions was enhanced even at W > 20, most probably due to the cosurfactant effect of the solute molecules. 3.2.1. Solubilization. Table 1 shows the solubilization of the amino acids at the initial (Ti, °C) and crystallization (Tc, °C) temperatures. It is seen that the solubilization of glycine, which is located within the water pools, decreased appreciably with decreasing temperature. By comparison, the difference of the solubilized amount of phenylalanine between Ti and Tc was not significant. 3.2.2. Droplet Sizes. The structural parameters of microemulsion droplets containing maximum amounts of glycine or phenylalanine, solubilized at Ti, are given in the preceding paper of this series (Table 3 in ref 19), except for the sizes of glycine-loaded microemulsion droplets measured at 35 °C, for which the water pool radius, R, is 1.51 (a), 1.92 (b), and 3.19 nm (c, d, e), respectively. (Note that a-e refer to the respective points in Figure 1). 3.3. Crystallization from Microemulsions. Crystallization of amino acids from microemulsions was a much (21) Zulauf, M.; Eicke, H. F. J. Phys. Chem. 1979, 83, 480.

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Yano et al. Table 1. Solubilization of Amino Acids in Microemulsions at the Initial Temperature, Ti (25 °C for Phenylalanine and 35 °C for Glycine), and at the Crystallization Temperature, Tc (5 °C) solubilized amino acid (mmol/100 g of solvent)

microemulsions W, isooctane-AOT-water [water]/ phenylalanine (wt %) [AOT] 5 °C 25 °C water a b c d e f g

Figure 3. (a, b) The X-ray diffraction patterns of needlelike (R) and platelike crystals (β) obtained from aqueous solution in the present study, together with the optical micrographs. In part b, the inset shows the diffraction pattern obtained from the well-crushed β sample. (c) The calculated X-ray powder diffraction pattern of the platelike crystal of which the crystal structure (c, right) was determined by Weissbuch et al.18,20 (d) The schematical representation of the present platelike crystal structure based on ref 20. In part b, the reflections indicated by arrows and dotted lines correspond to the even series of the (00l) reflections and some (hkl) reflections in part c, respectively.

60/35/5 70/25/5 85/10/5 55/30/15 70/20/10 55/25/20 70/15/15

3.5 4.9 12.2 12.2 12.2 19.8 24.4

8.9 6.1 6.2 3.8 11.7 7.4 11.7 7.4

17.0 6.9 6.5 3.8 13.1 8.1 13.7 8.5

glycine 5 °C 35 °C 246 370 0.6 13.7 1.6 14.4 10.7 18.0

slower process than from aqueous solutions, occurring on a time scale of several days to 1 or 2 weeks, rather than hours. 3.3.1. Glycine. The size and morphology of the glycine crystals and the rate of their growth depended on the initial degree of supersaturation, i.e., on ∆T (Ti - Tc), and the composition of the microemulsion chosen in a particular experiment. The crystallization temperature, Tc could not be lower than 5 °C, otherwise the microemulsion would fail. Furthermore, there was a distinct difference in crystal sizes and morphology when Ti was 35 °C or higher. At Ti > 35 °C, needlelike crystals of R, having a length of several hundred microns, grew within 1 day after sample preparation. In most of the present experiments, Ti and Tc were set at 35 and 5 °C, respectively. Under these conditions, crystals, detectable by optical microscopy, could not be observed even after 3 weeks; but in samples aged for 3 days, submicron-sized crystals were detected by electron microscopy in samples of the composition a and b, but not point c, marked in Figure 1 (for the composition of the respective microemulsions see Table 1). Electron micrographs of crystals observed after 1 week of aging at 5 °C are shown in Figure 4a. Rhombic and hexagonal-shaped crystals were observed and their sizes were relatively uniform, depending on the droplet size of the microemulsions. However, in all cases, crystal sizes exceeded by far the droplet sizes of microemulsions, ranging from several tens of nanometers to micrometers as the water pool radius of droplets changed from 1.5 (point a in Figure 1) to 1.9 nm (point b).19 We obtained the electron diffraction patterns of crystals having suitable thickness. Then the polymorphic form was determined by comparing the predicted diffraction patterns from the single-crystal structures of three known polymorphs (R, β, and γ).13-16 At both points a and b in Figure 1, the predominant diffraction pattern obtained from most of the crystals was of the γ-form, for which the zone axis is [0001] (Figure 4c,d). On rare occasions, the diffraction pattern of the R-form was also observed at point b, for which the zone axis is [110] (Figure 4e,f). 3.3.2. Phenylalanine. Depending on the experimental conditions, phenylalanine crystals appeared within several hours to several days. Figure 5 shows optical micrographs of the crystals grown from microemulsions having different water pool sizes (compositions designated as points a, b, c, e, and g in Figure 1), while X-ray powder diffraction patterns of crystals grown from microemulsions with W ) 4.9 (point b) and W ) 24.4 (point g) are given in Figure 6. Generally within 1 day crystals grew far larger than the order of magnitude of droplet sizes of the respective

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Figure 4. Electron micrographs of glycine crystals grown from the microemulsions at (a) point a and (b) point b in Figure 1. The scale bars show 1 µm length. (c) and (e) Show the electron diffraction patterns of glycine crystals and (d) and (f) show the assignments of the main diffraction spots observed in parts (c) and (e), respectively.

microemulsions, an exception being crystals grown from microemulsions designated as point a in Figure 1. In all cases, the only crystallographic form obtained from microemulsions was the β-polymorph (Figure 6), which, however, appeared in a wide variety of morphologies, mainly as mixtures of platelike and pyramidal-shaped crystals (Figure 5). The observed variety in morphologies seems to be a result of different crystal growth rates in microemulsions of different compositions. The crystal growth rates can be roughly estimated from the induction times, listed in Table 2. By comparing Table 2 and Figure 5, we see that from microemulsions of compositions e and g, in which crystals grew relatively quickly, predominantly platelike crystals appeared, while the population of deformed pyramidal

shapes increased as the induction time became longer (points a-c in Table 2 and Figure 5). As a consequence of the morphological changes, the intensities of some (hkl) reflections with h * 0 and/or k * 0 were enhanced as indicated by arrows in Figure 6a. In all cases, such pyramidal-shaped crystals tended to have numerous steps, parallel to the dominant (001) plane (Figure 5, c-2). The induction times of crystallization seem to be affected by both the sizes and number of droplets. We first consider points c, d, and e in Table 2 and Figure 1. In these microemulsions, the W value is the same (W ) 12.2), and therefore, droplets of these three microemulsions should be similar in size and shape. Since the main difference between these three microemulsions is the number of droplets, the significant difference in the induction time

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Figure 5. Optical micrographs of phenylalanine crystals grown for 4 days from microemulsions having different isooctaneAOT-water composition. The alphabetical symbols at the top left correspond to the points in Figure 1. The scale bars show 1 mm at a and c-2 and 0.25 mm at b, c-1, e, and g.

can be attributed to higher frequencies of droplet interactions in the order of points d > e > c. On the other hand, the induction time tended to become shorter as W increased from 3.5 to 24.4, i.e., with increasing water pool sizes (Table 2). This phenomenon seemed to be related to the stability of microemulsion droplets. 3.4. Structural Changes of Microemulsion Droplets during Crystallization. Structural changes of the microemulsion droplets during crystallization were monitored by SAXS measurements. Figure 7 shows changes in the pair distance distribution functions, P(r), of pure microemulsions and amino acid-loaded ones during cooling and crystallization. When pure microemulsions were cooled from 35 to 5 °C (Figure 7a), there were no detectable changes in the scattering profiles during the initial stage, but a reduction

of droplet sizes on the order of 0.1-0.2 nm in Rg value was observed after 1 week of standing at 5 °C. Therefore, we concluded that significant structural changes and/or aggregation of droplets, which might affect the crystallization of the amino acids, do not occur as a consequence of the cooling procedure. A similar behavior was observed in the glycine-loaded microemulsions (Figure 7b). At W < 5, the P(r) function measured after 1 week at 5 °C was slightly distorted as compared to the initial one, indicating that some structural changes or aggregation of droplets occurred, probably through the interaction of droplets. However, when W was 12.2, the symmetry of the P(r) curve did not change during 1 week, but the curve shifted to smaller dimensions in the same manner as observed in pure microemulsions (Figure 7a).


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4. Discussion

Figure 6. X-ray diffraction patterns of phenylalanine crystals grown from microemulsions: (a) point b (W ) 4.9) and (b) point e (W ) 24.4) in Figure 1. Arrows indicate some (hkl) reflections with h * 0 and/or k * 0. Table 2. Induction Time of Phenylalanine Crystallization in the Microemulsions microemulsions isooctane-AOT-water (wt %) a b c d e f g

60/35/5 70/25/5 85/10/5 55/30/15 70/20/10 55/25/20 70/15/15

W, [water]/ [AOT]

induction timea

3.5 4.9 12.2 12.2 12.2 19.8 24.4

4 days 1 day 2 days 10-15 h 15-20 h 10-15 h 15-20 h

a

Time until crystals are observed under the optical microscope (×400).

When the microemulsions were loaded with phenylalanine (Figure 7c), the symmetry of the P(r) curves was profoundly disturbed after keeping specimens at 5 °C for several hours, and the effect was larger at smaller W. Since similar structural changes did not occurr in the controls (Figure 7a), the result suggests that aggregation of droplets occurred during the crystallization process. The difference in magnitude of the structural changes between the two microemulsions may be explained as follows: At constant AOT (2 wt %), the main differences between two microemulsions of W ) 4.9 and 12.2 are the size, VW, and number, NW, of the droplets, (i.e. VW)4.9 < VW)12.2 and NW)4.9 > NW)12.2, respectively). As the frequency of interaction between droplets increases with increasing number of particles, it is reasonable to assume that the larger structural changes of droplets at smaller W, seen in Figure 7c, are a consequence of higher frequency of droplet interaction.

The above results show that the crystallization behavior of amino acids in microemulsions depends on their hydrophobicity, and, thus, on the localization of molecules within the microemulsions. We have investigated crystallization of two different molecules, (i) the hydrophilic glycine, which solubilizes only within the water pools, and (ii) the hydrophobic phenylalanine, which migrates mainly to the W/O interfacial area. In both cases, because of compartmentalization, crystallization from microemulsions was much slower than in aqueous solution and interactions between microemulsion droplets seem to be involved in the crystallization process. In the following, the two types of crystallization behavior will be discussed in more detail. 4.1. Glycine. During the crystallization of glycine in microemulsions, two phenomena, (1) the significant reduction in crystal size and (2) the predominant occurrence of the γ-form, were observed within the range of supersaturations investigated. With respect to the first point, however, crystals grew much larger than the sizes of individual microemulsion droplets, their actual sizes depending on the composition of the microemulsion. To explain this phenomenon, we will first examine the possibility of homogeneous nucleation within the microemulsion water pools, which is where most of the glycine molecules are located. To begin, the amount of glycine molecules which may exist in one microemulsion droplet was calculated from solubilization data. The volume of the water pool in one microemulsion droplet, Vw, is expressed as

Vw ) 4πrw3/3

(1)

where rw is the water pool radius of microemulsion droplets. For simplification, we assumed that the volume of one glycine molecule (vgly) is 7.7 × 10-2 nm3 and that of one water molecule (vw) is 3.0 × 10-2 nm3 without taking account of the different physical state of water in microemulsions.13-16,19 By assuming a monodispersed system, the number of microemulsion droplets in the system, Nd, is roughly estimated by

Nd ) (vwnw + vglyC)NA/Vdrop

(2)

where nw (mol/100 g of microemulsion) is the molar amount of water in the microemulsion, C is the solubilized amount of glycine in the system (mol/100 g of microemulsion), and NA is Avogadro’s constant. The number of glycine molecules per microemulsion droplet, Ngly/d, is then described by

Ngly/d ) CNA/Nd

(3)

In the next step, we consider the size of the critical homogeneous nucleus of glycine. In general, the radius of the critical nucleus, r*, is expressed as

r* ) 2vσ/∆µ

(4)

where v is the volume of one molecule, σ, the interfacial energy, and ∆µ, the driving force of crystallization. In eq 4, ∆µ is expressed as

∆µ ) kT ln(C/Ce)

(5)

where k is Boltzmann’s constant (J/K), T ) Tc is the crystallization temperature (K), and Ce is the equilibrium concentration of the solute (mol/100 g of solvent) at Tc.

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Figure 7. The pair distance distribution function, P(r), of (a) pure, (b) glycine-loaded, and (c) phenylalanine-loaded microemulsions (W ) 4.9, 12.2). All experiments were carried out at 2 wt % AOT (0.04 m). The P(r) curves 1 represented by the closed circles were obtained by SAXS measurements at Ti (25 °C for phenylalanine-loaded microemulsion and 35 °C for pure and glycine-loaded ones). The curves 2 and 3 were measured at Tc (5 °C) after times indicated in the figures. Table 3. Glycine Molecules in the Microemulsion Droplets and the Driving Force of Crystallization microemulsion ab cb

nw (mol/100 g water pool of microrw ∆µa Vw Nd emulsion (nm) (nm3) (×1020) Ngly/d (×10-21 J) 0.27 0.27

1.51 14.4 3.19 136.0

3.83 0.42

23 258

12.0 2.0

a Solubilization data in microemulsions at 35 °C (C) and 5 °C (Ce) (Table 1) were used for the calculation. b Microemulsions at point a (W ) 3.5) and point c (W ) 12.2) in Figure 1.

The number of molecules which compose one critical nucleus, Ngly/r* is then represented by

Ngly/r* )

4πr*3 1 3 v

(6)

Table 3 lists the calculated results of Ngly/d and ∆µ (eqs 3 and 5). Concerning the parameters of critical nuclei, precise values of r* and Ngly/r* were not obtained, since the

interfacial energy, σ, of glycine in water is not available. However, taking account of the literature,22 the value of σ for molecules such as glycine in aqueous solution would be on the order of several tens of mJ/m2. In this case, r* changes from being subnanometer in size to several nanometers, with decreasing ∆µ from point a to c (Table 3) and the Ngly/r* value changes from several molecules to several hundred molecules. Accordingly, comparison of Ngly/r* with Ngly/d suggests that the number of solute molecules in a water pool is sufficient to make a homogeneous nucleus in small droplets (i.e., Ngly/d > Ngly/r*), while for larger water pools, there is less possibility of making a nucleus in one droplet (Ngly/d < Ngly/r*). This is consistent with the crystallization phenomena observed in the present study: the number of crystals grown from different water pool sizes decreased in the order: point a > point b > point c in Figure 1. In fact, no crystals were obtained at point c. It is therefore conceivable that glycine (22) Mullin, J. W., Ed. Crystallization, 3rd ed.; Butterworth-Heinemann Ltd.: Oxford, 1993; pp 172-201.


crystals are formed by homogeneous nucleation in small droplets where W < 5. However, the predominant occurrence of the γ-form in microemulsions instead of the R-form strongly suggests an effect of interfacial structures in microemulsions. In addition, the crystals grown from microemulsions were far larger than the size of the droplets. Therefore, a much more likely mechanism of nucleation and subsequent crystal growth is as follows: Our solubilization studies19 have shown that the quantity of solubilized glycine in microemulsions is slightly higher than the value expected from the solubility in free water. It indicates that the bound water layer includes glycine molecules as well. We then assume that crystallization of glycine within microemulsions is initiated by heterogeneous nucleation. Heterogeneous nuclei are formed close to the W/O interface, in the bound water layer. Such nuclei would then grow by the addition of solute supplied by the interaction of many microemulsion droplets: indeed, the crystals observed under the electron microscope are several hundred nanometers in size. Since microemulsions are dynamic systems, such interactions with exchange of solute do not necessarily cause significant distortions in droplet shapes; however, the reduction in droplet sizes with the progress of crystallization observed by SAXS (Figure 7b) is quite conceivable under these conditions. One additional factor to consider is the significant difference between crystal growth rates in microemulsions, where 100-nm-sized crystals were formed within several days, as compared to crystallization rates in aqueous solutions at comparable degrees of supersaturation (Table 1), where millimeter-sized crystals grew within 1 day. We therefore postulate that crystallization of glycine is retarded by the compartmentalization of the glycine solution into small droplets and the growth rate is related to the frequency of interaction of microemulsion droplets. The significant change in crystal growth rates and morphology in the higher temperature range (Ti > 35 °C) has not been investigated further, but we assume that it is related to destabilization of the microemulsion as a consequence of “overloading” with glycine molecules at the crystallization temperature. Concerning the predominant occurrence of the γ-form from microemulsions, despite the occurrence of R in bulk solution, this may be explained by the presence of the bound water layer near the AOT headgroup. As shown in part I of this series,19 there is a certain amount of glycine molecules solubilized in this region. When heterogeneous crystallization occurs through the interaction of microemulsion droplets as described above, the interaction between glycine and water bound to the AOT headgroups may be the most important factor determining the nature of the polymorph. A recent study by Allen and Davey discussed the preferential occurrence of the γ-form in microemulsions by taking account of the different growth mechanisms of R and γ.23 They surmised that the anionic AOT molecules encourage the nucleation of the (103) face of γ, on which electrically positive amine groups are exposed, and then the carboxylic surfaces grow into the water pool. As the droplet sizes become larger, the effect of the interface monolayer becomes smaller and the possibility of the nucleation of R, whose growth unit is the hydrogen-bonded dimer, becomes higher inside the water pools.23 To better understand the effect of bound water for (23) Allen, K.; Davey, R. J. Proceedings of the 14th International Symposium on Industrial Crystallization; Cambridge, IChemE, Rugby, UK, 1999.

Langmuir, Vol. 16, No. 26, 2000 10013

the crystallization of glycine, we need further quantitative experiments concerning the relationship between droplet sizes of microemulsions and occurrence of glycine crystal polymorphs. 4.2. Phenylalanine. The crystallization of phenylalanine in microemulsions showed the following features: (i) Only the β-form crystallized from microemulsions. (ii) Crystals appeared as mixtures of two basic morphological forms, plates and pyramids, with their sizes unrelated to the sizes of the microemulsion droplets. A great number of steps, parallel to the dominant (001) plane, were observed in the pyramidal-shaped crystals. (iii) The population of the different morphological types within the mixtures seemed related to the crystal growth rates, which in turn depended on the number of microemulsion droplets and their water pool sizes (Table 2). (iv) The crystallization process induced significant structural changes in the microemulsion droplets (Figure 7c). All the above observations indicate an intimate involvement of the microemulsion droplets in the crystallization process. As shown by solubilization studies,19,24 solubilized phenylalanine molecules are located at the W/O interface. Thus it is reasonable to assume a mechanism by which nucleation of phenylalanine crystals occurs at the interface of the droplets and crystal growth proceeds by droplet interaction. Since the frequency of interaction of the microemulsion droplets depends on their number and size distribution, the dependence of the induction times on these parameters is readily explained. The fact that only the β-polymorph crystallizes from microemulsions can be explained by its crystal structure. As illustrated in Figure 2d, the β-form has a layered structure in which neighboring molecules are bonded by hydrogen bonds in the ab-plane.18 At the W/O interface, where the initial, heterogeneous nuclei are formed, phenylalanine molecules probably orient with their hydrophilic parts toward the water pools and the hydrophobic parts toward the oil phase, together with the adjacent AOT molecules. Such orientation would facilitate the formation of the layered molecular arrangements. Therefore, the predominant occurrence of the β-form is due to the two-dimensional orientation of phenylalanine molecules at the interfaces. The peculiar morphological features, especially the numerous steps within the crystals, could indicate that the crystals were formed via two-dimensional “building blocks”, rather than by gradual addition of solute molecules. Such building blocks probably consist of assemblages of phenylalanine molecules, oriented by the W/O interfaces of individual microemulsion droplets. This picture is consistent with the significant distortion that microemulsion droplets experience during the crystallization process. Another possible factor in this morphological change is the selective adsorption of AOT molecules to a certain growth plane of phenylalanine crystals, which results in growth inhibition. In the bulk solution, we could not observe the equivalent morphological changes caused by the addition of AOT molecules. However, we should not ignore this effect, since in the microemulsion the solubilized amount of AOT is much higher than in the bulk solution and therefore AOT molecules can closely contact the crystal nuclei. 5. Conclusions In the present study, we have demonstrated that, in the crystallization of both hydrophilic glycine and hy(24) Leodidis, B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6400.

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drophobic phenylalanine molecules from microemulsions, interaction of microemulsion droplets is involved. In the case of glycine, the microemulsion serves as a medium for compartmentalization, without intimate involvement of the W/O interface in the crystallization process. Crystals are initiated by heterogeneous nuclei and grow by the addition of solute molecules supplied through dynamic droplet interactions. In the crystallization of phenylalanine, however, the W/O interface is intimately involved. Crystals are initiated by heterogeneous nuclei, consisting of molecules which are preferentially oriented at the W/O interface. They subsequently grow through contact with interfaces of other droplets, which contain similarly oriented phenylalanine molecules. Under these conditions,

Yano et al.

both the crystal structure and morphology are controlled by the crystallizing environment. Acknowledgment. We are grateful to Ezra Rahamim, Faculty of Medicine, The Hebrew University of Jerusalem, as well as Abraham Willenz and Ruth Govrin, Life Science Institute, The Hebrew University of Jerusalem, for measuring the electron micrographs. We thank Dr. Frederic Cuisinier, Universite Louis Pasteur, Strasbourg, for discussions on the analyses of electron diffraction patterns. We also thank I. Weissbuch, Weizmann Institute of Science, for useful discussions concerning the crystal structures of amino acids. LA000615+

Crystallization of Organic Compounds in Reversed Micelles. II

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