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Probing the Initial Events in the Spontaneous Emulsification of trans-Anethole Using Dynamic NMR Spectroscopy David Carteau,† Isabelle Pianet,*,† Pascal Brunerie,‡ Bruno Guillemat,‡ and Dario M. Bassani*,† ISM/CNRS UMR 5255, UniVersite´ Bordeaux 1, F-33405, Talence, France, and Centre de Recherche Pernod Ricard, F-94015 Cre´ teil, France ReceiVed August 8, 2006. In Final Form: December 8, 2006 The spontaneous emulsification of alcoholic solutions of trans-anethole (t-A) in water is investigated using EXSY and DOSY NMR techniques. The system investigated (5-10 mM t-A in 5% EtOH/H2O solution) is exceptional in providing sharp, clearly resolved signals for both t-A that is dissolved in the aqueous phase (free t-A) and t-A that is incorporated in aggregates (3-6 nm diameter) thus allowing both fractions to be probed simultaneously. This feature is utilized to explore the initial events that occur during the spontaneous emulsification process. Upon mixing, the majority of the t-A (ca. 75%) undergoes nucleation to form small aggregates (ca. 10 nm diameter), while 15% (corresponding to [t-A] ) 7.5 × 10-4 M) is dissolved in the aqueous phase. The kinetic rates governing the exchange process between aggregated and free t-A are found to be time-dependent and slow on the NMR time scale (k ) 0.8-2 s-1). DOSY experiments indicate that the initially formed small aggregates undergo rapid coalescence to form larger droplets. Ostwald ripening of these droplets at the expense of the remaining small aggregates is responsible for the subsequent, slower time-evolution of the system.
Introduction With few exceptions, mixing two immiscible liquids in the absence of a suitable surfactant results in phase separation and the formation of two stable pure liquid phases. However, under the proper conditions, certain organic compounds are capable of spontaneous emulsification,1-5 a process sometimes referred to as the “ouzo effect”.6 Emulsification processes are of paramount importance for the chemical industry, where they are used in foods,7,8 paints,9 pharmaceutical and cosmetic products,10-14 and also to control polymerization reactions in aqueous-based systems.15-18 Yet another potential application, the formation of * To whom correspondence should be addressed. E-mail: d.bassani@ ism.u-bordeaux1.fr. Tel: +33 540 002827. Fax: +33 540 006158. E-mail:
[email protected]. Tel: +33 540 006448. Fax: +33 540 002623. † Universite ´ Bordeaux 1. ‡ Centre de Recherche Pernod Ricard. (1) Miller, C. A. Surf. Sci. Ser. 2006, 132, 107. (2) Lamaallam, S.; Bataller, H.; Dicharry, C.; Lachaise, J. Colloids Surf., A 2005, 270-271, 44. (3) Bouchemal, K.; Briancon, S.; Perrier, E.; Fessi, H. Int. J. Pharm. 2004, 280, 241. (4) Pautot, S.; Frisken, B. J.; Cheng, J.-X.; Xie, X. S.; Weitz, D. A. Langmuir 2003, 19, 10281. (5) Nishimi, T.; Miller, C. A. Langmuir 2000, 16, 9233. (6) Vitale, S. A.; Katz, J. L. Langmuir 2003, 19, 4105. (7) Clark, M. M.; Ahn, W.-Y.; Li, X.; Sternisha, N.; Riley, R. L. Langmuir 2005, 21, 7207. (8) Vaziri, A.; Warburton, B. J. Microencapsulation 1994, 11, 649. (9) Paints, Caotings and SolVents; 2nd ed.; Stoye, D., Freitag, W., Eds.; WileyVCH: Weinheim, Germany, 1998. (10) Ricci, M.; Blasi, P.; Giovagnoli, S.; Perioli, L.; Vescovi, C.; Rossi, C. Int. J. Pharm. 2004, 275, 61. (11) Bouchemal, K.; Briancon, S.; Perrier, E.; Fessi, H.; Bonnet, I.; Zydowicz, N. Int. J. Pharm. 2004, 269, 89. (12) Niwa, T.; Takeuchi, H.; Hino, T.; Kunou, N.; Kawashima, Y. J. Pharm. Sci. 1994, 83, 727. (13) Liu, M.; Dong, J.; Yang, Y.; Yang, X.; Xu, H. Eur. Polym. J. 2005, 41, 375. (14) Sonneville-Aubrun, O.; Simonnet, J. T.; L’Alloret, F. AdV. Colloid Interface Sci. 2004, 108-109, 145. (15) Alargova, R. G.; Bhatt, K. H.; Paunov, V. N.; Velev, O. D. AdV. Mater. 2004, 16, 1653. (16) Xu, J.; Jamieson, A. M.; Qutubuddin, S.; Gopalkrishnan, P. V.; Hudson, S. D. Langmuir 2001, 17, 1310.
nanoparticles and nanocapsules based on spontaneous emulsification phenomena, has been recently reviewed by Ganachaud and Katz.19 In contrast to the use of mechanical force, ultrasound, or the addition of surfactants, spontaneous emulsification is a process leading to an emulsion that is meta-stable over extended periods of time upon simple mixing of a solution of waterinsoluble compound in a hydrophilic solvent with water. Current environmental concerns have raised interest in lowering the large-scale use of organic solvents by employing aqueousbased emulsions, and probing the fundamental processes behind spontaneous emulsification has become increasingly important. The most well-known manifestation of this effect, and arguably the most popular, is the cloudy aspect obtained upon dilution of anis-flavored alcoholic beverages that are common in the Mediterranean countries. In this case, the organic compound is the essential oil obtained from star anis, trans-anethole (t-A), which undergoes spontaneous emulsification as the alcohol content of the hydro-alcoholic solution is dropped from 45% to ca. 5% V/V. In such systems, large fluctuations in the surface tension at the interface have been proposed to cause selfemulsification,20 but an alternative process, “diffusion and stranding”, has also been proposed.21-23 In the latter model, the sudden diffusion of the hydrophilic solvent (ethanol in the case of ternary t-A/ethanol/water systems) into the aqueous phase leaves the hydrophobic oil “stranded” in the aqueous phase as small droplets. Recently, however, this view has been challenged by Vitale and Katz,6 who proposed that the process is governed by the nucleation of the water-insoluble oil from the supersaturated (17) Bouchemal, K.; Briancon, S.; Fessi, H.; Chevalier, Y.; Bonnet, I.; Perrier, E. Mater. Sci. Eng., C 2006, 26, 472. (18) Salager, J.-L.; Forgiarini, A.; Marquez, L.; Pena, A.; Pizzino, A.; Rodriguez, M. P.; Rondon-Gonzalez, M. AdV. Colloid Interface Sci. 2004, 108-109, 259. (19) Ganachaud, F.; Katz, J. L. ChemPhysChem 2005, 6, 209. (20) Miller, C. A.; Scriven, L. E. J. Colloid Interface Sci. 1970, 33, 360. (21) Lopez-Montilla, J. C.; Herrera-Morales, P. E.; Pandey, S.; Shah, D. O. J. Dispersion Sci. Technol. 2002, 23, 219. (22) Quintanar-Guerrero, D.; Allemann, E.; Doelker, E.; Fessi, H. Colloid Polym. Sci. 1997, 275, 640. (23) Davies, J. T.; Rideal, E. K. Interfacial Phenomenae; Academic Press: New York, 1963.
10.1021/la062339q CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007
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aqueous solution formed upon dilution, a process that directly leads to the formation of small droplets that are stable over time. In spite of the growing interest in spontaneous emulsification, only few reports experimentally address the question of the processes involved in the initial stages of the emulsion process. Dynamic light scattering (DLS) techniques have been extensively employed in studying such systems and corroborate the formation of micrometer-sized droplets that are stable over time.24 In the case of t-A, recent small-angle neutron scattering (SANS) experiments have shown that the size of the initially formed aggregates, ca. 380 nm, is independent of the initial volume fraction of t-A and increases slowly over a period of several hours.25 However, to obtain information on the origin of the droplets formed at the initial stages of the emulsification process, it is necessary to employ techniques that are capable of directly monitoring the exchange between the free (dissolved) and the aggregated fractions of the immiscible component. For this, NMR spectroscopy is particularly well adapted as it is capable, in favorable cases when the individual components possess different spectral signatures, to directly monitor the exchange between molecules that are aggregated and molecules that are free in solution. This is indeed expected to be the case for t-A, as the environment within the aggregates or small droplets should be comparable to that of an aromatic solvent, much different from the local environment of t-A molecules dissolved in the aqueous phase. Additionally, through the use of diffusion ordered spectroscopy (DOSY), it is possible to determine the diffusion constant for each component, which is directly linked to the hydrodynamic radius of the species being monitored. DOSY NMR has been recently applied to the study of numerous supramolecular26-31 and polymeric systems32-34 and to the selfassembly of peptides into ordered aggregates.35,36 However, its application to emulsions remains largely unexplored and complicated by broad overlapping signals and the nonhomogeneity of the particle size distribution.37 In this work, we report the use of EXSY (exchange spectroscopy) and DOSY NMR spectroscopy to elucidate the initial dynamics of aggregation leading to the process of spontaneous emulsification in the t-A/ ethanol/water system. The unusually slow exchange rates in this system result in exceptionally clean NMR spectra, allowing a detailed analysis to be carried out. The results we obtain are consistent with a model in which t-A initially forms small (ca. 2 nm diameter) aggregates that are in pseudo-equilibrium with the small amount of t-A in the aqueous phase (Figure 1). Coalescence of these aggregates into larger droplets occurs on a longer time scale and leads to the formation of the meta-stable (24) Sitnikova, N. L.; Sprik, R.; Wegdam, G.; Eiser, E. Langmuir 2005, 21, 7083. (25) Grillo, I. Colloid Surf. A: Physicochem. Eng. Aspects 2003, 225, 153. (26) Cohen, Y.; Avram, L.; Frish, L. Angew. Chem. Int. Ed. 2005, 44, 520. (27) Schmuck, C.; Rehm, T.; Groehn, F.; Klein, K.; Reinhold, F. J. Am. Chem. Soc. 2006, 128, 1430. (28) Dalgarno, S. J.; Fisher, J.; Raston, C. L. Chem.-Eur. J. 2006, 12, 2772. (29) Johnstone, K. D.; Yamaguchi, K.; Gunter, M. J. Org. Biomol. Chem. 2005, 3, 3008. (30) Megyes, T.; Jude, H.; Grosz, T.; Bako, I.; Radnai, T.; Tarkanyi, G.; Palinkas, G.; Stang, P. J. J. Am. Chem. Soc. 2005, 127, 10731. (31) Rudzevich, Y.; Rudzevich, V.; Moon, C.; Schnell, I.; Fischer, K.; Boehmer, V. J. Am. Chem. Soc. 2005, 127, 14168. (32) Plummer, R.; Hill, D. J. T.; Whittaker, A. K. Macromolecules 2006, 39, 3878. (33) Dobrawa, R.; Lysetska, M.; Ballester, P.; Gruene, M.; Wuerthner, F. Macromolecules 2005, 38, 1315. (34) Gambs, C.; Dickerson, T. J.; Mahajan, S.; Pasternack, L. B.; Janda, K. D. J. Org. Chem. 2003, 68, 3673. (35) Martinek, T. A.; Hetenyi, A.; Fulop, L.; Mandity, I. M.; Toth, G. K.; Dekany, I.; Fulop, F. Angew. Chem. Int. Ed. 2006, 45, 2396. (36) Narayanan, S.; Reif, B. Biochemistry 2005, 44, 1444. (37) Poznanski, J.; Szymanski, J.; Basinska, T.; Somkowski, S.; Zielenkiewicz, W. J. Mol. Liq. 2005, 121, 21.
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Figure 1. Mechanism in which small (2-3 nm) aggregates are initially formed upon spontaneous emulsification of an ethanolic solution of t-A in water is proposed. These then rapidly coalesce to form small droplets which continue to grow by Ostwald ripening.
Figure 2. 1H NMR of a 5 mM t-A solution in 5% EtOD/95% D2O. The signals assigned to the free and aggregated t-A are sharp and well separated.
emulsion that has been previously investigated using DLS techniques.24
Results and Discussion Spontaneous emulsions of t-A are readily obtained by rapidly diluting a solution of t-A in ethanol with water, and, by employing ethanol-d6 and D2O, it is possible to directly follow the aggregation process by NMR spectroscopy. The 1H NMR spectrum of a solution of t-A (5 mM) in 5% C2D5OD/D2O is shown in Figure 2. The most striking features are the presence of two sets of signals for t-A, with the more intense signals located upfield by ca. 0.6 ppm with respect to the weaker set, and the fact that all of the signals are extremely sharp. These observations point to the existence of t-A in two different environments, which are in slow interconversion on the NMR time scale. Based on the observed chemical shifts and the low solubility of t-A in aqueous media, the weaker signals are assigned to t-A dissolved in the aqueous phase, whereas the stronger t-A signals at higher field are attributed to aggregated t-A (this is also confirmed by the diffusion constants associated to the two t-A signals, see below). Because the exchange of t-A between the two environments is slow on the NMR time scale, it can be probed using twodimensional NOE experiments to determine the exchange rate between the two sets of t-A signals (EXSY). The exchange rates between the free and aggregated t-A measured by observing magnetization transfer confirms that the two populations are slowly exchanging on the NMR time scale. However, the observed rates are themselves time-dependent, as shown graphically in Figure 3 for a solution of t-A (4 mM) such that the signals of free and aggregated species are roughly similar in intensity at the beginning of the experiment. Upon mixing, the initial exchange rates38 are 0.8 and 1.2 s-1 for k-a and ka, respectively, indicating that the t-A aggregates are rapidly growing in size. This process continues for the first hour, after which the relative rates invert and k-a > ka. During this time, larger aggregates that escape detection by NMR but whose formation (38) Identical diffusion rates were obtained from analysis of the aromatic or the alkyl portion of the spectra of the free or aggregated t-A.
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Langmuir, Vol. 23, No. 7, 2007 3563
Figure 3. Time dependence of k-a (filled circles) and ka (filled diamonds) for a 4 mM t-A solution in 5% EtOD/95% D2O.
has been established by DLS have formed. The process being monitored is therefore assigned to the Ostwald ripening of the larger droplets by slow dissolution of the smaller t-A aggregates, with the total amount of t-A dissolved in the aqueous phase remaining constant. After ca. 5 h, the system has reached a pseudo equilibrium that can last for several days, with the apparent dissolution rate (k-a) remaining somewhat higher than ka. The spectrum shown in Figure 2 contrasts sharply with the spectra generally obtained for dispersed aggregates, which tend to be broad and ill-defined. The reason for this is the very slow exchange between t-A molecules in the aqueous phase and t-A molecules located within the aggregates. In fact, the observed rates (1-2 Hz) are roughly 6 orders of magnitude slower than those generally associated with the exchange of surfactants in micelles.39,40 It is interesting to note that ka and k-a are actually quite similar in magnitude to the rate of exchange of a small aromatic molecule (benzene) between the aqueous phase and the interior of SDS micelles,41 suggesting that a similar activation barrier is present. The difference in chemical shift between the two environments (∆∂ ) 0.6 ppm) is large and contributes to rendering the exchange process slow on the NMR time scale. Whereas the spectrum assigned to free t-A is similar to that of t-A recorded in a nonpolar solvent such as CDCl3, the resonances of the t-A signals within the aggregates are all uniformly shifted upfield in agreement with their location in an environment more typical of an aromatic solvent.42 The time-dependence of rates of exchange of t-A between the isotropic aqueous phase and the aggregates suggest that the latter are slowly growing in volume, but do not provide a measure of their size. For this, diffusion oriented NMR spectroscopy (DOSY) provides direct access to the diffusion rate of a given species. Because the aggregated and free t-A are in slow interconversion, it is possible to measure the diffusion constant for each species simultaneously. The high field portion of the 1H NMR spectrum of an emulsion of t-A in 5% is shown in Figure 4 (top). The signals corresponding to the species with the highest diffusion constants decrease more rapidly than those associated to slowly diffusing species. Thus, it can be seen that the signals assigned to free t-A decrease concomitantly to the signal of the residual solvent peak, indicating similar diffusion constants. Conversely, the signals assigned to aggregated t-A are only slightly diminished over the same time period, indicating a much slower diffusion rate. From the field-gradient-dependence of the signal intensity, (39) Guo, W.; Brown, T. W.;Fung, B. M. J. Phys. Chem. B 1991, 95, 1829. (40) Frindi, M.; Michels, B.; Levy, H.; Zana, R. Langmuir 1994, 10, 1140. (41) Luo, R. S.; Mao, X. A. Chem. Phys. Lett. 1997, 270, 77. (42) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512.
Figure 4. (a) DOSY of a 5 mM t-A solution in 5% ethanol-d6 in D2O and (b) determination of the diffusion constants of the free (filled triangles) and aggregated (filled circles) t-A.
it is possible to extract the diffusion constant for each species. The semilogarithmic linearization of the data is shown in Figure 4b, with the larger (more negative) slope indicating higher diffusion rates.43 In the case of the above example, the free t-A is found to possess a diffusion rate similar to that of water, used as an internal standard to calibrate the field gradient. No additional signals indicative of ethanol trapped within the slowly diffusing aggregates were observed. The diffusion constants determined for the aggregates represent average diffusion rates for the ensemble of aggregates observed. By applying the Stokes-Einstein equation (eq 1, where D is the diffusion constant, k is Boltzman’s constant, η is the viscosity, and r is the Stokes radius of the species), one can estimate the average hydrodynamic radius of the free and aggregated t-A. The results are collected in Table 1 for a 10 mM solution of t-A.44 As mentioned above, the diffusion constant for free t-A is high and invariable over time and concentration. The diffusion constant of the aggregates is much slower, increasing over the first 5 h and then remaining relatively constant. In the case of a 5 mM t-A solution, the diffusion constants slowly decrease after the initial 5-h period. Quite naturally, the values thus (43) All plots were found to be linear, with the exception of the data collected immediately after emulsification. In the latter case, a small apparent contribution from a fast-diffusing species is detected in the echo decay of the t-A aggregate signal. Laplace transform of the data indicates that the diffusion constants for the smallest aggregates are only somewhat larger than those of free t-A (see the Supplementary Information). (44) The values determined for a 5 mM solution are collected in the Supplementary Information.
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Table 1. Diffusion Rates and Calculated Average Hydrodynamic Radius of Free and Aggregated t-A ([t-A] ) 10 mM)a time (h)
Dfree (1010 m2/s)
rfree (Å)
Dagg (1011 m2/s)
Ragg (Å)
0 4 8 12 16 20 24 28 32 36 40 44
5.10 5.90 5.65 5.60 5.69 5.65 5.58 5.71 5.56 5.69 5.74 5.61
3.4 3.0 3.1 3.1 3.1 3.1 3.1 3.1 3.2 3.1 3.1 3.1
11.90 6.68 5.93 6.34 5.89 6.13 5.65 6.40 6.32 7.22 6.49 6.18
14.74 26.26 29.58 27.66 29.78 28.61 31.04 27.40 27.75 24.29 27.02 28.38
a
In 5% ethanol-d6/D2O solution. Calculated according to eq 1.
Figure 5. Time evolution of the hydrodynamic radii determined from DOSY experiments using eq 1 for t-A aggregates (circles) and free t-A (triangles) at 5 (empty symbols) and 10 mM (filled symbols) total t-A concentration.
determined represent the average size of the sampled aggregates. The distribution of the diffusion rates (and hence hydrodynamic radii) can be visualized by applying a Laplace transformation to the data obtained from the DOSY experiments (see the Supporting Information).
D)
kT 6πηr
(1)
The hydrodynamic radius obtained for free t-A from DOSY experiments is determined to be 3.1 Å, very near to the Van der Waals radius calculated for the short axis of t-A (4.3 Å). This value remains constant over time and is independent of total t-A concentration, as would be expected for isolated t-A molecules dissolved in the aqueous phase. In contrast, the behavior of aggregated t-A is both time- and concentration-dependent, as shown in Figure 5 for t-A concentrations of 5 and 10 mM in 5% aqueous EtOH solution. Shortly after their preparation, the t-A aggregates that are formed are relatively small, ca. 2 and 3 nm in diameter for solutions that are 5 and 10 mM in total t-A concentration, respectively. These grow rapidly over the following 4-5 h to reach a maximum value of 4-6 nm, which then decreases slowly with time. In the case of the 5 mM solution, this decrease is characterized by a linear dependence of the aggregates’ volume with time, in agreement with an Ostwald ripening mechanism for the dissolution of the smaller aggregates in favor of the larger droplets detected by DLS techniques.45 The more concentrated (45) An Ostwald ripening constant of 4 Å3 min-1 (r2 ) 0,967) is determined for the larger droplets. See the Supporting Information.
Figure 6. Molar fraction of t-A present in aggregates visible by NMR (filled circles), free in solution (filled triangles) as a function of time after sample preparation (5 mM t-A in 5% ethanol-d6/D2O). The filled squares represent the molar fraction of t-A not visible by NMR (obtained from the mass balance of the sample and attributed to large droplets, see text). The solid lines are the best-fit using a monoexponential function for the aggregates (y ) Ae-kt, with k ) 11.5 min-1) and a linear regression for the free t-A.
10 mM solution, on the other hand, does not conform to the Ostwald ripening model over the time frame explored, indicating that coalescence of the aggregates is significant at higher concentrations. The ensemble of the spectroscopic data obtained from NMR spectroscopy suggest that small t-A aggregates are formed upon spontaneous emulsification of ethanolic t-A solutions. However, this information is inconclusive in the absence of a quantitative method to evaluate the proportion of t-A that is being probed by NMR. Indeed, the rapid formation of turbidity in t-A emulsions agrees with the DLS results indicating the formation of micronsized droplets. To probe this point, we proceeded to calibrate the t-A samples by adding a small quantity of undeuterated methanol as internal standard. Using the intensity of the methanol resonance at 3.34 ppm, it is possible to determine the absolute concentration of t-A in the free and aggregated forms observed by NMR. The results are presented in Figure 6, where it can be seen that the concentration of t-A dissolved in the aqueous phase remains constant at the saturation limit (0.7 mM in 5% EtOH). The fraction of t-A in small aggregates visible by NMR, in contrast, decreases rapidly with time to represent only ca. 10% of the total t-A in the emulsion after 1 h. This decrease, as shown in Figure 6, is pseudo-first order in t-A aggregates. Fitting to a monoexponential function allows the total t-A fraction present in the aggregates to be extrapolated to t ) 0, and it is estimated that ca. 70% of the total t-A is present at the onset of the spontaneous emulsification process as small aggregates, with an additional 15% actually dissolved in the aqueous phase. Together, this accounts for ca. 85% of the total t-A concentration deployed at the beginning of the experiment. The remaining t-A not visible by NMR (i.e., not present in the form of small aggregates or free in solution) is attributed to t-A contained in small droplets, whose presence has been previously detected by DSL and neutron (46) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, J. J. Chem. Phys. 1979, 71, 4546. (47) Marion, D.; Wu¨thrich, K. Biochem. Biophys. Res. Commun. 1983, 113, 967. (48) Perrin, C. L.; Dwyer, T. J. Chem. ReV. 1990, 90, 935. (49) Perrin, C. L.; Gipe, R. K. J. Am. Chem. Soc. 1984, 106, 4036. (50) Pianet, I.; Fouquet, E.; Pereyre, M.; Gielen, M.; Kayser, F.; Biesemans, M.; Willem, R. Magn. Reson. Chem. 1994, 32, 617. (51) Price, W. S. Concepts Magn. Reson. 1997, 9, 299. (52) Mills, R. J. Chem. Phys. 1973, 77, 685.
Spontaneous Emulsification of t-Anethole
scattering. Its proportion can be estimated by applying the equation for mass balance to the system, and the results are presented in Figure 6.
Conclusion The use of NMR spectroscopy has proven itself to be an invaluable tool to explore the initial events during the spontaneous emulsification of t-A in aqueous media. If the results obtained from the EXSY and DOSY experiments are combined with previous DLS and SANS investigations, it is possible to propose a relatively complete picture of the spontaneous emulsification process. As the alcoholic t-A solution is dispersed in water, the hydro-alcoholic phase becomes saturated in t-A. The vast majority of the excess t-A does not, as previously proposed, directly form micron-sized droplets. Instead, a large number of small (2-6 nm diameter) aggregates are formed, which immediately begin coalescence to generate the larger droplets responsible for the observed turbidity of t-A emulsions. The coalescence regime continues for about 30 min in the case of a 5 mM t-A emulsion in 5% EtOH (longer for a more concentrated t-A solution), after which the concentration of the small aggregates has decreased sufficiently to make this process inefficient. Beyond this time, Ostwald ripening depletes the small aggregates in favor of the larger droplets. The small aggregates thus are now undergoing a reduction in size, which is more pronounced for lower overall concentrations of t-A. Ultimately, this leads to continuous growth of the larger droplets, which quickly reach several microns in size. This state is now meta-stable and can last for several days before creaming occurs. It must be emphasized that the slow interconversion between free and aggregated t-A makes this a unique system for such a study. Indeed, the simultaneous observation of such clear signals in the NMR spectrum of the free and aggregated species is extremely rare. The formation of a stable emulsion strongly affects the photochemical properties of t-A, similarly to other conjugated alkenes placed in a locally condensed53 (or supramolecular54,55) environment. These results will be reported elsewhere in due course.56 Experimental Section Materials and Methods. trans-Anethole (>99% pure) was obtained from Aldrich and used as received. All samples were (53) Ramnath, N.; Ramamurthy, V. J. Org. Chem. 1984, 49, 2827. (54) Huang, C.-H.; Bassani, D. M. Eur. J. Org. Chem. 2005, 4041. (55) Bassani, D. M.; Sallenave, X.; Darcos, V.; Desvergne, J.-P. Chem. Commun. 2001, 1446. (56) Carteau, D.; Brunerie, P.; Guillemat, B.; Bassani, D. M. Photochem. Photobiol. Sci., accepted for publication.
Langmuir, Vol. 23, No. 7, 2007 3565 prepared using ethanol-d6 (HDO + D2O < 0.3%) and deuterium oxide (99.90% D) from Euriso-Top. Microemulsions were prepared using an identical procedure: a given amount of trans-anethole was completely dissolved in ethanol-d6. Subsequently, deuterium oxide was added to the homogeneous single-phase solution to reach a concentration of 5 or 10 mM in 5% C2D5OD/D2O. NMR experiments were performed at 298 K on a Bruker DPX 400 equipped with a 5 mm gradient inverse broad band probe. All proton chemical shifts are given with respect to TMS as an external reference. Two types of experiments were recorded: 1. Exchange Rate Measurements. The 2D 1H EXSY NMR spectra were recorded using the pulse sequence of Jeener et al.46 in the phase sensitive mode with time proportional phase incrementation.47 A total of 24 experiments were recorded at two successive mixing times (60 and 0 ms) every 15 min during the first hour and then every 30 min during the following 10 h. The auto and cross-peak volumes were determined after phase and baseline correction of the two dimensions using the Bruker UXNMR software. Rates exchange were obtained from these experiments based on the resonances of the methoxy or β-methyl groups (both gave identical values) by using the method of Perrin, Gipe, and Dwyer48,49 that allows extraction of rates from a single experiment recorded at one mixing time by resolving the matrix L ) (1/tm) ln(A) ) (1/tm)U ln(λ)U-1, where the elements of the matrix A are composed by intensity values of the EXSY spectra as previously described.50 2. Diffusion Measurements. Diffusion measurements were performed at different t-A concentrations using a 1H NMR pulsedgradient experiment: the stimulated spin-echo sequence51 which leads to the measurement of the translational self-diffusion coefficients D, where D is the slope of the straight line obtained when ln(I) is displayed against the gradient-pulse power’s square according to the following equation: ln(I) ) -γ2G2Dδ2(∆ - δ/3), where I is the relative intensity of a chosen resonance, γ is the proton gyromagnetic ratio, ∆ is the intergradient delay (150 ms), δ is the gradient pulse duration (1.3 ms), and G is the gradient intensity (10 values varying from 0.01 to 0.40 G/m were used). The diffusion constant of water (2.3 × 10-9 m2/s)52 was used to calibrate the instrument.
Acknowledgment. Support from the Region Aquitaine, ANRT, and the CNRS is gratefully acknowledged. Supporting Information Available: EXSY plots, data from DOSY experiments, diffusion constants and Laplace transform of DOSY data for 5 mM t-A solution, and graph showing Ostwald ripening of the t-A aggregates. This material is available free of charge via the Internet at http://pubs.acs.org. LA062339Q
