Microemulsions Based on Soybean Phosphatidylcholine and

Joubran, R. F.; Cornell, D. G.; Parris, N. Colloids Surfaces, A 1993, 80, .... Winsor, P. A. Solvent properties of amphiphilic compounds; Butterworth:...
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Langmuir 1997, 13, 5061-5070

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Microemulsions Based on Soybean Phosphatidylcholine and Triglycerides. Phase Behavior and Microstructure Christian von Corswant,*,† Sven Engstro¨m,‡ and Olle So¨derman§ Astra Ha¨ ssle AB, S-431 83 Mo¨ lndal, Sweden, Food Technology, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden, and Physical Chemistry 1, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden Received March 17, 1997. In Final Form: July 7, 1997X We have studied the phase behavior and microstructure of water/1-propanol/soybean phosphatidylcholine (SbPC)/triglyceride systems. Microemulsions are formed with both medium-chain triglycerides (MCT) and long-chain triglycerides. The amount of SbPC and 1-propanol needed to form a microemulsion increases as the chain length of the triglyceride increases. The MCT system was investigated in more detail and compared with a hexadecane system. The phase behavior and pulsed field gradient NMR data for the MCT system suggest that the microemulsions formed at low SbPC concentrations and 0 < R < 0.5, where R is defined as the weight fraction of oil/(oil + water), are of a bicontinuous type. The self-diffusion data for the microemulsions formed at higher SbPC concentrations clearly indicate that there is some structure also in these microemulsions with an oil-in-water droplet structure at the water rich side and a gradual change to a bicontinuous structure when the MCT concentration is increased. The bicontinuity appears to be preserved even at low water concentrations. The microstructure of the microemulsion in this part of the phase diagram is, however, less well-defined, most probably with large polydispersity and rapid fusion and fission of the oil and water domains. The hydrodynamic radius and aggregation number of the SbPC aggregates formed in a mixture of 22.5 wt % SbPC, 22.5 wt % 1-propanol, and 55 wt % water was calculated as 27.5 Å and 68 SbPC molecules/aggregate, respectively.

1. Introduction In recent years, microemulsions, which we define as systems of water, oil, and surfactant that are single, optically isotropic and thermodynamically stable solutions, have been identified as potential drug delivery systems for lipophilic drugs due to their translucent appearance, long-term stability, high solubilization capacity, and ease of preparation.1-4 Since the first microemulsion system was described by Shulman and Hoar,5 an extensive number of papers have been published in this area. Most of the systems described are, however, not suitable for pharmaceutical use. An ionic surfactant and a cosurfactant, often a short-chain alcohol, are frequently used together with an aliphatic or aromatic hydrocarbon. These components are not, however, accepted for pharmaceutical use due to their toxicity. A suitable oil phase for pharmaceutical use would be a vegetable oil, e.g., a triglyceride or a fatty acid ester such as isopropylmyristate or ethyloleate. Of these, triglycerides are preferred as they have been used in intravenous fat emulsions for more than 20 years and are consequently well characterized from the toxicological aspect.6 * To whom correspondence should be addressed: e-mail, [email protected]. † Astra Ha ¨ ssle AB. ‡ Food Technology, University of Lund. § Physical Chemistry 1, University of Lund. X Abstract published in Advance ACS Abstracts, September 1, 1997. (1) Attwood, D. In Colloidal Drug Delivery Systems; Kreuter, J., Ed.; Marcel Dekker: New York, 1994; Vol. 66; pp 31-71. (2) Mu¨ller, B. W.; Kleinbudde, P. Pharm. Ind. 1988, 50, 370-375. (3) Bhargava, H. N.; Narurkar, A.; Lieb, L. M. Pharm. Technol. 1987, 11, 46. (4) Malmsten, M. In Microemulsions: Fundamentals and applied aspects; Kumar, P., Mittal, K. L., Eds.; Marcel Dekker, Inc.: New York, 1996. (5) Hoar, T. P.; Schulman, J. H. Nature 1943, 152, 102. (6) Davis, S. S.; Hadgraft, J.; Palin, K. J. In Encyclopedia of emulsion technology; Becher, P., Ed.; Marcel Dekker: New York/Basel, 1983; Vol. 2, pp 159-238.

S0743-7463(97)00289-8 CCC: $14.00

Relatively few studies using triglycerides as the lipophilic phase in a microemulsion have been published.7-12 Wa¨rnheim and Alander8,9 have described partial phase diagrams of a medium-chain triglyceride with mainly C8 and C10 chains and a long-chain triglyceride with mainly C18:1 and C18:2 chains as the nonpolar phase. Both nonionic and anionic surfactants were studied. They concluded that triglycerides, in particular long-chain triglycerides such as peanut oil, are considerably more difficult to solubilize into microemulsions than hydrocarbons or alkyl esters. Joubran et al.10,11 have studied microemulsions of soybean oil, polyoxyethylene(40)sorbitanhexaoleate, and water-ethanol. They found that the extension of the water in oil (w/o) microemulsion regions were strongly dependent on temperature. This is the expected behavior since ethylene oxide-based surfactants are known to have a strong temperature dependence in their phase behavior. Moreover, water/ethanol ratios also affected the phase behavior. In these systems large amounts of the surfactant had to be used to form a microemulsion with equal parts of triglyceride and aqueous phase. There are very few surfactants that can be used in pharmaceutical formulations.1 In this respect, phospholipids, which are amphiphiles of a natural source and major components of membrane lipids, are an interesting group of surfactants. The ability of phospholipids to form microemulsions with alkanes has been studied by several authors.13-18 Shinoda et al. have shown that it is possible to form microemulsions with equal amounts of hexadecane (7) Kunieda, H. J. Colloid Interface Sci. 1989, 133, 237. (8) Alander, J.; Wa¨rnheim, T. J. Am. Oil Chem. Soc. 1989, 66, 16611665. (9) Alander, J.; Wa¨rnheim, T. J. Am. Oil Chem. Soc. 1989, 66, 16561660. (10) Joubran, R. F.; Cornell, D. G.; Parris, N. Colloids Surfaces, A 1993, 80, 153-160. (11) Trevino, S. F.; Joubran, R.; Parris, N.; Berk, N. F. Langmuir 1994, 10, 2547-2552. (12) Aboofazeli, R.; Patel, N.; Thomas, M.; Lawrence, M. J. Int. J. Pharm. 1995, 125, 107-16. (13) Shinoda, K.; Shibata, Y.; Lindman, B. Langmuir 1993, 9, 12547. (14) Shinoda, K.; Araki, M.; Sadaghiani, A.; Khan, A.; Lindman, B. J. Phys. Chem. 1991, 95, 989-93.

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and aqueous phase with only 2.5 wt % soybean phospholipid using 1-propanol as the cosurfactant,13-15 and Kalweit et al. have systematically studied the influence of the chain length of both the phospholipid and the hydrocarbon.16,17 Aboofazeli et al.12 and Leser et al.19 have studied microemulsions based on phospholipids and triglycerides. Aboofazeli et al. report partial phase diagrams of systems containing water, egg lecithin, propanol, and different polar oils such as Miglyol 812 and soybean oil, showing the influence of the oil and the ratio of egg lecithin and propanol on the microemulsion area, but no study of the microstructure was presented. Leser et al. have studied the phase behavior and microstructure of soybean phosphatidylcholine, medium-chain triglyceride, and different short-chain alcohols. In the present study, we have investigated the detailed phase behavior of microemulsions composed of SbPC (soybean phosphatidylcholine), water, 1-propanol, MCT (medium-chain triglyceride), and LCT (long-chain triglyceride) and compared it with a hexadecane system. We have also studied the microstructure of the systems containing MCT by measuring the self-diffusion coefficients of the different components in the microemulsion using the pulsed field gradient (PFG)-NMR technique in order to discriminate between molecularly dispersed solutions, droplet, or bicontinuous systems. The overall aim of our work is to formulate and characterize microemulsions which can be used for parenteral administration. In the present study, we have, however, used 1-propanol as the cosurfactant which clearly cannot be accepted for parenteral administration. The microemulsion system studied here should be regarded as a model system in order to study the behavior of systems based on triglycerides and SbPC in more detail. Our work on 1-propanol-free microemulsions will be published in the near future. 2. Experimental Section 2.1. Materials. Soybean phosphatidylcholine (Epicuron 200) was obtained from Lucas Meyer Co., Germany. The water content was 1.3-1.9% determined using the Carl-Fisher coulometric method. The chain-length distribution of the fatty acids in Epicuron 200 was according to Shinoda et al.14 as follows: C16:0 ) 13.3%, C18:0 ) 3%, C18:1 ) 10.2%, C18:2 ) 66.9%, and C18:3 ) 6.6%. The medium chain triglyceride, Miglyol 810N, was purchased from Hu¨ls, Germany. The chain-length distribution of the fatty acids was according to the specifications from the manufacturer: C6:0 e 2%, C8:0 ) 70-80%, C10:0 ) 18-28%, C12:0 e 2%. Soybean oil was purchased from Sigma. 1-Propanol (HPLC grade) and hexadecane (99+%) were purchased from Aldrich Chemical Co. All reagents were used as received. Water was of Milli-Q quality. For the NMR study, a mixture of 80 wt % D2O (99.8 atom % D, supplied by Dr. Glaser AG, Basel) and 20 wt % H2O was used. 2.2. Phase Diagrams. The partial phase diagrams in Figures 1-5 and 7 were constructed using a conventional titration technique. Typically, a stock solution of SbPC and 1-propanol at the appropriate weight ratio was made. This stock solution was then mixed with a certain amount of MCT in glass vials which were sealed with rubber stoppers and aluminum seals and placed in a water bath at 25 ( 0.05 °C. The actual temperature and temperature variations in the water baths were checked with a calibrated thermometer (ASL F25, S/N 1069-21, (15) Shinoda, K.; Kaneko, T. J. Dispersion Sci. Technol. 1988, 9, 555-9. (16) Kahlweit, M.; Busse, G.; Faulhaber, B. Langmuir 1995, 11, 157683. (17) Kahlweit, M.; Busse, G.; Faulhaber, B.; Eibl, H. Langmuir 1995, 11, 4185-7. (18) Schurtenberger, P.; Peng, Q.; Leser, M. E.; Luisi, P.-L. J. Colloid Interface Sci. 1993, 156, 43-51. (19) Leser, M. E.; Vanevert, W. C.; Agterof, W. G. M. Colloids Surf., A 1996, 116, 293-308.

von Corswant et al. Pentronic). This mixture was then titrated with water using a syringe equipped with an injection needle until the two-phase region was reached. The amount of water which was added was determined by weighting the glass vial before and after titration. Some additional stock solution of SbPC and 1-propanol was added, and the sample was titrated with water again. In this way, one sample was used to determine several points in the phase diagram. In the same way, samples containing water, 1-propanol, and SbPC were titrated with MCT for the water-rich region. The phase diagram in Figure 6 was constructed by weighing appropriate amounts of the components in glass vials which were sealed with rubber stoppers and aluminum seals. The samples were shaken by a vortex shaker (Vibrofix VF1, Janke & Kunkel, IKA Labortechnik) and placed in water baths at 25 ( 0.05 °C. The samples were allowed to equilibrate for at least 4 days before they were examined. The nature of the different phases was established using ocular and optical (crossed polarized filters) methods. The volume fraction of the different phases was measured with a height measuring instrument (Feinmesszeugfabrik Suhl GmbH). 2.2.1. Phase Notations. The different phases observed in all the presented phase diagrams were noted as follows: W, clear aqueous phase, consisting mainly of water and 1-propanol; We, turbid aqueous phase; Lc, a liquid crystalline phase; Me, microemulsion phase characterized by a low viscosity, optical isotropic behavior, and a translucent appearance, no distinction between Winsor I (o/w), II (w/o) or III (bicontinous) type microemulsion was made; O, clear oil phase consisting mainly of MCT and 1-propanol. 2.3. Oil-Water Partitioning of 1-Propanol. Samples of equal amounts (w/w) of water and MCT and with different amounts of 1-propanol were mixed in glass vials which were sealed with rubber stoppers and aluminum seals and equilibrated at 25 °C for at least 2 days. The 1-propanol concentration in both the MCT phase and the water phase was measured by highperformance liquid chromatography. The partitioning coefficient Ko/w was calculated as

Ko/w )

g of propanol in MCT phase/g of MCT g of propanol in water phase/g of water

The extrapolated value of Ko/w at zero 1-propanol concentration was determined as 0.18 and increased with increasing 1-propanol concentrations up to 0.43 at 12.5 wt % 1-propanol. 2.4. Density and Viscosity. The density of water-1propanol mixtures was measured at 25 ( 0.5 °C with a DMA 48 density meter manufactured by Anton Paar. The viscosity at 25 ( 0.5 °C was measured with an Ubbelohde viscometer from Schott Gera¨te. 2.5. Interfacial Tension. The interfacial tension was measured with a spinning drop interfacial tensionmeter, Kru¨ss SITE 04. 2.6. X-ray Diffraction. The X-ray diffraction measurements were performed using a dual detector camera for simultaneous small- and wide-angle measurements (M Braun Graz Optical Systems, Graz, Austria) as described previously.20 The X-ray radiation was generated by a Philips PW 1830/40 generator with a Cu KR-anode (λ ) 1.5418 Å). The samples were placed in a quartz capillary sealed mechanically at both ends to permit the evacuation of the camera tube down to 0.15 mbar. The accuracy in the Peltier temperature control device (Anton Paar) was better than (1 °C. 2.7. PFG-NMR Measurements. The pulsed field gradient nuclear magnetic resonance technique21,22 was used to determine the different self-diffusion coefficients of the components at 25 ( 0.5 °C by monitoring the 1H signal on a Varian 600 MHz spectrometer. In the experiments, the length of the gradient pulse was kept constant and the gradient strength was varied between 1 and 30 G/cm. The gradient strength was calibrated by measuring the self-diffusion coefficient of small amounts of H2O in D2O. A stimulated echo with a LED pulse sequence23 was used for SbPC, 1-propanol, and MCT to minimize j(20) Laggner, P.; Mio, H. Nucl. Instrm. Meth. Phys. Res. 1992. (21) Stilbs, P.; Moseley, M. E. Chem. Scr. 1980, 15, 176. (22) Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1987, 19, 1-45. (23) Gibbs, S. J.; Johnsson, C. S., Jr. J. Magn. Reson. 1991, 93, 391.

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3.1. Three-Component Systems. In Figure 1 the four different ternary phase diagrams of the system water/ 1-propanol/SbPC/MCT are shown as the unfolded phase tetrahedron. From the bottom triangle (water/1-propanol/ MCT) we infer that very small amounts of MCT (less than 0.05 wt %) were dissolved in water/1-propanol mixtures with a 1-propanol concentration below 60 wt % (the solubility of tricaprylin (a major part of the MCT) in water at 37 °C is as low as 8.5 × 10-7 M28). At the MCT rich side, water was dissolved in the 1-propanol/MCT mixture at much lower concentrations of 1-propanol. The phase behavior of the water/1-propanol/LCT system was essentially the same as with MCT, except for the fact that the maximum 1-propanol concentration of the twophase region was 87 wt % and the solubility of water in the LCT-rich phase was substantially lower. The main constituent of the binary phase diagram of water and SbPC is a lamellar liquid crystalline phase

with a water content of 7-35 wt %.29 This lamellar phase is destabilized by the addition of 1-propanol (Figure 1, left triangle). 18-25 wt % 1-propanol was needed to bring the SbPC into solution depending on the water/ SbPC ratio. These solutions were isotropic and fairly viscous. The solubility of SbPC in pure MCT was very low and MCT did not dissolve to any appreciable extent in the SbPC phase. Similar systems studied by others display the same behavior. The solubility of triolein in egg lecithin has thus been determined as 3.5 wt %30 and the solubility of LCT in egg lecithin was less than 0.1 wt %.31 Despite the low solubility of SbPC in pure MCT, only 2-3 wt % of 1-propanol was needed to form an isotropic solution of SbPC and MCT (Figure 1, right triangle). This was also true for LCT. The phase diagram of water/SbPC/MCT (Figure 1, middle triangle) was not studied in any detail, but a general pattern of the formation of stable emulsions at zero and low concentrations of 1-propanol was observed. The phase diagram of water/SbPC/LCT has been determined by Rydhag and Wilton32 and it consists mainly of two- and three-phase areas in which very stable emulsions are formed. 3.2. Four-Component Systems. 3.2.1. Water/1Propanol/SbPC/MCT. The complete phase diagram of the quaternary water/1-propanol/SbPC/MCT system is a tetrahedron with the four three-component phase diagrams from Figure 1 as its faces. We have limited our study to the construction of pseudo-ternary-phase diagrams with water, MCT, and different fixed 1-propanol/ SbPC ratios and water, MCT, and 1-propanol at a constant SbPC concentration, since our main interest is the microemulsion region. This produces a rough picture of the complete phase tetrahedron. As can be seen from Figure 2, the extension of the microemulsion region is increased when the ratio of 1-propanol/SbPC is decreased from 1.6 to 1.0. A further increase in the SbPC proportion promotes the formation of a liquid crystalline phase and the microemulsion region becomes smaller again. The phase diagram with a 1-propanol/SbPC ratio of 1.0 in Figure 2b has an interesting feature. Starting with a solution of 60 wt % water and 40 wt % 1-propanol/SbPC and moving toward the MCT corner, i.e., diluting this mixture with MCT, it is possible to incorporate about 38 wt % of MCT in the microemulsion. Any further addition of MCT causes an excess oil-phase to form on top of the microemulsion. If, from the indicated point × in Figure 2b, we instead move toward the 1-propanol/SbPC corner, oil is also expelled from the microemulsion phase until the upper two-phase border is reached. It is thus possible to make a one-phase microemulsion of almost equal volume fractions of water and MCT at around 25 wt % 1-propanol/ SbPC and for concentrations of 1-propanol/SbPC in excess of 40 wt %. In the former case, the formation of the microemulsion with R ≈ 0.5, where R is defined as the weight fraction of oil/(oil + water), occurs under somewhat stringent conditions with respect to the 1-propanol/SbPC concentration, indicating that the microstructure of the microemulsion in this case is more pronounced than that of the microemulsion formed at higher 1-propanol/SbPC concentrations. This is discussed in greater detail below. This behavior is far more pronounced with hexadecane,

(24) Stejskal, E. O.; Tanner, J. E. J. Chem. Phys. 1965, 42, 288-292. (25) Eriksson, P. O.; Lindblom, G. J. Phys. Chem. 1987, 91, 846. (26) So¨derman, O.; Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 445-482. (27) von Goldammer, E.; Hertz, H. G. J. Phys. Chem. 1970, 74, 3734. (28) Small, D. M. The physical chemistry of lipids, 2nd ed.; Plenum Press: New York, 1986; Vol. 4.

(29) Bergensta˚hl, B.; Fontell, K. Prog. Colloid Polym. Sci. 1983, 68, 48-52. (30) Miller, K. W.; Small, D. M. J. Colloid Interface Sci. 1982, 89 (2), 446-478. (31) Kabalnov, A.; Tarara, T.; Arlauskas, R.; Weers, J. J. Colloid Interface Sci. 1996, 184, 227-235. (32) Rydhag, L. Fette Seifen Anstrichm. 1979, 81, 168-173.

Figure 1. Unfolded phase tetrahedron at 25 °C with water/ SbPC/MCT triangle as the base and 1-propanol on top. Only the isotropic one-phase region is shown. modulation effects and phase errors due to eddy currents induced by the gradients. For the determination of the water diffusion coefficient, a standard stimulated echo pulse sequence was used. The self-diffusion coefficient was calculated by fitting the Stejskal-Tanner equation to the obtained peak heights.24 For a detailed description of the procedure, see, for instance, the work of Eriksson and Lindblom25 or So¨derman and Stilbs.26 The curve fitting was performed with Microcal Origin version 3.5 (Microcal Software Inc). The 1-propanol self-diffusion coefficient was measured through the peak heights of the 1-methylene group, the SbPC self-diffusion coefficient was measured through the peak heights of the choline group, and the MCT self-diffusion coefficient was measured through the peak heights of the protons from the glycerol backbone and the R-methylene group in the fatty acid in the triglyceride. For samples containing very little or no water, a sealed glass capillary with D2O was placed concentrically in the NMR tube to produce a lock signal. It should also be noted that, due to the fast exchange of protons between water and the hydroxyl group of 1-propanol, Dw was calculated according to the two-site model.27 The self-diffusion coefficients of water and 1-propanol in water/1-propanol mixtures were measured and appropriate concentrations were used as D0 values for water to calculate the relative self-diffusion coefficients.

3. Phase Behavior

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Figure 2. Pseudoternary phase diagrams at 25 °C of the water/1-propanol/SbPC/MCT system at different ratios of 1-propanol and SbPC: (a) 1-propanol/SbPC 1.6:1; (b) 1-propanol/SbPC 1:1; (c) 1-propanol/SbPC 1:1.6. Below the phase border on the water-rich side, a lamellar liquid crystalline phase appeared, but it was not investigated in any detail. Lines a and b and points p and × refer to the NMR self-diffusion experiments. See the text for details.

Figure 3. Pseudoternary phase diagram of the water/1propanol/SbPC/MCT system at a constant SbPC concentration of 12.5 wt % and 25 °C. In this cut, the microemulsion phase shows up as two separate regions.

see section IIc, but it is not seen at all with LCT. This behavior by MCT was not observed by Aboofazeli et al.12 Below the one-phase border, the microemulsion was in equilibrium with a lamellar liquid crystalline phase at low R and an excess MCT phase at high R. In Figure 3, the SbPC concentration was kept constant at 12.5 wt % and it can be seen that, in this phase cut, the bicontinuous microemulsion showed up as a separate area for 0.35 < R < 0.5. When R was further decreased, a lamellar phase appeared and at higher R the microemulsion was in equilibrium with an excess oil phase. 3.2.2. Water/1-Propanol/SbPC/LCT. The phase behavior of the LCT system resembled the MCT system to a great extent, but the microemulsion region was much smaller, as can be seen from Figure 4. More than 60 wt % of 1-propanol and SbPC was needed to form a microemulsion with equal amounts of water and LCT, and there was no microemulsion with a “well-defined” microstructure (see above). 3.2.3. Water/1-Propanol/SbPC/Hexadecane. With hexadecane, a narrow bicontinuous microemulsion is formed at low 1-propanol/SbPC concentrations and a wide range of R; see Figure 5. This is in accordance with the data published by Shinoda et al.13,14 The two-phase region above the bicontinuous microemulsion is also larger, which

indicates that variations in 1-propanol concentration affect the microstructure in the hexadecane system to a larger extent than the corresponding 1-propanol concentration variations in the MCT system. 3.3. Winsor Representation Diagrams. A convenient way of obtaining a qualitative understanding of the microstructure and the spontaneous curvature (H0) of the surfactant film of a microemulsion is to use a method first described by Winsor33 in which equal amounts of water and oil are mixed with a sufficient amount of surfactant. The spontaneous curvature is defined as positive when it curves around the oil. Winsor defined three different phase behaviors: Winsor I, a water-rich microemulsion in equilibrium with an excess oil phase (H0 > 0), Winsor II, an oil-rich microemulsion in equilibrium with an excess water phase (H0 < 0), and, finally, the Winsor III system with a bicontinuous microemulsion in simultaneous equilibrium with excess water and oil phases (H0 ≈ 0). In Figure 6, the volume fraction of the different phases in the water/1-propanol/SbPC/hexadecane and water/1propanol/SbPC/MCT systems is shown. Note that the concentration of 1-propanol is expressed as weight percent of 1-propanol and water according to ref 14. With hexadecane (Figure 6a), a w/o microemulsion is formed at low 1-propanol concentrations and, when the 1-propanol concentration is increased, the classical change from a Winsor II system via a Winsor III system to a Winsor I system can be observed although it should be remarked that the We phase is turbid and thus contains large structures (most likely constituted by dispersed lecithin lamellar phase). However, this is most probably a nonequilibrium situation, and when equilibrium is obtained the We phase should be a clear aqueous phase.14 With MCT, no w/o microemulsion was formed at low 1-propanol concentrations, Figure 6b; instead, a lamellar liquid crystalline phase containing all the water was in equilibrium with a clear oil phase. At higher 1-propanol concentrations, the lamellar phase was destabilized and a Winsor III system was formed, although there was always more water than oil in the middle phase. When the 1-propanol concentration was further increased, oil was expelled from the microemulsion and the aqueous phase was incorporated until a Winsor II system was formed with an o/w microemulsion in equilibrium with an excess oil phase. The phase behavior was also determined (33) Winsor, P. A. Solvent properties of amphiphilic compounds; Butterworth: London, 1954.

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Figure 4. Pseudoternary phase diagrams at 25 °C of water/1-propanol/SbPC/LCT at different ratios of 1-propanol and SbPC: (a) 1-propanol/SbPC 1.6:1; (b) 1-propanol/SbPC 1:1; (c) 1-propanol/SbPC 1:1.6. Below the phase border on the water-rich side, a lamellar liquid crystalline phase appeared, but it was not investigated in any detail.

Figure 5. Pseudoternary phase diagrams at 25 °C of water/ 1-propanol/SbPC/hexadecane at a 1-propanol/SbPC ratio of 1.68: 1.

at 37 °C, and it was found to be almost identical to the phase behavior at 25 °C. In the hexadecane case, the phase behavior is consistent with the following change in H0. At low 1-propanol concentrations, H0 is negative and, as the 1-propanol concentration is increased, H0 increases via the balanced state where H0 ) 0 to positive values. The change in H0 is mainly due to a decrease in the polarity of the water phase with the addition of 1-propanol.14 With MCT, a w/o microemulsion is not seen at low 1-propanol concentrations; instead, there is a water-swelled lamellar phase in equilibrium with the oil phase. When the 1-propanol concentration is further increased, the surfactant film become more flexible and a middle-phase microemulsion is formed (Winsor III system). Thus, the phase behavior of hexadecane and triglycerides is substantially different. A possible explanation for this behavior is the greater ability of hexadecane as compared with MCT to penetrate the surfactant “brush”31 and, to some extent, the different polarity of hexadecane and MCT, which affects the partitioning of the alcohol between the water and the oil phase. SbPC is known to be a nearly-balanced surfactant with a H0 of the monolayer at an oil-water interface close to zero and a high bending modulus of the surfactant film. The penetration of hexadecane into the hydrocarbon tails of the SbPC film decreases both the spontaneous curvature H0 and the bending modulus of the surfactant film. The formation of a microemulsion is favored by the low bending modulus of the surfactant film.

With the nonpenetrating MCT, the lamellar phase is stable at much higher 1-propanol concentrations, i.e., since MCT do not decrease the bending modulus of the surfactant film to the same extent as hexadecane, higher concentrations of 1-propanol are needed to achieve a flexible surfactant film and H0 appears to be positive even at low 1-propanol concentrations, since the microemulsion phase of the MCT Winsor III system always incorporates more water than oil. Hence, to reach a balanced microemulsion with MCT, it is necessary to decrease H0 still further, while maintaining the flexibility of the surfactant film, and one way of doing this is to replace 1-propanol with 1-butanol, which is known to decrease H0.16 When 1-butanol was used instead of 1-propanol, a w/o microemulsion was indeed formed with MCT. A proper mixture of 1-propanol and 1-butanol would therefore presumably also produce a balanced (H0 ) 0) microemulsion with MCT. When short-chain alcohols are used to tune H0, the fact that they also decrease the bending elasticity of the monolayer which promotes the formation of a microemulsion over the liquid crystalline phase34 and that they also increase the mutual solubility of the water and the oil phase must obviously be borne in mind. With LCT, no Winsor II or III system was formed with 1-propanol as the cosurfactant. Instead, the lamellar phase changed directly to an o/w microemulsion at high concentrations of 1-propanol. 3.4. Fish-Cut Diagrams. In Figure 7, the phase behavior as a function of the 1-propanol and SbPC concentration for different R, the so-called fish-cut, is shown. In Figure 7c the two-phase region separating the one-phase Me region and the three-phase O + Me + W region has not been observed. As a consequence, this twophase region which must exist on account of the phase rule, is narrow. In Figure 8, the minimum amount of SbPC needed to obtain a microemulsion phase is plotted against R. At R ) 0.2 and 0.3, the phase behavior resembles that observed for nonionic systems35 with a symmetrical three-phase body. As R is increased, H0 must decrease (in theory it should be zero at R ) 0.49 which corresponds to equal volume fractions of water and MCT), which means that the 1-propanol concentration must be decreased. On the other hand a certain addition of 1-propanol is required to destabilize the lamellar phase. At R ) 0.4, the 1-propanol concentration required to create the optimal H0 very close to the critical 1-propanol (34) De Gennes, P. G.; Taupin, C. J. Phys. Chem. 1982, 86, 2294. (35) Olsson, U.; Wennerstro¨m, H. Adv. Colloid Interface Sci. 1994, 49, 113-46.

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Figure 6. Volume fractions of different phases of the hexadecane system (a) and the MCT system (b), as a function of 1-propanol concentration in the aqueous phase at 25 °C. Note that the 1-propanol concentration is expressed as weight percent of the aqueous phase. The SbPC concentration was 3.0 wt % in (a) and 7.5 wt % in (b). The hexadecane system displays classical Winsor II f III f I behavior as the 1-propanol concentration is increased. This was not observed in the MCT system; instead a liquid crystalline phase appeared at low 1-propanol concentrations.

concentration needed to destabilize the lamellar phase and, at R ) 0.5, this concentration has been passed as the tail is no longer attached to the three-phase body of the fish. A close look at the phase diagram of R ) 0.4 shows that, at an SbPC concentration of 11.5 wt %, a microemulsion phase is obtained at about 13 wt % 1-propanol. When the 1-propanol concentration is further increased, a Winsor III system followed by a Winsor I system is unexpectedly obtained. This could be explained by the effect 1-propanol has on the bending modulus of the SbPC monolayer. Increasing the 1-propanol concentration decreases the bending modulus and, as a result, more surfactant is needed to incorporate all the water and oil.36 This effect can also be seen in Figure 6 where the middle phase shrinks slightly with increasing 1-propanol concentrations at the point where the Winsor III system has just formed and in Figure 8 where the minimum SbPC concentration needed to form a one-phase microemulsion passes a maximum. 4. Interfacial Tension Measurements It has been found in the case of several surfactant systems37 that the interfacial tension between the aqueous phase and the oil phase, denoted σwo, goes through a minimum of ultralow value at the point where the microemulsion is balanced. Typical values of σwo are in the order of 0.001 mN/m.13,37 In Figure 9, σwo of a system containing 3 wt % SbPC is plotted against the concentration of 1-propanol (expressed as weight percent of water + 1-propanol) for samples in the three-phase region (Figure 6b). σwo decreases with decreasing concentrations of 1-propanol down to the point at which the Winsor III system vanishes and the lamellar phase appears. The minimum value of σwo at this point is 0.01 mN/m. Thus, no minimum in σwo is found, indicating that a balanced microemulsion is not obtained. 5. SAXS and WAXS Measurements In Figure 10, the small- and wide-angle X-ray diffraction patterns of MCT and hexadecane at 25 °C are presented. The MCT shows a broad peak in the long-spacing area (36) Daicic, J.; Olsson, U.; Wennerstro¨m, H. Langmuir 1995, 11, 2451-8. (37) Strey, R. Colloid Polym. Sci. 1994, 272, 1005-1019.

with a maximum at d ) 18.3 Å. A peak of this kind is not observed for hexadecane. MCT therefore possesses some structure in the liquid state which is not present in the case of hexadecane. The intensity of the peak in the longspacing area is in the same range as the peak in the shortspacing area, and the diffraction must therefore evolve from the major part of the molecules in the liquid and not from some small fragment of dimers. This behavior was also observed when 1-propanol was added in concentrations corresponding to the studied microemulsions. The MCT which were used consist mainly of tricaprylin (7080%) and the β-crystalline form of pure tricaprylin has a long-spacing of 22.3 Å.28 Assuming that the corresponding value for our MCT is the same or very close to this, there is a decrease of about 15% in the long-spacing in the liquid phase. Larsson studied trimyristin38 and found a longspacing of 32 Å in the liquid state and 35.5 Å for the β-crystalline form. There is therefore a decrease in long spacing of 11% upon melting. It seems reasonable to suppose that the MCT behave in a similar way. Larsson has suggested that the liquid state of triglycerides not too far from the melting point consists of small pseudolamellar domains with a diameter of roughly 100-200 Å.38,39 Thus the triglycerides are structured in the pure liquid state. In this regard they are fundamentally different from hydrocarbons. In addition, Alander and Wa¨rnheim8,9 has pointed out that the molecular structure of triglycerides prevent them from penetrating the surfactant “brush”. These facts could explain the inability of triglycerides as compared to hydrocarbons to form microemulsions with a small amount of surfactant. 6. NMR Self-Diffusion Measurements The microstructure of a microemulsion is conveniently studied using the PFG-NMR technique, where the selfdiffusion coefficients, D, of all the components can be measured simultaneously without any need to label the compounds or add probe molecules.26,40 6.1. Size and Shape of SbPC Aggregates at r ) 0. At point P in Figure 2b (55 wt % water, 22.5 wt % SbPC, and 22.5 wt % 1-propanol), Dw ) 7.0 × 10-10 m2/s, D1-prOH (38) Larsson, K. Fette Seifen Anstrichm. 1972, 74, 136-142. (39) Larsson, K. J. Am. Oil Chem. Soc. 1992, 69, 835-6. (40) Lindman, B.; Shinoda, K.; Olsson, U.; Anderson, D. M.; Karlstro¨m, G.; Wennerstro¨m, H. Colloids Surf. 1989, 38, 205-24.

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Figure 7. Phase behavior of pseudoternary water/1-propanol/SbPC/MCT system at 25 °C and different R, where R is defined as the weight fraction of oil/(oil + water). Both 1-propanol and SbPC concentrations are expressed as weight percent of total mixture.

) 3.3 × 10-10 m2/s, and DSbPC ) 1.7 × 10-11 m2/s. The fact that DSbPC is about 1 order of magnitude lower than Dw and D1-prOH rules out the possibility of a molecularlydispersed solution. Instead, it is evident that the SbPC molecules form some sort of aggregates. By way of

comparison, DSbPC in a 5.0 wt % solution of SbPC in pure 1-propanol was measured and found to be 2.4 × 10-10 m2/s. The hydrodynamic radius RH of the SbPC aggregates can be calculated according to the Stokes-Einstein

5068 Langmuir, Vol. 13, No. 19, 1997

Figure 8. Minimum SbPC concentration required to obtain one-phase microemulsions in the water/1-propanol/SbPC/MCT system at 25 °C. The figures connected to every point are the corresponding 1-propanol concentrations in weight percent.

von Corswant et al.

Figure 10. X-ray diffraction patterns for MCT and hexadecane recorded at 25 °C: (a) small-angle X-ray; (b) wide-angle X-ray data. The small-angle X-ray data for MCT shows a broad peak with a maximum at 18.3 Å, which indicate some sort of structure in the liquid state. The peak at high d is an artifact from the primary beam.

η is the viscosity of the medium, and D the observed diffusion coefficient. At 25 °C, the viscosity of the water/ 1-propanol solution without SbPC was measured at 2.5 mPas. The Stokes-Einstein equation is, however, only valid for infinitely dilute spherical aggregates, and at finite aggregate concentrations the aggregate obstruction effects have to be considered. The aggregate obstruction effects can be estimated using the following equation41,42

D ) D0(1 - kφagg)

Figure 9. Interfacial tension between the oil phase and the water phase in the water/1-propanol/SbPC/MCT system at 25 °C. The 1-propanol concentration is given as weight percent of the aqueous phase (water + 1-propanol). SbPC concentration: 3 wt %.

equation:

RH )

kBT 6πηD

where kB is the Boltzmann constant, T is the temperature,

where φagg is the volume fraction of aggregates, D0 is the diffusion coefficient in an infinitely dilute system, and k is a constant. Some of the water is bound to the aggregates, and φagg was calculated assuming 15 water molecules/ SbPC molecule43 giving φagg ) 0.2975. The adsorption of 1-propanol molecules to the aggregates was not taken into account. The value of k depends on the shape of the particle and the degree of hydrodynamic interactions present. For hard spheres with no hydrodynamic interaction, k is equal to 2. Using this value of k, the hydrodynamic radius of the SbPC aggregates was calculated at 27.5 Å (excluding the bound water). Using the molecular volume of 1267 Å3 for SbPC,28 an aggregation number of 68 SbPC molecules/aggregate is obtained. Tinker and Saunders44 have studied the aggregate sizes in mixtures of 1.5-5 wt % egg lecithin, 20-25 wt % 1-propanol, and water at 23 °C by light scattering, viscosity and osmotic pressure and suggest that this part of the isotropic region is made up of aggregates of 12-14 (41) Wang, J. H. J. J. Am. Chem. Soc. 1954, 76, 4755. (42) Ohtsuki, T.; Okano, K. J. J. Chem. Phys. 1982, 77, 1443. (43) Balinov, B.; Olsson, U.; So¨derman, O. J. Phys. Chem. 1991, 95, 5931-6. (44) Tinker, D. O.; Saunders, L. Chem. Phys. Lipids 1968, 2, 316.

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molecules of egg lecithin, which is somewhat smaller than our findings. The water and the 1-propanol molecules are also obstructed by the SbPC aggregates, and the obstruction factor, i.e., the relative self-diffusion coefficients for water (and 1-propanol), contains information about the shape of the SbPC aggregates.45 However, since the concentration of SbPC is somewhat high, we cannot ignore the solvation effect46 where some of the solvent molecules are bound to the SbPC molecules and are slowed down. The solvation effect can be estimated by:

D ) pbDb + (1 - pb)D0 ≈ (1 - pb)D0 where D is the observed self-diffusion coefficient for water, pb the fraction of water molecules bound to the SbPC aggregates, and Db the diffusion coefficient for the aggregate. Since Db , D0, the first term can be ignored. Pb is given by

Pb ) nw,bound nSbPC/nw,tot where nw,bound is the number of water molecules bound to one SbPC molecule, nSbPC is the total number of SbPC molecules, and nw,tot is the total number of water molecules. As stated above, the value of nw,bound is assumed to be 15, which gives D/D0 ) 0.84. The measured value of D/D0 for water is 0.73, giving an aggregate obstruction factor of 0.86. For hard spheres, the obstruction factor for the water molecules can be calculated from the following equation:45

D ) D0

1 φagg 1+ 2

With φagg ) 0.2975, the calculated value of the aggregate obstruction factor for hard spheres is 0.87, which is almost identical to the observed value. From these calculations, a picture of the SbPC aggregates as rather small, aggregation number about 70, spherical objects appears. It should, however, be noted that the value of the aggregate obstruction factor is fairly dependent on the assumed value of nw,bound. 6.2. At Constant Water/SbPC/1-Propanol Ratio (along Line a in Figure 2b). The standard procedure of analyzing the microstructure from self-diffusion data is to plot the relative self-diffusion coefficients (D/D0) as a function of R. This is normally straightforward47 and, for an oil-in-water droplet structure, D/D0 for water is close to 1 and D/D0 for oil is usually below 0.1. For a water-in-oil droplet structure, the opposite situation holds true. D0 is the self-diffusion coefficient in the neat liquid. In the bicontinuous case, D/D0 is about 2/3 for both water and oil (at equal volumes of water and oil).40 In our case, the situation is slightly more complex. Firstly since Ko/w is relatively close to 1, the fact that the concentration of 1-propanol in the water phase and in the MCT phase will vary with R must be considered. Secondly, D0 for neat MCT is in the same range as the D values for the oil aggregates that could be expected in the o/w microemulsion structure and no large difference will therefore be observed (45) Jo¨nsson, B.; Wennerstro¨m, H.; Nilsson, P. G.; Linse, P. Colloid Polym. Sci. 1986, 264, 77-88. (46) Lindman, B.; Olsson, U. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 344-63. (47) Olsson, U.; Lindman, B. In NATO ASI Ser. C Struct. Dyn. Equilib. Prop. Colloidal Syst.; Bloor, D. M., Wyn-Jones, E., Eds.; Kluwer Academic Publishers:, 1990; Vol. 324, pp 233-42.

Figure 11. Self-diffusion coefficients for water (closed circles), SbPC (closed triangles), and MCT (open triangles) along line a in Figure 2b at 25 °C.

in the relative self-diffusion coefficient for MCT. Attention will therefore focus on the relative water self-diffusion. In Figure 11, the self-diffusion coefficients for water, SbPC, and MCT near the two-phase border along line a in Figure 2b are plotted as a function of R. The relative diffusion coefficient for water corrected for the solvation effect (assuming nw,bound ) 15) and the partitioning of 1-propanol between water and MCT is shown in Figure 13. It is evident that Dw remains high up to point × where maximum MCT is incorporated, and from Figure 13 it can be seen that D/D0 decreases slowly with increasing R and reaches a value of 0.6 at R ) 0.45. This is the expected behavior of a bicontinuous structure and thus further supports the conclusions from the phase studies. Anderson and Wennerstro¨m48 have calculated the geometrical obstruction factor of the self-diffusion of water in a bicontinuous microemulsion as a function of the waterto-oil ratio and an expansion around the balanced state gave to leading order

D/D0 ) 0.66 - β(Φo - 1/2) where Φo is the oil volume fraction. The expansion coefficient, β, depends on the coordination number of the structure. The lateral diffusion coefficient of SbPC in the lamellar phase is about 5 × 10-12 m2/s at 35 °C,49 and the diffusion coefficient for a molecule residing in the separating monolayer of a bicontinuous structure should theoretically48 be around 2/3 of the lateral diffusion in the lamellar phase, in our case about 3 × 10-12 m2/s. This is an order of magnitude lower than the measured values in our system. In order to investigate whether the observed high DSbPC could be explained by the effect of 1-propanol, DSbPC was measured in a balanced hexadecane system containing 4 wt % SbPC and 7.6 wt % 1-propanol and DSbPC was found to be 3 × 10-11 m2/s, which is of the same order as we have observed in the MCT system. Obviously, small amounts of 1-propanol have a major impact on the selfdiffusion coefficient (and thereby also the bending modulus) of SbPC in the surfactant monolayer. 6.3. At Constant SbPC/1-Propanol Concentrations (along Line b in Figure 2b). The self-diffusion coefficients for water, 1-propanol, SbPC, and MCT were measured in a series of samples with varying R, keeping (48) Anderson, D. M.; Wennerstro¨m, H. J. Phys. Chem. 1990, 94, 8683-94. (49) Lindblom, G.; Ora¨dd, G. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 483-515.

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in-oil droplet structure even at these high MCT concentrations. The self-diffusion data presented here clearly indicate that there is some structure in the microemulsion even at these high concentrations of SbPC and 1-propanol with an oil-in-water droplet structure at the water rich side, a gradual change to a bicontinuous structure when the MCT concentration is increased. The bicontinuity appears to be preserved even at low water concentrations. It is, however, important to note that since we have fairly high amounts of both SbPC and 1-propanol, which are known to destabilize micellar structures,50 the system is dynamic and less well-defined with large variations in size and shape, as well as rapid fusion and fission of the oil and water domains. Figure 12. Self-diffusion coefficients for water (closed circles), SbPC (closed triangles), and MCT (open triangles) along line b in Figure 2b at 25 °C.

7. Conclusions In this paper the differences in phase behavior and microstructure of triglycerides and hexadecane based water/1-propanol/SbPC/oil systems have been studied. For MCT a Lc + O-type two-phase region is present at low 1-propanol concentrations. With increasing 1-propanol concentration the Lc phase is destabilized and a Winsor III system (W + Me + O) is formed. At even higher 1-propanol concentrations the Winsor III system is transformed to a Winsor I system (Me + O). This behavior is different compared with the hexadecane system which is known to change from a Winsor II system (Me + W) via a Winsor III system to a Winsor I system at increasing 1-propanol concentration.

Figure 13. Relative self-diffusion coefficients, D/D0, for water from Figures 11 (open circles) and 12 (closed circles). D0 for water was determined for each point using the calculated actual 1-propanol concentration and corrected for solvation effects.

the concentrations of 1-propanol and SbPC constant at 22.5 wt %. This is illustrated by line b in Figure 2b. The measured D values as a function of R are shown in Figure 12. The relative diffusion coefficient for water corrected for the solvation effect (assuming nw,bound ) 15) and the partitioning of 1-propanol between water and MCT is shown in Figure 13. From Figure 13, it can be seen that D/D0 for water decreases almost linearly up to R ) 0.7. It was not possible to measure the water self-diffusion at lower water concentrations due to fast T2 relaxation of the water molecule. It should also be noted that, at low water concentrations, some water will be dissolved in MCT and the self-diffusion of water will be considerably affected by the hydration of the SbPC which makes an interpretation of the microstructure less relevant.46 At low MCT concentrations, DMCT was slightly higher than DSbPC but much lower than Dw, which indicates an oil-in-water droplet structure. At higher MCT concentrations, DMCT increased, while DSbPC decreased, which is interpreted as a gradual change to a bicontinuous structure. Since D0 for MCT is in the same range as D for the oil droplets, no drastic change in the DMCT at high MCT concentrations can be seen. Note that, even at R ) 0.70, DW is 1 order of magnitude higher than DSbPC, which excludes a water-

The phase behavior of the MCT system depends, to a large extent, on the influence of 1-propanol on H0 and the flexibility of the surfactant film and also on the inability of MCT to penetrate the surfactant “brush”. The concentration of 1-propanol needed to destabilize the Lc phase was found to be higher than the 1-propanol concentration corresponding to a balanced system, and as a consequence, H0 in the middle-phase microemulsion was found to be positive even at the phase boundary close to the Lc phase. The obtained D/D0 values for the MCT microemulsion formed at low SbPC concentrations and 0 < R < 0.5 are in good agreement with what is expected for a bicontinuous minimal surface structure. The microstructure of the microemulsions formed at higher SbPC concentrations was less well-defined, most probably with large polydispersity and rapid fusion and fission of the oil and water domains. Finally, it is concluded that the SbPC aggregates, in a mixture of water/1-propanol/SbPC (2/1/1 weight by weight), appears to be rather small spherical objects with an aggregation number of about 70. Acknowledgment. Professor K. Shinoda and K. Larsson are acknowledged for their valuable comments and discussions, Dr. P.-O. Eriksson for his invaluable assistance with the NMR measurements, Mr. T. PeterssonNorde´n for his assistance with the X-ray measurements, and S. Skov Christensen and P. Thore´n for technical assistance. LA9702897 (50) Stilbs, P. J. Colloid Interface Sci. 1982, 89, 547-554.