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Structure and Phase Behavior of Phospholipid-Based Micelles in Nonaqueous Media Rajan Gupta,*,† H. S. Muralidhara,† and H. T. Davis‡ Cargill, Inc., P.O. Box 5699, Minneapolis, Minnesota 55440, and Department of Chemical Engineering, University of Minnesota, Minneapolis, Minnesota 55419 Received March 12, 2001. In Final Form: June 7, 2001 The phase behavior of phospholipid aggregates in nonaqueous media of hexane and vegetable oil with a negligible aqueous core (water < 0.3 wt %) was investigated. It was found that inverse micellar structures formed by phospholipids in a hexane-oil mixture resulted in three principal domains of phase behavior: micellar solution, two-phase dispersion, and dense micellar solution. The dependence of the phases on temperature, water, and solvent/phospholipid concentration was also examined. As opposed to structures in aqueous media, nonaqueous structures were found to be weakly dependent on these parameters, with the exception of solvent composition. Micellar sizes (∼60 Å diameter) were determined using various experimental techniques. The principal techniques used in our studies were cryo-transmission electron microscopy, dynamic light scattering, and X-ray scattering. In our studies, we explored the limitations of these techniques and estimated size, shape, and phase nature of lipid aggregates.
1. Introduction Understanding surfactant aggregate formation, or selfassembly, in nonaqueous systems can be fascinating from a both scientific and technological point of view. A good understanding of the system can be applied to a wide variety of areas: in food applications (e.g., micellarenhanced separations from membranes), in detergent applications (e.g., in making liquid dispensable soaps), in specialty chemical applications (e.g., dispersion of organic compounds in polymeric matrices and as wetting agents for spreading herbicides on hydrophobic weeds), in pharmaceutical applications (e.g., drug delivery systems), and even in creation of electrooptical displays made of liquid crystal droplets. For optimal applications, it is highly desirable to understand the scientific aspects, such as phase behavior and microstructure of the system, stability, and interactions among various micellar aggregates. Though the experimental techniques to understand selfassembly in aqueous systems are very well established, applications of these techniques to nonaqueous systems are not so well established. In this paper we attempt to advance the understanding of phospholipids in hexane and vegetable oil mixture. Aggregates in a nonaqueous system can behave very differently from those in aqueous systems. In aqueous systems, these structures are significantly affected by the surfactant nature, solution charge, temperature, pH, surfactant concentration, and other parameters.1 Electrical double layers strongly influence micellar interactions for ionic surfactants, whereas molecular geometry and other parameters are important for nonionic surfactants.2,3 The orientation of the surfactant molecules in aqueous solutions is well understood; hydrophilic surfactant “head* To whom correspondence should be addressed. † Cargill, Inc. ‡ University of Minnesota. (1) Kumar, P.; Mittal, K. L. Handbook of Microemulsion Science and Technology; Marcel Dekker: New York, 1999, and references therein. (2) Evans, D. E.; Wennerstrom, H. The Colloidal Domain: Where Physics, Chemistry, Biology and Technology Meet; Wiley: New York, 1999. (3) Israeclachvili, J. N. Intermolecular and Surface Forces; Academic Press: San Diego, 1991.
groups” are aligned toward the bulk aqueous phase, and the hydrophobic “tails” “hide” inside a micelle. More complex systems, for example, block copolymers in aqueous solution4 and surfactants at solid-aqueous phase interfaces, are also relatively well understood.5 However, not much information is available in the open literature for nonaqueous systems. It is known though that in nonaqueous mixtures the hydrophilic headgroups of surfactant molecule go inside the micelle, and the tail groups are exposed to the nonaqueous bulk phase.6,7 Since the polar headgroup is inside the micellar core, electrical double-layer effects for ionic surfactants are not the same anymore. The effects of temperature, salt, cosurfactant, bulk solution, pH, etc., can also be different on the critical micelle concentration (cmc) and on a wide variety of inverse micellar and lyotropic mesophases (hexagonal, cubic, and lamellar).8 In this work we focus on phase behavior and aggregate formation in a bulk nonaqueous system at various temperature, solvent, surfactant, and (low) water levels. We also discuss techniques and methods for characterizing nonaqueous systems having a negligible aqueous phase. The system used in this study consisted of phospholipids, vegetable oil (triglycerides), hexane, and a limited quantity of water (50 °C). This not only changes the system properties (hexane-to-oil ratio) but also condenses drops on the inside wall of a DLS tube. These drops reenter into the system and induce a convective motion into the sample. This obviously is undesired as the natural movement of micelles in the bulk solution is disturbed. The overall motion does not remain purely diffusive, and the Stokes-Einstein equation used for analysis becomes invalid. To prevent this, samples were filled up to the top, leaving some headspace, and were then sealed. It was found that drop evaporation and condensation at the very top of the DLS sample tube had minimal impact on the lower portion of the tube, where DLS measurements were made. SAXS measurements had no high-temperature issues. However, our nonaqueous samples had weak signals and required long exposures to have a high signal-to-noise ratio. Now, conducting experiments for long time also induces more undesired parasitic scattering at small angles for SAXS instruments with a pinhole camera. We found that conducting experiments for 1 h gives us the reasonable results. Further, we also confirmed our results by using a Kratky camera, which has no parasitic scattering issues (but other smearing issues).
3. Results and Discussion 3.1. Phase Diagram of Soybean Oil-Phospholipid-Hexane System. As shown in Figure 2, three principal regions (micellar solution, two-phase dispersion, and dense micellar phase) exist in an oil-phospholipidhexane system. The details on the various phases are discussed in the sections below. A micellar phase consists of uniformly distributed reverse micelles in the bulk phase of hexane and oil. Since the concentration of phospholipids is low, these micelles do not interact strongly with each other and remain dispersed in the solution. As the concentration of phospholipids is increased, a smooth transition to dense micellar phase is observed for the mixtures with a hexaneto-oil ratio greater than unity. The number density of micelles increases, resulting in a dense micellar phase. Both micellar phase and dense micellar phase exist as single phases. However, a phase transition to a two-phase region from micellar phase is observed with an increase in phospholipid concentration for mixtures with a hexaneto-oil ratio less than unity. The two-phase region forms as large micron-size phospholipid structures dispersed in (15) Danino, D.; Gupta, R.; Talmon, Y., manuscript in preparation.
Phospholipid-Based Micelles
Figure 2. Phase diagram of the oil-phospholipids-hexane system. Broken lines should be taken as guides for approximate phase boundaries. Dotted line represents a smooth transition between dilute and dense micellar phases.
Figure 3. Estimated aggregate sizes (in Å) for micellar phase samples at 24 °C using DLS.
a continuous phase. These dispersed structures aggregate over time, resulting into two equilibrium phases (micellar and dense micellar phases). It must be mentioned here that phase boundaries presented in the phase diagram are diffuse, and no sharp transition was observed in our experiments. Further work may help in defining these boundaries better. Phase behavior was also investigated at higher temperatures. The maximum temperature for all experiments was 65 °C. As discussed in the following sections, temperature had a weak effect on phase behavior. The effect of small amounts of water in the micellar phase was also investigated. It was found that aggregate size is unaffected up to a water level of 1 wt %. Our findings are discussed in detail below. 3.2. Micellar Phase. This phase consists of reverse micelles in the bulk solvent. Samples in this region were first examined visually and microscopically. It was found that the phase is clear and isotropic, except for a trace of dust particles found in some of our samples. 3.2.1. Micellar Size. DLS measurements: Dynamic light scattering measurements were done for various phospholipid and solvent composition mixtures. The water level was kept constant at 0.3 ( 0.05%. Results on calculated sizes are presented in Figures 3 and 4 at 24 and 65 °C, respectively. Aggregate sizes were found to be in the range 50-92 Å. SAXS measurements: SAXS measurements on select samples were performed to compare and validate sizes obtained from the DLS technique. A comparison between
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Figure 4. Estimated aggregate sizes (in Å) for micellar phase samples at 65 °C using DLS.
Figure 5. Cryo-TEM image showing randomly distributed micelles. The image is a projection of several layers of micelles. Table 2. Aggregate Sizes As Calculated Using DLS and SAXS Techniques (Temperature ) 24 °C, Water
