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Freeze-Fracture Electron Microscopy of Sheared Lamellar Phase T. Gulik-Krzywicki,† J. C. Dedieu,† D. Roux,*,‡ C. Degert,§ and R. Laversanne§ Centre de Recherche Paul-Pascal CNRS, Avenue DR Schweitzer, 33600 Pessac, France, Centre de Ge´ ne´ tique Mole´ culaire, CNRS, 91198 Gif sur Yvette, France, and CAPSULIS S. A., Centre Condorcet, 162 Avenue Schweitzer, 33600 Pessac, France Received January 22, 1996. In Final Form: June 24, 1996X It has been shown recently that shearing of lyotropic lamellar phases may lead to the formation of relatively monodispersed multilayered vesicles named spherulites. Freeze-fracture electron microscopy analysis of such preparations, presented here, shows that their three-dimensional organization is of a space-filling polyhedral type, built up from very closely packed spherulites, without any visible additional water present neither in the center of the spherulites nor in between them. Dilution of these preparations leads to the separation of individual spherulites without appreciable changes of their internal structure (multilayered nature and spacing between the layers). Diluted spherulites become spherical and are separated by the water dispersions of much smaller vesicles, originating probably from the fragmentation of some external layers of concentrated spherulites.
Introduction Recently, it was observed that the shearing of a lyotropic lamellar phase leads to the formation of very interesting structures.1-3 Indeed, while at small or very high shear rates the lamellar phase is more or less oriented with the layers perpendicular to the direction of the velocity gradient, at intermediate shear rates the lamellar phase arranged itself into a concentrated phase of multilayered vesicles. One of the particularities of this phase is to form monodisperse vesicles the size of which is of the order of microns and varies continuously with the shear rate.1,2 When the shear rate is fixed to a given value, it can take from a few minutes to a few hours for the multilamellar vesicles to reach a steady state. Light scattering under shear conditions shows a diffraction peak at an angle corresponding to the spherulite size. When the shear is stopped very rapidly the spherulite organization is preserved. The sample has the consistency of a cream and can be removed from the shear cell without destroying the structure. It is also possible to dilute this concentrated phase of spherulites and to obtain a suspension of microcapsules similar to liposomes. First discovered with a quaternary system,2 this new instability has been found with many different types of surfactants.1-4 This method is a very efficient way of preparing well controlled multilayered vesicles.4 Multilayered vesicles have also been described in systems which are supposed to be in equilibrium5-7 but in a much less reproducible way. It is possible that, in the latter case, the simple shearing applied during the preparation of the samples may be sufficient to give rise to the formation of some spherulites, but with the shear * To whom correspondence should be addressed. † Centre de Ge ´ ne´tique Mole´culaire. ‡ Centre de Recherche Paul-Pascal. § CAPSULIS S. A., Centre Condorcet. X Abstract published in Advance ACS Abstracts, September 1, 1996. (1) Diat, O.; Roux, D. J. Phys. II 1993, 3, 9. (2) Diat, O.; Roux, D.; Nallet, F. J. Phys. II 1993, 3, 1427. (3) Roux, D.; Nallet, F.; Diat O. Europhys. Lett. 1993, 24, 53. (4) Roux, D.; Diat, O. French Patent 9204108. Roux, D.; Diat, O.; Laversanne, R. Patent WO 9319735. (5) Dubois, M.; Zemb, Th. Langmuir 1991, 7, 1352. (6) Van der Linden, E.; Droge, J. H. M. Physica A 1993, 193, 439. (7) Boltenhagen, Ph.; Lavrentovich, O. D.; Kle´man, M. Phys. Rev. A 1992, 46, 1743.
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rate being less controlled and the steady state not reached the monodispersity in size is not as homogeneous as for the samples prepared under controlled shear. Freeze-fracture electron microscopy has been used for many years to study lipid-water phases, and the analysis of images of properly frozen samples was shown to be in excellent agreement with the structures determined by X-ray diffraction.8-10 In the present article, we used this method to study the structure of sheared lamellar phases. Material and Methods Preparation of Spherulites. Two lamellar phases, containing respectively 20% AOT (from Sigma) and 80% brine (10 g/L of NaCl in water containing 30% glycerol) for the first one and 60% Sochamine 35 (a high molecular weight imidazoline from Witco Chemicals) and 40% water (containing 30% of glycerol) for the second, were studied. The glycerol was added in order to achieve the best preparation of the sample structure upon cryofixation. We have checked, both with X-ray diffraction and light microscopy (texture), that the replacement of water by water-glycerol mixtures did not modify the sample structure. Each lamellar phase has been placed in a homemade Couette cell and sheared at a constant shear rate for 4-5 h in order to obtain spherulites. Light scattering was used to follow the spherulite formation and to estimate their size;1 the spherulite size measured is on the order of 1 µm. The preparation is removed from the cell and kept in a closed vessel before cryofixation. Dilute samples were obtained by the dispersion of this preparation in a water-30% glycerol solution. Regular and phase contrast microscopy showed that these diluted samples form stable colloidal suspensions of spherical particles which can be kept for weeks without any appreciable change. Freeze-Fracture Electron Microscopy. A thin layer of the sample (20-30 µm) was placed on a thin copper holder and then rapidly quenched in liquid propane. The frozen sample was fractured at -125 °C, in a vacuum better than 10-6 Torr, with the liquid nitrogen cooled knife in a Balzers 301 freezeetching unit. The replication was done using unidirectional shadowing, at an angle of 35°, with platinum-carbon and 1-1.5 nm of mean metal deposit. The replicas were washed with organic solvents and distilled water and were observed in a Philips 301 electron microscope. (8) Gulik-Krzywicki, T. Biochim. Biophys. Acta 1975, 415, 1-28. (9) Gulik-Krzywicki, T.; Aggerbeck, L. P.; Larsson, K. In Surfactant in Solution; Mittal, K. L., Lindman, B., Eds.; Plenum Press: New York, 1984; pp 237-257. (10) Gulik-Krzywicki, T.; Delacroix, H. Biol. Cell 1994, 193-201.
© 1996 American Chemical Society
Electron Microscopy of Sheared Lamellar Phase
Figure 1. Electron Micrograph Photo of Replica of Samples of Lamellar Phases Prepared under Shear. (A) AOT sample sheared at 4 s-1. The typical size of the multilayered vesicles is on the order of 3 µm. Note the regularity of the vesicle size. (B) SOCHAMINE 35 sample sheared at 20 s-1. In this case due to the different nature of the surfactant some vesicles are cut through revealing the multilayered nature of the samples. The typical size is on the order of 1 µm.
Results and Discussion The low magnification views of freeze-fractured sheared lamellar phases of AOT and SOCHAMINE 35 are shown in Figure 1. In both cases one observes an assembly of very closely packed, relatively monodisperse, spherulites. The mean size of AOT spherulites (about 3 µm) is in excellent agreement with the value obtained from the lightscattering analysis of the same preparation. The main difference between AOT and SOCHAMINE 35 spherulites, apart from the values of their mean diameters due to the different shearing rates (4 s-1 for AOT and 20 s-1 for SOCHAMINE 35), appears to be related to their fracture properties. Only a very few cross-fractured spherulites are observed in the case of AOT as compared to the almost
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Figure 2. Photos of two samples prepared at different shear rates: (A) 13 s-1; (B) 20 s-1. The typical sizes corresponding to the samples are respectively 1.4 and 1 µm.
all cross-fractured SOCHAMINE 35 spherulites. The latter type of preparations are then more interesting for the study of spherulite morphology and will be used all throughout this study. The analysis of many crossfractured, closely packed, spherulites indicates that the external bilayers are often covering several neighboring spherulites. This indicates that the structure of the sheared lamellar phases is not formed by just the close packing of individual, independent spherulites but is a somewhat more complex, foamlike type structure. All space is occupied by spherulites and bilayers, and there is no indication of the presence of any additional water layer in between the spherulites. Consequently, we may estimate that the spherulites fill up nearly 100% of the total volume. Figure 2 shows electron micrographs of the samples prepared at different shear rates respectively of 13 s-1 (Figure 2A) and 20 s-1 (Figure 2B). The mean diameters of spherulites for these shearing rates respectively of 1.4
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Figure 3. Enhancement of one multilayered vesicle among its neighbors. This photo reveals the hexagonal shape of the vesicles packed together. It also reveals that the layers exist until the core of the vesicles.
Figure 5. Vesicles diluted with extra water: (A) about 10% vesicles; (B) less than 1%. The basic multilayered structure is preserved. Smaller vesicles, probably unilamellar, appear. They are due to some external layers that have been torn off during the dilution process. Figure 4. Ensemble of vesicles packed together. It is clear that the vesicles fill up space and consequently that the structure is probably foamlike.
and 1 µm. micron, are in excellent agreement with the scaling law observed previously on the AOT system,1 (the diameter has been shown to vary as the inverse of the square root of the shear rate). Figure 3 shows a higher magnification image of a crossfractured spherulite. The stacked layers are clearly seen and their separation, as measured from the optical diffraction (about 60 Å), is very close to that measured by X-ray diffraction. It is possible to follow the layers down to the center of the spherulite (see also the cross-fractured spherulites in Figure 5A). This indicates that there is no water core at the center or, more exactly, that the water core has a size of the order of the d-spacing, which is expected from the theoretical estimates of the core size.11
Figure 4 shows a large number of closely packed crossfractured spherulites. Since, as already mentioned, the shape of fractured spherulites is not spherical but close to hexagonal or pentagonal in the cut-plane, we may assume that the three-dimensional shape of the spherulites is of polyhedral type. In fact, neutron scattering analysis on well ordered (crystallized) spherulites has shown that the shape corresponds very closely to the Wigner-Seitz cell of a body centered cubic structure and consequently is tetrakaidecahedral.12 It is clear from this image that the layer curvatures follow exactly the spherulite shape. In fact this assembly of spherulites can be described as a three-dimensional lattice (with no long (11) Roux, D.; Prost, J.; Leibler, L. To be published. (12) Sierro, P.; Panizza, P.; Roux, D.; Nallet, F. To be published. (13) Roux, D.; Safinya, C. J. Phys. (Paris) 1988, 49, 307.
Electron Microscopy of Sheared Lamellar Phase
range order) of crossed disclinations. Consequently, this sheared lamellar phase can be alternatively (and probably more accurately) described as a lamellar phase with a finite density of defects. The characteristic size between these defects will determine the spherulite size. The appearance of the instability under shear leading to the structure observed here is a very efficient way of producing a well controlled array of defects (disclinations). This property may be very useful to study the effect of defects (controlling their nature and density) on liquid crystalline properties. The concentrated phase of spherulites can be dispersed in water or water-glycerol solutions. Figure 5 shows freeze-fracture images of such dispersions at different dilution (10× for Figure 5A and 100× for Figure 5B). Upon dilution, the basic structure of spherulites is conserved, but they now exhibit a spherical shape and are well separated from each other. The appearance of smaller vesicles (probably unilamellar) may be due to the tearing off, followed by the fragmentation, of the most external layers (probably those common to many spherulites) during the dilution process. One obtains, in fact, an emulsion of lamellar phase dispersed in water, the spherical shape being due (like for classical emulsions) to the surface tension which develops at the surface of each spherulite.
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Conclusions Freeze-fracture electron microscopy of sheared lamellar phases allowed us to confirm the previous description of their structure based on light scattering and X-ray diffraction,1-3 in particular the absence of any appreciable water core inside the spherulites as well as the absence of water layers in between the spherulites. The threedimensional structure of sheared lamellar phases seems to be of polyhedral type (foamlike). The overall structure of spherulites (namely their multilayered nature and the spacing between the bilayers) is conserved upon dilution, but they are spherical and separated from each other by the solvent and smaller vesicles. The latter are probably due to the fragmentation of the external layers of concentrated spherulites. Acknowledgment. The authors would like to acknowledge some earlier tests done with O. Diat, R. Strey, and P. Jahn, who have demonstrated the feasibility of the cryofracture technique. They also want to thank Mrs. Fiquet from Witco for the gracious gift of the surfactant. LA960069D
