Langmuir 2000, 16, 9113-9116
9113
A Sponge Morphology in an Elementary Coacervate F. M. Menger,*,† A. V. Peresypkin,† K. L. Caran,†,‡ and R. P. Apkarian‡ Department of Chemistry and Integrated Microscopy and Microanalytical Facility, Emory University, Atlanta, Georgia 30322 Received July 26, 2000. In Final Form: October 3, 2000 A zwitterionic gemini surfactant forms a coacervate which is “elementary” in the sense that it consists of a single solute as opposed to the multicomponents (e.g., cetylpyridinium chloride/hexanol/water/NaCl) common in the coacervate literature. The gemini dissolves in water but then quickly separates as oily droplets which, despite the high water content of 83 wt %, are immiscible with water. Cryogenic temperature high-resolution scanning electron microscopy (cryo-HRSEM) examination of the droplets shows a distinct “sponge” structure. Although previously proposed for coacervate phases, a sponge morphology has never before been clearly depicted. The absence of previous electron microscopy (EM) pictures of the coacervate network is attributed to artifacts associated with transmission electron microscopy (TEM) methods, to the fragility of the coacervate toward physical perturbations, and to possible compositional changes with complex mixtures during sample preparation. When the gemini coacervate was exposed to mild shear, the honeycomb structure disappeared and was replaced by a lamellar phase.
In his classic book1 The Origin of Life, A. I. Oparin wrote, “The formation of coazervates [sic] was a most important event in the evolution of the primary organic substance and in the process of autogeneration of life.” Almost 6 decades later coacervates remain among the most esoteric of the colloidal systems. The word “coacervate” is derived from the Latin “co” (together) and “acerv” (a heap). The term implies that a colloidal solution separates into two immiscible aqueous phases: a coacervate layer which is rich in colloidal material, and the so-called equilibrium liquid which is usually poor in colloidal material. Thus one is confronted with a remarkable situation in which two aqueous layers, one with up to 95% water and the other with 99-100% water, do not freely mix.2,3 Common wisdom, namely that the colloidal material in the coacervate phase adopts a “sponge” morphology (Figure 1),4 is based on three measurement techniques (conductivity, self-diffusion, and neutron scattering)3,5-11 plus indirect observation by freeze-fracture transmission electron microscopy (FF-TEM).4,12,13 The few electron optical images that are available show rough surfaces resembling the topology of bilayer cubic phases, but they fail to depict clearly the proposed “sponge” * To whom correspondence and requests for materials should be addressed. E-mail:
[email protected]. † Department of Chemistry. ‡ Integrated Microscopy and Microanalytical Facility. (1) Oparin, A. I. The Origin of Life; Dover Publications: New York, 1953. (2) Menger, F. M.; Sykes, B. M. Langmuir 1998, 14, 4131. (3) Anderson, D.; Wennerstro¨m, H.; Olsson, U. J. Phys. Chem. 1989, 93, 4243. (4) Strey, R.; Jahn, W.; Porte, G.; Bassereau, P. Langmuir 1990, 6, 1635. (5) Gazeau, D.; Bellocq, A. M.; Roux, D.; Zemb, T. Europhys. Lett. 1989, 9, 447. (6) Balinov, B.; Olsson, U.; So¨derman, O. J. Phys. Chem. 1991, 95, 5931. (7) Snabre, P.; Porte, G. Europhys. Lett. 1990, 13, 641. (8) Maldonado, A.; Urbach, W.; Langevin, D. J. Phys. Chem. B 1997, 101, 8069. (9) Pieruschka, P.; Olsson, U. Langmuir 1996, 12, 3362. (10) Hyde, S. T. Langmuir 1997, 13, 842. (11) Roux, D.; Coulon, C.; Cates, M. E. J. Phys. Chem. 1992, 96, 4174. (12) Hoffmann, H.; Thunig, C.; Munkert, U. Langmuir 1992, 8, 2629. (13) Strey, R.; Jahn, W.; Skouri, M.; Porte, G.; Marignan, J.; Olsson, U. In Structure and Dynamics of Strongly Interacting Colloids and Supramolecular Aggregates in Solution; Chen, S.-H., et al., Eds.; Kluwer Academic Publishers: Netherlands, 1992; pp 351-363.
Figure 1. Schematic drawing of the sponge phase. Adapted from ref 4.
morphology.4,12,13 Herein we specifically observe a coacervate’s three-dimensional network as obtained by cryohigh-resolution scanning electron microscopy (cryoHRSEM). The work is unique in another way: Previous studies of coacervates involve complicated ternary or quaternary systems such as tetradecyldimethylamine oxide/heptanol/water12 or cetylpyridinium chloride/hexanol/water/NaCl.4 We, on the other hand, worked with a binary system in which a simple zwitterionic “gemini surfactant”,14,15 drawn below, forms an “elementary” coacervate when added to water.14
Since the coacervate contains a single solute and no volatile organics, the possibility of composition changes and resulting artifacts during the EM manipulations were minimized. A Dutch chemist, Bungenberg de Jong, began studying coacervates in the 1930s and actually coined the word.16 Coacervation is generally divided into two classes: simple (14) Peresypkin, A. V.; Menger, F. M. Org. Lett. 1999, 1, 1347. Includes preparation and characterization details. (15) Menger, F. M.; Keiper, J. S. Angew. Chem., Int. Ed. 2000, 39, 1906.
10.1021/la0010626 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/03/2000
9114
Langmuir, Vol. 16, No. 24, 2000
Letters
Figure 2. Schematic representation of coacervation of a gemini surfactant.
and complex.2,16-21 Simple coacervation is achieved by addingsaltsand/oralcoholstoaqueoussurfactantsolutions.16-18 Complex coacervation occurs upon mixing two oppositely charged macromolecules.16-18,20 In recent times, the term “coacervate” has been often replaced with “L3 phase”,6,11 “anomalous phase”,6 “sponge phase”,11,22 “blue I phase”,22 and “plumber’s nightmare”.4 Whether these terms all refer to an identical morphology, or even whether all systems designated by a given term are identical, is not clear. Laughlin, in a review of the sponge phase,22 also expressed uncertainty on the subject: “Viewed from a rigorous perspective, it must be admitted that our knowledge of the phase behavior of systems that display the L3 phase is primitive at this time.” In our case, we prefer to retain the more euphonious and noncommittal word “coacervate” for the oily droplets that separate from water, ultimately to coalesce into a distinct lower layer, upon dissolving the gemini surfactant in water (Figure 2). When solid gemini surfactant was added to water (0.05 M, 20 mL), immiscible droplets separated from the equilibrium water. Figure 3 shows a light microscopy image of these droplets which varied from 10 to 300 µm in diameter. The droplets coalesced into a layer of 2.5 mL. Although the coacervate volume increases with the amount of compound added to the water, the composition of the coacervate remains constant at ca. 17 wt % surfactant (the equilibrium liquid possessing less than 1 wt % surfactant). Adding water to the coacervate/equilibrium liquid mixture increases the volume of the equilibrium liquid without affecting the coacervate. Likewise, addition of up to 14 mol equiv NaCl has little effect upon our coacervate, in contrast to most simple coacervates (which are stabilized by salt) and to complex coacervates (whose formation is impeded by salt).16 Insensitivity to salt by the gemini surfactant is likely attributable to its zwitterionic structure where associations with external ions play little role. Our coacervates were always prepared at room temperature (25 °C) and were found to be relatively stable to higher or lower temperatures (5-80 °C). (16) Bungenberg de Jong, H. G. In Colloid Science; Kryut, H. R., Ed.; Elsevier: Amsterdam, 1949; pp 232-258. (17) Vassiliades, A. E. In Cationic Surfactants; Jungermann, E.; Ed.; Marcel Dekker, Inc.: New York, 1970; pp 387-415. (18) Burgess, D. J. In Macromolecular Complexes in Chemistry and Biology; Dubin, Bock, Davis, Schultz, Thies, Eds.; Springer-Verlag: Berlin, Heidelberg, 1994; pp 285-300. (19) Newton, D. W. In Coacervation: Principles and Applications; Tarcha, P. J.; Ed., CRC Press: Boca Raton, FL, 1991; pp 67-81. (20) Burgess, D. J.; Carless, J. E. J. Colloid Interface Sci. 1984, 98, 1. (21) Luzzi, L.; Palmieri, A. In Biomedical Applications of Microencapsulation; Franklin, L., Ed.; CRC Press: Boca Raton, FL, 1984; pp 2-155. (22) Laughlin, R. G. In Micelles, Microemulsions, and Monolayers; Shah, D. O., Ed.; Marcel Dekker: New York, 1998; pp 73-99.
Figure 3. Light microscopy image of coacervate droplets (bar ) 100 µm).
There have been several reports in which L3 phases have been examined by electron microscopy. 4,12,13,23,24 All of these studies have utilized freeze fracture transmission electron microscopy for the imaging. This procedure involves rapid freezing of the sample, fracturing, replication of the fracture face, and cleaning of the carbonstabilized platinum replica which is then observed at ambient temperature by transmission electron microscopy. This technique has distinct disadvantages: (a) It is a laborintensive process resulting in an indirect observation of the fracture face. (b) The sample is forced into a copper grid prior to freezing. In our experience, the resulting shear stress can disrupt the long-range order within the fragile coacervate network. (c) Discrimination between artifacts and reality is difficult when large sections of the replicas are damaged by fractures, phase separations, etc.4,13 We thus relied upon the recently developed cryoHRSEM whose elegant yet simple preparation procedure has produced high-resolution images (1-10 nm).25,26 Moreover, for the first time we have cryo-imaged both the suspended coacervate droplets and the separated coacervate layer that is created by coalescence of the droplets, samples that unexpectedly show striking differences. Cryo-HRSEM experiments were carried out by rapid freezing (ca. 10 000 K/s) of the suspended coacervate droplets or separated layer in liquid ethane, fracturing of the specimen, coating with a 1 nm layer of Cr, and directly observing the frozen coacervate in the upper stage of a (23) Jahn, W.; Strey, R. J. Phys. Chem. 1988, 92, 2294. (24) Gustafsson, J.; Nylander, T.; Almgren, M.; Ljusberg-Wahren, H. J. Colloid Interface Sci. 1999, 211, 326. (25) Apkarian, R. P.; Caran, K. L.; Robinson, K. A. Microsc. Microanal. 1999, 5, 197. (26) Menger, F. M.; Caran, K. L.; Apkarian, R. P. Langmuir 2000, 16, 98.
Letters
Langmuir, Vol. 16, No. 24, 2000 9115
Figure 4. (a) Coacervate droplet of the gemini surfactant as detected by cryo-HRSEM (bar ) 2.5 µm). (b) Higher magnification reveals the porous morphology of the surface (bar ) 667 nm).
Figure 5. (a) Fractured coacervate droplet as detected by cryo-HRSEM (bar ) 667 nm). (b) Fractured droplet with projections corresponding to the pores from image a (bar ) 667 nm).
field emission scanning electron microscope.25 The electron microscopy images of the droplet surface at 4000× and 15000× instrumental magnification (panels a and b of Figure 4, respectively) show clearly a spongelike morphology with pore diameters ranging from 25 to 125 nm. Fractured coacervate droplets detected by cryo-HRSEM are given in Figure 5. A honeycomb interior, with 100200 nm pores, is apparent. The image in Figure 5B represents a droplet in which a large portion of the honeycomb has been removed during fracturing, leaving regions of vitreous ice that presumably occupy the volume inside the pores. As in a lock and key, the pores in Figure 5A correspond qualitatively in size and shape to the projections in Figure 5B. For comparison purposes, we applied the cryo-HRSEM method to droplets of AOT/NaCl/water, a classical coacervate that we and many others have examined in the past.2,13,27,28
(27) Acharya, R.; Ecanow, B.; Balagot, R. J. Colloid Interface Sci. 1972, 40, 125. (28) Fontell, K. J. Colloid Interface Sci. 1973, 44, 318.
Figure 6. Coacervate droplet of AOT/NaCl/water as detected by cryo-HRSEM (bar ) 250 nm).
Figure 6 shows an image of such a droplet at a 40000× instrumental magnification. Surface pores, if they exist at all, fall below the resolution of the experiment. Clearly, the network structure of a coacervate is highly system dependent. As seen in Figure 7A, the coacervate layer of the gemini surfactant displays a morphology quite different from that of its droplets. A directionality, caused by the effects of shear when the sample was expelled from a syringe during the sample preparation, is evident in the image. In other words, the coacervate network, presumably composed of interconnected bilayers, is quite fluid and easily deformed. An L3 to LR phase transition, in which a coacervate
9116
Langmuir, Vol. 16, No. 24, 2000
Letters
Figure 7. (a) Coacervate layer as detected by cryo-HRSEM. Notice the effect of shearing on the bilayer network of the coacervate (bar ) 667 nm). (b) Stretched bilayer stacks demonstrate the effect of shearing on a mixture of coacervate layer and “equilibrium liquid” (bar ) 500 nm).
transforms into a simple lamellar phase, has been previously reported.4,12,13 We ourselves observed what appears to be a lamellar phase when a sheared sample of our coacervate was viewed under a polarized microscope. In Figure 7B, interconnections between the bilayers in the coacervate layer have been broken, and the bilayer stacks partially aligned, as a result of hand-shaking the coacervate with equilibrium liquid. The delicate nature of the coacervate structure, and the artifacts imparted by metal replication and etching procedures using FF-TEM, may account in part for the absence of previous images accurately depicting the sponge morphology. In the beginning of this paper we remarked how Oparin believed that life began in the coacervate. Although no evidence currently exists for his theory, it is clear that coacervates are commercially important (e.g., in the encapsulation of drugs)21,29-31 and that their morphology has been something of a mystery to colloid chemists. Our cryo-HRSEM pictures of a particularly simple (“elemen(29) Madan, P. L. Drug Dev. Ind. Pharm. 1978, 4, 95. (30) Burgess, D. J.; Carless, J. E. Int. J. Pharm. 1985, 27, 61.
tary”) coacervate system strongly support the presence of a fragile spongelike network that resists miscibility with its own solvent. As a final thought, we speculate that Alexander Pope, who wrote the verse below, might have been less negative about microscopy had he seen SEM pictures of a coacervate! Why has not Man a microscopic eye? For this plain reason, Man is not a fly. Say what the use, were finer optics giv’n, T’inspect a mite, not comprehend the heav’n? Acknowledgment. This work was supported by the National Institutes of Health. We thank Dr. M. Ko¨lbel for assistance with the polarized microscope and helpful discussions. LA0010626 (31) Nixon, J. R.; Ed. Microencapsulation; Marcel Dekker: New York and Basel, 1976.
