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The relationship between a dried and native steroid gel
The Relationship between a Dried and Native Steroid Gel P. TERECH *,1 AND R. H. WADE~ D~partement de Recherche Fondamentale, *Groupe Physico-Chimie Moldculaire, Service de Physique, and t Laboratoire de Biologie Structurale, L. Bio., 85 X, 38041 Grenoble C3dex, France Received July 24, 1987; accepted November 19, 1987 Steroid/cyclohexane gels are dried and observed by the shadowing replication electron microscopy technique. Two very different kinds of images are obtained: quasi-infinite connected filaments and isolated small filaments. In both cases, helical pitches and diameters of twisted fibers are respectively ~ 5 8 and 25 nm. Results are compared with those of the native gels from freeze-etching replication electron microscopy and SANS techniques which show helical filaments about 9 nm in diameter. The modification in the filament structure of the dried gel is interpreted as being an intrinsic property of the chiral native filaments. © 1988 Academic Press, Inc.
1. INTRODUCTION
A gel behaves in many ways like a solid although it has in fact two distinct components; the major component volumewise is a liquid while the second component is a solid which forms a network running throughout the sample. The related sol phase behaves as a liquid. The sol is converted to a gel, via a critical transition which is characterized by the divergence of certain physical properties such as the viscosity. The thermo-reversibility of the transition depends on the nature of the cohesive interactions involved in the establishment of the solid network within the gel. An example of the so-called chemical gels, which are irreversible, is the polymerization of acrylamide, in the presence of a bisacrylamide reticulating agent, which leads to the formation of a threedimensional covalently linked solid network (1). In other cases the gel is thermo-reversible. This is the category of the physical gels for which weak interactions (hydrogen bonds, van der Waals interactions) are involved in establishing the solid network. Examples are col1 Present address: Insfitut Laue-Langevin, 156 X, 38042 Grenoble C&lex, France. Author to whom correspondence should be addressed.
0021-9797/88 $3.00 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
lagen (2) and some small amphilic molecules (3). Gel systems are becoming increasingly important in a wide range of fields: medicine, pharmacology, cosmetics, photography, and the food and petroleum industries. The solgel process is used to obtain special coatings which involve the dried gel (or xerogel). This process can be used to obtain, at low temperatures, glasses, ceramics, coatings, and paints from inorganic sols such as SIO2, A1203, or W2Os (4). High-modulus fibers can be obtained from polymer solutions (5). Such materials have useful and interesting optical, electronic, or ionic properties (6) and constitute a new solid-state form. Both from the applied and fundamental points of view it is important to characterize the structural aspects of these new materials. We have recently described observations of the gel network formed by a two-component system which because of its simplicity is an extremely useful model system. It consists of cyclohexane and an amphilic steroid molecule (molecular weight 390) derived from cholesterol, D-homosteroidal nitroxide (hereafter referred to as the steroid). The present article describes a structural study of xerogels oh-
542 Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
STEROID GELS tained by evaporation ofcyclohexane from the native gels. Work such as this depends on having a sound knowledge of the initial gel structure. In the present case this has already been studied both by electron microscopy (7) and by small-angle neutron scattering (8) and good agreement was found between these independent methods. It is on this foundation that we present our electron microscope observations of xerogel structures and give tentative interpretations of the important structural differences between the xerogel and the gel states. 2. M A T E R I A L S
AND
METHODS
The synthesis of the steroid (9) and the preparation of the native gel samples (7) have been described previously. The preparation of replicas of dried gel samples for electron microscopy was carried out by sandwiching several drops of warm solution between two freshly cleaved mica sheets. The solution was allowed to gel at, or somewhat below, room temperature. The sandwich was cleaved in air by separating the two mica sheets and the gel adhering to the mica sheets was allowed to dry; see Figs. 1 and 3 for specific conditions. One of the mica sheets was transferred to a vacuum evaporation system and its gel-covered surface was first rotary shadowed with a tantalum-tungsten alloy evaporated from an electron beam-heated rod and then carbon replicated. The replicas were separated from the substrate either by floating the carbon film onto water or by immersion in a water-ethanol mixture which dissolves the steroid layer beneath the replica. The replica fragments were picked up on 3-mm-diameter 400-mesh copper electron microscope grids. The replicas were examined at 80 keV in a JEOL 100 C or 100 CX electron microscope. 3. R E S U L T S
3.1. NATIVEGEL We summarize briefly the conclusions drawn from a previous electron microscope study of the steroid gel (7).
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This gel, which has also been characterized by rheological experiments (10, 11), contains a solid infinitely connected filamentary network with an average filament diameter of 9.1 nm. From stereoscopic views an average mesh size of about 300 nm is found in the case of gels formed at room temperature from a solution of initial concentration about 3 X 10-2 M. Individual filaments are at least 5/~m long. A statistical analysis shows the most frequently occurring filament diameter to be 9.1 nm and also reveals the presence of finer filaments some 4.6 nm in diameter. We deduced that at least two of these finer filaments, protofilaments, are required to form the 9.1-nm-diameter filaments which also show 5-nm pitch striations. The connections between the filaments were found to consist of fusion zones produced by exchange of protofilaments, filaments bodily entwining one about the other, or of juxtaposed filaments. 3.2. DRIED GEL STRUCTURES For the sake of clarity the images obtained from dried gels are divided into two main classes, those showing filaments of quasi-infinite length to diameter ratio and those showing filaments of finite length to diameter ratio. The first class appears similar to the network structures shown by the native gel and has very few loose filament ends. The second type, by contrast, shows few interconnected filaments and numerous loose ends.
3.2.1. Quasi-infinite Filaments Images of this class, in Fig. 1, typically show a continuous network with filaments many microns in length similar in overall appearance to the native gel. The filament diameters have an average value of 26 nm which is of the order of three times those found in the native gel. Details of the many interfilament contact zones are revealed clearly in less densely populated regions such as those shown in Figs. 2a and 2b. A notable feature is that the contacts are similar to those in the native gels, princiJournal of Colloid andlnterfaceScience, Vol. 125, No. 2, October 1988
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FIG. 1. Replica of a dried gel. The quasi-infinitely connected filament network is clearly visible; the filament diameters are about 26 nm. The images of Figs. 1, 2, 5, and 6 were obtained from specimens prepared as follows. Three drops of a steroid solution (concentration ~3 X 10-2 M) were sandwiched between two mica sheets and allowedto gel at room temperature. Two hours later the sheets were separated and the xerogelwas replicated as described in Section 2.
pally parallel juxtaposed filaments and entwined filaments. A very pronounced helical structure is apparent and has a mean pitch of about 58 nm.
3.2.2. Finite Filaments Images in this class can be subdivided into those showing filaments for which the lengths, diameters, and pitches are approximately constant (constant geometry case) and those for which these parameters vary considerably (variable geometry case). Images corresponding to the constant geometry case are shown in Fig. 3 for different lengths and concentrations of filaments. The filaments have a mean diameter of about 25 n m and have a less pronounced helical structure than was the case for the quasi-infinite filaments. The filaments either develop from a c o m m o n center or are isolated; both cases Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
are dearly visible in Fig. 4. These micrographs represent specimens obtained from dilute solutions by solvent evaporation before the critical gelation threshold was reached and they show structures obtained from the sol phase. In the variable geometry case a wide range of lengths, diameters, and pitches can be observed in the same image (Figs. 5 and 6). In these images the filament diameters range up, wards from about 20 n m to over 100 nm in cases where two or more filaments can be seen to twist together. 4. DISCUSSION In the experiments described here the diameters of the filamentary structures in the quasi-infinite connected networks, Figs. 1 and 2, and the finite fibers, Figs. 3 to 6, are of the order of 25 n m which is about three times the
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FIG. 2. Dried gel replica showing quasi-infinite filaments and details of the filament interactions. (a) Overall view of entwined filaments (A) and of parallel juxtaposed filament regions (B). (b) Detail Of a twisted filament zone showing the 26-nm-diameter and 58-nm-pitch filaments.
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FIG, 3. Replicas of dried gelling solutions showing finite-length filaments with approximately fixed geometrical parameters. The filament helicities are less pronounced than in the case of the dried gels. Filament diameters are about 26 n m and helical pitch is 58 nm. (a) Short disconnected filaments; (b) longer filaments which are still not interconnected. The images of Figs. 3 and 4 were obtained from a dilute steroid solution sandwiched between two mica sheets for 15 min. W h e n the mica sheets were separated, the inside layer was still liquid. After solvent evaporation the surface was replicated as described.
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FIG. 4. Replica of a dried gelling solution showing finite-length filaments which are either isolated or which grow from a common germination center. Filament diameters and pitches are respectivelyabout 26 and 58 rim. value found in the native gels (7). The filaments in Figs. 1 and 2 are distinctly helical with a pitch of about 58 n m whereas those in Fig. 4 are m u c h more rod-like. In the case of Fig. 6 the majority of filaments are interconnected but some are broken and give m u c h thicker structures made up o f overtwisted filaments. The breaks m a y have occurred during specimen preparation or m a y have resulted from the strong tendency of the filaments to twist during the drying out of the gel. The overtwisting can occur equally well for a broken filament twisting back on itself as for an unbroken filament with reticulation points which have become closer as the mesh size decreases. Figure 5 shows the same type of effect in filament fragments. In these cases (variable geometry ) there is a wide distribution in the observed helical pitches and filament diameters. It would appear that these broken and overtwisted structures can be considered as structural defects of the ideal xerogel network.
In order to understand the relationship between the native gel and the xerogel, two important questions must be answered. 1. What happens to the steroid remaining in solution as the cyclohexane evaporates? In attempting to answer this question we should note that the speed at which the cyclohexane/air interface moves through the gel sample will determine whether the steroid molecules remaining in solution will have time to associate as filaments or crystals or whether they will be precipitated from solution in some other form, The governing parameters are the diffusion rate of the steroid molecule in solution (v = 2 X 10 4 n m sec -t for D ~ 10 -6 cm 2 sec -1) compared to the velocity of the air/liquid interface, about 300 n m sec-1 (12 ), and the measured kinetic growth rate, 1 min -1 (13), for usual gelling solutions (concentration ~ 1 0 -2 M, T = 20°C). These parameters indicate that the dried gel should be mainly made up of filaments which grow following Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
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FiG. 5. Finite-length filaments of variable geometry showing folded and overtwisted filaments at different magnifications. A wide distribution of diameters and pitches is observed (for example, in (b) the minimum diameter is 26 n m and the maximum diameter is over 100 nm). In (c) the two filament pitches are about 300 and 150 nm.
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1 0 0 nm
I~G. 5.--Continued.
the same process as in the initial native gel. The xerogel network structures depicted by the quasi-infinite filament distributions of Figs. 1 and 2 support this conclusion since we never observe any structural forms other than the steroid filaments. 2. How is the gel network modified by the passage of the solvent meniscus? An obvious consequence of solvent evaporation is that the overall volume of the gel decreases. The gel network will react to this constraint by shrinking in a manner depending on the filament elasticity (see, for example, the introductory paper by Tanaka (1)). It is possible to imagine several mechanisms for this process which is governed by the osmotic pressure of the gel. Two of these are shown in Fig. 7, where just three interfilament contact points are considered. If the filaments conserve their initial length they must fold (Fig. 7b) as the contact points become closer. This folding process can assume various forms and may lead to collapse
of filaments onto one another or onto themselves, or a given filament may twist around itself. Another possibility shown in Fig. 7c is that as the contact points approach each other the filaments remain tightly stretched between them as if they have a built-in elasticity. Such elastic filaments must increase in diameter as their length decreases. This type of effect could explain a consistent feature of all our images of dried gel systems, namely that observed filament diameters are systematically greater than those found in the native gel (about three times greater, in fact), whereas if the mechanism of Fig. 7b were operating we would expect to observe in addition to many collapsed or entwined filaments some filaments having the diameter of those in the native gel. This is never the case (see Figs. 1 to 4). It would be tempting to argue at this stage that the filament diameters increase simply because of steroid deposition from solution. It is difficult to imagine how this could take place with complete uniformity for all filaments. Moreover in order to at least double the diameters of all Journal of ColloM and Interface Science, Vol. 125, No. 2, October 1988
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FIG. 6. Infinite-filament network of a dried gel sample. In addition to the connected filament network similar to Fig. I, broken, folded, and overtwisted filaments give finite fragments o f variable geometry as in Fig. 5.
filaments three times the quantity of steroid involved in the filament formation of the native gel is required. At the working concentrations this amount of material is simply not available in the remaining solution. We are consequently obliged to accept the diameter increase as an intrinsic property of the filaments themselves. This interpretation is supported by our observations that the same filament diameters are found both for the finite length filaments in the sol-phase domains (Figs. 3 and 4) and for the connected filaments of the dried gel networks (Figs. 1 and 2). 5. CONCLUSIONS
A comparison of the electron micrographs obtained from the gel (7) and xerogel of the steroid cyclohexane system leads to two significant conclusions concerning the xerogel structure. In the first place, since only the filamentary form of the steroid is observed in Journal of Colloid and Interface Science, Vol. 125,No. 2, October1988
the xerogel images, as the steroid concentration increases due to solvent evaporation the excess steroid must come out of solution as filaments which are incorporated into the original gel network. Then as the solvent meniscus sweeps through the gel network the filaments change their conformation compared to the native gel. They thicken considerably and become strongly helical. In this context it can be noted that there are numerous published examples of enantiomorphic molecules for which either optical isomer gives chiral superstructures. Examples are lithium 12-hydroxy-octadecanoate (14, 15), PBLG (16), and substituted valines (17). In addition, supertwisting is frequently encountered in systems made up of long filaments, for example, deoxycholic acid (18), sodium deoxycholate (19), starch (20), and certain liposomes (21). Striking structural modifications have also been observed in chiral bilayers formed from double-chain ammonium glutamic acid derivatives (22-24).
STEROID GELS
÷
÷ c
FIG. 7. Idealized representation of two possible structural modifications (situations b and c) when a gel network with three contact points (situation a) shrinks by solvent evaporation. (b) The points get closer and the slack filaments are free to fold or to overtwist. (c) The filaments contract elastically and the resulting filament diameters are increased. The crosses in b and c indicate the initial contact point positions.
ACKNOWLEDGMENTS The authors are grateful to Drs. R. Ramasseul and F. Volino for helpful discussions and continued interest. We thank C. Closse for technical assistance at various stages of this work. REFERENCES 1. Tanaka, T., Scientific American January, 110 (1981). 2. Hausch, A., Actuality Chim. 40 (1978). 3. Hermans, P. H., in "Colloid Science," Vol. II, "Reversible Systems" (H. R. Kruyt, Ed.). Elsevier, Amsterdam, 1969. 4. Livage, J., J. SolidState Chem. 64, 322 (1986).
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5. Frost, H. H., Cohen, Y., and Thomas, E. L., in "Reversible Polymeric Gels and Related Systems" (P. S. Russo, Ed.), Chap. 12, ACS Symposium Series No. 350. Amer. Chem. Soc., Washington, D.C., 1987. 6. Mukherjee, S. P., J. Non-Cryst. Solids42, 477 (1980). 7. Wade, R. H., Terech, P., Hewat, E. A., Ramasseul, R., and Volino, F., J. Colloid Interface Sci. 114, 442 (1986). 8. Terech, P., Volino, F., and Ramasseul, R., J. Phys. (Orsay, Fr.) 46, 895 (1985). 9. Martin-Borret, O., Ramasseul, R., and Rassat, A., Bull. Soc. Chim. Fr. H401 (1979). 10. Callec, G., Gauthier-Manuel, B., Terech, P., and Ramasseul, R., C. R. Acad. Sci. Paris, SYr, H 293, 99 (1981). 11. Gauthier-Manuel, B., Allain, C., and Guyon, E., C. R. Acad. Sci. Paris, S~r. H296, 217 (1983). 12. Terech, P., Thesis, Grenoble, France, 1983. 13. Terech, P., J. Colloid Interface Sci. 107, 244 (1985 ). 14. Tachibana, T., and Kambara, H., Bull. Chem. Soc. Jpn. 42, 3422 (1969). 15. Tachibana, T., Kayama, K., and Takeno, H., Bull. Chem. Soc. Jpn. 45, 415 (1972). 16. Tachibana, T., and Kambara, H., Kolloidn. ZI. 219, 40 (1967). 17. Hidaka, H., Murata, M., and Onai, T., J. Chem. Soc., Chem. Commun. 562 (1984). 18. Ramanathan, N., Currie, A. L., and Ross Colvin, J., Nature (London) 4778 (1961). 19. McCrea, J. F., and Angerer, S., Biochim. Biophys. Acta 42, 355 (1960). 20. Heyn, A. N. J., TextileRes. J. 29, 366 (1959). 21. Lin, K. C., Wets, R. H., and McConnell, H. M., Nature (London) 296, 164 (1982). 22. Kunitake, T., Okahata, Y., Shimomura, M., Yasunami, S. I., and Takarabe, K., J. Amer. Chem. Soc. 103, 5401 (1981). 23. Nakashima, N., Asakuma, S., and Kunitake, T., J. Amer. Chem. Soc. 107, 509 (1985). 24. Yamada, K., Ihara, H., Ide, T., Fukumoto, T., and Hirayama, C., Chem. Lett. 1713 (1984).
Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
1. INTRODUCTION
A gel behaves in many ways like a solid although it has in fact two distinct components; the major component volumewise is a liquid while the second component is a solid which forms a network running throughout the sample. The related sol phase behaves as a liquid. The sol is converted to a gel, via a critical transition which is characterized by the divergence of certain physical properties such as the viscosity. The thermo-reversibility of the transition depends on the nature of the cohesive interactions involved in the establishment of the solid network within the gel. An example of the so-called chemical gels, which are irreversible, is the polymerization of acrylamide, in the presence of a bisacrylamide reticulating agent, which leads to the formation of a threedimensional covalently linked solid network (1). In other cases the gel is thermo-reversible. This is the category of the physical gels for which weak interactions (hydrogen bonds, van der Waals interactions) are involved in establishing the solid network. Examples are col1 Present address: Insfitut Laue-Langevin, 156 X, 38042 Grenoble C&lex, France. Author to whom correspondence should be addressed.
0021-9797/88 $3.00 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
lagen (2) and some small amphilic molecules (3). Gel systems are becoming increasingly important in a wide range of fields: medicine, pharmacology, cosmetics, photography, and the food and petroleum industries. The solgel process is used to obtain special coatings which involve the dried gel (or xerogel). This process can be used to obtain, at low temperatures, glasses, ceramics, coatings, and paints from inorganic sols such as SIO2, A1203, or W2Os (4). High-modulus fibers can be obtained from polymer solutions (5). Such materials have useful and interesting optical, electronic, or ionic properties (6) and constitute a new solid-state form. Both from the applied and fundamental points of view it is important to characterize the structural aspects of these new materials. We have recently described observations of the gel network formed by a two-component system which because of its simplicity is an extremely useful model system. It consists of cyclohexane and an amphilic steroid molecule (molecular weight 390) derived from cholesterol, D-homosteroidal nitroxide (hereafter referred to as the steroid). The present article describes a structural study of xerogels oh-
542 Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
STEROID GELS tained by evaporation ofcyclohexane from the native gels. Work such as this depends on having a sound knowledge of the initial gel structure. In the present case this has already been studied both by electron microscopy (7) and by small-angle neutron scattering (8) and good agreement was found between these independent methods. It is on this foundation that we present our electron microscope observations of xerogel structures and give tentative interpretations of the important structural differences between the xerogel and the gel states. 2. M A T E R I A L S
AND
METHODS
The synthesis of the steroid (9) and the preparation of the native gel samples (7) have been described previously. The preparation of replicas of dried gel samples for electron microscopy was carried out by sandwiching several drops of warm solution between two freshly cleaved mica sheets. The solution was allowed to gel at, or somewhat below, room temperature. The sandwich was cleaved in air by separating the two mica sheets and the gel adhering to the mica sheets was allowed to dry; see Figs. 1 and 3 for specific conditions. One of the mica sheets was transferred to a vacuum evaporation system and its gel-covered surface was first rotary shadowed with a tantalum-tungsten alloy evaporated from an electron beam-heated rod and then carbon replicated. The replicas were separated from the substrate either by floating the carbon film onto water or by immersion in a water-ethanol mixture which dissolves the steroid layer beneath the replica. The replica fragments were picked up on 3-mm-diameter 400-mesh copper electron microscope grids. The replicas were examined at 80 keV in a JEOL 100 C or 100 CX electron microscope. 3. R E S U L T S
3.1. NATIVEGEL We summarize briefly the conclusions drawn from a previous electron microscope study of the steroid gel (7).
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This gel, which has also been characterized by rheological experiments (10, 11), contains a solid infinitely connected filamentary network with an average filament diameter of 9.1 nm. From stereoscopic views an average mesh size of about 300 nm is found in the case of gels formed at room temperature from a solution of initial concentration about 3 X 10-2 M. Individual filaments are at least 5/~m long. A statistical analysis shows the most frequently occurring filament diameter to be 9.1 nm and also reveals the presence of finer filaments some 4.6 nm in diameter. We deduced that at least two of these finer filaments, protofilaments, are required to form the 9.1-nm-diameter filaments which also show 5-nm pitch striations. The connections between the filaments were found to consist of fusion zones produced by exchange of protofilaments, filaments bodily entwining one about the other, or of juxtaposed filaments. 3.2. DRIED GEL STRUCTURES For the sake of clarity the images obtained from dried gels are divided into two main classes, those showing filaments of quasi-infinite length to diameter ratio and those showing filaments of finite length to diameter ratio. The first class appears similar to the network structures shown by the native gel and has very few loose filament ends. The second type, by contrast, shows few interconnected filaments and numerous loose ends.
3.2.1. Quasi-infinite Filaments Images of this class, in Fig. 1, typically show a continuous network with filaments many microns in length similar in overall appearance to the native gel. The filament diameters have an average value of 26 nm which is of the order of three times those found in the native gel. Details of the many interfilament contact zones are revealed clearly in less densely populated regions such as those shown in Figs. 2a and 2b. A notable feature is that the contacts are similar to those in the native gels, princiJournal of Colloid andlnterfaceScience, Vol. 125, No. 2, October 1988
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FIG. 1. Replica of a dried gel. The quasi-infinitely connected filament network is clearly visible; the filament diameters are about 26 nm. The images of Figs. 1, 2, 5, and 6 were obtained from specimens prepared as follows. Three drops of a steroid solution (concentration ~3 X 10-2 M) were sandwiched between two mica sheets and allowedto gel at room temperature. Two hours later the sheets were separated and the xerogelwas replicated as described in Section 2.
pally parallel juxtaposed filaments and entwined filaments. A very pronounced helical structure is apparent and has a mean pitch of about 58 nm.
3.2.2. Finite Filaments Images in this class can be subdivided into those showing filaments for which the lengths, diameters, and pitches are approximately constant (constant geometry case) and those for which these parameters vary considerably (variable geometry case). Images corresponding to the constant geometry case are shown in Fig. 3 for different lengths and concentrations of filaments. The filaments have a mean diameter of about 25 n m and have a less pronounced helical structure than was the case for the quasi-infinite filaments. The filaments either develop from a c o m m o n center or are isolated; both cases Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
are dearly visible in Fig. 4. These micrographs represent specimens obtained from dilute solutions by solvent evaporation before the critical gelation threshold was reached and they show structures obtained from the sol phase. In the variable geometry case a wide range of lengths, diameters, and pitches can be observed in the same image (Figs. 5 and 6). In these images the filament diameters range up, wards from about 20 n m to over 100 nm in cases where two or more filaments can be seen to twist together. 4. DISCUSSION In the experiments described here the diameters of the filamentary structures in the quasi-infinite connected networks, Figs. 1 and 2, and the finite fibers, Figs. 3 to 6, are of the order of 25 n m which is about three times the
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FIG. 2. Dried gel replica showing quasi-infinite filaments and details of the filament interactions. (a) Overall view of entwined filaments (A) and of parallel juxtaposed filament regions (B). (b) Detail Of a twisted filament zone showing the 26-nm-diameter and 58-nm-pitch filaments.
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FIG, 3. Replicas of dried gelling solutions showing finite-length filaments with approximately fixed geometrical parameters. The filament helicities are less pronounced than in the case of the dried gels. Filament diameters are about 26 n m and helical pitch is 58 nm. (a) Short disconnected filaments; (b) longer filaments which are still not interconnected. The images of Figs. 3 and 4 were obtained from a dilute steroid solution sandwiched between two mica sheets for 15 min. W h e n the mica sheets were separated, the inside layer was still liquid. After solvent evaporation the surface was replicated as described.
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FIG. 4. Replica of a dried gelling solution showing finite-length filaments which are either isolated or which grow from a common germination center. Filament diameters and pitches are respectivelyabout 26 and 58 rim. value found in the native gels (7). The filaments in Figs. 1 and 2 are distinctly helical with a pitch of about 58 n m whereas those in Fig. 4 are m u c h more rod-like. In the case of Fig. 6 the majority of filaments are interconnected but some are broken and give m u c h thicker structures made up o f overtwisted filaments. The breaks m a y have occurred during specimen preparation or m a y have resulted from the strong tendency of the filaments to twist during the drying out of the gel. The overtwisting can occur equally well for a broken filament twisting back on itself as for an unbroken filament with reticulation points which have become closer as the mesh size decreases. Figure 5 shows the same type of effect in filament fragments. In these cases (variable geometry ) there is a wide distribution in the observed helical pitches and filament diameters. It would appear that these broken and overtwisted structures can be considered as structural defects of the ideal xerogel network.
In order to understand the relationship between the native gel and the xerogel, two important questions must be answered. 1. What happens to the steroid remaining in solution as the cyclohexane evaporates? In attempting to answer this question we should note that the speed at which the cyclohexane/air interface moves through the gel sample will determine whether the steroid molecules remaining in solution will have time to associate as filaments or crystals or whether they will be precipitated from solution in some other form, The governing parameters are the diffusion rate of the steroid molecule in solution (v = 2 X 10 4 n m sec -t for D ~ 10 -6 cm 2 sec -1) compared to the velocity of the air/liquid interface, about 300 n m sec-1 (12 ), and the measured kinetic growth rate, 1 min -1 (13), for usual gelling solutions (concentration ~ 1 0 -2 M, T = 20°C). These parameters indicate that the dried gel should be mainly made up of filaments which grow following Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988
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FiG. 5. Finite-length filaments of variable geometry showing folded and overtwisted filaments at different magnifications. A wide distribution of diameters and pitches is observed (for example, in (b) the minimum diameter is 26 n m and the maximum diameter is over 100 nm). In (c) the two filament pitches are about 300 and 150 nm.
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1 0 0 nm
I~G. 5.--Continued.
the same process as in the initial native gel. The xerogel network structures depicted by the quasi-infinite filament distributions of Figs. 1 and 2 support this conclusion since we never observe any structural forms other than the steroid filaments. 2. How is the gel network modified by the passage of the solvent meniscus? An obvious consequence of solvent evaporation is that the overall volume of the gel decreases. The gel network will react to this constraint by shrinking in a manner depending on the filament elasticity (see, for example, the introductory paper by Tanaka (1)). It is possible to imagine several mechanisms for this process which is governed by the osmotic pressure of the gel. Two of these are shown in Fig. 7, where just three interfilament contact points are considered. If the filaments conserve their initial length they must fold (Fig. 7b) as the contact points become closer. This folding process can assume various forms and may lead to collapse
of filaments onto one another or onto themselves, or a given filament may twist around itself. Another possibility shown in Fig. 7c is that as the contact points approach each other the filaments remain tightly stretched between them as if they have a built-in elasticity. Such elastic filaments must increase in diameter as their length decreases. This type of effect could explain a consistent feature of all our images of dried gel systems, namely that observed filament diameters are systematically greater than those found in the native gel (about three times greater, in fact), whereas if the mechanism of Fig. 7b were operating we would expect to observe in addition to many collapsed or entwined filaments some filaments having the diameter of those in the native gel. This is never the case (see Figs. 1 to 4). It would be tempting to argue at this stage that the filament diameters increase simply because of steroid deposition from solution. It is difficult to imagine how this could take place with complete uniformity for all filaments. Moreover in order to at least double the diameters of all Journal of ColloM and Interface Science, Vol. 125, No. 2, October 1988
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FIG. 6. Infinite-filament network of a dried gel sample. In addition to the connected filament network similar to Fig. I, broken, folded, and overtwisted filaments give finite fragments o f variable geometry as in Fig. 5.
filaments three times the quantity of steroid involved in the filament formation of the native gel is required. At the working concentrations this amount of material is simply not available in the remaining solution. We are consequently obliged to accept the diameter increase as an intrinsic property of the filaments themselves. This interpretation is supported by our observations that the same filament diameters are found both for the finite length filaments in the sol-phase domains (Figs. 3 and 4) and for the connected filaments of the dried gel networks (Figs. 1 and 2). 5. CONCLUSIONS
A comparison of the electron micrographs obtained from the gel (7) and xerogel of the steroid cyclohexane system leads to two significant conclusions concerning the xerogel structure. In the first place, since only the filamentary form of the steroid is observed in Journal of Colloid and Interface Science, Vol. 125,No. 2, October1988
the xerogel images, as the steroid concentration increases due to solvent evaporation the excess steroid must come out of solution as filaments which are incorporated into the original gel network. Then as the solvent meniscus sweeps through the gel network the filaments change their conformation compared to the native gel. They thicken considerably and become strongly helical. In this context it can be noted that there are numerous published examples of enantiomorphic molecules for which either optical isomer gives chiral superstructures. Examples are lithium 12-hydroxy-octadecanoate (14, 15), PBLG (16), and substituted valines (17). In addition, supertwisting is frequently encountered in systems made up of long filaments, for example, deoxycholic acid (18), sodium deoxycholate (19), starch (20), and certain liposomes (21). Striking structural modifications have also been observed in chiral bilayers formed from double-chain ammonium glutamic acid derivatives (22-24).
STEROID GELS
÷
÷ c
FIG. 7. Idealized representation of two possible structural modifications (situations b and c) when a gel network with three contact points (situation a) shrinks by solvent evaporation. (b) The points get closer and the slack filaments are free to fold or to overtwist. (c) The filaments contract elastically and the resulting filament diameters are increased. The crosses in b and c indicate the initial contact point positions.
ACKNOWLEDGMENTS The authors are grateful to Drs. R. Ramasseul and F. Volino for helpful discussions and continued interest. We thank C. Closse for technical assistance at various stages of this work. REFERENCES 1. Tanaka, T., Scientific American January, 110 (1981). 2. Hausch, A., Actuality Chim. 40 (1978). 3. Hermans, P. H., in "Colloid Science," Vol. II, "Reversible Systems" (H. R. Kruyt, Ed.). Elsevier, Amsterdam, 1969. 4. Livage, J., J. SolidState Chem. 64, 322 (1986).
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5. Frost, H. H., Cohen, Y., and Thomas, E. L., in "Reversible Polymeric Gels and Related Systems" (P. S. Russo, Ed.), Chap. 12, ACS Symposium Series No. 350. Amer. Chem. Soc., Washington, D.C., 1987. 6. Mukherjee, S. P., J. Non-Cryst. Solids42, 477 (1980). 7. Wade, R. H., Terech, P., Hewat, E. A., Ramasseul, R., and Volino, F., J. Colloid Interface Sci. 114, 442 (1986). 8. Terech, P., Volino, F., and Ramasseul, R., J. Phys. (Orsay, Fr.) 46, 895 (1985). 9. Martin-Borret, O., Ramasseul, R., and Rassat, A., Bull. Soc. Chim. Fr. H401 (1979). 10. Callec, G., Gauthier-Manuel, B., Terech, P., and Ramasseul, R., C. R. Acad. Sci. Paris, SYr, H 293, 99 (1981). 11. Gauthier-Manuel, B., Allain, C., and Guyon, E., C. R. Acad. Sci. Paris, S~r. H296, 217 (1983). 12. Terech, P., Thesis, Grenoble, France, 1983. 13. Terech, P., J. Colloid Interface Sci. 107, 244 (1985 ). 14. Tachibana, T., and Kambara, H., Bull. Chem. Soc. Jpn. 42, 3422 (1969). 15. Tachibana, T., Kayama, K., and Takeno, H., Bull. Chem. Soc. Jpn. 45, 415 (1972). 16. Tachibana, T., and Kambara, H., Kolloidn. ZI. 219, 40 (1967). 17. Hidaka, H., Murata, M., and Onai, T., J. Chem. Soc., Chem. Commun. 562 (1984). 18. Ramanathan, N., Currie, A. L., and Ross Colvin, J., Nature (London) 4778 (1961). 19. McCrea, J. F., and Angerer, S., Biochim. Biophys. Acta 42, 355 (1960). 20. Heyn, A. N. J., TextileRes. J. 29, 366 (1959). 21. Lin, K. C., Wets, R. H., and McConnell, H. M., Nature (London) 296, 164 (1982). 22. Kunitake, T., Okahata, Y., Shimomura, M., Yasunami, S. I., and Takarabe, K., J. Amer. Chem. Soc. 103, 5401 (1981). 23. Nakashima, N., Asakuma, S., and Kunitake, T., J. Amer. Chem. Soc. 107, 509 (1985). 24. Yamada, K., Ihara, H., Ide, T., Fukumoto, T., and Hirayama, C., Chem. Lett. 1713 (1984).
Journal of Colloid and Interface Science, Vol. 125, No. 2, October 1988