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Letters A Cryogenic Transmission Electron Microscopy Study of Counterion Effects on Hexadecyltrimethylammonium Dichlorobenzoate Micelles L.J. Magid’ and J. C. Gee Department of Chemistry, University of Tennessee, Knoxuille, Tennessee 37996-1600
Y. Talmon Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa, Israel 32000 Received May 7,1990
We have used the controlled environment vitrification system to prepare vitrified TEM specimens of aqueous solutions of hexadecyltrimethylammonium 3,5-dichlorobenzoate (CTA-3,5-C1Bz) and hexadecyltrimethylammonium 2,6-dichlorobenzoate(CTA-2,6ClBz)at various concentrations. We report direct images of the cylindrical micelles of CTA-3,5CIBz at all concentrations studied. For the CTA-2,6CIBz solutions, we present a series of micrographs showing an evolution of spheres to cylinders as a function of concentration, as well as a micrograph for one solution that shows coexisting spheres and cylinders. Introduction Recent progress in cryogenic transmission electron microscopy has made possible the direct imaging of micelles. Introduced by Dubochet and co-workers for preparation of biological specimens,’-4 this technique has recently been modified to elucidate microstructure in colloidal systems such as vesicular and micellar dispersions. Cryo-TEM images of cylindrical micelles first appeared in the literature in 1986: and images of spherical micelles first appeared in 1987.6 Additional images of cylinders and spheres have appeared more recently?-’O but the application of this technique to micellar systems is still in the early stages. The procedure has distinct advantages over the use of conventionalstaining techniques,”-16 because it introduces (1) Dubochet. J.; Lepsult, J.; Freeman, R.; Berriman, J. A,; Homo, J.C. J. Mierose. 1982,128,219-237. (2) Lepault, J.; Booy, F. P.; Dubwhet, J. J. Mierosc. 1983, 129, 89-
102. (3) Adrian, M.: Dubochet, J.; Lepault, J.; McDowall. A. W. Nature 1984, 308.34-36. (4) Dubochet. J.: Adrian, M.; Chang, J.; Homo, J.; Lepault. J.; McDowall. A. W.; Schultz. P. Q. Re”. Biophys. 1988.21, 129-228. ( 5 ) Bellare, J. Proe. 11th Int. Congress on EM, Kyoto 1986.1, 367. (6) Burns, J. L.; Talmon, Y. Ploe. of the 45th Annuol Meeting of the EM Society of America 1987, 500. ( 7 )Ness,J. N.; Moth. D. K. J . Colloid Znterfoee Sei. 1988, 123,546-
547.
(8)Vinson, P. K.; Talmon, Y. J. Colloid Interface Sei. 1989,133,28& “00 L.33.
(9) Miller. D. D.; Bellare, J. R.; Kaneko, T.; Evans, D. F. Langmuir 1988,4, 1363-1367. (10) Vinson, P. K.; Talmon. Y.; Walter, A. Biophys. J. 1989,565,669681. (11) Imae, T.: Kamiya, R.; Ikeda,S. J. Colloid Interface Sei. 1984,99, 3W301. (12) Sakaiguchi, Y.; Shikata, T.; Urakami, H.: Tamura, A.; Hirata, H. corrotd Pdym. Sci. 1987,265.75~753. (13) Imae, T.;Kamiya. R.; Ikeda, S. J. Colloid Interface Sei. 1985,108. 215-225. (14) Hirsta, H.; Ssto, M.; Sakaiguchi, Y.; Katsube, Y. Colloid Polym. Sei. 1988,266,862-864. (15) Shikata, T.: Sakaiguchi, Y.: Uragami, H.; Tamura, A,; Hirata, H. J. Colloid Znterfme Sei. 1987, 119, 291-293.
0743-7463/90/2406-1609$02.50/0
Figure 1. Cylindrical micelles of aqueous 2.1 m M CTA3,5CIRz in t h i n layer of vitreous ice. Dark spots are frost contamination deposited during sample transfer into microscope (bar
=
500 nm).
no foreign staining substances and avoids drying that actually perturbs the structure of the systems under investigation. Instead, solutions are cooled rapidly enough t o vitrify them and leave t h e micelles intact. T h e micrographs therefore show micelles as they were a t the instant of vitrification; there are no staining or drying artifacts.’? We have used this technique to study the effects of two different dichlorobenzoate counterions on the shapes of micelles formed by hexadecyltrimethylammonium(cetyl TA, CTA) surfactants. We have obtained images of both spherical and cylindrical micelles, and we have followed the evolution of spheres t o cylinders as a function of concentration in one binary surfactant/water system. Especially noteworthy is the observation of coexisting spheres and cylinders in one of the solutions. (16) Hrata, H.; Sskaiguchi, Y. J. ColloidZnterfoee Sei. 1989,127,589591. (17) Kilpatriek, P. K.;Miller, W. G.; Talmon. Y. J. Colloid Interface Sei. 1985, 107, 146-158.
0 1990 American Chemical Society
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Figure 2. (a) Arrows point nut small, spherical micelles of aqueous 10 mM CTA-Z,6CIRz. Sample preparation has concentrated micelles near edge of hole in carhon film. (b) Coexisting spherical and cylindrical micelles are visible in aqueous 40 mM CTA-Z,6CIBz. (c) A t 80 mM, CTA-2.6ClBz forms long, cylindrical micelles indistinguishablefrom those of CTA-3,5ClBz. The shear applied during sample preparation has evidently induced a sphere to cylinder transition in the more concentrated solutions of CTA-Z,6CIBz(bar = 500 nm). Solutions of cetyltrimethylammonium 3,5-dichlorohenzoate (CTA-3,5CIBz) in the concentration range of 0.10.5 wt % (2.1-10.5 mM) are clear and viscoelastic. Scatteringexperiments have indicated that these solutions consist of long, cylindrical micelles. In contrast, cetyltrimethylammonium 2,6-dichlorohenzoate (CTA-2,6CIBz) gives solutions that are of low viscosity, and scattering measurements have indicated that, helow concentrations of about 5 wt 9; (100 mM), these solutions consist of spherical rather than long cylindrical micelles.'S
The variation of micelle shape with a change in counterion is well d o c ~ m e n t e d . 8 JIn ~ this ~ ~ case, conductimetry measurements show a higher percentage of counterion binding for the CTA-3,5CIBz than for t h e CTA2,6CIBz. Furthermore, NMR data indicate t h a t t h e
(18) Msgid, L. J. The Effect of Aromatic Counterions on Micelles and Bilayers. In Orderingond Owniring in Ionic Solutions;World Scientific Publishing: Singapore, 1988.
W . J . Phys. Chem. 1986,90.1853-1859. (24) Gamboa, C.; Rim, H.: Sepulveda,L.J. Phys. Chem. 1989.93.55W
(19) Anacker, E. W.: Ghose, H. M. J. Phys. Chem. 1963,657, 17131116. (20)Anaeker, E. W.; Ghose, H. M. J. Am. Chem. Soe. 1968,90.31613166.
( 2 1 ) Anacker, E. W.; Underwood, A. L. J. Phys. Chem. 1981,85.24632466. (22) Underwood. A. L.; Anscker, E. W. J. Colloid Interface Sei. 1984, 7M 17R-lRT. . .., . ._ (23) Brady. J. E.;Evans, D.F.;Wan, G. G.: Grieser, F.; Ninham, B. 5543.
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Figure 3. (a) Micrograph shows empty vitreous ice. Dark spots are frost deposited during sample transfer into microscope. (b) Note the grainy appearance uf the spherical micelles of 2 C ( w j w ) CTAB (55 mM). The spheres are clearly visible in this concentrated solution (bar = 500 nm). orientation of binding is different for t h e two counterions; the 3.5CIRz intercalates f u r t h e r into the head. than doesthe 2 , 6 ~ 1which ~ ~ , decreasesheadgroup repulsions and promotes micelle groWth.*5
group
E x p e r i m e n t a l Section Synthesis of Cetyltrimethylammonium Dichlorobenzoates. The two surfactants were synthesized by using the same procedure. The sodium dichlorobenzoate salts were prepared by titrating the corresponding acids with 3 N NaOH, and the resulting aqueous salt solutions were trented with an ex. cess of silver nitrate dissolved in a little water. The silver salts precipitated and were collected and washed w i t h water and dried. Methanolic solutions of cetyltrimethylammonium bromide (CTAB) were then stirred with the desired silver dichlo-
robenzoate salts for 3 h. The AgBr was removed by filtration, and the methanolicsolutions were stirred overnight with decol. orizing charcoal, filtered, and evaporated to dryness. The solid CTA dichlorobenzoate surfactant8 were recrystallized from acetonitrile, collected, and dried under vacuum. Cryo-TEM Imaging. We have utilized the previously described controlled environment vitrification system (CEVS).m The device provides us with a controlled environment of constant temperature (iO.1 "C) and humidity, in which we can prepare a thin film of surfactant solution on a standard TEM grid covered hv B holev carbon film. We can then vitrifv the sam~, ~~~~, ple by rapidly plunging the grid into liquid ethane at it.9 freezing p0int.n Unlike crystalline ice, vitreous ice does not obscure the structural details of micelles emhedded in it. nor are the mi~~
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1261 Bellare. J. R.:,Davis. H.T.: Scriven. L. ~, E.:Talmon. Y.J. Efeetmn ~~, Mierose. Tech. 1988,10,81-111. (27) Talmon, Y. Colloids Surf. 1986,19,237-248. ~
(25) Weber, R.Personal communication.
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micrographs, minimization of this flow during sample preparation is technically very difficult. AU concentrations of CTA-3,5ClBz gave images like those in Figure 1; a network of entangled cylinders was clearly visible. In all the solutions, the blotting procedure concentrated the micelles at the hole edges; a quantitative analysis of micelle length as a function of concentration is therefore not feasible with this system. But the micelle diameter is easily measured for each concentration, and it remains approximately 5 nm, as expected, considering the tail length of the CTA surfactant. This value is in good agreement with values obtained from other
technique^.^^
Figure 4. (a) Shear applied during sample preparation has evidently induced a complete sphere to cylinder transition in aqueous 100 mM CTA-2.6CIBz. (b) More cylinders in aqueous 100 mM CTA-2,6ClBz. Arrows point out some micelle end points that are clearly visible (bar = 500 nm). celles disrupted by crystallization of surrounding water, and no precipitates form during the thermal fixation. Preparation of the thin film involves placing a drop of solution on a standard TEM grid (covered by a holey carbon film) in the CEVS, blotting away most of the liquid with filter paper, and then plunging the grid into liquid ethane. Once a sample is prepared, we transfer the grid under liquid nitrogen to a Gatan work station, where we mount the grid onto a Gatan 626 cold stage specimen holder and insert it into an Hitachi H-800 transmission electron microscope. After about 30 min of equilibration, the specimen rod maintains the grid at -171 'C. We examine the grids at 100-kV accelerating voltage and 20000x magnification. By employingthe proper amount of defocus (about -4 pm). we obtain enough phase contrast to resolve the micelles. We record micrographs on Kodak SO-163 EM film and develop in full strength Kodak D-19 developer for 12 min.
Results a n d Discussion Cetyltrimethylammonium 3,5-Dichlorobenzoate. We examined 2.1, 4.2, 6.3, and 10.5 mM solutions; all specimens were quenched from 25 "C. The 10.5 mM solution was extremely viscous and therefore difficult to thin, but we have obtained some images of structure, although the ice was largely hexagonal rather than vitreous. Figure 1shows long, entangled, cylindrical micelles in the 2.1 mM solution. Long micelles like the ones pictured are believed to be responsible for the viscoelasticity of these solutions.28 Note how the fluid flow during the blotting procedure has pushed the structures toward the edge of the hole in the carbon film. As is evident from these (28, Hoffmann.H.; Platz. C.; Rehage, H.: Schorr. W.: Ulbncht. W. BPI. Rumen-Crr. Ph>s. Chem. 1981.85.255.877.
Cetyltrimethylammonium 2,6-Dichlorobenzoate. For the CTA-2,6CIBz, we examined several solutions in the concentration range 10-100 mM. These were considerably more concentrated than the solutions we examined for the CTA-3,5ClBz. We could work with these higher concentrations, because the solutions of the CTA2.6CIBz are not viscoelastic and therefore were more manageable with out sample preparation techniques then the extremely viscous CTA-3,5CIBz solutions. To avoid proximity to a Krafft boundary, we also quenched from 30 "C. Figure 2a shows a micrograph of the 10 mM CTA2,6CIBz solution. Small images of spheres are just visible and are similar to images of spherical micelles reported by Bellare et a1.9 The structure in this micrograph is easily discernible from the featureless vitreous ice in Figure 3a. Diameters of the spheres are measured to be about 5 nm, providing more evidence that these images are not artifads or grains in the paper. As a comparison, Figure 3b shows spherical micelles in a 2% (w/w) CTAB solution (55 mM), which are clearly visible in this more concentrated solution. Figure 2b shows the structure of a 40 mM solution. One can see spheres identical with those found in the 10 mM solution, but upon close inspection, one also sees structures that resemble short cylinders. In some cases, these appear to be spheres that are closely packed, but some of these structures are undoubtedly short, cylindrical micelles. To our knowledge, this is the first documented case of spherical and cylindrical micelles coexisting in an aqueous isotropic micellar solution containing a single surfactant, although similar images have been reported for surfactant mixtures.'O At 80 mM, we discovered a surprising phenomenon (see Figure 24. We found only cylindrical micelles similar t o those observed in the solutions of the CTA-3,5CIBz. This observation is not in agreement with static SANS data on unsheared solutions,'8 yet we have repeatedly found cylinders and no spheres a t 80 mM. On inspection of the micrographs, we see, as in the case of the CTA-3,5CIBz, that these cylinders are pressed to the hole edges. We conclude that the shear applied during sample preparation has induced a sphere to cylinder transition in this solution. In fact, additional work has even revealed cylindrical structures at concentrations as low as 10 mM, when higher shear (Le., vigorous blotting) has been applied during sample preparation. Such shear-induced transitions have previously been reported by Kalus et al.," who performed SANS measurements on shear-aligned samples of hexadecyloctyldimethylammonium bromide. In some cases, the effect may also be the result of an extremely high local (29) Magid, L. J. Unpublished results. (30)Kalus, J.; Hoffmann, H.;Chen. S.-H.; Lindner, P. J. Pkw. Chem. 1989,93,42674276.
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Letters concentration of surfactant near the hole edges, as reported by Burns et al. during investigations of the nonionic surfactant HCO-60.31 In light of our findings for the 100 mM CTA-2,6ClBz, this transition cannot be solely t h e result of local concentration effects. Figure 4 shows extremely long cylinders a t 100 mM. Furthermore, these cylinders are not just visible a t hole edges; they are present throughout the vitreous ice and immediately give the distinct impression of fingerprints. The transition has occurred in the entire specimen, not just at concentrated areas a t hole edges. These particular micrographs are a fine demonstration of the potential of this technique. The ice has thinned beautifully, and the structure is clear throughout the hole.
Conclusions Our cryo-TEM micrographs have supported t h e cylindrical structure of CTA-3,5ClBz micelles and the spherical structure of CTA-2,6ClBz micelles a t low (31)Burns, J. L.;Cohen, Y.; Talmon, Y. J . Phys. Chem. In Press.
concentration, as indicated by earlier scattering data. We have seen no evidence to support the idea, proposed by Anet and others, that the cylindrical micelles are merely tightly packed sphere^;^^^^^ our micrographs clearly show single, cylindrical structures. We have also discovered that the systems of CTA-2,6CLBzspherical micelles are probably sensitive to shear and/or local concentration effects and will transform into systems of cylindrical micelles, especially at higher concentrations.
Acknowledgment. Magid and Gee acknowledge the Sun Co. and the National Science Foundation (NSF Grant Number CHE86-11586) for financial support for this work. Y. Talmon’s research in cryo-TEM has been supported by a grant from the US.-Israel Binational Science Foundation (BSF), Jerusalem. Registry No. CTA-3,5ClBz, 117932-67-9; CTA-2,6ClBz, 128779-78-2.
(32)Anet, F. A. J . Am. Chem. SOC.1986,108,7102-7103. (33)Manohar, C.;Rao, U. R. K.; Valaulikar, B. S.;Iyer, R. M. J.Chem. SOC.,Chem. Commun. 1986,379-381.
