Langmuir 1992,8, 2200-2205
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Cryogenic Electron Microscopy of Rodlike or Wormlike Micelles in Aqueous Solutions of Nonionic Surfactant Hexaethylene Glycol Monohexadecyl Ether 2.Lin, L. E. Scriven, and H. T. Davis* Department of Chemical Engineering & Materials Science, University of Minnesota, 421 Washington Avenue S.E.,Minneapolis, Minnesota 55455 Received February 7, 1992. In Final Form: April 13, 1992 Cryogenic transmission electron micrographs of the hexaethylene glycol monohexadecyl ether (CI&)/
D20 system, with and without the presence of an electrolyte, NaSCN or NaC1, are present as a function
of the temperature. The micelles are about 60 A in diameter and the lengths of micelles vary from several hundred angstroms to more than a micrometer. The micelles are often curved, bent, and looped and sometimes even form rings and polygonal structures. The magnitude of the micelle diameter estimated from cryo-TEM agrees with small angle neutron scattering (SANS) results but neither the magnitudes of the micellar lengths nor their trends with temperature appear to agree with the SANS results.
Introduction The geometry of micelles in aqueous solution of poly(oxythylene) nonionic surfactants has been explored by Cummins et al.1-3 using small-angle neutron scattering (SANS). In particular, they have adduced evidence that rodlike micelles occur in solutions of hexaethylene glycol monohexadecyl ether ( C I A )and deuterated water (DzO). Assuming that the micelles are cylindrical rigid rods, they deduce from their SANS data that the rods have a diameter of about 60 A and a length ranging from 1600 to 4000 A, depending on temperature and concentration of added salt (NaC1 or NaSCN). An attractive aspect of SANS is that it represents a precise quantitative tool for determining the geometric parameters of supramolecularmicrostructures such as micelles, liquid crystals, and ultrafine dispersed solids. A disadvantage, however, is that a scattering technique determines microstructure only indirectly, Le., one must assume a microstructural model and test its validity by comparison of predicted and measured scattering pattern. It is, therefore, desirable to visualize microstructure directly in order to determine its qualitative nature and to use this information as a guide to quantitative analysis with scattering techniques. This is particularly true when scattering predictions are available for only one of two or more microstructures that should be considered, as in the case of elongated micelles that may be more wormlike than rodlike. Transmission electron microscopy (TEM) is suitable for visualization of microstructure of the size scale of micelles. However, samplesmust be observed under vacuum in TEM and so some method of fixation must be used to prepare samples for TEM studies. The method of choice
Table I. Lengths of Micelles Deduced by Cumminr et al. from Small Angle Neutron Scattering (SANS). composition (wt % in D2O)
1% Cl& 1% clsEs/o.6 M NaSCN
3% Cl& 3% clsEs/0.5 M NaCl
temp (OC)
apparent micelle length (A)
30 34 38 30 34 40 48 28 34 38 28 34
33w 2950 1600 1800 3050 3800 2400 3700 2850 2100 3200 1800
0 They conclude that in all the solutions the rods are circular cylinders with diameters of about 60 A. Interpolated from the data.
*
presently is c ~ ~ o - T E M with ; ~ this method one prepares thin fiis of a liquid sample in a controlled environment of constant temperature (fO.l "C) and humidity, in which one freezes the sample so fast the water is vitrified so that the structural details of micelles embedded in it will not be obscured,and observes the microstructure in an electron microscope equipped with a cold stage that keeps the sample near the temperature of liquid nitrogen. By this method, one can accurately control the temperature and humidity of the system prior to rapid freezing. There are no staining or drying a r t i f a ~ t s . ~ The cryo-TEM method has been used recently to study a variety of micelles, including rodlike or wormlike micelles of the cationic surfactant cetyltrimethylammonium chloride.6 Our purpose here is to report the results of applying the technique to the Cl& systems studied by Cummins et al. The results shed light on the validity of their geometric model of the micelles.
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(1) Penfold, J.; Staples, E.; Cummins, P. G. Small angle neutron scattering investigation of rodlike micelles aligned by shear flow. Adu. Colloid Interface Sci. 199t,34, 451-476. ( 2 ) Cummins,P. G.; Staples,E.; Penfold, J.; Heenan, R. K. Geometry of micellesof the poly(oxyethy1ene)nonionic surfactantsCl& and C& in the presence of electrolyte, Langmuir 1989,5,11961199. (3) Cummine, P. G.;Hayter, J. B.; Penfold, J.; Staples, E. A smallangle neutron scattering investigation of shear-alignedhexaethlene glycolmonohexadecylether (C&) micelles as a function of temperature. Chem. Phys. Lett. 1987,138,436440.
(4) Vinson, P. K.; Bellare, J. R;Davis, H. T.; Miller, W. G.; Scriven, L. E. Direct imagiug of surfadant micelles, vesicles, discs and ripple phase structures by cryo-trammiasionelectron microscopy. J. Colloid
Interface Sei. 1991, 142, 74-91. (5) Kdpatrick, P. K.; Miller, W. G., Talmon Y. Staining and dryinginduced artifads in electron microscopy of surfactant dispersions. J. Colloid Interface Sci. 1985,107, 146-158. (6) Clawen, T.; V i o n , P. K.; Minter, J. R.; Davis, H. T.;Talmon, Y.; Miller, W. G. Viscoelastic micellar solutions: microscopy and rheology, J. Phys. Chem. 1992,96,474-4&4.
0743-7463/92/2408-2200$03.00/00 1992 American Chemical Society
Cryo-TEM of Micelles
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Figure 1. Cryo-TEM of 1% C 1 a smicelles in D20. (a) At 30 "C. Although it is hard to trace one individual micelle from end-to-end, the segment between two arrows is 4000 A. (b) At 34 "C. There are short rodlike micelles (A, 590 A), loops (L), bends (B), rings (R), and polygonal structures (P). (c) At 38 "C. There are loops (L), curves (C), bends (B), and rings (R). Notice there are many more curves than bends compared to the 34 "C case and that the micelle (M) a t the center of the micrograph is more than 1.45 pm long. (d) At 30 "C. Here micelles have been aligned around the thinner region of the sample and the lengths of micelles are longer than 8000 A. Bar = 0.1 pm.
Materials and Methods Hexaethylene glycol monohexadecylether (C1,&) was obtained from Nikko Chemical Co., Ltd. (Japan), and was used without further purification. Sodium thiocyanate (NaSCN, Analytical Reagent) was obtained from Mallinckrodt. Sodium chloride (NaC1,>99.5 % ) was obtained from Fisher Scientific. Deuterium oxide (D2O >99%) was obtained from CIL. c1&6was dissolved in D20. Solutionswere made by adding D2O to weighed quantities of surfactant and salts, then put in the water bath at 35 "C for several hours. Cryo-TEM samples were prepared in the Controlled Environment Vitrification System, or CEVS, which is described in detail el~ewhere.~ In the CEVS, temperature was controlled to within &O.l "C. Before the sample was introduced into the CEVS, the environmental chamber was brought to steady-state at the desired temperature and near saturation of D20 (95-99 % relative humidity). The humidificationof the chamber was accomplished with porous sponges extending upward from liquid reservoirs. The air inside the chamber was recirculated across the sponges to reduce temperature and composition gradients in the vapor. The high relative humidity within the chamber reduced evaporation of D20 from the sample and prevented the artifacts that result from drying. (7) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. Controlled environment vitrification system (CEVS): An improved sample preparation technique J. Electron Microsc. Tech. 1988, 10, 87-111.
Thin films of sample were formed by placing a 3-pL drop of the liquid on a holey polymer support film which had been coated with carbon and mounted on the surface of a standard 200-mesh TEM grid.8 The drop was blotted with filter paper (a process accompanied by deformation of the remaining liquid) so that thin (10-500 nm) fiis of the sample remained, and these spanned the 2-10 pm holes in the support film. About 20-30 s after the blotting, the assembly was then vitrified by rapidly plunging it through a synchronousshutter at the bottom of the environmental chamber and into liquid ethane situated immediately beneath. The 20-30-9 delay was used in view of the finding by previous investigators6 that shear alignment from blotting of wormlike micelles relaxed within this time period. Next the sample was transferred to the sample holder under liquid nitrogen and stored in liquid nitrogen for electron microscope examination. Vitrified specimens were examined at 100 kV in the conventional TEM mode of an analytical electron microscope (Model CM30, Philips) with a cryoholder (Model 626, Gatan, Inc., Warrendale, PA). The cryoholder temperature was maintained below -165 "C during imaging. Images were recorded on SO-163 film and developed in full-strength D-19 developer (Eastman Kodak Co., Rochester, NY) for 12 min. Images were recorded at 4 pm nominal underfocus of the microscope objective lens to (8) Vinson, P. K. The preparation and study of a holey polymer film. Proceedings of the 45th Annual Meeting of the Electron Microscopy Society of America;Bailey,G.W . ,Ed.; San FranciscoPress: San Francisco, CA, 1987; pp 644-645.
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Figure 2. Cryo-TEM of 1%Cl&d0.5 M NaSCN micelles in DzO. (a) At 30 "C. The micelles here are much shorter than those in pure C1&6. There is frost (F)due to the sample transfer. (b) At 34 "C. (c) At 40 "C. The length of micelles is polydispersed. F denotes frost. (d) 48 "C. Notice numerous bends and some frost artifacts (F). Bar = 0.1 pm. provide phase contrast, which is mainly responsible for gradients of optical density in the images. Images were recorded at 31000X (A5% ) and photographically enlarged.
Summary of Findings of Cummins et al. In their SANS studies of the geometry of elongated micelles of in D20 and in salt-DaO solutions, Cummins et al. subjected the solutions to a shear rate of 5000 in Couette flow. This was done to align the micelles in order to simplify the deduction of rod sizes from SANS data. The authors remarked that higher shear rates can apparently degrade micelles, i.e. reduce the mean size. Some of their results are summarized in Table I. To obtain the value of the micelle diameter and lengths presented in Table I, Cummins et al. assumed that the micelles are monodisperse, rigid rods of circular cross section. They found that the rod diameter is insensitive to temperature or 0.5 M concentrations of NaSCN (a "salting in" salt) or NaCl (a "salting out" salt). The rod lengths they deduced appeared to fall with rising temperature in the salt-free solutions and in the solution with added NaC1. The rod lengths deduced for the salt-free and added NaCl solutions can be reduced approximately to a common curve by plotting rod length versus T,- T,where Tcis the solution cloud point. With added NaSCN, the rod lengths go through a maximum at about 40 "C. The lengths in 0.5 M NaSCN solutions at 1 and 3 w t % C&6 also fall approximately on a common curve versus T,- T.
Results and Discussion
Micrographs of micelles in the solutions represented in Table I are presented in Figures 1-4. Because of the defocusing needed for image contrast, the diameters of the micelles cannot be determined precisely from the micrographs. Nevertheless, in agreement with the conclusion of Cummins et al., the images of the rods appear to have about the same width in all solutions. This indicates that the rods are indeed circular cylinders with a diameter that is independent of temperature, surfactant concentration, and presence of NaCl or NaSCN. From the micrographs we estimate a rod diameter of about 65 A, which, given the defocusing distortion, is in satisfactory agreement with the value of 60 A found by SANS. From the micrographs it is immediately apparent that after the treatment in cryomicroscopy the micelles are not linear and there are numerous strands that are longer than the apparent rod lengths deduced from SANS. The micelles are often curved, bent, or looped (see for example the features marked in Figure lb,c), as might be expected of association structures with aspect ratios of 70 to 100. In fact, instead of rodlike, the words threadlike and wormlike more accurately reflect the images of the structures observed in most of the TEM micrographs. Although it is hard to tell where a micelle begins and ends in many of the micrographs, examples abound of continuous strands that are much longer than the 2000-4000 A
Cryo- TEM of Micelles
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Figure 3. Cryo-TEM of 3% Cl& micelles in DzO. (a) At 28 "C. (b) At 34 "C. There are loops (L) and a lot of curved micelles. The micelles appear more wormlike or threadlike than rodlike. (c) At 38 "C. (d) 34 "C. Like Figure Id, here the micelles align around the thin region, the segment of the micelle marked M between two arrow8 is about 1.1 pm long. Bar = 0.1 pm.
lengths deduced from SANS using a rod model. For instance, in 1% c1& a t 30 "C and in 3 % cl& a t 34 "C (Figures I d and 3d) there are micelles longer than 7000 that have been aligned around a region where the film was thinned-and no doubt sheared-during the sample preparation. The ends of these aligned micelles lie off the edges of the micrograph; 7000 or 8000 A long micelles are easily found in 1% C16E6 a t 38 OC as well as in several of the other solutions. Neither the magnitudes of the micellar lengths nor their trends with temperature appear to agree with the SANS results. In addition to the rodlike or wormlike micelles, we observe a few ringlike micellar structures (see for example Figure lb,c). The perimeters of these rings are much smaller than the typical lengths of the rodlike or wormlike micelles. In the SANSexperiment, the micelles are shear-aligned at about 5000 s-l. This is perhaps the reason for the discrepancy between the finding of Cummins et al. that the micelles are linear and our cryo-TEM results which show that they are curved, bent, and looped. The micelles in the cryo-TEM samples are presumably in configurations more representative of equilibrium (albeit in a constrained thin film geometry) since we waited 20-30 s for on-grid equilibration after blotting. From previous studies of wormlike micelles of aqueous solutions of cetyltrimethylammonium chloride, we know that blotting induced alignment relaxes in a few seconds.6 Rheologically mea-
sured relaxation times in these solutions ranged from tenths of seconds to a few seconds. It is not so easy to explain why the micelles in the SANS experiment appear nearly monodisperse. The shear perhaps breaks the wormlike micelles into shorter pieces: Cummins et al. remark that shear degradation occurs at rates above 5000 s-l. Why, however, are the pieces nearly of the same length? The temperature dependence of the SANSdetermined lengths are also difficult to understand in the light of our results. In the temperature sequence for 1% C&6, there appear to be more loops and bends in a micellar strand as temperature increases. The samplepreparation for the cryo-TEM can introduce some artifacts. The blotting procedure could concentrate micelles at the hole edges; a quantitative analysis of micelle length as a function of concentration is thus rendered difficult. However, there is no evidence that a phase change is induced in the systems studied here. The blotting also introduces shear and extensional force to the liquid film. As demonstrated by Clausen et al.: this force tends to align the micelles along the shear field. By letting micelles relax, this artifact can be overcome. To do this, as mentioned above, we wait a few seconds, typically 2030 s after blotting and then, freeze the specimen.6 The observed flexibility of micelles is unlikely to be due to the sample preparation. It is also important to recognize that usually the thin
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Figure 4. Cryo-TEM of 3% ClsEs/0.5 M NaCl micelles in DzO:(a) at 28 "C; (b) at 34 "C. At both temperatures, micelles form loops (L), rings (R), and wormlike structures more than 7000 A in length. F denotes frost. Bar = 0.1 pm.
liquid film has biconcave shape; at the center of the hole, the film is very thin, whereas along the edge, near the
holey polymer support,the film is much thicker. The fact that electronmicrographs are two-dimensional projections
Cryo-TEM of Micelles of three-dimensional objects then makes the concentration of micelles look more concentrated at the hole edges. The apparent micelle concentration in individual micrograph depends on the area chosen and does not have a simple relationship to the micelle concentration. One might resolve the differences between the cryoTEM and SANS results by interpreting the length determined in the SANS experiment as a persistence length (the average length below which a segment of the micelle can be considered as straight and rigid).+ll Unfortunately, because in our sample preparation we confiie the wormlike micellesto thin films whose thickness (1000-2000A in the region of the sample where we usually take our micrographs) is comparable to or less than the length of the micelles, the cryo-TEM images cannot be used reliably to estimate the persistence length of the micelles. The confinement is unlikely to cause the bends, loops, and rings observed because their characteristic lengths are generally small compared to the film thickness. The full three-dimensional structure of the micelles must of course be partially suppressed by the confinement, although it is difficult to guess just how it is affected. (9) Cummins, P. G.; Staples, E.; Hayter, J. B.; Penfold, J. A smallangleneutronscatteringinvestigationof rod-likemicelles alignedby shear flow. J. Chem. SOC.,Faraday Trans. 1 1987,83,2773. (IO)Porte,G.;Marignan,J.;Bassereau;May,R. Shape transformations of the aggregates in dilute surfactant solutions: a small-angle neutron scattering study. J. Phys. (Paris) 1988, 49, 511. (11)Appell, J.; Mariguan, J. Structure of giant micelles: a small angle neutron scattering study. J.Phys. II 1991,1, 1447.
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In most of the micrographs there are dark spots that appear along the micellar chains. We judge that at least some of these are bends or joints in the micelle that are oriented orthogonally to the plane of the thin film of solution. We cannot eliminate the possibility, however, that some of these spots are small globular micelles. It is desirable at this point to look at other experiments, e.g., shear-dependent NMR, rheology, and cryo-TEM of just-sheared samples, to try to gain further insight into the origin of the discrepancies between the SANS and cryo-TEM results and to better understand the supramolecular structure and dynamics of wormlike micelles. Perhaps also theory, along the lines initiated by Cates,12 can help in understanding these systems. The C ~ ~ E ~ / D Z O system is an especiallyattractive one for studying wormlike micelles since it is a binary system with a relatively simple phase diagram. Added salts or cosurfactants are not needed to induce the formation of rodlike or wormlike micelles.
Acknowledgment. This work was financially supported through the NSF Center for InterfacialEngineering. We thank Dr. Gordon Tiddy’s group at Unilever for furnishing us with a partial phase diagram for the Cl86 and HzO solutions. Registry No. CI&, 5168-91-2; NaSCN, 540-72-7; NaCl, 764714-5. (12) Cabs, M. E. Dynamics of living polymers and flexible surfactant micelles: scaling laws for dilution. J. Phys. (Paris) 1988,49, 1593.
