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water interface
Electron Microscopic Studies of Monolayers at the Air/Water Interface 1,2 SAEED FERESHTEHKHOU,
RONALD
D. N E U M A N , 3'4 AND R A F A E L O V A L L E
Department of Forest Products, University of Minnesota, St. Paul Minnesota 55108
Received September 24, 1984; accepted May 22, 1985 The question of artifact formation associated with electron microscopic examination of the structure of monomolecular films was investigated. The effects of mode of film compression, surface energy of solid substrate, and surface roughness of solid substrate onto which fatty acid and phospholipid monolayers were transferred by the Langmuir-Blodgett technique from the air/water interface were systematically examined. Aberrations in monolayer structure can occur when low-energy solid surfaces such as Formvar and collodion are used for monolayer transfer below some specific surface pressure which is slightly different for each solid substrate. The observed structures bear similarity to the observations reported in other studies. However, when the monolayers are transferred onto mica substrates, the artifacts previously observed by electron microscopy appear to be eliminated and surface films existing in the liquid-expanded and liquid-condensed states exhibit a structure indicative of single-phase behavior, i.e., they are smooth, continuous, and homogeneous in agreement with current theories on monolayer structure. These results are discussed and compared with those of earlier microscopic studies. © 1986AcademicPress,Inc.
INTRODUCTION
a n d the q u e s t i o n o f artifacts occurring d u e to
T h e structure o f " i n s o l u b l e " o r sparingly soluble m o n o m o l e c u l a r films has b e e n in disp u t e for m a n y years. Electron m i c r o s c o p y has b e e n one a p p r o a c h for e x a m i n i n g the fine structure o f these films. T h e L a n g m u i r - B l o d gett t e c h n i q u e (1, 2), w h e r e b y m o n o l a y e r s are transferred f r o m the a i r / w a t e r interface to a solid substrate being w i t h d r a w n slowly t h r o u g h the surface film, usually has b e e n a n interm e d i a t e step in p r e p a r i n g the m o n o l a y e r s for o b s e r v a t i o n b y electron m i c r o s c o p y . T h e r e h a v e b e e n discrepancies, however, a m o n g the e x p e r i m e n t a l findings o f various investigators,
the transfer o f these films has never been fully clarified. Ries a n d K i m b a l l (3, 4) in their classical 1955 study transferred n - h e x a t r i a c o n t a n o i c acid m o n o l a y e r s , after c o m p r e s s i o n with a m o t o r - d r i v e n barrier, o n t o c o l l o d i o n - c o v e r e d electron m i c r o s c o p e screens o r grids. T h e i r m i c r o g r a p h s o f the l o n g - c h a i n (36-carbon ato m s ) fatty a c i d m o n o l a y e r at various stages o f c o m p r e s s i o n showed t h a t small " i s l a n d s " or d o m a i n s irregular in size a n d shape were present at 15 m N / m . A t 25 m N / m , the m o n o l a y e r b e c a m e the c o n t i n u o u s phase a n d the areas w h i c h at low pressure a p p e a r e d to be uncovered o r b a r e n o w were discontinuous. A c o n t i n u o u s h o m o g e n e o u s m o n o l a y e r was observed o n l y w h e n the surface pressure was raised to still higher pressures (39 m N / m ) j u s t b e l o w c a t a s t r o p h i c collapse. T h e results o b t a i n e d b y Ries a n d K i m b a l l have b e e n d i s c o n c e r t i n g o v e r the years to m a n y investigators because the prevailing view o f m o n o l a y e r structure is t h a t such films
Published as Scientific Journal Series Paper 14,142 of the University of Minnesota Agricultural Experiment Station. 2 Presented in part at the State-of-the-Art Review: Monomolecular Films Symposium at the Annual Meeting of the American Institute of Chemical Engineers, November 14-19, 1982. Los Angeles, Calif. 3Present address: Department of Chemical Engineering, Auburn University, Auburn, Alabama 36849. 4 To whom correspondence should be addressed. 385
0021-9797/86 $3.00 Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
Copyright © t986 by Academic Press, Inc. All fights of reproduction in any form re,fred.
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should have been continuous and homogeneous at the surface pressures investigated rather than exhibit two-phase behavior. Accordingly, Sheppard et al. (5) examined a variety of surfactant monolayers deposited on glass and Parlodion-covered microscope slides by the Langmuir-Blodgett method and obtained smooth surfaces when viewed under the electron microscope. Spink (6) similarly observed featureless surfaces when he transferred stearic acid monolayers onto mica, glass, and collodion substrates. Whereas Sheppard et al. used a constant weight apparatus, Spink employed piston oils to compress and transfer the monolayer onto solid substrates. Importantly, Spink's observations, as well as those of Sheppard et al., were in disagreement with the findings of Ries and Kimball. Neuman (7) reported in 1975 that monolayer deposition on collodion surfaces was surface pressure dependent. Although collodion (and Formvar) films are commonly employed in conventional microscopic studies, caution was recommended in the use of these low-energy solid surfaces in studies utilizing transferred monolayers due to the likelihood of only a partial monolayer being deposited on such substrates at low and intermediate surface pressures. Neuman (8), in order to examine the process of monolayer collapse by electron microscopy, transferred stearic acid monolayers at high surface pressure (31 mN/ m) onto mica and collodion substrates using an automatic film balance. At about the same time, Ries and co-workers in another series of studies observed island structures, once again, in cholesterol monolayers (9) and, additionally, a pronounced porous structure in lecithin monolayers (10). Although Ries believed that the monolayers transferred quantitatively onto the solid surfaces so employed (11), he did not claim a one-to-one correspondence between the film structures which were observed in the electron micrographs with those that exist on the water surface. Nevertheless, Ries maintained that the observed sequence of structural changes must reflect similar changes in the monolayer during Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
compression on the water surface of the film balance. 5 Ries and his co-workers in the aforementioned series of papers again did not employ more than one solid substrate, but rather they continued to transfer monolayers solely onto a single low-energy surface--Formvar--now being substituted for the collodion used in their earlier investigations (3, 4, 15-17). Since many of their studies, including more recent investigations (18-21), were interested in the structure of collapsed films at high surface pressures, the use of Forrnvar or collodion should not have created too serious problems at least with respect to the transfer process. 6 Berg (22) recently attempted to eliminate any artifact formation that might be caused by transfer ofa monolayer from the water surface onto a solid substrate by piercing the monolayer with a chilled plug and freezing the monolayer and subsolution beneath in situ. The frozen surface was shadowed, and results similar to those found by Ries and Kimball were obtained. No further reports have been published presumably due to experimental difficulties associated with this novel approach. Our analysis of previous research indicates that there are three major variables which may influence the structure of transferred monomolecular films: (a) mode of film compression, (b) surface energy of solid substrate, and (c) surface roughness of solid substrate. The objective of this investigation was to compare the different procedures used by previous investigators and attempt to resolve the differences in their experimental conclusions. In this study, we examined the effects of compressing and transferring stearic acid, pentadecanoic acid, and L-a-dipalmitoyl phosphatidylcholine 5 It should be mentioned that surface potential (12, 13) and radiotracer investigations (14) of monolayers at the air/water interface have found island structures at very low pressures corresponding to the surface vapor pressure of the monomolecular film under study. 6 However, as we will show in this paper, there still remain serious questions concerning the stability of transferred films on low-energy surfaces both before and during the replication procedures.
MONOLAYER
MICROSCOPIC
STRUCTURE
387
Wilhelmy plate (sandblasted glass) technique employing a Cahn RG electrobalance. Monolayer transfer was accomplished by lowering prepared solid surfaces (7.5 × 2.5 cm) into the clean subsolution prior to spreading and compressing the monolayer and then slowly withdrawing the solid substrate up through the monolayer. The prepared surfaces were (a) freshly cleaved Brazilian ruby mica MATERIALS AND METHODS slides (United Mineral and Chemical), (b) Although octadecanoic (stearic) acid was Formvar slides prepared by dipping clean glass emphasized because it is the classical surfac- or freshly cleaved mica slides into either 0.25 tant of study in monolayer investigations, or 0.5% Formvar [poly(vinyl-formal)] in dipentadecanoic acid and DPPC also were stud- chloroethylene, (c) collodion slides prepared ied. Stearic and pentadecanoic acid (Applied by casting 2% collodion (nitrocellulose) in Sciences, purity 99+%) were deposited from amyl acetate on distilled water followed by de2 m M n-hexane spreading solutions onto 0.01 position onto clean glass or mica slides, and NHC1 subsolutions (EM Laboratories, Supra- (d) film-covered electron microscope grids pur). Synthetic DPPC (purity 99+%), obtained prepared by floating Formvar or collodion from Avanti Polar Lipids, was dissolved in a films on distilled water, lowering grids (2009:1 (vol/vol) n-hexane/ethanol solution and or 300-mesh) of copper, gold, nickel, or nylon spread on distilled water subsolutions. The onto the floating film, and then sandwiching subsolutions were prepared from distilled wa- the grids between the film and a clean glass or ter previously purified by reverse osmosis. All mica support. The Formvar and collodion soexperiments were conducted in a dust-shielded lutions were supplied by Ernest F. Fullam. environment, and the water temperature was Direct carbon replicas of the deposited maintained at 21-23°C. The materials, ex- monolayers were prepared within 30 min after perimental apparatus, and procedures are dis- transfer in a Kinney KSE-2A-M vacuum cussed in greater detail elsewhere (23). evaporator. The samples were preshadowed The monolayers were compressed to a given with palladium (Pd) wire at an angle of 14 °, surface pressure either (a) by use of a piston and the source-to-specimen distance for the oil expanding against a flexible 3-rail FEP Pd (7 mg on W filament) evaporant was 100 monofilament thread or (b) by moving a rigid ram. The use of a heat shield having a small Teflon compression barrier forward manually. diameter (6 ram) aperture for passage of the Tricresyl phosphate, castor oil, and oleic acid evaporant permitted very thin evaporant films were used as piston oils to generate equilib- (ca. 1 nm) to be deposited which, thereby, rium surface pressures of 9, 17, and 31 mN/ avoids masking of the monolayer fine strucm, respectively. Piston oils having spreading ture. The preshadowed replicas on mica, which pressures lower than 9 mN/m or at other in- could not be directly stripped in contrast to termediate pressures were obtained by mixing the replicas on Formvar and collodion slides, ethyl laurate and n-hexadecane in varying were first dipped into dilute NaOH solution proportions; these mixtures, however, were not to loosen the tenacious replicas before being as convenient to use because the surface pres- floated onto water and collected on grids. The sure had to be continually monitored to ensure development of this simple procedure greatly a "constant" pressure to within ~<0.2 mN/m facilitates the use of mica slides in electron during the transfer process. The surface pres- microscopic studies of monolayer strucsures of manual compression and selected pis- ture; previously, the time-consuming shadow ton oil experiments were measured by the transfer technique was employed whenever (DPPC) monolayers by the use of piston oils and by the use of a compression (manual) bartier. In addition, we compared the effects of mica, Formvar, and collodion substrates, onto which the monolayers were transferred, as well as Formvar-covered and collodion-covered microscope grids.
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FERESHTEHKHOU, NEUMAN, AND OVALLE
preshadowed carbon replicas on mica substrates were to be examined (24). Nevertheless, there were still occasional difficulties in removing the preshadowed carbon replicas from the underlying mica substrates. The prepared replicas were examined, and representative views were photographed typically at a magnification of about 40,000 diameters in a Philips 300 transmission electron microscope. The deposited monolayers also were examined using radiotracer techniques. [ 1-14C] stearic acid (59 mCi/mM) was purchased from New England Nuclear and was further purified by dissolving in purified n-hexane and then extracting with 0.01 N HCI and several portions of best quality double-distilled water. Radioactive monolayers were spread, compressed, and transferred under identical experimental conditions onto the three (mica, Formvar, and collodion) substrates, as well as onto glass, using the piston oil technique. The preparation of the solid substrates was as previously noted except that their size was 7.5 × 5 cm. The transfer ratios on Formvar, collodion, and mica were calculated from radioactivity measurements relative to those on glass using procedures similar to those reported previously (7). Autoradiographs were obtained by contacting Dupont MRF-31 X-ray film against the radioactive monolayer-covered substrates in an aluminum exposure cassette. Stearic acid monolayer deposition was also characterized by withdrawal contact angle measurements. The withdrawal contact angle (0w) formed between the monolayer-covered subsolution surface and the emerging solid slide was photographed using a Minolta X700 35-mm camera fitted with a Macro 100mm F4 lens. The withdrawal contact angles, after being enlarged 6 times, were measured with a contact angle tangentometer. RESULTS AND DISCUSSION
Surfactant monolayers such as stearic acid appear to be fragile systems. Artifacts can be induced in the monolayer structure by heretofore believed innocuous techniques. These artifacts have not previously been revealed beJournal of Colloid and Interface Science, Vol. 109,No. 2, February 1986
cause of crossmasking and similarity to known structures. While a difference due to the mode ofmonolayer compression was observed in the electron micrographs we believe, as will be discussed in the next section, that it is not an artifact in the true sense. But rather, the main cause for aberrations of monolayer morphology is transfer onto low-energy solid surfaces.
Compression Effects Figure 1 shows electron micrographs of stearic acid monolayers. The micrographs, it should be noted, are printed with shadows in white. The blank sample shown in Fig. 1a was obtained when a mica slide was raised through a clean air/water interface before the monolayer was spread. Stearic acid monolayers were compressed from the two-phase gaseous/liquid-condensed transition region and deposited on mica at different surface pressures using the piston oil technique. The electron micrographs of stearic acid monolayers in the liquid-condensed state at 0.5, 2, and 4.8 mN/m appear to be essentially identical and generally are uniformly smooth, continuous, and homogeneous as shown in Fig. lb. Stearic acid monolayers in the liquidcondensed state at 9 and 17 mN/m also were examined. At these intermediate surface pressures the stearic acid monolayer is above its equilibrium spreading pressure (ESP), which is about 5.2 mN/m (25). Close examination of the electron micrographs reveals the presence of particles previously described by Neuman (8) as "crystallites" as shown in Figs. lc and d. On the other hand, below the ESP, these molecular aggregates were not observed (Fig. lb) as should be the case. Interestingly, however, there were some differences in the number and size of molecular aggregates depending upon the mode of film compression, i.e., whether the stearic acid monolayer was compressed manually with a rigid barrier or by the use of a piston oil. For example, the number of aggregates decreased by about 40-90% when the monolayers were compressed with a barrier. Although the diameter of the aggregates ranged from 10 to 42
MONOLAYER MICROSCOPIC STRUCTURE
389
FIG. 1. Electron micrographsof stearic acid monolayers deposited on mica. (a) Blank showing the mica substrate with no film (×40,000); stearic acid monolayer transferred at (b) 2 mN/m (X40,000), (c) 9 raN/ m (X80,000), (d) 17 mN/m (X80,000), (e) and (f) 31 mN/m (X10,250). n m in the monolayers compressed by piston oils and ranged from 20 to 32 n m in the monolayers compressed manually, the average diameter, nevertheless, appeared to be about the same for both compression modes. However, the average height of the aggregates was higher in barrier compressed monolayers (3.4
nm) compared to that in piston oil compressed films (2.6 nm). No significant differences, furthermore, were observed between the aggregates formed at 9 and 17 m N / m . Whereas the final pressure is reached almost instantaneously during compression with piston oils, the final pressure in barrier compresJournal of Colloid and Interface Science, Vol. !09, No. 2, February 1986
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FERESHTEHKHOU, NEUMAN, AND OVALLE
sion experiments, in marked contrast, is not reached for as long as 5-15 min. Typically 10 min still pass before the slide is completely withdrawn from the subsolution. If instead the monolayer is transferred very quickly (ca. 30 s) after compression with piston oils, little, if any, molecular aggregates are formed. In general, the molecular aggregates which form during barrier compression are fewer in number but larger than those resulting from piston oil compression. The trend in the number and size of molecular aggregates appears analogous to that which occurs in the seeding and growth of crystals from supersaturated bulk solutions. The nature of these structures, however, is yet to be defined. Nevertheless, the molecular aggregates have formed in response to compressing the monolayer above its ESP. Similar structures were observed in the collapsed regions as well as in the monolayer at high pressure. It is intriguing, therefore, to speculate whether these interfacial microstructures are homogeneous nuclei formed during monolayer collapse (8, 25). Figures l e and f show that stearic acid monolayers compressed to 31 mN/m undergo the slow collapse process observed earlier where there are regions of "collapsed phase" material, typically multiples of a bilayer structure, distributed about the regular monolayer (solid-condensed) state (8), It is very difficult to show a single micrograph which is representative of monolayer collapse. Two electron micrographs, at least, are required. Figure 1e is illustrative of the collapse structures existing between or outside the collapsed regions whereas Fig. lfshows the film structure within a collapsed region. This slow collapse is to be distinguished from the catastrophic collapse occurring when the surface pressure is so high that the monolayer fails by a folding-over or fracture process (4, 26). Similar results were obtained when stearic acid monolayers were compressed stepwise with a rigid barrier.
Solid Substrate Effects In the preceding section the stearic acid monolayers were transferred onto mica subJournal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
strates exclusively. Herein, the use of smooth Formvar and collodion slides in monolayer studies is examined. It is to be expected that the blank Formvar slide shown in Fig. 2a has a slightly rougher texture than mica. Blank collodion slides have a similar texture. Subtle discrepancies were evident in the "structure" of stearic acid monolayers transferred onto Formvar and collodion slides. At high pressure (31 mN/m), collapse structures are present on Formvar, and Figs. 2e and f show they are similar to those which were observed when the monolayer was deposited on mica substrates. On the other hand, Fig. 2c shows that the electron micrographs of stearic acid monolayers transferred onto Formvar at 9 mN/m appear essentially smooth, continuous, and homogeneous just as do those transferred onto Formvar at pressures below the ESP (see Fig. 2b). Significantly, however, the molecular aggregates previously seen in Fig. lc, when stearic acid monolayers were transferred onto mica at 9 mN/m, were not observed in Fig. 2c. Instead, the monolayer-covered Formvar slides have the same general appearance (albeit a somewhat rougher texture) as the blank Formvar in Fig. 2a. Stearic acid monolayers transferred onto collodion-covered slides give similar results. Therefore, monolayer deposition on Formvar and collodion is different from that on mica. Autoradiographs clearly show a difference in the deposition behavior of monomolecular films on mica, Formvar, and collodion substrates. For example, stearic acid monolayers generally appear to transfer completely at high surface pressure (31 mN/m) onto both mica and Formvar as evidenced by essentially similar autoradiographs. In contrast, at lower surface pressures, e.g., 9 mN/m, the activity of the deposited monolayer on mica is still high whereas the activity of the transferred monolayer on Formvar appears to be barely above background as shown in Fig. 3. Similar behavior was exhibited by stearic acid monolayers transferred onto collodion substrates. However, contrary to Spink (6), we did not observe any uniform distribution of small
MONOLAYER MICROSCOPIC STRUCTURE
391
lOG. 2. Electron micrographsof stearic acid monolayersdeposited on Formvar-coveredslides. (a) Blank showing the Formvar substrate with no film (X40,000); stearic acid monolayertransferred at (b) 2 mN/m (X40,000), (c) 9 mN/m (X40,000), (d) 17 mN/m (X40,000), (e) and (f) 31 mN/m (X10,250).
holes in the autoradiographs of stearic acid monolayers on mica (and glass) when the transfer was performed very carefully. The results obtained on Formvar and collodion can be explained by a partial monolayer transferring onto both low-energy substrates
at low surface pressures as originally demonstrated by Neuman (7) for monolayer deposition on collodion. The stearic acid molecules do not quantitatively transfer with the same packing they had on water but rather their surface density now is some small fraction of Journal of Colloid and Interface Science, Vol. 109, No, 2, February 1986
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F E R E S H T E H K H O U , N E U M A N , A N D OVALLE
I/
FIG. 3. Autoradiographs of stearic acid monolayers transferred onto (a) mica at 31 m N / m , (b) mica at 9 m N / m, and (c) Formvar at 9 m N / m . Dark areas are regions of higher activity such as monolayer collapse.
that on the subsolution as evidenced by the autoradiographs. As some monolayer molecules do transfer onto these surfaces, the longchain molecules are either distributed uniformly across the solid surface or in small aggregates that are below the resolution of the electron microscope. Since at high surface
pressure the monolayer appears to quantitatively transfer onto these low-energy solid surfaces, there should exist a critical pressure corresponding to a transition between the two deposition regimes. Direct evidence that monolayers transferred onto low-energy solid surfaces can yield a discontinuous structure is shown in Fig. 2d. This micrograph shows a monolayer of stearic acid which has been transferred onto Formvar at a surface pressure of 17 mN/m. This structure is to be compared, once again, with that of the monolayer transferred onto mica at this same pressure (see Fig. 1d). There are two possibilities for the discontinuous structure. It will be shown later in Table I that the withdrawal contact angle between the monolayer-covered subsolution and the
TABLE I Transfer of Stearic Acid Monolayers onto Solid Substrates
Substrate
Surface pressure (naN/m)
Net activity (counts/rainy
5c 7 6 8
Transfer ratiob
Withdrawal contact angle (degree)
Unit3/ Unity Unity Unity
Zero Zero Zero Zero
0.98 1.01 1.01 0.99
Zero Zero Zero Zero
Glass
9 17 21.5 31
262 271 367 402
+ + + +
Mica e
9 17 21.5 31
258 274 372 397
+ 3 + 1 ___8 _+ 7
Collodion r
9 17 21.5 31
69 + 3 193 _+ 4 -392 + 8
0.26 0.71 -0.97 s
40 26 22 Zero
Formvar f
9 17 21.5 31
54 185 274 390
0.21 0.68 0.75 0.97 ~
42 28 24 Zero
+ 6 + 7 +__6 +_ 6
a The activities at 21.5 and 31 m N / m are higher than anticipated on the basis of~r-A isotherms. The marked increases arise from the significant n u m b e r of microscopic collapsed regions present in these highly compressed films. b Calculated from radioactivity measurements relative to the radioactivity on glass. c Standard deviation of activities. d Spink (6) and N e u m a n (7) have demonstrated that p = 1 for stearic acid monolayer deposition on glass. e The backscattering of/3 radiation from mica and glass appears to be virtually identical (6). YAssumed 0.03-~m-thick collodion and Formvar films contribute negligibly to the backscattering of the collodionand Formvar-covered glass substrates. g It was difficult to obtain reproducible and accurate measurements due to the presence of significant monolayer collapse and voids in the deposited monolayers. Within experimental error, however, p = 1. Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
MONOLAYER
393
MICROSCOPIC STRUCTURE
Formvar slide is acute at this surface pressure and the stearic acid monolayer does not transfer completely onto the Formvar. The monolayer may transfer uniformly onto the Formvar, but because it is only partially transferred, it subsequently may undergo a rearrangement after the deposition or during the replication procedures. Alternatively, whenever the local contact angle becomes zero, due possibly to vibrations or a slight change of rate of withdrawal of the slide, the stearic acid monolayer transfers completely. This intermittent transfer then would yield a patchy or discontinuous film. However, on the basis of the withdrawal contact angle results shown in Table I, this explanation appears unlikely. On the other hand, some evidence for such a mechanism is provided by the occasional observation of thin bands of increased activity parallel to the threephase line interface on the autoradiographs of stearic acid monolayers transferred onto collodion. It is of interest to note that Spink (6) has also observed a similar deposition anisotropy on flat silver surfaces when the transfer ratio was less than unity. In any event, the shadow lengths observed in Fig. 2d correspond to a film thickness of about 2.1 nm and, thus, demonstrate that a stearic acid monolayer is present on the Formvar substrate. The monolayer-covered surface shows a slightly rougher texture than the apparently bare or uncovered surface. Examination of the micrograph in Fig. 2d also reveals the presence of molecular aggregates. Shadow-width and shadow-length measurements indicate that the aggregates free of monolayer are lenticular in shape. It is not too difficult to envision that these aggregates are similar to those observed in Fig. ld except the aggregates on mica substrates have their lower portion (not visible) embedded in the basic monolayer structure. The shape of these microstructures at the air/water interface may, in fact, be spherical; it is possible, however, that they have deformed upon transfer to the solid support. Figure 4 shows that the critical pressures for the deposition of monolayers on the Formvar
1.0 ~ ~ ?
~+ o.~
o
0.~
A 0.7 40
50
60
SURFAOETENSION,YLV' mNlm
70
FIG. 4. Wettability of Formvar and collodion by stearic
acid monolayer-covered0.01 N HCI subsolutions. (O) Collodion and (A) Formvar substrates. and collodion films used in this study are approximately 27 and 26 mN/m, respectively. Formvar, therefore, is slightly less wettable than collodion. At higher surface pressures, 0w = 0 ° and below these pressures, 0w > 0 °. The view that only a partial monolayer of stearic acid is present on Formvar and collodion slides below a specific transition pressure is confirmed by transfer ratio measurements. The transfer ratio, 0, is defined as the ratio of the monolayer area removed from the subsolution surface to the geometrical area of the solid substrate. Ideal transfer occurs when p = 1. Table I clearly shows that below the critical pressures the transfer ratios on Formvar and collodion slides are less than unity and only partial monolayers transfer. The structures of these partial monomolecular films often are below the resolution of the electron microscope, thus explaining why micrographs of these monolayer-covered surfaces appear smooth even at 9 mN/m. At 31 m N / m or surface pressures higher than the critical pressure, Table I shows that o = 1 within experimental error, and quantitative film transfer occurs onto Formvar and collodion just as it does onto mica substrates. We have confirmed that, at least for stearic acid monolayer deposition onto film-free solids, # = 1 when 0w = 0 ° and 0< lwhen0w>0% The question of the stability of the deposited monolayers, i.e., loss and/or rearrangement of Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
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monolayer molecules during the various stages of replication for microscopic examination, was examined using radiotracer techniques. Deposited monolayers were stored under ambient conditions, and their activities were monitored as a function of time. In addition, the stability of deposited monolayers when subjected to the high vacuum conditions (4 × 10-5 mm Hg) which they experience during the replication procedure was investigated. Table II shows that the loss in air of stearic acid monolayers on mica at room temperature was 3% after 7 days and 5% after 14 days while about 8% would appear to be lost during preparation of preshadowed replicas under vacuum. For stearic acid monolayers on glass, 26% is lost during replication. The observed stability behavior is in good agreement with that found by Spink (6). The loss for monolayers of stearic acid on Formvar is still higher, 31% under vacuum conditions. It was not uncommon that the counts at a given location sometimes increased with time when the monolayers were aged on glass and Formvar substrates; it was as though molecular migration or a structural rearrangement was occurring on glass and, to a lesser extent, on Formvar. Electron microscopic examination, how-
TABLE II Stability of Deposited Stearic Acid Monolayers Percentage lossin air
Substrate~
7 days
14 days
Percentage loss in vacuumb
Mica Glassc Formvar c
3.0 0.1 4.1
5.3 -0.7 4.2
7.8 26.2 30.8
ever, failed to reveal any structural changes in the samples upon aging for 2 weeks. Mica, thus, is seen to be a preferred solid substrate for microscopic study because the monolayer transfers completely or quantitatively (i.e., the transfer ratio is unity) at all surface pressures. In addition, the deposited monolayers are very stable on mica, clean surfaces are easily obtainable by cleavage, and the surfaces are atomically smooth.
Roughness Effects This section examines the use of Formvarcovered and collodion-covered grids. It will be recalled that electron microscope grids have been inserted under a Formvar or collodion film, and the monolayer to be examined was deposited on this composite surface. The advantage of this technique, and presumably why it has been often employed in the past, is that it simplifies the steps involved in obtaining replicas for microscopic examination. Stearic acid monolayers were transferred at 9 and 17 mN/m onto Formvar-covered grids. The electron micrographs were similar to those shown in Fig. 2c and d for monolayers transferred onto smooth Formvar-covered substrates. Stearic acid monolayers transferred onto collodion-covered grids exhibit a similar behavior. Thus, we conclude that the Formvar-covered and collodion-covered grid surfaces do not have any significant effect on monolayer deposition over and above that of the smooth Formvar and collodion slides.
DPPC and Pentadecanoic Acid Monolayers
a Monolayer samples were transferred at 9 m N / m and duplicated. b Deposited monolayer exposed to vacuum for about 15 rain. Maximum vacuum was 4 × 10-5 m m Hg. c Molecular migration apparently occurred such that the local counting rates often increased on glass and Forrnvar when the deposited monolayers were aged under ambient conditions, thus masking the expected increase in film loss with time. Journal of Colloid and Interface Science, Vol. 109,No. 2, February 1986
The electron micrographs of DPPC monolayers are smooth, continuous, and homogeneous when compressed and transferred onto mica at surface pressures of 2 and 17 mN/m. DPPC, under the experimental conditions employed, is in the liquid-expanded state at 2 mN/m, whereas it is in the liquid-condensed state at 17 mN/m. DPPC monolayers, thus, are continuous and uniform in both the liquidexpanded phase (Fig. 5a) and the liquid-con-
MONOLAYER MICROSCOPIC STRUCTURE
395
FiG. 5, Electron micrographs of DPPC monolayers deposited on mica at (a) 2 m N / m and (b) 17 m N / m (×40,000).
densed phase (Fig. 5b). Even though DPPC was compressed to 31 m N / m - - a pressure much higher than its ESP--it is of interest to note that molecular aggregates were not observed in the micrographs. The electron micrographs of DPPC monolayers transferred onto mica did not show the marked microporosity observed by Ries et al. (10). However, if DPPC monolayers were transferred at 17 mN/m onto Formvar or collodion substrates, then structures similar to those observed by Ries and his co-workers were evident, presumably due to the transfer of a partial monolayer as discussed earlier (manuscript in preparation). For the sake of completeness, it should be mentioned that the structure of the transition region between the liquid-expanded and liquid-condensed phases was recently examined using electron microscopic techniques (23). Two-phase behavior was observed; this finding is consistent with the interpretation of a firstorder phase transition occurring between the liquid-expanded and liquid-condensed states in monomolecular films. Pentadecanoic acid monolayers showed similar trends as DPPC with the exception that the structural detail of the micrographs was much finer.
RELATIONSHIP TO PREVIOUS STUDIES
The results of past research on monomolecular films deposited on Formvar and collodion substrates must be reconsidered in view of our findings. Below a given surface pressure, slightly different for Formvar and collodion, a partial monolayer transfers onto these surfaces. This critical pressure was approximately 27 and 26 mN/m for the Formvar and collodion films, respectively, used in this study. Both Sheppard et al. (5) and Spink (6) probably prepared and observed partial monolayers on collodion, but incorrectly interpreted these structures as normal monolayers which they mimic. Neuman (8) did not observe partial monolayers in his earlier microscopic studies because they were conducted above the critical surface pressure for collodion. Our results confirm the pressure dependence of the transfer process first observed by Neuman (7) and also show the same phenomenon occurs on Formvar surfaces. We also were able to duplicate some of the results of Ries and his co-workers in this study. Discontinuous and heterogeneous films of stearic acid and DPPC were seen under the electron microscope when the monolayers were transferred onto Formvar and collodion Journal of Colloid and Interface Science, Vol. 109,No. 2, February1986
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FERESHTEHKHOU, NEUMAN, AND OVALLE
substrates at intermediate (17 mN/m) surface pressures. However, we have shown that this is due to the monolayers transferring onto a low-energy solid surface rather than a highenergy surface such as mica. Pankhurst's concerns (27) that the island structures observed by Ries and Kimball were an experimental artifact introduced during the transfer process were justified. Berg's (22) effort to study the structure of monolayers generated results similar to those first published by Ries and Kimball (3, 4). The monolayer was probably disturbed prior to complete thermal fixation by either the shearing action of the cooling plug passing through the monolayer or the abrupt change in surface interactions due to the freezing of the subsolution. More attention should be given to eliminating or modifying steps which could cause monolayer artifacts with this procedure. The formation of molecular aggregates appears to be a more general phenomenon than realized with very important implications. These film aggregates may play an important role in the hysteresis behavior of spread monolayers. The physical nature of these interfacial aggregates and the conditions under which they form certainly warrant further investigation. The liquid-condensed state of stearic acid monolayers was examined microscopically over a wide range of surface pressures. The electron micrographs of the basic monolayer structure are smooth, continuous, and homogeneous when the stearic acid monolayers were compressed with piston oils (or compression barriers) and transferred onto mica substrates. In this respect, our findings agree with Sheppard et al. (5) who saw smooth surfaces on glass and Spink (6) who also observed featureless surfaces on glass and mica. In addition, we have shown that monolayers such as DPPC in the liquid-expanded (and liquidcondensed) state are smooth, continuous, and homogeneous. These results are in agreement with current theories on monolayer structure in that amphipathic molecules should form a single phase in these surface states. Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
CONCLUSIONS
Experimental artifacts can be induced during the transfer of monomolecular films for observation by electron microscopy. Partial monolayers transfer onto Formvar and collodion surfaces (and, in general, low-energy solids) at low and intermediate surface pressures. At sufficiently high pressures, however, quantitative transfer takes place. Furthermore, deposited monolayers, e.g., stearic acid, are relatively unstable on these surfaces, and structural rearrangements may occur. On the other hand, when mica is employed, the artifacts previously observed appear to be eliminated. Indeed, a monolayer structure which is in agreement with current views on ordering in surface films can be obtained when the monolayer is compressed and transferred onto mica substrates by either the piston oil or compression barrier technique. ACKNOWLEDGMENTS This research was supported by the Office of Basic Energy Sciences, Division of Chemical Sciences, Department of Energy under Contract DE-AC02-81ER10859 and, in part, by the University of Minnesota Agricultural Experiment Station. A Graduate and Professional Opportunities Program (GPOP) Fellowship received by R. Ovalle is acknowledged. The authors also wish to thank M. Agrawal and T. Tompkins for their assistance in some monolayer experiments. REFERENCES 1. Langmnir, I., Trans. Faraday Soc. 15, 62 (1920). 2. Blodgett, K. B., J. Amer. Chem. Soc. 56, 495 (1934); 57, 1007 (1935). 3. Ries, H. E., Jr., and Kimball, W. A., aT.Phys. Chem. 59, 94 (1955). 4. Ries, H. E., Jr., and Kimball, W, A., "Proceedings Second International Congress on Surface Activity," London Vol. 1, p. 75, 1957. 5. Sheppard, E., Bronson, R. P., and Tcheurekdjian, N., J. ColloidSci. 19, 833 (1964); 20, 755 (1965). 6. Spink, J. A., J. Colloid Interface Sci. 23, 9 (1967). 7. Neuman, R. D., ar. Colloid Interface Sci. 50, 602 (1975). 8. Neuman, R. D., J. Colloid InteoCace Sci. 56, 505 (1976). 9. Ries, H. E., Jr., Matsumoto, M., Uyeda, N., and Suito, E., .I. Colloid Interface Sci. 57, 396 (1976).
MONOLAYER MICROSCOPIC STRUCTURE 10. Ries, H. E., Jr., Matsumoto, M., Uyeda, N., and Suito, E., Adv. Chem. Set. 144, 286 (1975). 11. Ries, H. E., Jr., and Swift, H., Jr. Cotloidlnterface Sci. 64, 111 (1978). 12. Harkins, W. D., and Fischer, E. K., J. Chem. Phys. 1, 852 (1933). 13. Middleton, S. R., and Pethica, B. A., Faraday Symposium 16, 109 (1981). 14. Cook, H. D., and Ries, H. E., Jr., J. Phys. Chem. 60, 1533 (1956). 15. Ries, H. E., Jr., Cook, H. D., and Loane, C. M., "Symposium of Steam Turbine Oils." ASTM Spec. Techn. Publ. No. 211, 55 (1957). 16. Ries, H. E., Jr., and Kimball, W. A., Nature (London) 181, 901 (1958). 17. Ries, H. E., Jr., and Walker, D. C., J. ColloidSci. 16, 361 (1961).
397
18. Ries, H. E., Jr., Colloids Surf. 10, 283 (1984). 19. Ries, H. E., Jr., and Swift, H., J. Co[loid Interface Sci. 89, 245 (1982). 20. Ries, H. E., Jr., J. ColloidlnterfaceSci. 88, 298 (1982). 21. Ries, H. E., Jr., Nature (London) 281, 287 (1979). 22. Berg, J. C., in "Proceedings of the Workshop on Interfacial Phenomena: Research Needs and Priorities" (J. C. Berg, Ed.), p. 89, NTIS, U. S. Dept. Commerce, Springfield, IU. 1979. 23. Neuman, R. D., Fereshtehkhou, S., and Ovalle, R., J. Colloid lnterface Sci. 101, 309 (1984). 24. Neuman, R. D., J. Microscopy 105, 283 (1975). 25. Smith, R. D., and Berg, J. C., J. ColloM Interface Sci. 74, 273 (1980). 26. Smith, T., J. Colloid Interface Sci. 25, 443 (1967). 27. Pankhurst, K. G. A., J. Phys. Chem. 59, 480 (1955).
Journal of Colloid and Interface Scier~ce,Vol. 109, No. 2, February 1986
RONALD
D. N E U M A N , 3'4 AND R A F A E L O V A L L E
Department of Forest Products, University of Minnesota, St. Paul Minnesota 55108
Received September 24, 1984; accepted May 22, 1985 The question of artifact formation associated with electron microscopic examination of the structure of monomolecular films was investigated. The effects of mode of film compression, surface energy of solid substrate, and surface roughness of solid substrate onto which fatty acid and phospholipid monolayers were transferred by the Langmuir-Blodgett technique from the air/water interface were systematically examined. Aberrations in monolayer structure can occur when low-energy solid surfaces such as Formvar and collodion are used for monolayer transfer below some specific surface pressure which is slightly different for each solid substrate. The observed structures bear similarity to the observations reported in other studies. However, when the monolayers are transferred onto mica substrates, the artifacts previously observed by electron microscopy appear to be eliminated and surface films existing in the liquid-expanded and liquid-condensed states exhibit a structure indicative of single-phase behavior, i.e., they are smooth, continuous, and homogeneous in agreement with current theories on monolayer structure. These results are discussed and compared with those of earlier microscopic studies. © 1986AcademicPress,Inc.
INTRODUCTION
a n d the q u e s t i o n o f artifacts occurring d u e to
T h e structure o f " i n s o l u b l e " o r sparingly soluble m o n o m o l e c u l a r films has b e e n in disp u t e for m a n y years. Electron m i c r o s c o p y has b e e n one a p p r o a c h for e x a m i n i n g the fine structure o f these films. T h e L a n g m u i r - B l o d gett t e c h n i q u e (1, 2), w h e r e b y m o n o l a y e r s are transferred f r o m the a i r / w a t e r interface to a solid substrate being w i t h d r a w n slowly t h r o u g h the surface film, usually has b e e n a n interm e d i a t e step in p r e p a r i n g the m o n o l a y e r s for o b s e r v a t i o n b y electron m i c r o s c o p y . T h e r e h a v e b e e n discrepancies, however, a m o n g the e x p e r i m e n t a l findings o f various investigators,
the transfer o f these films has never been fully clarified. Ries a n d K i m b a l l (3, 4) in their classical 1955 study transferred n - h e x a t r i a c o n t a n o i c acid m o n o l a y e r s , after c o m p r e s s i o n with a m o t o r - d r i v e n barrier, o n t o c o l l o d i o n - c o v e r e d electron m i c r o s c o p e screens o r grids. T h e i r m i c r o g r a p h s o f the l o n g - c h a i n (36-carbon ato m s ) fatty a c i d m o n o l a y e r at various stages o f c o m p r e s s i o n showed t h a t small " i s l a n d s " or d o m a i n s irregular in size a n d shape were present at 15 m N / m . A t 25 m N / m , the m o n o l a y e r b e c a m e the c o n t i n u o u s phase a n d the areas w h i c h at low pressure a p p e a r e d to be uncovered o r b a r e n o w were discontinuous. A c o n t i n u o u s h o m o g e n e o u s m o n o l a y e r was observed o n l y w h e n the surface pressure was raised to still higher pressures (39 m N / m ) j u s t b e l o w c a t a s t r o p h i c collapse. T h e results o b t a i n e d b y Ries a n d K i m b a l l have b e e n d i s c o n c e r t i n g o v e r the years to m a n y investigators because the prevailing view o f m o n o l a y e r structure is t h a t such films
Published as Scientific Journal Series Paper 14,142 of the University of Minnesota Agricultural Experiment Station. 2 Presented in part at the State-of-the-Art Review: Monomolecular Films Symposium at the Annual Meeting of the American Institute of Chemical Engineers, November 14-19, 1982. Los Angeles, Calif. 3Present address: Department of Chemical Engineering, Auburn University, Auburn, Alabama 36849. 4 To whom correspondence should be addressed. 385
0021-9797/86 $3.00 Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
Copyright © t986 by Academic Press, Inc. All fights of reproduction in any form re,fred.
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FERESHTEHKHOU, NEUMAN, AND OVALLE
should have been continuous and homogeneous at the surface pressures investigated rather than exhibit two-phase behavior. Accordingly, Sheppard et al. (5) examined a variety of surfactant monolayers deposited on glass and Parlodion-covered microscope slides by the Langmuir-Blodgett method and obtained smooth surfaces when viewed under the electron microscope. Spink (6) similarly observed featureless surfaces when he transferred stearic acid monolayers onto mica, glass, and collodion substrates. Whereas Sheppard et al. used a constant weight apparatus, Spink employed piston oils to compress and transfer the monolayer onto solid substrates. Importantly, Spink's observations, as well as those of Sheppard et al., were in disagreement with the findings of Ries and Kimball. Neuman (7) reported in 1975 that monolayer deposition on collodion surfaces was surface pressure dependent. Although collodion (and Formvar) films are commonly employed in conventional microscopic studies, caution was recommended in the use of these low-energy solid surfaces in studies utilizing transferred monolayers due to the likelihood of only a partial monolayer being deposited on such substrates at low and intermediate surface pressures. Neuman (8), in order to examine the process of monolayer collapse by electron microscopy, transferred stearic acid monolayers at high surface pressure (31 mN/ m) onto mica and collodion substrates using an automatic film balance. At about the same time, Ries and co-workers in another series of studies observed island structures, once again, in cholesterol monolayers (9) and, additionally, a pronounced porous structure in lecithin monolayers (10). Although Ries believed that the monolayers transferred quantitatively onto the solid surfaces so employed (11), he did not claim a one-to-one correspondence between the film structures which were observed in the electron micrographs with those that exist on the water surface. Nevertheless, Ries maintained that the observed sequence of structural changes must reflect similar changes in the monolayer during Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
compression on the water surface of the film balance. 5 Ries and his co-workers in the aforementioned series of papers again did not employ more than one solid substrate, but rather they continued to transfer monolayers solely onto a single low-energy surface--Formvar--now being substituted for the collodion used in their earlier investigations (3, 4, 15-17). Since many of their studies, including more recent investigations (18-21), were interested in the structure of collapsed films at high surface pressures, the use of Forrnvar or collodion should not have created too serious problems at least with respect to the transfer process. 6 Berg (22) recently attempted to eliminate any artifact formation that might be caused by transfer ofa monolayer from the water surface onto a solid substrate by piercing the monolayer with a chilled plug and freezing the monolayer and subsolution beneath in situ. The frozen surface was shadowed, and results similar to those found by Ries and Kimball were obtained. No further reports have been published presumably due to experimental difficulties associated with this novel approach. Our analysis of previous research indicates that there are three major variables which may influence the structure of transferred monomolecular films: (a) mode of film compression, (b) surface energy of solid substrate, and (c) surface roughness of solid substrate. The objective of this investigation was to compare the different procedures used by previous investigators and attempt to resolve the differences in their experimental conclusions. In this study, we examined the effects of compressing and transferring stearic acid, pentadecanoic acid, and L-a-dipalmitoyl phosphatidylcholine 5 It should be mentioned that surface potential (12, 13) and radiotracer investigations (14) of monolayers at the air/water interface have found island structures at very low pressures corresponding to the surface vapor pressure of the monomolecular film under study. 6 However, as we will show in this paper, there still remain serious questions concerning the stability of transferred films on low-energy surfaces both before and during the replication procedures.
MONOLAYER
MICROSCOPIC
STRUCTURE
387
Wilhelmy plate (sandblasted glass) technique employing a Cahn RG electrobalance. Monolayer transfer was accomplished by lowering prepared solid surfaces (7.5 × 2.5 cm) into the clean subsolution prior to spreading and compressing the monolayer and then slowly withdrawing the solid substrate up through the monolayer. The prepared surfaces were (a) freshly cleaved Brazilian ruby mica MATERIALS AND METHODS slides (United Mineral and Chemical), (b) Although octadecanoic (stearic) acid was Formvar slides prepared by dipping clean glass emphasized because it is the classical surfac- or freshly cleaved mica slides into either 0.25 tant of study in monolayer investigations, or 0.5% Formvar [poly(vinyl-formal)] in dipentadecanoic acid and DPPC also were stud- chloroethylene, (c) collodion slides prepared ied. Stearic and pentadecanoic acid (Applied by casting 2% collodion (nitrocellulose) in Sciences, purity 99+%) were deposited from amyl acetate on distilled water followed by de2 m M n-hexane spreading solutions onto 0.01 position onto clean glass or mica slides, and NHC1 subsolutions (EM Laboratories, Supra- (d) film-covered electron microscope grids pur). Synthetic DPPC (purity 99+%), obtained prepared by floating Formvar or collodion from Avanti Polar Lipids, was dissolved in a films on distilled water, lowering grids (2009:1 (vol/vol) n-hexane/ethanol solution and or 300-mesh) of copper, gold, nickel, or nylon spread on distilled water subsolutions. The onto the floating film, and then sandwiching subsolutions were prepared from distilled wa- the grids between the film and a clean glass or ter previously purified by reverse osmosis. All mica support. The Formvar and collodion soexperiments were conducted in a dust-shielded lutions were supplied by Ernest F. Fullam. environment, and the water temperature was Direct carbon replicas of the deposited maintained at 21-23°C. The materials, ex- monolayers were prepared within 30 min after perimental apparatus, and procedures are dis- transfer in a Kinney KSE-2A-M vacuum cussed in greater detail elsewhere (23). evaporator. The samples were preshadowed The monolayers were compressed to a given with palladium (Pd) wire at an angle of 14 °, surface pressure either (a) by use of a piston and the source-to-specimen distance for the oil expanding against a flexible 3-rail FEP Pd (7 mg on W filament) evaporant was 100 monofilament thread or (b) by moving a rigid ram. The use of a heat shield having a small Teflon compression barrier forward manually. diameter (6 ram) aperture for passage of the Tricresyl phosphate, castor oil, and oleic acid evaporant permitted very thin evaporant films were used as piston oils to generate equilib- (ca. 1 nm) to be deposited which, thereby, rium surface pressures of 9, 17, and 31 mN/ avoids masking of the monolayer fine strucm, respectively. Piston oils having spreading ture. The preshadowed replicas on mica, which pressures lower than 9 mN/m or at other in- could not be directly stripped in contrast to termediate pressures were obtained by mixing the replicas on Formvar and collodion slides, ethyl laurate and n-hexadecane in varying were first dipped into dilute NaOH solution proportions; these mixtures, however, were not to loosen the tenacious replicas before being as convenient to use because the surface pres- floated onto water and collected on grids. The sure had to be continually monitored to ensure development of this simple procedure greatly a "constant" pressure to within ~<0.2 mN/m facilitates the use of mica slides in electron during the transfer process. The surface pres- microscopic studies of monolayer strucsures of manual compression and selected pis- ture; previously, the time-consuming shadow ton oil experiments were measured by the transfer technique was employed whenever (DPPC) monolayers by the use of piston oils and by the use of a compression (manual) bartier. In addition, we compared the effects of mica, Formvar, and collodion substrates, onto which the monolayers were transferred, as well as Formvar-covered and collodion-covered microscope grids.
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FERESHTEHKHOU, NEUMAN, AND OVALLE
preshadowed carbon replicas on mica substrates were to be examined (24). Nevertheless, there were still occasional difficulties in removing the preshadowed carbon replicas from the underlying mica substrates. The prepared replicas were examined, and representative views were photographed typically at a magnification of about 40,000 diameters in a Philips 300 transmission electron microscope. The deposited monolayers also were examined using radiotracer techniques. [ 1-14C] stearic acid (59 mCi/mM) was purchased from New England Nuclear and was further purified by dissolving in purified n-hexane and then extracting with 0.01 N HCI and several portions of best quality double-distilled water. Radioactive monolayers were spread, compressed, and transferred under identical experimental conditions onto the three (mica, Formvar, and collodion) substrates, as well as onto glass, using the piston oil technique. The preparation of the solid substrates was as previously noted except that their size was 7.5 × 5 cm. The transfer ratios on Formvar, collodion, and mica were calculated from radioactivity measurements relative to those on glass using procedures similar to those reported previously (7). Autoradiographs were obtained by contacting Dupont MRF-31 X-ray film against the radioactive monolayer-covered substrates in an aluminum exposure cassette. Stearic acid monolayer deposition was also characterized by withdrawal contact angle measurements. The withdrawal contact angle (0w) formed between the monolayer-covered subsolution surface and the emerging solid slide was photographed using a Minolta X700 35-mm camera fitted with a Macro 100mm F4 lens. The withdrawal contact angles, after being enlarged 6 times, were measured with a contact angle tangentometer. RESULTS AND DISCUSSION
Surfactant monolayers such as stearic acid appear to be fragile systems. Artifacts can be induced in the monolayer structure by heretofore believed innocuous techniques. These artifacts have not previously been revealed beJournal of Colloid and Interface Science, Vol. 109,No. 2, February 1986
cause of crossmasking and similarity to known structures. While a difference due to the mode ofmonolayer compression was observed in the electron micrographs we believe, as will be discussed in the next section, that it is not an artifact in the true sense. But rather, the main cause for aberrations of monolayer morphology is transfer onto low-energy solid surfaces.
Compression Effects Figure 1 shows electron micrographs of stearic acid monolayers. The micrographs, it should be noted, are printed with shadows in white. The blank sample shown in Fig. 1a was obtained when a mica slide was raised through a clean air/water interface before the monolayer was spread. Stearic acid monolayers were compressed from the two-phase gaseous/liquid-condensed transition region and deposited on mica at different surface pressures using the piston oil technique. The electron micrographs of stearic acid monolayers in the liquid-condensed state at 0.5, 2, and 4.8 mN/m appear to be essentially identical and generally are uniformly smooth, continuous, and homogeneous as shown in Fig. lb. Stearic acid monolayers in the liquidcondensed state at 9 and 17 mN/m also were examined. At these intermediate surface pressures the stearic acid monolayer is above its equilibrium spreading pressure (ESP), which is about 5.2 mN/m (25). Close examination of the electron micrographs reveals the presence of particles previously described by Neuman (8) as "crystallites" as shown in Figs. lc and d. On the other hand, below the ESP, these molecular aggregates were not observed (Fig. lb) as should be the case. Interestingly, however, there were some differences in the number and size of molecular aggregates depending upon the mode of film compression, i.e., whether the stearic acid monolayer was compressed manually with a rigid barrier or by the use of a piston oil. For example, the number of aggregates decreased by about 40-90% when the monolayers were compressed with a barrier. Although the diameter of the aggregates ranged from 10 to 42
MONOLAYER MICROSCOPIC STRUCTURE
389
FIG. 1. Electron micrographsof stearic acid monolayers deposited on mica. (a) Blank showing the mica substrate with no film (×40,000); stearic acid monolayer transferred at (b) 2 mN/m (X40,000), (c) 9 raN/ m (X80,000), (d) 17 mN/m (X80,000), (e) and (f) 31 mN/m (X10,250). n m in the monolayers compressed by piston oils and ranged from 20 to 32 n m in the monolayers compressed manually, the average diameter, nevertheless, appeared to be about the same for both compression modes. However, the average height of the aggregates was higher in barrier compressed monolayers (3.4
nm) compared to that in piston oil compressed films (2.6 nm). No significant differences, furthermore, were observed between the aggregates formed at 9 and 17 m N / m . Whereas the final pressure is reached almost instantaneously during compression with piston oils, the final pressure in barrier compresJournal of Colloid and Interface Science, Vol. !09, No. 2, February 1986
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sion experiments, in marked contrast, is not reached for as long as 5-15 min. Typically 10 min still pass before the slide is completely withdrawn from the subsolution. If instead the monolayer is transferred very quickly (ca. 30 s) after compression with piston oils, little, if any, molecular aggregates are formed. In general, the molecular aggregates which form during barrier compression are fewer in number but larger than those resulting from piston oil compression. The trend in the number and size of molecular aggregates appears analogous to that which occurs in the seeding and growth of crystals from supersaturated bulk solutions. The nature of these structures, however, is yet to be defined. Nevertheless, the molecular aggregates have formed in response to compressing the monolayer above its ESP. Similar structures were observed in the collapsed regions as well as in the monolayer at high pressure. It is intriguing, therefore, to speculate whether these interfacial microstructures are homogeneous nuclei formed during monolayer collapse (8, 25). Figures l e and f show that stearic acid monolayers compressed to 31 mN/m undergo the slow collapse process observed earlier where there are regions of "collapsed phase" material, typically multiples of a bilayer structure, distributed about the regular monolayer (solid-condensed) state (8), It is very difficult to show a single micrograph which is representative of monolayer collapse. Two electron micrographs, at least, are required. Figure 1e is illustrative of the collapse structures existing between or outside the collapsed regions whereas Fig. lfshows the film structure within a collapsed region. This slow collapse is to be distinguished from the catastrophic collapse occurring when the surface pressure is so high that the monolayer fails by a folding-over or fracture process (4, 26). Similar results were obtained when stearic acid monolayers were compressed stepwise with a rigid barrier.
Solid Substrate Effects In the preceding section the stearic acid monolayers were transferred onto mica subJournal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
strates exclusively. Herein, the use of smooth Formvar and collodion slides in monolayer studies is examined. It is to be expected that the blank Formvar slide shown in Fig. 2a has a slightly rougher texture than mica. Blank collodion slides have a similar texture. Subtle discrepancies were evident in the "structure" of stearic acid monolayers transferred onto Formvar and collodion slides. At high pressure (31 mN/m), collapse structures are present on Formvar, and Figs. 2e and f show they are similar to those which were observed when the monolayer was deposited on mica substrates. On the other hand, Fig. 2c shows that the electron micrographs of stearic acid monolayers transferred onto Formvar at 9 mN/m appear essentially smooth, continuous, and homogeneous just as do those transferred onto Formvar at pressures below the ESP (see Fig. 2b). Significantly, however, the molecular aggregates previously seen in Fig. lc, when stearic acid monolayers were transferred onto mica at 9 mN/m, were not observed in Fig. 2c. Instead, the monolayer-covered Formvar slides have the same general appearance (albeit a somewhat rougher texture) as the blank Formvar in Fig. 2a. Stearic acid monolayers transferred onto collodion-covered slides give similar results. Therefore, monolayer deposition on Formvar and collodion is different from that on mica. Autoradiographs clearly show a difference in the deposition behavior of monomolecular films on mica, Formvar, and collodion substrates. For example, stearic acid monolayers generally appear to transfer completely at high surface pressure (31 mN/m) onto both mica and Formvar as evidenced by essentially similar autoradiographs. In contrast, at lower surface pressures, e.g., 9 mN/m, the activity of the deposited monolayer on mica is still high whereas the activity of the transferred monolayer on Formvar appears to be barely above background as shown in Fig. 3. Similar behavior was exhibited by stearic acid monolayers transferred onto collodion substrates. However, contrary to Spink (6), we did not observe any uniform distribution of small
MONOLAYER MICROSCOPIC STRUCTURE
391
lOG. 2. Electron micrographsof stearic acid monolayersdeposited on Formvar-coveredslides. (a) Blank showing the Formvar substrate with no film (X40,000); stearic acid monolayertransferred at (b) 2 mN/m (X40,000), (c) 9 mN/m (X40,000), (d) 17 mN/m (X40,000), (e) and (f) 31 mN/m (X10,250).
holes in the autoradiographs of stearic acid monolayers on mica (and glass) when the transfer was performed very carefully. The results obtained on Formvar and collodion can be explained by a partial monolayer transferring onto both low-energy substrates
at low surface pressures as originally demonstrated by Neuman (7) for monolayer deposition on collodion. The stearic acid molecules do not quantitatively transfer with the same packing they had on water but rather their surface density now is some small fraction of Journal of Colloid and Interface Science, Vol. 109, No, 2, February 1986
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F E R E S H T E H K H O U , N E U M A N , A N D OVALLE
I/
FIG. 3. Autoradiographs of stearic acid monolayers transferred onto (a) mica at 31 m N / m , (b) mica at 9 m N / m, and (c) Formvar at 9 m N / m . Dark areas are regions of higher activity such as monolayer collapse.
that on the subsolution as evidenced by the autoradiographs. As some monolayer molecules do transfer onto these surfaces, the longchain molecules are either distributed uniformly across the solid surface or in small aggregates that are below the resolution of the electron microscope. Since at high surface
pressure the monolayer appears to quantitatively transfer onto these low-energy solid surfaces, there should exist a critical pressure corresponding to a transition between the two deposition regimes. Direct evidence that monolayers transferred onto low-energy solid surfaces can yield a discontinuous structure is shown in Fig. 2d. This micrograph shows a monolayer of stearic acid which has been transferred onto Formvar at a surface pressure of 17 mN/m. This structure is to be compared, once again, with that of the monolayer transferred onto mica at this same pressure (see Fig. 1d). There are two possibilities for the discontinuous structure. It will be shown later in Table I that the withdrawal contact angle between the monolayer-covered subsolution and the
TABLE I Transfer of Stearic Acid Monolayers onto Solid Substrates
Substrate
Surface pressure (naN/m)
Net activity (counts/rainy
5c 7 6 8
Transfer ratiob
Withdrawal contact angle (degree)
Unit3/ Unity Unity Unity
Zero Zero Zero Zero
0.98 1.01 1.01 0.99
Zero Zero Zero Zero
Glass
9 17 21.5 31
262 271 367 402
+ + + +
Mica e
9 17 21.5 31
258 274 372 397
+ 3 + 1 ___8 _+ 7
Collodion r
9 17 21.5 31
69 + 3 193 _+ 4 -392 + 8
0.26 0.71 -0.97 s
40 26 22 Zero
Formvar f
9 17 21.5 31
54 185 274 390
0.21 0.68 0.75 0.97 ~
42 28 24 Zero
+ 6 + 7 +__6 +_ 6
a The activities at 21.5 and 31 m N / m are higher than anticipated on the basis of~r-A isotherms. The marked increases arise from the significant n u m b e r of microscopic collapsed regions present in these highly compressed films. b Calculated from radioactivity measurements relative to the radioactivity on glass. c Standard deviation of activities. d Spink (6) and N e u m a n (7) have demonstrated that p = 1 for stearic acid monolayer deposition on glass. e The backscattering of/3 radiation from mica and glass appears to be virtually identical (6). YAssumed 0.03-~m-thick collodion and Formvar films contribute negligibly to the backscattering of the collodionand Formvar-covered glass substrates. g It was difficult to obtain reproducible and accurate measurements due to the presence of significant monolayer collapse and voids in the deposited monolayers. Within experimental error, however, p = 1. Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
MONOLAYER
393
MICROSCOPIC STRUCTURE
Formvar slide is acute at this surface pressure and the stearic acid monolayer does not transfer completely onto the Formvar. The monolayer may transfer uniformly onto the Formvar, but because it is only partially transferred, it subsequently may undergo a rearrangement after the deposition or during the replication procedures. Alternatively, whenever the local contact angle becomes zero, due possibly to vibrations or a slight change of rate of withdrawal of the slide, the stearic acid monolayer transfers completely. This intermittent transfer then would yield a patchy or discontinuous film. However, on the basis of the withdrawal contact angle results shown in Table I, this explanation appears unlikely. On the other hand, some evidence for such a mechanism is provided by the occasional observation of thin bands of increased activity parallel to the threephase line interface on the autoradiographs of stearic acid monolayers transferred onto collodion. It is of interest to note that Spink (6) has also observed a similar deposition anisotropy on flat silver surfaces when the transfer ratio was less than unity. In any event, the shadow lengths observed in Fig. 2d correspond to a film thickness of about 2.1 nm and, thus, demonstrate that a stearic acid monolayer is present on the Formvar substrate. The monolayer-covered surface shows a slightly rougher texture than the apparently bare or uncovered surface. Examination of the micrograph in Fig. 2d also reveals the presence of molecular aggregates. Shadow-width and shadow-length measurements indicate that the aggregates free of monolayer are lenticular in shape. It is not too difficult to envision that these aggregates are similar to those observed in Fig. ld except the aggregates on mica substrates have their lower portion (not visible) embedded in the basic monolayer structure. The shape of these microstructures at the air/water interface may, in fact, be spherical; it is possible, however, that they have deformed upon transfer to the solid support. Figure 4 shows that the critical pressures for the deposition of monolayers on the Formvar
1.0 ~ ~ ?
~+ o.~
o
0.~
A 0.7 40
50
60
SURFAOETENSION,YLV' mNlm
70
FIG. 4. Wettability of Formvar and collodion by stearic
acid monolayer-covered0.01 N HCI subsolutions. (O) Collodion and (A) Formvar substrates. and collodion films used in this study are approximately 27 and 26 mN/m, respectively. Formvar, therefore, is slightly less wettable than collodion. At higher surface pressures, 0w = 0 ° and below these pressures, 0w > 0 °. The view that only a partial monolayer of stearic acid is present on Formvar and collodion slides below a specific transition pressure is confirmed by transfer ratio measurements. The transfer ratio, 0, is defined as the ratio of the monolayer area removed from the subsolution surface to the geometrical area of the solid substrate. Ideal transfer occurs when p = 1. Table I clearly shows that below the critical pressures the transfer ratios on Formvar and collodion slides are less than unity and only partial monolayers transfer. The structures of these partial monomolecular films often are below the resolution of the electron microscope, thus explaining why micrographs of these monolayer-covered surfaces appear smooth even at 9 mN/m. At 31 m N / m or surface pressures higher than the critical pressure, Table I shows that o = 1 within experimental error, and quantitative film transfer occurs onto Formvar and collodion just as it does onto mica substrates. We have confirmed that, at least for stearic acid monolayer deposition onto film-free solids, # = 1 when 0w = 0 ° and 0< lwhen0w>0% The question of the stability of the deposited monolayers, i.e., loss and/or rearrangement of Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
394
FERESHTEHKHOU, NEUMAN, AND OVALLE
monolayer molecules during the various stages of replication for microscopic examination, was examined using radiotracer techniques. Deposited monolayers were stored under ambient conditions, and their activities were monitored as a function of time. In addition, the stability of deposited monolayers when subjected to the high vacuum conditions (4 × 10-5 mm Hg) which they experience during the replication procedure was investigated. Table II shows that the loss in air of stearic acid monolayers on mica at room temperature was 3% after 7 days and 5% after 14 days while about 8% would appear to be lost during preparation of preshadowed replicas under vacuum. For stearic acid monolayers on glass, 26% is lost during replication. The observed stability behavior is in good agreement with that found by Spink (6). The loss for monolayers of stearic acid on Formvar is still higher, 31% under vacuum conditions. It was not uncommon that the counts at a given location sometimes increased with time when the monolayers were aged on glass and Formvar substrates; it was as though molecular migration or a structural rearrangement was occurring on glass and, to a lesser extent, on Formvar. Electron microscopic examination, how-
TABLE II Stability of Deposited Stearic Acid Monolayers Percentage lossin air
Substrate~
7 days
14 days
Percentage loss in vacuumb
Mica Glassc Formvar c
3.0 0.1 4.1
5.3 -0.7 4.2
7.8 26.2 30.8
ever, failed to reveal any structural changes in the samples upon aging for 2 weeks. Mica, thus, is seen to be a preferred solid substrate for microscopic study because the monolayer transfers completely or quantitatively (i.e., the transfer ratio is unity) at all surface pressures. In addition, the deposited monolayers are very stable on mica, clean surfaces are easily obtainable by cleavage, and the surfaces are atomically smooth.
Roughness Effects This section examines the use of Formvarcovered and collodion-covered grids. It will be recalled that electron microscope grids have been inserted under a Formvar or collodion film, and the monolayer to be examined was deposited on this composite surface. The advantage of this technique, and presumably why it has been often employed in the past, is that it simplifies the steps involved in obtaining replicas for microscopic examination. Stearic acid monolayers were transferred at 9 and 17 mN/m onto Formvar-covered grids. The electron micrographs were similar to those shown in Fig. 2c and d for monolayers transferred onto smooth Formvar-covered substrates. Stearic acid monolayers transferred onto collodion-covered grids exhibit a similar behavior. Thus, we conclude that the Formvar-covered and collodion-covered grid surfaces do not have any significant effect on monolayer deposition over and above that of the smooth Formvar and collodion slides.
DPPC and Pentadecanoic Acid Monolayers
a Monolayer samples were transferred at 9 m N / m and duplicated. b Deposited monolayer exposed to vacuum for about 15 rain. Maximum vacuum was 4 × 10-5 m m Hg. c Molecular migration apparently occurred such that the local counting rates often increased on glass and Forrnvar when the deposited monolayers were aged under ambient conditions, thus masking the expected increase in film loss with time. Journal of Colloid and Interface Science, Vol. 109,No. 2, February 1986
The electron micrographs of DPPC monolayers are smooth, continuous, and homogeneous when compressed and transferred onto mica at surface pressures of 2 and 17 mN/m. DPPC, under the experimental conditions employed, is in the liquid-expanded state at 2 mN/m, whereas it is in the liquid-condensed state at 17 mN/m. DPPC monolayers, thus, are continuous and uniform in both the liquidexpanded phase (Fig. 5a) and the liquid-con-
MONOLAYER MICROSCOPIC STRUCTURE
395
FiG. 5, Electron micrographs of DPPC monolayers deposited on mica at (a) 2 m N / m and (b) 17 m N / m (×40,000).
densed phase (Fig. 5b). Even though DPPC was compressed to 31 m N / m - - a pressure much higher than its ESP--it is of interest to note that molecular aggregates were not observed in the micrographs. The electron micrographs of DPPC monolayers transferred onto mica did not show the marked microporosity observed by Ries et al. (10). However, if DPPC monolayers were transferred at 17 mN/m onto Formvar or collodion substrates, then structures similar to those observed by Ries and his co-workers were evident, presumably due to the transfer of a partial monolayer as discussed earlier (manuscript in preparation). For the sake of completeness, it should be mentioned that the structure of the transition region between the liquid-expanded and liquid-condensed phases was recently examined using electron microscopic techniques (23). Two-phase behavior was observed; this finding is consistent with the interpretation of a firstorder phase transition occurring between the liquid-expanded and liquid-condensed states in monomolecular films. Pentadecanoic acid monolayers showed similar trends as DPPC with the exception that the structural detail of the micrographs was much finer.
RELATIONSHIP TO PREVIOUS STUDIES
The results of past research on monomolecular films deposited on Formvar and collodion substrates must be reconsidered in view of our findings. Below a given surface pressure, slightly different for Formvar and collodion, a partial monolayer transfers onto these surfaces. This critical pressure was approximately 27 and 26 mN/m for the Formvar and collodion films, respectively, used in this study. Both Sheppard et al. (5) and Spink (6) probably prepared and observed partial monolayers on collodion, but incorrectly interpreted these structures as normal monolayers which they mimic. Neuman (8) did not observe partial monolayers in his earlier microscopic studies because they were conducted above the critical surface pressure for collodion. Our results confirm the pressure dependence of the transfer process first observed by Neuman (7) and also show the same phenomenon occurs on Formvar surfaces. We also were able to duplicate some of the results of Ries and his co-workers in this study. Discontinuous and heterogeneous films of stearic acid and DPPC were seen under the electron microscope when the monolayers were transferred onto Formvar and collodion Journal of Colloid and Interface Science, Vol. 109,No. 2, February1986
396
FERESHTEHKHOU, NEUMAN, AND OVALLE
substrates at intermediate (17 mN/m) surface pressures. However, we have shown that this is due to the monolayers transferring onto a low-energy solid surface rather than a highenergy surface such as mica. Pankhurst's concerns (27) that the island structures observed by Ries and Kimball were an experimental artifact introduced during the transfer process were justified. Berg's (22) effort to study the structure of monolayers generated results similar to those first published by Ries and Kimball (3, 4). The monolayer was probably disturbed prior to complete thermal fixation by either the shearing action of the cooling plug passing through the monolayer or the abrupt change in surface interactions due to the freezing of the subsolution. More attention should be given to eliminating or modifying steps which could cause monolayer artifacts with this procedure. The formation of molecular aggregates appears to be a more general phenomenon than realized with very important implications. These film aggregates may play an important role in the hysteresis behavior of spread monolayers. The physical nature of these interfacial aggregates and the conditions under which they form certainly warrant further investigation. The liquid-condensed state of stearic acid monolayers was examined microscopically over a wide range of surface pressures. The electron micrographs of the basic monolayer structure are smooth, continuous, and homogeneous when the stearic acid monolayers were compressed with piston oils (or compression barriers) and transferred onto mica substrates. In this respect, our findings agree with Sheppard et al. (5) who saw smooth surfaces on glass and Spink (6) who also observed featureless surfaces on glass and mica. In addition, we have shown that monolayers such as DPPC in the liquid-expanded (and liquidcondensed) state are smooth, continuous, and homogeneous. These results are in agreement with current theories on monolayer structure in that amphipathic molecules should form a single phase in these surface states. Journal of Colloid and Interface Science, Vol. 109, No. 2, February 1986
CONCLUSIONS
Experimental artifacts can be induced during the transfer of monomolecular films for observation by electron microscopy. Partial monolayers transfer onto Formvar and collodion surfaces (and, in general, low-energy solids) at low and intermediate surface pressures. At sufficiently high pressures, however, quantitative transfer takes place. Furthermore, deposited monolayers, e.g., stearic acid, are relatively unstable on these surfaces, and structural rearrangements may occur. On the other hand, when mica is employed, the artifacts previously observed appear to be eliminated. Indeed, a monolayer structure which is in agreement with current views on ordering in surface films can be obtained when the monolayer is compressed and transferred onto mica substrates by either the piston oil or compression barrier technique. ACKNOWLEDGMENTS This research was supported by the Office of Basic Energy Sciences, Division of Chemical Sciences, Department of Energy under Contract DE-AC02-81ER10859 and, in part, by the University of Minnesota Agricultural Experiment Station. A Graduate and Professional Opportunities Program (GPOP) Fellowship received by R. Ovalle is acknowledged. The authors also wish to thank M. Agrawal and T. Tompkins for their assistance in some monolayer experiments. REFERENCES 1. Langmnir, I., Trans. Faraday Soc. 15, 62 (1920). 2. Blodgett, K. B., J. Amer. Chem. Soc. 56, 495 (1934); 57, 1007 (1935). 3. Ries, H. E., Jr., and Kimball, W. A., aT.Phys. Chem. 59, 94 (1955). 4. Ries, H. E., Jr., and Kimball, W, A., "Proceedings Second International Congress on Surface Activity," London Vol. 1, p. 75, 1957. 5. Sheppard, E., Bronson, R. P., and Tcheurekdjian, N., J. ColloidSci. 19, 833 (1964); 20, 755 (1965). 6. Spink, J. A., J. Colloid Interface Sci. 23, 9 (1967). 7. Neuman, R. D., ar. Colloid Interface Sci. 50, 602 (1975). 8. Neuman, R. D., J. Colloid InteoCace Sci. 56, 505 (1976). 9. Ries, H. E., Jr., Matsumoto, M., Uyeda, N., and Suito, E., .I. Colloid Interface Sci. 57, 396 (1976).
MONOLAYER MICROSCOPIC STRUCTURE 10. Ries, H. E., Jr., Matsumoto, M., Uyeda, N., and Suito, E., Adv. Chem. Set. 144, 286 (1975). 11. Ries, H. E., Jr., and Swift, H., Jr. Cotloidlnterface Sci. 64, 111 (1978). 12. Harkins, W. D., and Fischer, E. K., J. Chem. Phys. 1, 852 (1933). 13. Middleton, S. R., and Pethica, B. A., Faraday Symposium 16, 109 (1981). 14. Cook, H. D., and Ries, H. E., Jr., J. Phys. Chem. 60, 1533 (1956). 15. Ries, H. E., Jr., Cook, H. D., and Loane, C. M., "Symposium of Steam Turbine Oils." ASTM Spec. Techn. Publ. No. 211, 55 (1957). 16. Ries, H. E., Jr., and Kimball, W. A., Nature (London) 181, 901 (1958). 17. Ries, H. E., Jr., and Walker, D. C., J. ColloidSci. 16, 361 (1961).
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18. Ries, H. E., Jr., Colloids Surf. 10, 283 (1984). 19. Ries, H. E., Jr., and Swift, H., J. Co[loid Interface Sci. 89, 245 (1982). 20. Ries, H. E., Jr., J. ColloidlnterfaceSci. 88, 298 (1982). 21. Ries, H. E., Jr., Nature (London) 281, 287 (1979). 22. Berg, J. C., in "Proceedings of the Workshop on Interfacial Phenomena: Research Needs and Priorities" (J. C. Berg, Ed.), p. 89, NTIS, U. S. Dept. Commerce, Springfield, IU. 1979. 23. Neuman, R. D., Fereshtehkhou, S., and Ovalle, R., J. Colloid lnterface Sci. 101, 309 (1984). 24. Neuman, R. D., J. Microscopy 105, 283 (1975). 25. Smith, R. D., and Berg, J. C., J. ColloM Interface Sci. 74, 273 (1980). 26. Smith, T., J. Colloid Interface Sci. 25, 443 (1967). 27. Pankhurst, K. G. A., J. Phys. Chem. 59, 480 (1955).
Journal of Colloid and Interface Scier~ce,Vol. 109, No. 2, February 1986