Atomic Force Microscopy of Emulsion Droplets: Probing Droplet

Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242. .... N. C. Woodward , A. P. Gunning , A. R. Mackie , P. J. Wilde a...
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Langmuir 2004, 20, 116-122

Atomic Force Microscopy of Emulsion Droplets: Probing Droplet-Droplet Interactions A. P. Gunning,* A. R. Mackie, P. J. Wilde, and V. J. Morris Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Received May 14, 2003. In Final Form: September 2, 2003 A method has been developed for attaching oil (tetradecane) droplets to the end of an atomic force microscopy (AFM) cantilever and for immobilizing droplets on a glass substrate. This approach has permitted the monitoring of droplet-droplet interactions in aqueous solution as a function of interdroplet separation. Coating the droplet surfaces with added proteins or surfactants has allowed the production of model emulsions. We demonstrate that AFM measurements of droplet deformability are sensitive to interfacial rheology by modifying the interfacial film on a pair of droplets in situ. For droplets coated with the anionic surfactant sodium dodecyl sulfate, screening of the double layer has been found to facilitate coalescence. Direct imaging of the droplets has revealed the presence of regularly spaced concentric rings on the droplet surfaces. Careful experimental studies suggest that these structures may be imaging artifacts and are not perturbations of the droplet surface determined by the composition of the interface.

Introduction The interactions between colloidal particles are important for determining the structure and stability of dispersed systems.1 The surface force apparatus (SFA) has made it possible to measure the interactions between solid surfaces in various liquids.2,3 However, the SFA is limited to measuring the interaction between flat surfaces or crossed cylinders in order to simulate the geometry of the interaction between a sphere and a flat surface.4 Since the advent of atomic force microscopy (AFM), another method has been developed which allows the interaction between single colloidal particles to be measured.5,6 This so-called “colloid probe technique” involves glueing a solid colloidal particle, usually a silica sphere, onto the end of an AFM cantilever. Most of these studies have concentrated on the interaction between rigid particles and a rigid flat surface6-9 or on the interactions between a pair of rigid colloidal particles.10 The interactions between one rigid and one deformable colloidal particle have also been explored. For example, the technique has been used to study the deformation of bubbles under liquid11,12 and of oil droplets in aqueous solutions.13,14 These experiments * Corresponding author. Tel: +44 1603 255201. Fax: +44 1603 507723. E-mail: [email protected]. (1) Gregory, J. Water Sci. Technol. 1993, 27, 1. (2) Tabor, D.; Winterton, R. H. S. Proc. R. Soc. London 1969, A312, 435. (3) Israelachvili, J. N.; Tabor, D. Proc. R. Soc. London 1972, A312, 435. (4) Claesson, P. M.; Ederth, T.; Bergeron, V.; Rutland, M. W. Adv. Colloid Interface Sci. 1996, 67, 119. (5) Ducker, W. A.; Senden, T. J.; Pashely, R. M. Nature 1991, 353, 239. (6) Ducker, W. A.; Senden, T. J.; Pashely, R. M. Langmuir 1992, 8, 1831. (7) Drummond, C.; Senden, T. Colloids Surf., A 1992, 87, 217. (8) Rabinovich, Y. I.; Yoon, R.-H. Colloids Surf., A 1994, 93, 263. (9) Larson, I.; Drummond, C. J.; Chan, D. Y. C.; Grieser, F. J. Phys. Chem. 1995, 99, 2114. (10) Li, Y. Q.; Tao, N. J.; Pan, J.; Garcia, A. A.; Lindsey, S. M. Langmuir 1993, 9, 637. (11) Butt, H.-J. J. Colloid Interface Sci. 1994, 166, 109. (12) Ducker, W. A.; Xu, Z.; Israelachvili, J. N. Langmuir 1994, 10, 3279. (13) Mulvaney, P.; Perera, J. M.; Biggs, S.; Grieser, F.; Stevens, G. W. J. Colloid Interface Sci. 1996, 183, 614. (14) Snyder, B. A.; Aston, D. E.; Berg, J. C. Langmuir 1997, 13, 590.

have been extended in order to allow the development of quite detailed models of the behavior of the deforming interface.15-18 In terms of complexity, perhaps the most challenging colloidal systems to understand at a fundamental level are those where the colloidal particles are deformable, as is the case for emulsions. An emulsion consists of two immiscible liquids where one phase is present as stabilized small droplets contained within a bulk phase of the other component. Coalescence of the droplets, a phenomenon that eventually leads to phase separation of the immiscible liquids, is prevented by the presence of surfactant or protein molecules, which coat the liquid-liquid interface and generate a repulsive interaction between the droplets. The interactions between the droplets will be moderated by the deformation of the droplets. The detail of the effect of interfacial structure on the interactions between deformable droplets that leads to their coalescence is still poorly understood. Understanding coalescence in emulsions is of both academic and industrial importance. This paper describes a method for attaching oil droplets to the end of AFM cantilevers under aqueous solutions. This has allowed the forces acting in a deformable colloidal system to be probed using an atomic force microscope. Although the deformable nature of the droplets makes complete calculation of force-distance curves difficult, due to the problem encountered in defining the point of contact (or perhaps more realistically for a deformable system, the threshold at which interaction begins) between droplets,15 the method does allow comparative measurements on real systems. That is, the interaction forces acting between two chemically identical droplets can be measured directly as a function of the properties of the bulk aqueous phase and the interfacial composition at the droplet surface. This “proof of principle” paper demonstrates the possibility of monitoring these interactions by AFM. The (15) Hartley, P. G.; Grieser, F.; Mulvaney, P.; Stevens, G. W. Langmuir 1999, 15, 7282. (16) Aston, D. E.; Berg, J. C. J. Colloid Interface Sci. 2001, 235, 162. (17) Aston, D. E.; Berg, J. C. Ind. Eng. Chem. Res. 2002, 41, 389. (18) Dagastine, R. R.; White, L. R. J. Colloid Interface Sci. 2002, 247, 310.

10.1021/la034835+ CCC: $27.50 © 2004 American Chemical Society Published on Web 11/25/2003

Atomic Force Microscopy of Emulsion Droplets

forces present in these systems will come from three main sources. Oil-water interfaces bear a significant negative zeta potential in aqueous liquids which will give rise to repulsive electrostatic interactions in the absence of added electrolyte.19 The second source of interaction between colloidal particles arises from hydrodynamics: when two colloidal particles are brought together in aqueous solution, there will be a liquid film between them which, if not given sufficient time to escape the closing gap, can exert a force on the particles even before they make contact. This effect has been studied in some detail by colloid probe AFM experiments on n-hexadecane drops and polystyrene or glass microspheres.16,17 For emulsion droplets, the size of the thin aqueous film and the rate at which it drains as the droplets are forced together are determined by both the charge on the droplet surfaces and the nature of the stabilizing interfacial film. If two colloidal particles which bear significant like charges are forced together, then the electrostatic repulsion between them means that the intervening aqueous film can be quite stable. The result for soft colloidal particles, such as oil droplets, is that deformation of the droplets occurs as they approach each other preventing thinning and rupture of the aqueous film, which in turn will prevent coalescence.15 The third important interaction in colloidal systems comes from attractive van der Waals forces, although these only become significant at very short distances. If the distance between two colloidal particle surfaces can be made small enough, then the van der Waals forces may overcome any repulsive electrostatic or steric interactions and will cause rupture of the intervening aqueous film. If this occurs in an emulsion, the result will be coalescence. Experimental Section Materials. n-Tetradecane was obtained from Sigma Chemicals, Poole, U.K. (T-4633; purity, minimum 99%), and was used without further purification. Ultrapure water was used throughout this study (surface tension γ ) 72.6 N m-1 at 20 °C; conductivity, 18 MΩ; Elga, High Wycombe, U.K.). The nonionic water-soluble surfactant Tween-20 (polyoxyethylene sorbitan monolaurate) was obtained from Pierce (Surfactamps-20; Pierce, Rockford, IL) as a 10% w/v solution in water. Aliquots of the Tween-20 solutions were added to the liquid cell in order to achieve the concentrations described in the text. The milk whey protein β-lactoglobulin (L-0130, Sigma Chemicals) was dissolved to a concentration of 100 µM in water, and an aliquot of this solution was added to the liquid cell of the atomic force microscope in order to achieve the desired concentration. Sodium dodecyl sulfate (SDS) and sodium chloride were obtained from Sigma Chemicals and dissolved to stock concentrations of 10% w/w. Aliquots of these solutions were added to the liquid cell in order to achieve the desired concentrations for the coalescence experiments of 1 mM SDS and 40 mM NaCl. Dynospheres were obtained from Agar Scientific (diameter, 20.1 µm ( 0.87%; Cambridge, U.K.). Instrumentation. The atomic force microscope used in this study was a TM Lumina (Veeco Inc., U.S.A.). This consists of an AFM head mounted above an inverted optical microscope (Olympus IX-70). The AFM head is fitted with a small CCD camera that views the sample from one side, which, in combination with the liquid scanner, permits observation of the sample under liquid. For the studies of the variation of force with distance between oil droplets, oil droplets were attached to cantilevers without tips (200 µm long thin variety of Nanoprobe NP0; Veeco Instruments Inc.) in order to ensure that the measurements represented droplet-droplet interactions and not interactions between a droplet and a protruding apex of a silicon nitride tip. For imaging purposes, the complementary lever with a tip attached (200 µm long thin variety of NPS; Veeco) was used unless otherwise stated. The atomic force microscope was (19) Anderson, P. J. Trans. Faraday Soc. 1959, 55, 1421.

Langmuir, Vol. 20, No. 1, 2004 117 operated in contact mode, and the scan rates were typically 1-2 Hz. Approach and retract speeds during force versus distance cycles were 0.5 µm s-1. Both cantilever types have quoted typical force constants of 0.06 N m-1. All AFM data presented were captured under liquid (water or buffer). Preparation of Oil Droplets. An ad hoc “sprayer” was used to prepare tetradecane droplets in the following manner: The tetradecane was sucked from a small reservoir into a 200 µL pipet (Gilson). A pipet tip, connected to an air line, was held at 90° to the aperture of the oil-filled pipet tip in order to create a fine spray of tetradecane droplets which was directed over a clean, dry glass microscope slide (BDH SuperFrost). It was found that the best method was to direct the mist over the top of the slide, which was placed approximately 10 cm from the sprayer tip, and to allow some of the droplets to fall onto the slide. This resulted in a fairly uniform coverage of the slide with discrete 10-50 µm diameter oil droplets. Pointing the sprayer tip directly at the glass slide simply led to coalescence of the droplets on the glass slide, producing a thin film of oil. Glass surfaces are hydrophilic, and the oil droplets were easily displaced when water was placed on top of them prior to AFM measurements. The technique used for wetting the glass was found to be critical. A small volume of water was squeezed out of the tip of a Gilson pipet, held approximately 3-4 mm above the glass surface, to form a drop hanging on the end of the pipet tip. The drop was then made to fall onto the glass slide by squeezing a fraction more water out of the pipet. The majority of the oil drops on the slide were displaced during the addition of water, and they formed a film of tetradecane on the water surface, together with some free floating oil droplets. However, some oil droplets remained attached to the glass slide (typically 10-15). The adherent droplets were generally found to be positioned in the middle of the wetted region of the glass slide. These droplets could be distinguished from floating oil patches on the water surface by their more spherical shape and by their lack of mobility in the visual field when the microscope was tapped. The AFM head was then lowered down onto the slide in order to sandwich the water between the liquid scanner and the glass slide. The remainder of the measured volume of water in the pipet was added dropwise onto the side of the liquid cell. This avoided the flow and shear effects associated with discharging the pipet directly into the sandwiched drop of water, which was found to displace the attached oil drops from the glass slide. Addition of the various surfactant and polymer solutions was carried out in the same manner at appropriate times during the course of the experiments. Attachment of Oil Droplets onto the AFM Cantilever. The AFM head was positioned using lateral micrometers so that the end of the tip-less cantilever sat directly above a droplet on the surface of the glass slide. The cantilever was then driven down into the target droplet using the fine approach motor on the atomic force microscope. Engulfment of the lever into the drop was observed via the instruments’ onboard camera. Once engulfment had been achieved, the AFM head and cantilever were retracted away from the surface of the slide, whereupon the oil droplet was found to detach itself from the slide surface. This method allowed the droplets to be placed with high precision at the end of the cantilever. Droplets attached to the cantilevers were typically in the size range of 20-60 µm. Once droplets had been attached to the cantilever, then surfactant or protein, required to stabilize the droplet, was added to the liquid cell of the atomic force microscope. It was found that this had to be done afterward; otherwise droplet attachment to the cantilever was found to be impossible, as the cantilever could not be made to penetrate the droplets. Droplet transfer to cantilevers was found to be only possible for oil droplets that had been deposited onto glass slides. For more hydrophobic surfaces, such as polystyrene (plastic culture dishes), the droplets were anchored too firmly to the substrate to allow transfer to the cantilever. The cantilever consists of silicon nitride which is nominally hydrophilic, although presumably less so than the glass surface. This explains why the oil droplets can be transferred onto the cantilevers from a glass surface. In fact, there is a delicate balance involved; if the cantilever was too hydrophobic, then the oil droplets would simply wet onto the lever rather than form a drop.

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Figure 1. Optical micrographs illustrating the attachment of oil droplets to a tip-less AFM cantilever under water. (a) Engulfment of the cantilever into a tetradecane drop. (b) The cantilever pulled away from the glass surface with an attached oil droplet (note change of focus). (c) Relocation of the cantilever with an attached droplet. Monitoring “Force-Distance” Interactions. The cantilever with the attached oil droplet was positioned directly over a second droplet, which was still attached to the glass slide, and the variation of the interaction force with separation was monitored and recorded. Pairs of droplets of approximately equal size were chosen for the measurements described in this study. Cantilever deflection is quantified by the photodiode detector of the atomic force microscope in terms of electrical output. This was converted into real distances by determination of the inverse optical lever sensitivity (Invols) of the particular lever used in each measurement.20 This involved performing a force-distance curve on the rigid surface of the glass slide using the bare cantilever. Since there is no sample deformation in this case, the gradient for the resulting cantilever deflection versus distance graph provides a scale factor for the conversion. Finally the data were scaled to a nominal point of zero cantilever deflection; this was defined as the point of furthest droplet separation distance in each graph. Droplet Imaging. Droplets were prepared by spraying tetradecane onto the surface of plastic culture dishes (polystyrene). The exact procedure for the addition of water is not as critical in this case, due to the much stronger adhesion of the tetradecane droplets onto the hydrophobic polystyrene surface. The force set point of the atomic force microscope was kept to minimal values (i.e., typically 5 nA above the null point of the cantilever) to prevent engulfment of the cantilever into the droplet. On uncoated oil droplets, the tip was quite frequently “sucked into” the droplet, particularly when attempting to obtain large scans, where the tip was forced to traverse the entire drop. It was found to be impossible to image tetradecane droplets attached to glass with a standard tipped cantilever; the scanning process simply swept the droplets away. However, it was found to be possible to capture AFM images of oil droplets attached to glass by using a tip-less cantilever that had a tetradecane droplet attached in place of the tip. The fact that it was possible to image the weakly attached drops on glass is interesting: This is presumably because deformation of the droplets acted to reduce the lateral forces on the droplets during scanning. Although image resolution is compromised, this approach may have application in the imaging of extremely delicate samples in water. Furthermore, attaching smaller oil drops to the cantilever could reduce losses in image resolution, although this will inevitably involve a tradeoff in terms of drop deformability.

Results Figure 1a-c shows a series of video images captured through the 10× objective of the Olympus microscope, illustrating the process of engulfment of a tip-less AFM cantilever by a tetradecane droplet, and the subsequent (20) Meyer, G.; Amer, N. M. Appl. Phys. Lett. 1988, 53, 1045.

Figure 2. Deflection versus distance graphs. A tip-less cantilever driven toward a glass surface (dotted line). The cantilever with a tetradecane droplet attached driven toward a glass surface (solid line). The cantilever with droplet attached driven toward a second tetradecane droplet attached to a glass surface (gray line).

transfer of the droplet onto the end of the cantilever. In the first image in the series (Figure 1a), the initial stage of engulfment of the cantilever into an oil droplet on the glass surface is shown. Note that the cantilever and several other droplets, which are attached to the glass, are in focus. Several other blurred features can be seen in the image; these are oil drops which have detached during the addition of the water to the glass slide and are floating on the surface of the water. The second image in the series (Figure 1b) shows that the droplet has remained attached to the lever as it was pulled away from the glass; the image is focused on the cantilever, and hence those drops that are still attached to the glass surface are now out of focus. The third image in the series (Figure 1c) demonstrates that the attached droplet is stable and that the cantilever can be repositioned and used to monitor droplet-droplet interactions. A video of the entire attachment process is available from the authors. Figure 2 shows a series of curves monitoring cantilever deflection versus distance for a tip-less cantilever and the same cantilever containing an attached droplet of tetradecane. The dotted line illustrates the interaction between a tip-less cantilever and the surface of the glass slide. An oil drop was then attached to this cantilever using the procedure described above. The oil drop on the cantilever (and any others in the imaging cell) was then coated with the protein β-lactoglobulin, by adding the protein to the surrounding water to produce a bulk concentration of 24 µM and allowing an interfacial protein film to form. The cantilever containing the attached, protein-coated oil droplet was then driven in toward the

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Figure 3. The effect of droplet-droplet approach speed on the magnitude of the gradient of the measured cantilever deflection versus distance curve, for a pair of uncoated tetradecane drops in water.

surface of the glass slide (Figure 2, solid black line). Finally, the oil drop-cantilever assembly was retracted from the glass surface and then positioned directly over a second tetradecane droplet (which would be expected have an identical interfacial protein coat) attached to the glass surface, and a “deflection-distance” measurement was performed (Figure 2, solid gray line). The most obvious feature of the data displayed in Figure 2 is the difference in gradient of the three curves in the region of constant compliance. It is suggested that these differences are largely determined by the relative degrees of deformation of the oil droplets. In this case, the largest degree of deformation would be expected when two droplets interact. The largest deformation would correspond to the shallowest gradient in the deflection versus distance data, and it is seen that this occurs when the two protein-coated tetradecane droplets were forced together. The experimental curves were found to be highly reproducible, suggesting that the oil droplets remained in their respective positions, attached to the cantilever and glass surface, throughout the measurements. This was later verified by retracting the cantilever and visually checking that neither droplet had become detached. The effect that the speed of approach has on the measured interaction between pairs of droplets was examined in order to rule out the possibility that the AFM data simply reflected hydrodynamic effects and little else. A graph of the gradient of cantilever deflection versus the approach speed between pairs of uncoated tetradecane droplets in water is shown in Figure 3. For the range of approach speeds of 0.01-2 µm s-1 used, the data obtained show that any changes in the measured gradient fall within the range of instrumental error and are therefore insignificant. Since all of the AFM measurements presented in this study were within this range of approach speeds (all measurements were made at 0.5 µm s-1), the present experiments will not be limited by hydrodynamic effects. Indeed, simple order of magnitude calculations show that both the fluid inertia and the drag force from the fluid motion can be neglected in these experiments, as they will generate forces in the pN range. The deformability of the droplets may be affected by different factors including droplet viscosity, interfacial tension, and interfacial rheology. The droplet viscosity is the same in all of these systems, so it can be discounted. Interfacial tension is, by definition, the surface free energy required to increase the surface area by a unit amount. Therefore reducing the interfacial tension will allow the surface area to be increased more easily; that is, the droplet becomes more deformable. The radius (r) of a droplet and its excess internal pressure (∆P) are therefore related to the interfacial tension (γ) according to Laplace’s law:

∆P )

2γ r

(1)

Figure 4. Effect of interfacial rheology on droplet deformation. Each data point represents the modulus of the gradient of the constant compliance region for AFM deflection vs distance measurements with time, for a pair of tetradecane droplets with varying interfacial composition. The droplets were initially coated with β-lactoglobulin, and then Tween-20 was added at the time points marked by arrows: (a) 2 µM (b) 4 µM, (c) 6 µM, and (d) 9 µM. Note: These data were not scaled using the Invols conversion factor since only the changes in cantilever deflection gradient were needed.

Droplet deformability may also be modified by changing the interfacial elasticity. The interfacial elasticity is, by definition, the restoring force of the interfacial layer that will resist a change in area and hence deformation of the interface. Both of the above factors may act to modify the cantilever displacement versus distance curves, but the data shown in Figure 4 suggest that the interfacial rheological factor appears to be dominant for the present system. Figure 4 charts changes in the gradient of the constant compliance region of deflection-distance curves obtained for two tetradecane droplets forced together sequentially as their interfacial composition was varied. The first data point, at time zero, represents a proteinstabilized system. The measurement was taken 30 min after addition of the milk protein β-lactoglobulin to the liquid cell to produce a bulk concentration of 2 µM. The two subsequent data points track changes in the gradients of deflection versus distance curves in the region of constant compliance obtained for the system at later time points. The notable feature of the data is that the modulus of the gradient is increasing with time (in fact all of the recorded gradients were negative, but we have plotted the modulus for clarity). It is known from previous studies that adsorption of β-lactoglobulin to a tetradecane-water interface will alter the interfacial tension and elasticity over a period of tens of minutes to hours.21 However, the changes introduced by these two contributions are opposite in character; while interfacial tension drops, interfacial elasticity increases over this period of time. The data shown in the first three points (after time zero) in Figure 4 demonstrate that the trend for the AFM measurements, obtained on the protein-coated droplets, correlates with the changes in interfacial elasticity of the system, since the increasing magnitude of the gradient values indicates that the droplets are becoming less deformable. This would not be the case if the only factor at play in the system was the lowering of interfacial tension as, in the absence of any other changes, this would act to reduce the surface free energy of the droplets and hence increase their deformability as discussed above. Further evidence that interfacial elasticity dominates the AFM measurements of drop deformability was provided by changing the nature of the interfacial film. Low molecular weight surfactants compete for space at interfaces with proteins.21 If the surfactant is present in sufficient concentration in the bulk, it will displace the interfacial protein film completely, given sufficient time to colonize the interface.21 This has (21) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 2242.

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provided a means of dramatically altering the interface on the tetradecane droplets, from a strongly elastic system, in the case of the protein-coated interface, to a much less elastic system after the protein at the interface has been replaced by a low molecular weight surfactant. The nonionic surfactant Tween-20 was added to the liquid cell in order to produce a bulk concentration of 2 µM (the time of addition is indicated by arrow a in Figure 4). The effect of adding Tween 20 was to reduce the magnitude of the gradient value for the subsequent deflection versus distance measurements. With time, the gradient value reduces in magnitude still further until a pseudoplateau is reached after approximately 5000 s. Beyond these times, further additions of surfactant (arrows b-d) did not significantly reduce the magnitude of the gradient. This is despite the fact that the addition of surfactant will have further reduced the interfacial tension (since the concentration of Tween-20 is well below its cmc value of 59 µM). Taken together, the data shown in Figure 4 provide compelling evidence that the AFM measurements of droplet deformability, presented here, predominantly reflect the interfacial elasticity of the droplets and do not simply correlate with changes in the interfacial tension as previously assumed.22 However, two other factors may influence these data. The first is the effect that a small change in droplet shape may have on the measurements. Both droplets are attached to a surface (glass or silicon nitride), and a lowering of the interfacial tension will result in a change of contact angle for the droplets and hence a change in shape. The second factor which will vary with interfacial structure is the behavior of the thin film of aqueous liquid present between the droplets. The drainage of this film, as the droplets are brought together, is influenced by the mobility of the interface. For surfactants which form highly mobile interfacial films, drainage occurs quickly and evenly. Protein-stabilized interfaces are essentially immobile, and the drainage of the intervening aqueous film is slowed by drag and will occur unevenly.23 Drainage is faster at the edges of two approaching oil droplets, resulting in a region of trapped water left in the central region between the droplets, which forms a dimple that drains away much more slowly.23 However, the correlation between the present deformability measurements and interfacial elasticity is consistent with the conclusions of a previous AFM study on the deformation of an SDS-coated drop of n-decane by a silica sphere.15 Manipulation of the electrostatic interactions between droplets is also amenable to study using the present method. We have examined the effect of adding additional electrolyte and thus additional counterions to a system comprising a pair of tetradecane droplets coated with the anionic surfactant SDS. The data shown in Figure 5a are those obtained for the pair of droplets forced together in a solution of 1 mM SDS. These data reveal that the droplets could not be forced to coalesce under those conditions. The stabilizing effect of SDS is due to the electrostatic repulsion between the like charges at the SDS-coated droplet surfaces. At an SDS concentration of 1 mM, the experiment was conducted below the critical micelle concentration (cmc) of SDS (2.3 mM). This system will have a Debye length of 5.6 nm. If additional counterions are introduced into the solution, then the charges on the droplet surfaces are screened, reducing the size and extent (22) Attard, P.; Miklavcic, S. J. Langmuir 2001, 17, 8217. (23) Clark, D. C.; Coke, M.; Wilde, P. J.; Wilson, D. R. Molecular diffusion at interfaces and its relation to disperse phase stability. In Food Polymers, Gels and Colloids; Dickinson, E., Ed.; Royal Society Special Publication No. 82; Royal Society of Chemistry: Cambridge, 1991; pp 272-278.

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Figure 5. Droplet coalescence: effects of screening. Deflection versus distance data representing the interaction between two tetradecane droplets. Approach (solid line) curves and retraction (gray line) curves measured in the presence of (a) 1 mM SDS and (b) 1 mM SDS, 40 mM NaCl.

of the barrier to close approach of the droplets. By increasing the screening of the surface charge, the droplets should be able to approach more closely, thus raising the chances of coalescence. The data shown in Figure 5b were obtained for two tetradecane droplets forced together in a solution of 1 mM SDS containing 40 mM NaCl. Under these conditions, the Debye length is reduced to 1.52 nm. The approach portion of the deflection versus distance curve initially climbs, once the region of constant compliance has been reached as before, but at a piezo extension distance of approximately -1000 nm the curve drops abruptly to the baseline level. The retraction part of the curve does not retrace the approach curve, as normally occurs, but simply follows the baseline, confirming that the drops have coalesced during the measurement. In principle, the experiment could be repeated with an even higher concentration of counterions present, further enhancing the prospect of coalescence. In practice, however, it was found to be difficult to monitor the interdroplet interactions at higher salt levels because the oil drops would coalesce as soon as feedback was established, before any measurements could be obtained. If we return to Figure 2, a second more subtle feature of the data is the difference in the interaction range of the onset of lever deflection. In the case of the bare lever, shown by the dotted line, the turning point is relatively sharp by comparison with the data obtained on the oil droplets. The longer range required to reach constant compliance seen in the other two curves is due principally to the deformable nature of the droplets, making longrange effects, such as electrostatic interaction between the droplets, difficult to resolve. This is particularly true for the approach part of the measurement where weak repulsive forces may well be impossible to detect, since they might simply lead to a small deformation of the oil drop with no measurable deflection of the cantilever.15 Weak attractive forces between the droplets may well also be masked upon approach for the same reason. This makes it difficult to define the actual point of contact between the oil drops.15 In fact, definition of a point of contact between deformable particles is a rather moot point since,

Atomic Force Microscopy of Emulsion Droplets

Figure 6. AFM images of a tetradecane droplet in water. (a) Topography image; scan size, 80 × 80 µm. (b) Error-signal mode image; scan size, 20 × 20 µm.

for repulsive interactions, it is possible that the droplets never come into contact; the particles could simply flatten while remaining at a finite separation. Imaging Oil Drops. In addition to studying the interaction between oil droplets, some preliminary experiments on imaging their surfaces were carried out. Figure 6a shows a typical AFM image of a droplet of tetradecane attached to a polystyrene surface in water. It was found that the attachment to a plastic substrate was necessary for imaging tetradecane droplets with a normal cantilever, as the scanning process swept away droplets attached to glass surfaces. The surface of the droplet appears smooth and featureless in this lowmagnification topography image. However, upon closer examination of the droplet surfaces, all were found to display a series of concentric rings, and a typical example of this is shown in the error signal image presented in Figure 6b. This sort of effect has been observed previously by others on the surface of an air bubble imaged under liquid.24 The pattern of rings was tentatively attributed to standing waves generated from the capillary waves which will be present at the surface of a deformable sphere (24) Knebel, D.; Sieber, M.; Reichelt, R.; Galla, H.-J.; Amrein, M. Biophys. J. 2002, 82, 474.

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Figure 7. AFM images. (a) Error-signal image of a 20 µm latex particle scanned in air; scan size, 9.95 × 9.95 µm. (b) Error-signal image of a tetradecane droplet in water imaged with an oil-drop cantilever assembly; scan size, 20 × 20 µm.

in liquid, since the calculated frequency of the capillary waves themselves was beyond the bandwidth of a typical AFM measurement.24 The frequency of capillary waves is proportional to the interfacial tension. Therefore, changes in interfacial tension should affect the spacing of the concentric rings seen in the images if they arise from capillary waves. Measurements of the distance between the concentric rings in AFM images obtained on a tetradecane droplet whose interfacial tension was altered by the addition of the surfactant Tween-20 produced very similar values of around 1 micron before and after addition of surfactant. Furthermore, this peak-to-peak spacing value was found to be sample invariant; it was reproduced in several different AFM images of droplets of different size, and drop dimensions ought to affect the frequency of capillary waves. This suggests that the concentric circles seen in the AFM images of the oil droplets are not due to capillary waves. Another possibility is that they represent some form of standing wave pattern induced by the perturbative action of the AFM tip. The AFM image in Figure 7 seems to discount even this possibility. Figure 7a is an AFM image of a solid latex particle of similar

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dimensions to the oil droplets studied here (20 µm). Although the surface of the latex particle is rougher than the surface of the oil droplets, which slightly masks the more subtle details, a pattern of concentric rings is visible in the image. These cannot arise due to standing waves as the latex particle is solid. Finally, the AFM image presented in Figure 7b shows a droplet of tetradecane attached to a glass surface, which has been imaged with an “oil-drop cantilever” (a tip-less cantilever with an attached oil drop). Once again, the familiar pattern of concentric rings is visible. This latter example demonstrates that the concentric ring pattern cannot arise from local differences in the topography of the droplet surface, because the contact area between the two oil drops will be very large. Since the oil droplet which was attached to the cantilever effectively acted as the AFM tip in the scan, this places a fundamental limitation on the image resolution; laterally it cannot be less than the contact area between the oil drops. Taken together, these observations suggest that the ring pattern seen in the AFM images of the oil droplets is more likely to be some form of imaging artifact. One possibility is that it may be caused by the assignment of height levels to a highly curved surface. There is a second and more likely possibility that may account for the concentric ring patterns seen in the AFM images. They might arise from optical interference effects; some of the laser light which spills over the edges of the AFM cantilever will be reflected by the sample, and a proportion of this will hit the photodiode detector. For flat surfaces, such “stray” reflection can give rise to predictable low-frequency undulation of the image background for large scans, but when the sample surface is highly curved the effect is less easy to predict. This scenario may also explain the apparent periodic features seen on some of the deflection versus distance curves, which incidentally have also been attributed to capillary waves in early studies.13 These obviously do not arise from plane fitting

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errors as the data are unprocessed in a deflection versus distance graph. It was found that these periodic features were often present in the cantilever deflection versus distance data obtained from one set of droplets in a given system but then completely absent for data collected on a different pair in the same experiment. Conclusions A method has been developed for attaching oil droplets to tip-less AFM cantilevers. These assemblies have been used to monitor interactions between protein-coated droplets attached to the cantilever and protein-coated droplets attached to flat glass surfaces. Differences in the interaction between droplets and interactions between a droplet and the flat glass surface have been attributed to deformation of the droplets. Furthermore, by changing the interfacial film on a pair of droplets in situ, data have been obtained which demonstrate that the deformation measurements are sensitive to interfacial rheology. The procedures developed for measuring droplet interactions have been used to monitor coalescence of the droplets. For droplets coated with the ionic surfactant SDS, screening of the electrical double layer has been found to promote coalescence. Finally, AFM images have been obtained of the droplets that exhibit concentric ring patterns. Similar patterns have also been observed on solid latex particles of similar dimensions, suggesting that the origin of the rings may lie in imaging artifacts, rather than standing capillary wave structures. Acknowledgment. The authors thank Gary Barker, David Hibberd, Andrew Kirby, Andrew Watson and Geoff Brownsey for helpful discussions. This research was supported by the BBSRC through its core strategic grant to IFR. LA034835+