Dynamic forces between emulsified water drops coated with Poly-Glycerol-Poly-Ricinoleate (PGPR) in canola oil

Accepted Manuscript Dynamic forces between emulsified water drops coated with Poly-GlycerolPoly-Ricinoleate (PGPR) in Canola oil Srinivas Mettu, Chu Wu, Raymond R. Dagastine PII: DOI: Reference:

S0021-9797(18)30122-X https://doi.org/10.1016/j.jcis.2018.01.104 YJCIS 23262

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

28 September 2017 29 January 2018 29 January 2018

Please cite this article as: S. Mettu, C. Wu, R.R. Dagastine, Dynamic forces between emulsified water drops coated with Poly-Glycerol-Poly-Ricinoleate (PGPR) in Canola oil, Journal of Colloid and Interface Science (2018), doi: https://doi.org/10.1016/j.jcis.2018.01.104

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Dynamic forces between emulsified water drops coated with PolyGlycerol-Poly-Ricinoleate (PGPR) in Canola oil Srinivas Mettu1,2, Chu Wu1,3, and Raymond R. Dagastine*1,2 1 Particulate Fluids Processing Centre (PFPC), 2 Department of Chemical Engineering 3 Department of Mathematics and Statistics, The University of Melbourne, Parkville VIC-3010, Australia. *Corresponding author: [email protected] Department of Chemical Engineering, The University of Melbourne, Parkville VIC-3010, Australia. Graphical abstract

Abstract The dynamic collision of emulsified water drops in the presence of non-ionic surfactants plays a crucial role in many practical applications. Interaction force between water drops coated with non-ionic food grade surfactants is expected to exhibit rich dynamic behavior that is not yet explored. The collision forces between immobilized water drops in canola oil in the presence of a well-known food grade surfactant polyglycerol polyricinoleate (PGPR) are measured at concentrations well below typically used to form stable emulsions. An extension or kink, attributed to a short-range attractive interaction due to PGPR bridging between the drops, was observed in the retract portion of the force curves at higher applied forces or slower collision velocities. The Stokes-Reynolds-Young-Laplace (SRYL) model was used to calculate theoretical force curves. For higher collisions velocities, the agreement between the calculated and experiment data was acceptable, but the SRYL model failed to describe the extension or kink feature observed at slower velocities below. Both the AFM data and the

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comparison to the model calculation indicated the presence of a short-range attractive force, not of a hydrodynamic origin, attributed to the bridging and extension of PGPR molecules on the surface of water drops below saturation of the interface. Key Words: PGPR, Interfacial Tension, Adsorption Isotherm, Water-in-Oil Emulsion Stability, Atomic Force Microscopy (AFM), Steric Force.

1. Introduction The stability of emulsions plays an important role in the formulation of food products such as mayonnaise, salad dressing, ice cream, butter and margarine [1]. Food emulsions are often classified as Oil-in-Water (O/W) emulsions with a continuous aqueous phase, such as mayonnaise, salad dressing and ice cream, or Water-in-Oil (W/O) emulsions with a continuous oil phase, such as butter and margarine. Food emulsions are generally made by addition of external energy to a two-phase system through homogenization and high shear mixing. In the absence of stabilizing surfactants, the emulsions tend to phase separate to reach thermodynamic equilibrium[2] via a number of mechanisms including Ostwald ripening[3], creaming or settling, flocculation and coalescence through drop collisions. Emulsion drops are stabilized against coalescence by the addition of a surfactant or a mixture of surfactants often in combination with additives to control the viscosity, microstructure and rheology of the continuous phase to achieve a long-term shelf life [1]. The amphiphilic nature of the surfactant can be classified by a hydrophilic head group type or a lyophobic tail group composition, but a more general approach for practitioners in choosing food grade surfactants for either O/W or W/O emulsions is based on the hydrophilic-lipophilic balance (HLB). The HLB number[4] of food grade surfactants typically varies from 1 to 20. Low HLB (<6) surfactants are more soluble in

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oil hence they favor the formation of W/O emulsions whereas high HLB (>8) surfactants are more soluble in water and hence favor the formation of O/W emulsions[5]. Examples of low HLB surfactants are polyglycerol polyricinoleate (PGPR), fatty acid esters and sorbitol fatty acid esters[5]. Typical high HLB surfactants are phospholipids (Lecithin), proteins, sodium and potassium acid salts of alcohols[5]. In the food industry, due to stringent regulations, the choice of surfactants is very limited. Unlike the personal care products and detergent industries where the use of ionic surfactants is wide spread, the food industry relies on few non-ionic plant or animal derived surfactants. Ionic surfactants present in detergents and cosmetics provide stability against coalescence of emulsion through electrostatic double layer repulsive forces. Whereas, non-ionic surfactants readily adsorb onto the oil-water interface giving rise to stability against the coalescence of emulsion drops through a steric repulsive force. Traditionally, the stability of emulsions as function of time tracked by either video microscopy or light scattering measures of drop size distributions. However, an inherent drawback of these tests is that an instantaneous or direct measure of the stability between individual emulsion droplets must be inferred. Another drawback is that the fundamental understanding of the relative contribution of different surface forces, such as electrostatic and steric repulsion, to stability cannot be measured creating challenges in surfactant selection and concentration. Recently developed experimental techniques[6],[7] ,[8] ,[9],[10-13] with Atomic Force Microscope (AFM) overcome such limitations. AFM can be used to directly measure the interaction forces between colliding emulsion drops in presence of various surfactants[6], [8] and salt solutions[14],[15]. The theoretical analysis[16, 17],[18],[19],[20],[21] that incorporates the interplay of surfaces forces, deformation of

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drops and drainage of liquid between them has been used to analyze the experimental data. Such an analysis helps to obtain a fundamental understanding of the forces acting either to stabilize or destabilize the emulsions. The theory is well developed and been validated with experiments in a large number of scenarios[22], including using independent measures of the drop separation by different research groups[23],[12]. Thus far, almost all direct force measurement studies between drops have been used to study O/W emulsions with the exception of two studies that are not in the food area. The first by Vakarelski et al. [24] where they examined the anomalous coalescence of water drops in alkane liquids in the absence of stabilizers and a study with toluene as the continuous phase[25]. Food systems have been studied using this AFM method over the last ten years, led by the group of Gunning and Wilde, in water continuous systems[9],[10]. The direct force measurements between oil drops in the presence of biopolymers as SBP[9] (Sugar Beet Pectin) or coated with other food proteins[10] have been studied. In contrast to well-studied water continuous emulsion systems, in the current study, the AFM direct force measurement method has been extended to oil continuous food emulsions. In our study, water drops are emulsified in canola oil phase in the presence of an oil soluble surfactant. This work is timely as W/O emulsions are in a great focus in personal care products, cosmetics[26],[27] as well as for fat replacement in fat continuous processed foods to reduce the cost as well as calorie content[28],[29],[30],[31-33],[34]. Encapsulation of water soluble active ingredients, flavors or and nutrients can be achieved through W/O emulsions as well. In these applications, controlling the stability of the emulsion is crucial. To understand the emulsifying properties of what is one of the most popular food grade surfactants, PGPR

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(Poly-Glycerol-Poly-Ricinoleate), we measured the repulsive and attractive dynamic interaction forces between immobilized water drops coated with PGPR in canola oil, schematically shown in figure 1. PGPR is a fat soluble non-ionic surfactant widely used in processed foods with a hydrophilic-lipophilic balance (HLB) number between 2 and 4, making it a common choice for stabilizing water-in-oil in food emulsions. It is manufactured by the esterification of condensed castor oil fatty acids with polyglycerol[35] and it is digested in a similar manner to fatty acids from vegetable oils[35]. PGPR has a polyglycerol hydrophilic section and polyricinoleate hydrophobic section[26] where, when present in water-in-oil emulsions, PGPR adsorbs at the water-oil interface by orienting the polyglycerol hydrophilic section into the water phase and extending the polyricinoleate hydrophobic chain into oil phase[26]. The macroscopic effect of PGPR concentration on the overall stability of W/O emulsions is very well characterized in literature[36-45] as well as in Water-in-Oil-in-Water (W/O/W) double emulsions[46]. Yet, emulsion studies often require stable emulsions for characterization, whereas the AFM methods developed to probe the interaction between micro-droplets allow for the study of both the attractive and repulsive interactions. The AFM method can be used to study both the stable emulsions at high surfactant concentrations and, meta-stable/unstable emulsions at low surfactant concentrations. This approach provides insights into the mechanism of stability as well as the causes of coalescence as a function of solution concentration of PGPR and surface adsorption.

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Figure 1. Side view of a schematic (not to scale) of the measurement of the interaction forces between water drops coated with PGPR in canola oil using atomic force microscopy (a), a water drop picked up by custom made rectangular hydrophilic cantilever and water drop immobilized on hydrophobic gold coated glass surface, (b), (c), where the radii of the drops on cantilever and surface were 45 and 56 μm, a schematic of the thin film between colliding water drops in canola oil with adsorbed PGPR at interface (not to scale) (d).

2. Materials and methods 2.1

Materials Poly-Glycerol-Poly-Ricinoleate (PGPR-4125 manufactured by Palsgaard Inc

(Denmark)) was donated by Hawkins Watts Inc (Mulgrave, Australia) and Mondelez Inc (Ringwood, Australia). Canola oil (home brand) from Woolworths Inc, Australia was used as recieved. Decane-thiol and MUA (11-Mercapto-Undecanoic-Acid) are used as received from Sigma-Aldrich. Sodium Nitrate (Analytical Reagent grade 99%) was obtained from Ajax fine chemicals and used without further purification. All glassware used in the experiments was cleaned using Ajax detergent, soaking in Nitric Acid followed cleaning in base (NaOH), rinsed thoroughly with Milli-Q water and dried.

2.2

Interfacial Tension

The interfacial tension of canola oil (containing various amounts of PGPR) and water was measured via pendant drop tensiometry using a Dataphysics OCA20 tensiometer. A rectangular quartz cuvette is first filled with Milli-Q water. Then, a glass syringe

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(Hamilton) with custom made upward bent needle filled with pure canola oil was positioned inside water in quartz cuvette. A drop of oil was slowly grown inside water by driving the syringe. The profile of oil drop was continuously monitored by image acquisition and profile detection software was used to determine the interfacial tension. Then pure canola oil in the syringe was replaced with canola oil containing known amounts of PGPR and the measurement was repeated. All the canola oil solutions with various amounts of PGPR were freshly made and used in the experiments. As PGPR starts adsorbing onto oil-water interface the interfacial tension decreases slowly and reached steady state after about 30 to 60 minutes. Then, the oil and water were discarded and syringe along with quartz cuvette were cleaned and dried thoroughly before a new experiment with higher concentration of PGPR in canola oil was started.

2.3

Force between deformable surfaces coated with PGPR 2.3.1 Force between water drops An MFP3D AFM from Asylum Research (Santa Barbara, CA) was used to measure

the interaction forces between water drops coated with PGPR in canola oil as shown schematically in figure 1 (a). In order for the water drops to be picked up by a custom made rectangular cantilever, the drops first had to be immobilized on a circular glass disk, used as the bottom in MFP3D fluid cell. To easily pick up the oil droplet using the cantilever, the relative hydrophobicity of the cantilever and the substrate were controlled using self-assembled monolayer of alkene thiols similar to the procedure developed by Lockie et al.[47] for oil drops in water. In this instance, the cantilever surface is made more hydrophilic than the substrate. Thus, one side of the glass disk was coated with a thin layer of chromium (adhesion promoter) and followed by gold using sputter coater (Emitech Sputter Coater). Then the gold coated glass disks were functionalized with freshly made decane-thiol in ethanol solution by soaking the disks

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in solution overnight. The glass disk was then washed with excess ethanol, dried using ultrapure nitrogen gas and then assembled into the fluid cell. Custom made rectangular cantilevers with 45 to 55 μm gold disk at the end were used to pick up water drops for the force measurements. The gold disk at the end of rectangular cantilever was made hydrophilic by immersing in it MUA (11-Mercapto-Undecanoic-Acid) in ethanol solutions overnight. The spring constant of rectangular cantilevers were measured using Hutter and Bechhoefer method[48] and ranged from 0.10 to 0.25 N/m. The water drops were immobilized onto hydrophobic glass disks by spraying with a custom made bent needle. Then 1.5 ml of canola oil was slowly added to fluid cell. With addition of oil to the fluid cell, most of the water drops were displaced from the bottom of fluid cell while a few of them remain stuck to the glass (figure 1 (c)). Then MFP3D AFM head loaded with a custom-made cantilever was slowly lowered into the fluid cell containing water drops in canola oil in order to ensure that no air bubbles were trapped on the cantilever. The optical lever sensitivity of the cantilever was measured in canola oil by bringing the bare glass bottom of fluid cell in contact with the cantilever without an immobilized drop until a constant compliance was achieved. The constant compliant region was used to calibrate the optical lever sensitivity. An individual water drop of an appropriate size (60-150 μm) was located on the glass bottom and the cantilever was positioned over it. Then, the cantilever was slowly lowered to contact the drop with the hydrophilic gold disk on the cantilever. The cantilever was then slowly lifted up to pick up the drop which detaches from glass bottom and attaches to cantilever due to higher hydrophilicity of the gold disk on the cantilever compared to the glass bottom of the fluid cell. The water drop attached to cantilever as shown in figure 1 (b), was then aligned with another larger water drop on the substrate (figure 1 (c)). The diameter of water drop on cantilever and substrate was measured by recording an optical image

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with inverted microscope (40X objective, Nikon Eclipse Ti-U). Then, canola oil in the fluid cell was exchanged with canola oil containing increasing amounts of PGPR using syringe pumps. Approximately 12 ml of solution were exchanged using two syringe pumps (Adelab Scientific, Model NEW ERA-NE-1000 Pump) where one pump was used to empty the fluid cell while other pump was used to simultaneously fill the fluid cell with canola oil with known amounts of PGPR. A small flow rate of about 0.5 ml/minute was used for solution exchange to ensure the water drops do not dislodge from the cantilever due to flow. The solution was then left to equilibrate for 20 minutes before carrying out the force measurement. Forces between the drops were recorded by driving the two drops together at a velocity ranging from 20 to 500 nm/s. At least 6 forces curves for each velocity were recorded for a number of drop pairs. The experiments were then repeated at various bulk concentrations of PGPR in canola oil.

3. Results and discussion 3.1 Adsorption of PGPR The measured equilibrium interfacial tension of canola oil and water as a function of bulk concentration of PGPR in oil is shown in figure 2 (a). The interfacial tension of pure canola oil and Milli-Q water was measured to be 22±2 mN/m (not shown in the figure). Several previous studies[39],[49] have focused on the adsorption of PGPR onto the oil-water interface. Marze[49] measured the interfacial tensions of sunflower oil and water in presence of various amounts of PGPR and found that pure sunflower oil had some surface active components. Hence, the interfacial tension of pure oil with water showed time dependence due to adsorption of surface active compounds in oil. Marze determined the equilibrium interfacial tension of pure sunflower oil with water to be 20mN/m. Marze also measured the interfacial tension of Florisil (150-250μm (Merck)), column purified sunflower oil with water and found that

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the interfacial tension was same as that from unpurified oil. We also observed that the interfacial tension of pure canola oil showed a time dependence which can be attributed to surface active components reaching a steady state interfacial tension of 22±2 mN/m. This value is reasonable agreement with the literature values[39],[49],[50]. The interfacial tension of canola oil and water decreased from 22 mN/m to 4 mN/m as the bulk concentration of PGPR in oil increased from 0 to 1 wt%. These interfacial tension are in good agreement with that measured by Marze and others[39],[49] for similar oils.

Figure 2. (Color online and in print) The interfacial tension of the water-canola oil interface as a function of the concentration of PGPR in the canola oil (a). Red filled circles are the experimental data and the solid black line is a fit to the Frumkin adsorption isotherm with the following parameters: adsorption rate constant b = 17.4 l/mmol, Area, ωo = 6.5*105 m2/mol and interaction parameter α = 0.01. Surface excess of PGPR at the canola oil-water interface calculated using the

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Frumkin isotherm (b). The inset in shows that that surface of oil-water interface becomes mostly covered with PGPR at approximately 0.20 wt%. In order to describe the adsorption of PGPR molecules from bulk of canola oil to the interface and predict the decrease in interfacial tension of oil-water with an increase in PGPR concentration, we used the Frumkin adsorption isotherm as this was the simplest common model that still allows for attractive or repulsive interaction between adsorbed molecules at the oil-water interface. Isofit® Software developed by Aksenenko and Miller[51] was used to fit surface tension data to Frumkin isotherm. The interfacial tension data of PGPR at the oil-water interface was well described by the Frumkin as shown in figure 2(a) where the Frumkin model is represented by a solid black line, where the three fit parameters used where: the adsorption rate constant b = 17.5 l/mmol, the area per molecule of PGPR at saturated adsorption, ωo = 6.5*105 m2/mol and the molecular interaction parameter α = 0.01. The surface excess (interfacial concentration) of PGPR calculated using the Frumkin isotherm is shown figure 2(b) as the solid blue line. The saturated adsorption amount of PGPR at the oil water was calculated to be 1.66 mg/m2 which is in reasonable agreement with that of 1.2 mg/m 2 measured by Marze[49] for sunflower oil and water interface. A range of concentrations have been reported, varying from 2 to 8 wt% of PGPR, required for stable water-in-oil emulsions[43],[49, 52],[52] [53],[54]. For sunflower oil, Marze[49] observed that the sunflower oil-water interfaces became saturated at a PGPR concertation of at 0.074 wt% yet water-in-oil emulsions (30% Water) made with a PGPR concentration of below 0.10 wt% were unstable after 5 months of storage at 4 o C. Thus, stability was not necessarily correlated with interface saturation. As shown in the inset of figure 2 (b), the interface approaches saturation at about a concentration of 0.20 wt%PGPR, consistent with Marze observation that stable emulsions required a PGPR

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concertation above 0.10 wt%. In practice, emulsions made for food applications often have an excess concentration of surfactant in order for the emulsions to be stable and survive high shear and temperature changes encountered during processing. Hence most of the water-in-oil emulsion stability studies involving PGPR available in the literature use high concentrations of PGPR that are above 2 wt%. Thus, by using AFM to study the forces between drop collisions at both low and high concentrations of surfactants, one can probe the stability of meta-stable water drops and the mechanisms behind the attractive interactions that lead to drop coalescence.

Figure 3. (Color online and in print) (a) Small force regime for the measured force between water drops with PGPR concentrations of 0.02, 0.05 and 0 .10 wt% in

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canola oil (at 200 nm/s approach velocities). (b). Forces between the same drop pair (at 200 nm/s approach velocities) were measured in at a PGPR concentration of 0.05 wt% with successive increases in the maximum applied force successively ranging from 1 to 4 nN. The different colours corresponds to the force curves with differing maximum applied forces. The radii of the drops on cantilever and surface were 58 and 79 μm, respectively. For both (a) and (b) the filled and open circles correspond to forces measured during approach and retract, respectively. The inset shows the retract portion of the force curves in the region where the force minima show additional structure from short-range attractive surface forces.

3.2

Forces between water drops as a function of PGPR concentration

The forces were measured between water drops in canola oil in presence of 0.02, 0.05 and 0.10 wt% of PGPR, shown in Figure 3. In the absence of PGPR, the water drops did not show any repulsion and coalesced immediately in coming close contact with each other (data not shown). This observation clearly indicates that in the absence of stabilizing surfactants, such as PGPR, the emulsified water drops are not stable and coalesce when in close contact. However, in presence of PGPR, the adsorbed PGPR molecules at the oil-water interface provide stability against coalescence through steric repulsion (detailed modeling discussed below). As observed, there is a large hysteresis between approach and retract curves at all the concentrations. This hysteresis is due to in part to hydrodynamic drainage effects from the higher viscosity of canola oil (~70 cP) which is approximately 70 times that of water (~1 cP). In order to eliminate the effects of drainage of oil film between water drops and viscosity on the force curves, the drops would have to be driven at velocities so slow they are inaccessible via AFM[55]. However, even with the significant hydrodynamic drainage effects at all concentrations of PGPR the following observations can be made of the data shown in figure 3 (a). The approach curve is a combination of a hydrodynamic drainage effect leading to a

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repulsion as the film drains as well as the result of surface forces, in this case from steric forces, and drop deformation. The retract curve shows an attractive well or minima. Based on previous work[55], [56], [8], this is often from a combination of surface forces and hydrodynamic drainage forces arising from a film suction between the drops as they are separated. Three things stand out for the data shown in figure 3 (a). First, even at such low concentrations of PGPR (0.02 wt%), the drops did not coalescence. Second, the overall slope of the force curve decreases with an increase in concentration, for a similar maximum force of interaction. This is largely attributed to the change in the interfacial tension and discussed in detail in the Supplementary Material. Third, the magnitude of the attractive well decreases with an increase in concentration, but the functional form of the force curves deviates from what is expected from the presence of a hydrodynamic drainage effects and a repulsive steric force (where force behavior typical to these two mechanisms is discussed in detail in [51]). In figure 3(a), for PGPR concentrations of 0.05 and 0.10 wt%, the functional form of the force curve appears qualitatively similar to previous studies where hydrodynamic drainage and a repulsive surface force can describe the system [8], [47],[58]. Yet, the for the PGPR concentration of 0.02 wt%, the force curve appears to have a much shaper attractive well that is not normally attributed solely to hydrodynamic drainage effects. This functional form is similar to force behavior that has been observed in the interactions between oil drops in water where specific ions effects have led to anomalous short range attractive forces[14],[15]. Force measurements between rigid hydrophilic interfaces (see Supplementary Material, Figure S1) in the presence of PGPR in canola oil indicate the presence of a steric force resulting from the adsorbed PGPR on hydrophilic surfaces at the same PGPR concentrations studied between water drops. Yet, the AFM data between drops in figure 3(a) indicates there is a short-range attraction. This is most

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likely associated with bridging of the PRPG as the oil-water interfaces come into close proximity since, as shown in figure 1 (b), the oil-water interface is not saturated with PGPR at this concentration. Interestingly, many experimental studies in the literature[49],[52] on water-in-oil emulsions made with a PGPR concentration of 0.02 wt% are not stable as the drops are prone to coalescence due to insufficient interfacial coverage of PGPR. Thus, these AFM observations may be related to the same attractive forces in bulk system yet, in AFM measurement, coalescence is not observed. This can be attributed to a combination of both the steric forces as well as the hydrodynamic drainage forces on approach stabilizing the drops from coalescence. It has been observed in AFM measurements previously where a repulsive hydrodynamic drainage force can stabilize drops where the surface forces are weakly attractive, where a detailed discussion can be found in [57]. Forces between the same drop pair (at 200 nm/s approach velocities) were measured in at a PGPR concentration of 0.05 wt% with successive increases in the maximum applied force successively ranging from 1 to 4 nN, shown in figure 3(b). The hysteresis between the approach and retraction curves increased with an increase in the maximum applied force between the drops. The magnitude of the attractive minimum increased with the increase in the applied force where the functional form exhibited a distinct change at 4 nN. This is shown in more detail in the inset, where during the retract phase there is a kink in the force curves and sudden jump out after the drops are pushed to a maximum force of 4 nN (Open red circles), yet the drops do not coalescence. The kink in the retraction is unlikely to be attributed to solely hydrodynamic drainage

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effects based on previous AFM studies. It is also worth noting, this occurs when the drops are held together for a longer time and at a higher applied force.

Figure 4. (Color online and in print) An attractive well, attributed to the bridging and extension of the PGPR, was also observed during pull off forces between water drops in canola oil at PGPR concentrations of 0.10 and 0.20 wt%. The filled and open circles correspond to forces measured during approach and retract, respectively. The radii of the drops on cantilever and surface were 59 and 85 μm, respectively. The drop collision velocity was 200 nm/s. The inset shows the retract portion of the force curves in the region where the force minima show additional structure from short-range attractive surface forces. Collisions between water drops at PGPR concentrations of 0.10 and 0.20 wt% at both low and high applied forces are shown in figure 4. Note that the slopes for 0.10 and 0.20 are different because of the difference in interfacial tension. At lower applied forces the kink in the retract curve is not observed, but the functional form of the attractive minimum appears to be sharper in nature compared to a system governed solely by hydrodynamic interactions and repulsive surface forces. Again, when large applied forces are reached, the attractive well and additional kink in the force curves on retract were also observed, also shown in the inset. Similar behavior was observed for a PGPR

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concentration of 0.2 wt% with 1 wt % of CaCl2 was added to water phase, data shown in the Supplementary Information (Figure S4). The behavior observed from the kink in the retract force curves, seen at PGPR concentrations from 0.02 to 0.20 wt%, is similar to polymer or single molecule extension or bridging between two surfaces on retract[59] and is unlikely to be caused by a hydrodynamic drainage effect based on previous dynamic force studies[8, 22, 55]. Drop coalescence is not observed with PGPR concentrations from 0.02 to 0.20 wt%, which is attributed to the presence of steric stabilization between the water drops even though the droplet interfaces are not at complete surface coverage (see Fig 2 b). PGPR is a large molecular surfactant, with high poly-dispersity[60] and large hydrophobic tail and a hydrophilic head group. At low surface coverage, it is plausible that the PGPR provides a steric barrier at lower applied forces, but as the drops are driven together at large forces, it is possible that the film thins and the PGPR layers come into reasonable inter-digitation or contact, leading to entanglement and bridging between the two interfaces. This process could then result in the observed extensions or bridging of PGPR molecules between water drops. These observations are unlikely to reflect single chain entanglements due to the magnitude of the forces, much larger than the typical 50 to 100 pN typical of single molecular behavior[61]. It would be interesting to probe if the additional features in the retract curves persisted at higher PGPR concentrations, but the drops fail to adhere to the substrate or cantilever.

3.3

Effect of velocity on the force curves

The effect of the collision velocity was also explored in a range from 50 nm/s to 200 nm/s at a PGPR concentration of 0.02 wt%( figure 5). The hysteresis between the approach and retract force curves increase with the increase in velocity, consistent with

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hydrodynamic drainage effects. The higher viscosity (70 times that of water) makes this occur at velocities that are much lower than previous AFM studies in aqueous systems ([22], [8]). PGPR is expected to form a steric layer where the presence of the steric layer may also affect the drainage behavior or at least that drainage of the thin film between the drops may occur both inside and outside the steric layer. This has previously been observed for Pluronic stabilized oil droplets [58] in aqueous solution from AFM measurements. In order to quantify the steric layer impact on the hydrodynamic drainage behavior and to confirm that the observed extension behavior in the retract force is not an hydrodynamic drainage effect, quantitative modeling of these drop collisions, using the model developed previously by the authors ([56], [8], [21], [58]), is used as shown in detail in Supplementary Information.

Figure 5. (Color online and in print) The force between water drops as a function of the velocity of collision at a PGPR concentration 0.02 wt% in canola oil. The radii of the drops on the cantilever and the surface were 44 and 64 μm, respectively. The filled and open markers correspond to forces measured during approach and retract, respectively.

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3.4 Stokes-Reynolds-Young-Laplace Model The Stokes-Reynolds-Young-Laplace (SRYL) model[16],[18],[20],[21] was used to quantitatively model the force curves measured between PGPR coated water drops at a range of velocities. In this case, the hydrodynamic pressure resulting from the drainage of thin oil film between water drops contributes to the force of interaction as does the disjoining pressure resulting from surface forces, steric in this instance, discussed below. The SRYL-model is comprised of two governing equations as given below[20],[21]

(1) (2)

where

is the function describing the spatial and temporal evolution of the thin oil

film, is the viscosity of oil, defined by

where

and surface, respectively forces and

is the interfacial tension of oil-water drop interface, and

is

are the radii of the drop on the cantilever

, is the disjoining pressure resulting from steric

is the hydrodynamic pressure resulting from flow of oil between the

approaching water drops. Equation 1 is the radial component of Stokes equation and it characterizes the fluid flow within the thin oil film between water drops. Equation 2 is derived from the Young-Laplace equation by assuming the drop deformation is limited to a very narrow interaction zone that brackets the apex of the two drops (see Figure S5

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for details). A full derivation of these two equation and the boundary condition used to solve these equations is given in Chan et al. [20],[21]. More details on the model and boundary conditions are given in Supplementary Material. The SRYL-model is a seminumerical method that confines the numerical calculations to an inner region that encompasses the interaction zone whilst the outer region is described analytically using an approximation obtained from the Young-Laplace equation. By solving this system of equations numerically, we calculated the shape of the film, the total pressure. The force for the interaction between water drops is calculated using the equation:

(3)

where the details of calculation are included in the Supplementary Material. In order to completely define equation 2 and 3, it is necessary to specify the detailed form of the disjoining pressure

between the two drops. Due to the

presence of adsorbed PGPR molecules at oil-water interface, the interaction forces between the drops are dominated by steric force that are modelled using Alexander-de Gennes scaling[62],[63] given by.

for

Here

(4)

is an experimental coefficient as used in Manor et al.[58] and Luckham[64, 65],

is the distance between PGPR molecules on the drop surface (figure 1 (d)) and

is the

brush thickness which was measured through fitting a series of AFM force curves as shown in the Supplementary Material. The repulsive force due to steric disjoining

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pressure when the film thickness

between the approaching drops falls below

, i.e.

when the adsorbed brush layers of PGPR on the drops come into contact. The brush thickness increases with increase in PGPR concentration as shown in the Supplementary Material. Electrical double layer and van der Waals forces are treated as negligible due to the size of the polymer brush ranging from 22 nm to 46 nm and the low dielectric constant of the continuous phase.

4. Dynamic force model As a consistency check of these force data, prior to applying a detailed quantitative analysis, we analyzed the repulsive force curves where the force curve has a pseudo-linear region (Figure S3). We found a good agreement between the experimental and theoretical slopes for PGPR concentrations of 0.02, 0.05 and 0.10 wt%. This agreement motivates a more detailed analysis into these data in order to predict the complete force curves (approach and retraction).

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Figure 6. (Color online and in print) Comparison of experimentally measured force to theoretically calculated force between water drops coated with PGPR in canola oil at PGPR concentration of 0.05wt%. The radii of the drops on cantilever and surface were 58 and 80 μm respectively. The velocity of approach between the drops is 200 nm/s.

4.1

Flow through PGPR brushes

The extension or bridging of the PGPR feature is unlikely to be modeled using the hydrodynamic drainage and surface forces model defined in equations 1 to 4, yet the agreement with the high force formula with these force data in Figure S3 suggests that the deformation of the drop can be described using the Laplace equations. As a first step in a quantitative analysis, the force data used for comparison to theory did not exhibit the kink attributed to extension or bridging behavior, shown in figures 3(b), and 4. The force data used to compare to the model the still exhibit a hysteresis between the approach and the retract portion of the force curves that is clearly velocity dependent and may be more consistent with the functional form of previous AFM studies of hydrodynamic drainage forces. In figure 6, we show the comparison of experimental and theoretical force curves of colliding water drops at a velocity of 200 nm/s in canola oil with a PGPR concentration of 0.05 wt%. To construct the steric force law, in equation 4, we used an experimentally determined brush thickness (Lo) of 34 nm and a distance between surface contact points of s =3.2 nm where these values were measured for the interactions between hydrophilic rigid surfaces coated with PGPR detailed in the Supplementary Material. We also used

, fit to these data, as an

experimental coefficient similar to that was used in Manor et al. ([58]) and Luckham [64, 65], data and fitting shown in the Supplementary Material. The theoretical force curves were calculated by solving equations 1 to 4 using the boundary conditions listed in Supplementary Material. Equations 1 to 4 are valid for the case where oil-water

23

interface is immobile and the shear plane is located at the edge of the adsorbed brush layer of the surfactant on the drop. A number of AFM studies have previously explored the boundary condition of the oil-water interface in both the presence[58],[47] and absence[47],[66],[67],[7] of surfactants, and found that the hydrodynamic drainage behavior is well described with an immobile boundary condition at the oil-water interface, for a further discussion can be found in[20],[21]and [22], [56]. Initial modelling of the AFM experiments with this boundary condition predicted force curves that were greater than the experimental force curves (figure 7). This mismatch could be due to thick adsorbed brush layers of PGPR on water drops where drainage of liquid may occur. In the case of adsorption of non-ionic surfactants at the oil-water interface, even though the adsorbed surfactants are immobile, the thickness of adsorbed brushes is larger compared to ionic surfactants. Hence, during the drainage of thin liquid film between these interfaces, there is a possibility of flow developing through adsorbed brush layers. Klein ([68, 69]) Manor et al.[58] assumed that adsorbed non-ionic large molecular weight surfactants are in brush regime where there is a flow within an outer region of the brush and that here is a region with no flow close to the substrate or drop. This is modelled using the modified version of the SRYL-model (equation S6 and is consistent with methods utilized in previous studies [20],[21],[58]). The shear plane was set within the brushes and it was assumed that flow outside this plane offered no resistance. The length of shear planes used in the theoretical calculations ranged from 9 to 13 nm that increased with increase in velocity of approach. The modified SRYL-model and was fitted to match theory with experiments by changing only the initial separation of the drops, shown in Figure 7.

24

Figure 7. (Color online and in print) Comparison of experimentally measured forces and theoretically calculated forces between water drops coated with PGPR in canola oil at PGPR concentration of 0.05wt%. The radii of the drops on cantilever and surface were 58 and 80 μm respectively. The velocity of approach between the drops ranged from 50-500 nm/s. Panel (a) shows only the experimental data whereas panel (b) shows only the theoretical prediction for clarity. Panel (c) shows the comparison of experiments(points) with theory (solid lines) for low velocities whereas panel (d) shows the comparison of experiments (points) with theory (solid lines) for low for high velocities. In figure 7 we present an experimental and theoretical comparison broken into sections starting with a suit of experimental measurements in figure 7(a) of colliding water drops at velocities ranging from 50 to 500 nm/s in canola oil with a PGPR concentration of 0.05 wt% PGPR by an analogous suit of theoretical force curves in figure 9(b), calculated using the SYRL model with using a steric force with the shear plane located in the brush. Direct comparisons of these experimental data to the theoretical calculations are presented in figure 9(c) for 50 and 100nm/s velocities and

25

in figure 9(d) for 200 and 500 nm/s. The comparison between theory and experiment for collision velocities of 50 nm/s and 100 nm/s in figure 7(c) show reasonable agreement on the approach force curve, but a large deviation between the theory calculations and the experimental measurements in the retraction force curve. The measured attraction is significantly larger than the theoretical one predicted based on a combination hydrodynamic drainage effects and a repulsive steric force. This is consistent with the discussion of the force measured in figures 3(a), and 4 where even at low applied forces, the attractive minima observed in these data seemed to differ in functional form to what is expected from hydrodynamic drainage forces with a repulsive surface force. However, for the drop velocities of 200 and 500 nm/s, the data compares more favorably to theoretical calculations based on hydrodynamic drainage shown in figure 9(d). It is expected the film thickness at these velocities is larger as the film has less time to drain at the higher velocities, thus the short-range attraction associated with bridge and extension of the PGPR molecules was not sufficiently sampled to affect the agreement at the minima. These comparisons show that the kink observed in the retraction force curve cannot be described by a hydrodynamic film drainage effect and that the presence of a short-range attraction, most likely associated with bridging and extension of the PRPG, is present and affects the force behavior at slower collision velocities, even in the absence of the kink feature. Incorporating the effects of bridging and adhesion of polymers into SRYL model is not trivial as the functional forms for these types of forces with separation remain elusive and are difficult to incorporate into an interfacial continuum approach used in the SRYL model.

26

5. Conclusions To probe the meta-stability of emulsified water drops coated with PGPR for concentrations from 0.02wt% to 0.20 wt%, the forces between colliding water drops in canola oil were measured as a function of PGPR concentration and collision velocities. The viscosity of the continuous phase (about 70 times that of water) meant that hydrodynamic drainage effects were present at all drop collision velocities accessible with the AFM. Thus, it was not possible to probe equilibrium surface independently of hydrodynamic drainage forces. The hysteresis between the approach and retraction portions of the forces curves increased with increase with velocity as expected, but exhibited two phenomena not attributed to hydrodynamic drainage forces in the retraction force curves. A sharper and larger magnitude minimum in the attractive portion of the retraction force curve was observed than for what was expected from hydrodynamic drainage forces as well as an additional kink feature in the force curve extending the minimum in the retraction force curve. These features were attributed to a short-range attractive interaction because of the PGPR bridging between the drops when brought into close contact, as the PGPR did not fully stature the oil-water interface at these concentrations. By systematically varying both collision velocities and the applied force between the drops, the features related to a short-range attraction where not observed when the applied force between the drops where restricted to small applied loads or higher collision velocities. This was attributed to a combination of either the presence of a repulsive steric force where the surfaces where not in sufficient contact to allow for the PGPR to interdigitate leading to bridging or in the case of higher velocities, incomplete drainage of the film between the drops, preventing close contact between the drops.

27

To further support the proposed qualitative analysis of these data, a SYRL model, developed to describe the AFM force measurement in the presence hydrodynamic drainage effects, surface forces, in this case from steric forces, and drop deformation was used to calculate theoretical force curves to compare to these AFM data. This model accounted for the presence of a steric force as well as flow of the solvent through the steric brush. Even with this detailed analysis, there was mismatch between the experiments and SRYL theory in the retract phase of drop collisions when the collision velocities were below 200 nm/s where the additional features in the retraction force curve cannot be described by hydrodynamic drainage effects. This further confirms that the additional short-range attraction can be attributed to the bridging and extension of PGPR molecules on the surface of water drops. The observations and analysis support the idea that this is the main surface force mechanism for how these emulsion drops may aggregate in solution and ultimately lead to emulsion destabilization. This is consistent with a number of bulk emulsions studies focused on determining the critical concentration of PGPR to form stable emulsions [43],[49, 52], [53],[54], where the AFM was able to probe the surface forces at PGPR concentrations that would have formed unstable emulsions. These force data and earlier emulsions studies also show that the critical concentration from either emulsion stability data or force measurements requires a high degree of PRPG drop surface coverage. Future studies may be able to use this approach to probe the force behavior between water drops with other oil or fat-soluble food surfactant systems.

6. Acknowledgements This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF). This work

28

was performed in part at the Materials Characterization and Fabrication Platform (MCFP) at the University of Melbourne and the Victorian Node of the Australian National Fabrication Facility (ANFF). We thank the Australian Research Council (ARC), The University of Melbourne and Mondelez Australia for providing funding through Industrial Transformation Research Hub (ITRH, Project ID: IH120100053) and the PFPC for providing infrastructure support.

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