tering of droplets on the surface (but prefer-ably not a continuous film). Next, the slide wasplaced onto the sample stage of the AFM anda 100 µl drop of water was deposited on itssurface. Although most of the oil drops weredisplaced by the addition of water, someremained attached to the glass slide (Figure 1).

Atomic Force MicroscopyForce spectroscopy experiments were per-formed using an Asylum MFP-3D BIO atomicforce microscope (Asylum Research, Santa Bar-bara, CA, USA). The cantilevers used wereNanoprobe (NP) silicon nitride levers with aquoted force constant of 0.04-0.08 N m-1. Atthe start of each experiment, force curvesbetween the cantilever and a clean glass slidewere acquired to determine the sensitivity ofthe optical response to cantilever bending.The cantilever spring constant was subse-quently measured using the thermal tunefunction of the MFP-3D software.The MFP-3D AFM head, containing a pre-

wetted V-shaped cantilever (k~0.04 N m-1),was placed onto the sample so that it sand-wiched the water on the slide. Then, the AFMhead was positioned so that the cantilever tipwas just over a droplet. Attachment of thedroplet was done by simply lowering the AFMhead using the thumbwheel so that the can-tilever pushed into the target drop until ajump-to-contact was observed. When thisoccurred the thumbwheel was reversed to pullthe tip and droplet away from the glass (Figure1). The lever/droplet assembly could now bepositioned over a second droplet and theexperiment began. Tip engagement was car-ried out in the normal manner under feedbackcontrol in dc mode, with a moderate set pointof ~200 mV greater than the null-point for thelever. Note that addition of surfactants, salts,polymers etc. should be done after dropletcapture onto the tip.

B IOGRAPHYAxel Gromer obtained aPhD in physics from theUniversity Louis Pasteur(Strasbourg, France) in2007. From 2007 to 2010,he was a postdoctoralfellow at the Institute ofFood Research. His scientific interestsencompass different branches of soft matterphysics including polymer physics, colloidand interface science, and biological physics.

ABSTRACTAtomic force microscopy (AFM) has beenused to probe the effect that both adsorb-ing and non-adsorbing polymers have onthe force interactions between oil dropletssuspended in aqueous solution. At low poly-mer concentrations depletion effects werefound to predominate in both cases. Athigher polymer concentrations steric effectstook over which could cause both repulsiveand attractive interactions between thedroplets. We show that such apparentlyparadoxical behaviour is rooted in thedeformable nature of the oil droplets, illus-trating the advantages of studying soft mat-ter systems using AFM which allows the useof realistic samples.

KEYWORDSatomic force microscopy, force spectroscopy,emulsions, oil droplets, sugar beet pectin,polystyrene sulphonate, depletion forces,black spot formation, thin liquid films, stericrepulsion, deformable surfaces

ACKNOWLEDGEMENTSWe thank our co-workers Robert Penfold,Andrew Kirby and Victor Morris. NicolaWoodward, Mike Ridout, and Julia Maldon-ado-Valderrama are acknowledged foradvice and experimental assistance. Thiswork was funded by the UK's Biotechnologyand Biological Sciences Research Council(BBSRC grant BB/E002153/1).

AUTHOR DETA I L SAxel Gromer and A. Patrick Gunning, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UKTel: +44 (0)1603 255000Email: axel.gromer@bbsrc.ac.uk patrick.gunning@bbsrc.ac.uk

Microscopy and Analysis 25(1):9-12 (AM), 2011

AFM OF OIL DROPLETS

I N TRODUCT IONUnderstanding the interactions between thecolloidal particles found in emulsions is impor-tant to a range of applications from the foodand pharmaceutical industries through to oilrecovery and mineral flotation. The interac-tions which occur between emulsion dropletsare of huge importance in determining thefunctional properties of such systems. Theseinteractions can be modified by the adsorp-tion at the oil-water interface of surface-activespecies such as small molecule surfactants, pro-teins or polymers. However, the physical inter-actions which occur between emulsified oildroplets have traditionally been a difficultarea to study, with work historically being car-ried out on model rigid colloidal particles [1].This has changed recently following the

development of methods to attach oil dropletsto atomic force microscope (AFM) cantilevers.These methods have demonstrated that themeasurements are sensitive to the nature ofthe interfacial film [2] and have alloweddetailed study of the force interactionsbetween single pairs of droplets [3], includingrecent mathematical modelling [4]. In thisapplication note we will illustrate the advan-tages of studying a real fluid droplet system,capturing effects in the AFM data which areunique to deformable particles with a mobileinterfacial layer.

MATER IALS AND METHODSSample PreparationThe oil used was n-tetradecane, treated toremove any surface-active impurities by pass-ing through a Florisil column (both obtainedfrom Sigma-Aldrich, St. Louis, MO, USA).To attach droplets to the AFM cantilever,

they first need to be sprayed onto a glass slide(which has to be thoroughly pre-cleaned).Using an ad-hoc sprayer, the oil was sprayedfleetingly over the slide to leave a close smat-

Atomic Force Spectroscopy of Interactionsbetween Oil Droplets in EmulsionsAxel Gromer and A. Patrick Gunning, Institute of Food Research, Norwich, UK

Figure 1: Oil droplets attached to glass under water (a),and attached to the AFM cantilever (b).

MICROSCOPY AND ANALYSIS JANUARY 2011 9

a b

RESULTSFigure 2 shows comparative force versus dis-tance data obtained between a bare cantileverand glass (inset) and data for the interactionbetween two tetradecane oil droplets. Thedroplet data show several interesting features.The first and most obvious is that the mea-surement is sensitive to the deformability ofthe droplets, since the slope in the contactregion is clearly much less than that obtainedwhen the bare lever was pressed against arigid surface. The second is that there wasnotable adhesion upon retraction. The smoothshape of this adhesive event reveals that itoriginated predominantly from the hydrody-namic interaction between the droplets andthe liquid medium in which they sit. As theywere forced together a water film becametrapped between the approaching droplets.This can only drain out at a finite rate, causingrepulsion if the approach speed between thedroplets is too great. When they were pulledapart again the opposite occurred and waterhad to rush back into the newly thinned film.As before, this can only happen at a finite rate,leading to an attractive force between thedroplets if they are pulled apart faster thanthe water can refill the gap. Both of theseeffects can be modified by adding material tothe medium in which the droplets are bathed(in emulsions this is usually referred to as thecontinuous phase) and the following sectionsdiscuss the effect of adding surface-active andnon-surface-active polymers.

Sugar Beet PectinSugar beet pectin (SBP) is a plant-derived pro-tein-polysaccharide complex capable of coat-ing oil droplets to form highly stable emulsions[5-7]. We have used the AFM to probe themechanisms occurring between tetradecanedroplets in the presence of SBP. Figure 3 compares the force interaction

between a pair of oil droplets in the presenceof a relatively low concentration of SBP (3a)and the interaction between the same pair ofdroplets after removal of the SBP by rinsing(3b). In the presence of the SBP the curvesexhibit a small repulsive term appearing uponapproach, followed immediately by a smallattractive term (this is seen on both theapproach (red) and retract (blue) data – thedata sets overlay almost perfectly at thisspeed). After rinsing, the curves simply showmonotonic repulsion with separation. Thisextra feature seen in the presence of thepectin is the characteristic hallmark of aneffect known as depletion.Depletion is caused by the osmotic pressure

exerted when non-adsorbing polymers aresqueezed out of the thin aqueous film thatexists between two close-packed colloidal par-ticles [8]. Work is required to exclude thesolute polymer from a region of the solutiondue to the entropic cost of de-mixing whichgives rise to the initial repulsive term seen inFigure 3a. Once the droplets are separated bypure solvent the converse is true, the particlesare now held together by an effective forcegenerated by the energy required to pullsolute polymers out of the bulk solution and

Figure 4: Approach curves for bare droplets (red) and sugar beet pectin-coated droplets (blue).

Figure 2: Force distance data for two tetradecane oil droplets. Inset: Data for the bare cantilever against glass.

MICROSCOPY AND ANALYSIS JANUARY 201110

Figure 3: Droplet interaction dataobtained in the presence (a)and absence (b) of sugarbeet pectin.

a

b

AFM OF OIL DROPLETS

back into the depleted aqueous film. This givesrise to the adhesive term observed at close sep-aration in the AFM data in Figure 3a. Note thatin order to observe this effect, which is verysmall in magnitude (typically ~70 pN), thedroplets have to be driven together at rela-tively low speeds to minimize the (potentiallymuch larger) hydrodynamic term discussedabove. Thus the data shown in Figure 3 revealthat at low speeds and polymer concentrationthe dominant effect on droplet behaviour inthe presence of SBP came from the non-adsorbed fraction present in solution ratherthan the adsorbed fraction coating the dropletsurfaces. Indeed we know from AFM imagingthat at low concentration the polymer chainslay flat on the droplet surfaces [9]. If weincreased the concentration of SBP on thedroplet interface by incubating at higher SBPconcentration (followed by rinsing to removenon-adsorbed polymer), the force data lookquite different; the interaction is purely repul-sive and the range of interaction extendsmuch further (Figure 4 blue curve) than if thedroplets are uncoated (Figure 4 red curve).At elevated interfacial concentration, the

polymeric part of the SBP coating had no roomto lie flat and stuck out away from the dropletsurface into the aqueous phase. Additionally,at high bulk concentration, the SBP can formaggregates in solution which can adsorb at theinterface – the repulsion observed could alsobe due to steric repulsion between such aggre-gates. Whichever of these two predominates,the force data shown in Figure 4 neverthelessreflect the steric exclusion that the extendedpectin chains caused as the droplets werepushed together. Such long-range steric exclu-sion explains why SBP is such a good stabiliser.We know that these long-range effects aredue to polymer and not simple electrostaticrepulsion because they remain even in thepresence of salt. However, by manipulatingthe experimental conditions it was possible topromote inter-chain association of the pectin(Figure 5). In the presence of calcium ions,events characteristic of single molecule poly-mer stretching are observed in the retractiondata at points well beyond droplet separation(Figure 5a, blue curve). Samples where thepectin chains have been chemically stripped oftheir charged ester groups by alkali treatmentshowed even greater inter-chain association:when droplets coated with alkali treated SBPwere studied, the retract data (Figure 5b, bluecurve) reveal multiple polymer stretchingevents following droplet separation.

Polystyrene SulphonateFinally, we explored the depletion effectsbetween droplets further using a non-adsorb-ing polyelectrolyte polymer, polystyrenesulphonate (PSS). This allowed us to work athigher polymer concentrations without theconcern of masking depletion effects with thesteric repulsion seen for SBP. Furthermore, therelatively low molecular weight and randomcoil nature of PSS means that it does not affectthe viscosity of the continuous phase as muchas the SBP. At low concentration of PSS, theforce data obtained exhibited almost identical

MICROSCOPY AND ANALYSIS JANUARY 2011 11

Figure 5: Association of pectin chainsupon retraction of coateddroplets in: (a) 4 mM CaCl2 solution;(b) de-esterified pectin.

Figure 6: Droplet interaction data in the presence of 2% polystyrene sulphonate. Note that for clarity an arbitrary distance offset has been added to separatethe data sets.

behaviour to the SBP data, namely a smallrepulsive peak followed by an attractive peakupon approach. At higher PSS concentration ajump-in feature appears on the approach partof the force curves (Figure 6) and an adhesivepeak upon droplet separation can be seen inthe retract curves. Two interesting effects areseen in these data. The position of the jump-inmoves progressively up the approach curvewith increasing approach speed whilst at thesame time the magnitude of the adhesionpeak reduces. This is contrary to the normallyexpected hydrodynamic behaviour of colloidalparticles, as discussed earlier, where fastervelocity produces greater adhesion uponretraction. When the PSS concentration was increased

still further the effect becomes more pro-nounced (Figure 7) and the jump-in moves allthe way onto the retract portion of the data.This seems counter-intuitive but can beexplained when one considers that the parti-cles being pushed together are deformable. Ifyou think of the droplets as a pair of balloons,one in each hand, the effect will be easier to

conceptualize: as you push them together theballoons will deform and store elastic energy.Now imagine slowly pulling them apart; theforce you feel against your hands immediatelyreduces (this represents the approach-retractturning point in our force data) but the regionwhere the balloons are touching continues tobe squeezed until the point at which the bal-loons have relaxed back to their undeformedshape. In just the same way the thin aqueousliquid film trapped at the point of contactbetween the droplets will continue to besqueezed, and therefore thin-out for sometime as they are pulled apart, explaining howjump-in events can occur even during theretraction part of the force-distance cycle. Thiseffect is of course unique to deformable parti-cles and has been confirmed by theoreticalmodelling [9]. Interestingly, careful examina-tion of the data reveals that there is a correla-tion between the position of the jump-in andthe magnitude of the adhesion when thedroplets were finally pulled apart. If we plotthe force data against time, this correlationbecomes more obvious.

a

b

MICROSCOPY AND ANALYSIS JANUARY 201112

Figure 8 shows that the longer the timeduration following jump-in, the larger is thesubsequent pull-off required to separate thedroplets. Our interpretation for these effects isthat they result from the formation of a regiondevoid of polymers in the thin liquid filmbetween the drops (an analogue of this effectis the formation of so-called ‘black films’between the lamellae of draining soap films).Neutron scattering studies have providedexperimental evidence that such ‘black films’can also occur between adhesive emulsiondroplets [10]. Figure 9 summarises the different steps of

the process. As the droplets are forcedtogether in the polymer solution (position 1)the liquid film between them is thinned (posi-tion 2) leaving less room for the polymer mol-ecules, which require a finite volume of sol-vent to remain hydrated. At a certain pointpolymer molecules are forced out of the clos-ing gap creating a very thin region betweenthe droplets (position 3). At this point thedroplet surfaces spontaneously jump closertogether causing the jump-in events seen inthe force curves. Once created, this thin regionquickly expands with time (position 4) pushingout polymer solute as it does so. This expan-sion of the very thin region can continue evenwhilst the drops are being pulled apart since,for a while at least, the region is still being sub-jected to a squeezing force by the deformeddroplets. The work required to separate thedroplets now becomes dominated by the areaof this very thin film, because the hydrody-namic suction created by this capillary-like filmis very large. This explains the correlationbetween the jump-in point and the magnitudeof the final pull-off adhesion seen in the forcedata; i.e. a slower approach speed allows thepolymer to escape from the closing gapbetween the droplets earlier in the cycle, andsubsequent expansion of this polymer-depleted region is given more time to proceed,resulting in a greater force being required tofinally separate the droplets.

CONCLUS IONSIn this article we have demonstrated how AFMcan be used to probe different types of inter-actions between oil droplets in aqueousmedia. As we show, the results of such experi-ments can reveal new effects specific to thecase of deformable surfaces which are impor-tant in understanding droplet interactions inreal emulsions.

REFERENCES1. Butt, H. J. et al. Surf. Sci. Rep. 59:1, 2005.2. Gunning, A. P. et al. Langmuir 20:116, 2004.3. Dagastine, R. R. et al. J. Colloid Interface Sci. 273:339, 2004.4. Carnie, S. L. et al. Langmuir 21:2912, 2005.5. Leroux, J. et al. Food Hydrocolloids 14:455, 2003.6. Williams, P. A. et al. J. Agric. Food Chem. 53:3592, 2005.7. Funami, T. et al. Food Hydrocolloids 21:1319, 2007.8. Asakura, S., Oosawa, F. J. Chem. Phys. 22:1255-1256, 1954.9. Gromer, A. et al. Soft Matter 6:3957, 2010.10. Poulin, P. et al. Physical Review Letters 77:3248, 1996.

©2011 John Wiley & Sons, Ltd

Figure 9: Interpretation of the hysteresis effect observed in the interaction between oil droplets inside a polymer solution. 1. When the droplet surfaces are farapart they don’t interact. 2. As the droplet surfaces come sufficiently close together, they start to deform; polymers remain in the thin liquid film betweenthem. 3. As the polymers diffuse away from a region of the film, a ‘jump in’ effect occurs which corresponds to the formation of a black spot; i.e. thelocal lamella thickness has reached the dimension of common black films. 4. The black spot expands with time and it keeps expanding even as thedroplets are pulled apart, until a ‘snap out’ is observed which corresponds to the droplets being suddenly disconnected. 5. After the droplets have beenseparated, the droplet surfaces recover their initial shape.

Figure 8: Chronology of droplet interaction data in the presence of 3% polystyrene sulphonate.

Figure 7: Droplet interaction data in the presence of 3% polystyrene sulphonate. Note that for clarity an arbitrary distance offset has been added to separatethe data sets.