Surface forces in model oil-in-water emulsions stabilized by … · 2007-04-30 · phase, the surfactants lower the interfacial tension much more than the proteins (consider 13–15

Advances in Colloid and Interface Science 108–109(2004) 73–86

0001-8686/04/$ - see front matter� 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.cis.2003.10.003

Surface forces in model oil-in-water emulsions stabilized by proteins

Tatiana D. Dimitrova *, Fernando Leal-Calderon , Theodor D. Gurkov , Bruce Campbella, b c d

Max-Planck-Institute for Polymer Research, Ackermannweg 10, Mainz D-55128, Germanya

Institut des Sciences et Techniques des Aliments de Bordeaux, Av. des facultes, Talence cedex 33405, Franceb ´Laboratory of Chemical Physics Engineering, Faculty of Chemistry, Sofia University, 1 James Bourchier Ave., Sofia 1164, Bulgariac

Kraft Foods, Inc., Technology Center, 801 Waukegan Road, Glenview, IL 60025, USAd

Abstract

We have employed two complementary techniques, namely, the magnetic chaining technique(MCT) and a variant of theMysels cell to obtain data concerning the repulsive interaction profiles between protein layers formed at liquid–liquid interfaces.For BSA-stabilized systems, a long-ranged repulsion is operative. It is not of an electrostatic origin, but originates most probablyfrom the formation of multiple protein layers at the interface. The interactions betweenb-casein layers formed at the wateryoilinterface are governed by electrostatic repulsion. Due to the relatively large final thickness of approximately 20 nm, the van derWaals contribution to the total disjoining pressure is inferior. The oscillatory component is also negligible for the studied proteinconcentration of 0.1 wt.%. For both proteins, the extracted information describes the situation where the protein-covered surfacesare approachedymanipulated in a quasi-static manner. We observe a very good agreement between the data obtained from MCTand Mysels cell. The comparison of our results with literature data from surface force apparatus(SFA) experiments reveals asubstantial difference in the force laws existing between protein-stabilizedliquid droplets and mica surfaces covered by proteins.We explain this discrepancy in terms of the different protein absorption on solid and liquid interfaces. We also measured thethreshold force necessary to induce irreversible flocculation inb-casein andb-lactoglobulin(BLG) stabilized emulsions. Undersimilar conditions, the threshold flocculation force is higher forb-casein than for BLG stabilized droplets. The flocs formed fromBLG covered droplets are tight and remain without visible change for at least 48 h. We speculate that the flocculation is due toformation of protein aggregates between the approaching droplets.� 2003 Elsevier B.V. All rights reserved.

Keywords: Protein-stabilized emulsions; Surface forces; Flocculation; Protein aggegation; Specific interactions

1. Introduction

1.1. Proteins as emulsion stabilizers

A simple definition of an ‘emulsion’ states that it isa heterogeneous, metastable system, consisting of twoimmiscible fluids, often referred to as ‘water’ and ‘oil’.One of the phases is dispersed into the other as dropletsof colloidal size (i.e. below 100mm). The long-termstability is achieved through the coverage of the oil–water interface with amphiphilic molecules. Those aresupposed to ensure the kinetic barrier, which preventsthe decomposition of the emulsion into water and oil.Protein-stabilized emulsions find a variety of applica-tions in food, cosmetic, and pharmaceutical industry and

*Corresponding author. Tel.:q31-512-592-298; fax:q31-512-592-561.

E-mail address: [email protected](T.D. Dimitrova).

provoke considerable scientific interest. The peculiarfeatures of these systems arise mainly from the specificinterfacial properties of the stabilizing molecules, i.e.proteins.Here we would like to emphasize the difference

between the stabilization mechanisms operative in thecase of low molecular weight surfactants and proteins.The simple emulsifiers significantly lower the interfacialtension and further actw1x via the Marangoni effect,involving lateral diffusion at the interface. The lateralinteractions between the adsorbed molecules themselvesare considered minimal. The low molecular weightsurfactants used in some formulations are non-ionic andgenerally soluble in both water and oil. As a rule, theyare initially dissolved in the continuous phase, andimmediately after emulsification a redistribution of thesolute between the two phases takes place. This mass-transfer opposes the thinning of the thin liquid films,

74 T.D. Dimitrova et al. / Advances in Colloid and Interface Science 108 –109 (2004) 73–86

formed between the emulsion droplets, thus ensuringshort-run stabilityw2x of the systems. The stabilizingmechanisms mentioned above are not always operativein the case of protein-stabilized emulsionsw3x. It isnoteworthy, that proteins do not lower the interfacialtension as much as simple surfactants do, but effectivesaturation of the surface in this case is reached at molarprotein concentrations 10 –10 times lower than for the3 4

simple surfactants. At higher concentrations in the bulkphase, the surfactants lower the interfacial tension muchmore than the proteins(consider 13–15 mNym as thelowest limit of the interfacial tension of protein layers),due to the better packing of the small amphiphiles inthe vicinity of the Gibbs dividing plane.In their bulk solutions, protein molecules maintain a

tightly packed structure. Their adsorption at fluid–fluidinterface is accompanied by a gradual unfolding, whichmeans that the bio-molecule loses its secondary andhigher structure in the adsorbed state. This happensbecause the presence of a hydrophobic fluid phase(i.e.oil or air) gives the molecule the possibility to minimizethe configurational free energy. The adsorbed speciesare often referred to as denatured or unfolded. Thisconformation state makes the adsorbed molecules verymuch different from their non-adsorbed analogues. Theprocess of unfolding uncovers the different segments ofadsorbed species and extremely facilitates the lateralinteraction between two or more adsorbed molecules.Various interactions are possible, for example ionic,hydrophobic, covalent(disulfide bridging) or hydrogenbonding. As a result, the adsorbed species form a rigidnetwork and the protein layers, which protects thedroplets against coalescence are ofnanometer thickness,viscoelastic, and almost always tangentially immobile.This is a substantial difference with respect to simplesurfactants. The large variety of possible interactions aswell as the their different time scale make the propertiesof the protein layers largely dependent on the samplehistory. This feature is frequently termed ‘aging effects’.Different workersw4,5x argued that conformation of

the protein molecule at the airywater interface differssubstantially from that at the oilywater interface. Furtherstudies demonstrated differences in the state of theadsorbed proteins at hydrocarbonywater and at triglicer-ideywater interfacesw3x. Apparently, the adsorption ofproteins at liquid interfaces cannot be considered as asimple occupation of surface sites, and the lucid andconvenient concept of ‘end-on’y’side-on’ orientation,which is frequently used in case of adsorption at solidsurfaces, is irrelevant. It becomes also problematic topostulate equilibrium adsorption, because of at leastthree reasons. Firstly, the existence of many adsorptionsites per molecule makes the hypothesis of instantaneousdesorption unlikely. Secondly, the denaturation uponadsorption is not reversible, i.e. desorbed species neverregain the conformation and structure they obeyed prior

to adsorption. The last, but not the least: it is anexperimental fact that reaching constant interfacial ten-sion does not mean reaching constant adsorption. Theprotein adsorption can continue via multilayer formationw4,6x. The multilayers are constituted by reversiblyadsorbed molecules and the surface coverage can beaugmented up to 5–10 mgym .2

1.2. Modeling of protein stabilized emulsions

The particularities of protein adsorption layers makethe examination of the protein-stabilized emulsionstricky, because the real control on the investigatingobject is not always achieved. The model experimentson emulsions can be divided in two main groups, whichwe shortly term as macroscopic and microscopic meth-ods. In the first group fall all the model experimentsdone on bulk quantities of emulsions as, for example,rheology, w7,8x stability against coalescence,w9x sedi-mentation studies,w10x etc. The advantage is that oneobtains useful data for the overall emulsion behaviorand evaluates the influence of different factors over sizegrowth, creaming, viscosity, flocculation etc. For exam-ple, data from this type of experiment helped forexplaining the influence of the total casein content onthe resistance of model emulsions against flocculationw11x. Experiments on batch emulsions stabilized withmixed casein allowed a better understanding the role ofthe protein interfacial composition on the stability offood-type emulsions,w12x etc. The main drawback ofthis class of methods is that the observed effects resultfrom a sophisticated interplay between various phenom-ena at different length and time scales. Therefore, aquantitative physical modeling of the behavior of anemulsion is possible only for some special cases.The experiments from the second group are designed

to mimic the interaction betweensingle drops. Theobject under study is the thin liquid film, which separatestwo droplets. One obtains lifetime(s), drainage proper-ties, disjoining pressure–thickness isotherms, criticalcapillary pressure necessary for breakage of the film,etc. The idea behind this is that the overall stability ofa real emulsion against coalescence andyor flocculationis related to the stability of the single filmsw1x. Otheroverall properties like sedimentation stability or rheolog-ical behavior are strongly dependent on the extent offlocculation, the tightness on the flocs, and droplet meandiameter, all those being directly related to the propertiesof the films as well.When (protein-stabilized) emulsions are mimicked,

there are at least three main reasons, which makeinvestigation of emulsion films(instead of foam ones)necessary.

i. As already discussed, the protein adsorbs in differentways at airywater and oilywater interfaces. This can

75T.D. Dimitrova et al. / Advances in Colloid and Interface Science 108 –109 (2004) 73–86

influence substantially the surface aggregation andinter-particle interactions between the adsorbed moi-eties. As a consequence, the surface rheologicalproperties(both dilatational and shear ones) in thetwo cases differ significantlyw13,14x. The rheologyof the film’s surfaces is important for the lifetimeand thinning ratew15x.

ii. The Hamaker constant for air bubbles interactingacross an aqueous medium is approximately 10 timeslarger than the Hamaker constant for hydrocarbonlayers interacting across the same water film. There-fore, the stability of the films may change substan-tially, especially for small thickness, where the vander Waals interactions become important. This argu-ment is valid for all emulsion systems, regardlessthe stabilizing agent.

iii. The emulsion films provide the unique opportunityto probe the influence of the mass transfer acrossthe interfaces(i.e. non-equilibrium effects) on thebehavior of the studied systems. Even this effect isnot supposed to play a major role in systems stabi-lized solely by proteins; it could be of considerableimportance when non-ionic surfactant is present.

Below we shortly consider several types of experi-ments that allow forming and reliably manipulatingmodel emulsion films. The referenced literature doesnot cover the whole body of available data, we restrictourselves to the experimental developments with a(possible) application to protein-stabilized systems.

1.2.1. Scheludko cellOriginally designed for studying foam films, the

technique has been extended for emulsion films as well.The film is made by sucking the liquid from a biconcavemeniscus. The set-up is relatively easy to operate, allowsprecise determination of the contact angles(i.e. inter-action energy between the studied droplets) and offersthe possibility video microscopy. The major drawbackis the low attainable capillary pressure—approximately100 Pa. In real emulsions it is much higher – aboveseveral thousand Pascals. The capillary pressure,P , isc

an important parameter because it determines both theradius of the formed film,r , and the rate of its thinning,f

V . For films with circular symmetry one obtains:w1xRe

2gRcP s , (1)c 2 2R y r coswc f

32 h PcV s , (2)Re 23mrf

whereR denotes the radius of the capillary cell,g isc

the interfacial tension,w is the three phase contact angle(glassyfilm phaseyhydrophobic phase), h is the filmthickness, andm is the viscosity of the film phase. Eq.

(2) is the well-known Reynolds equation, which is validfor the case of film with non-deformable surfacesw16x.The Scheludko cell may be combined with FRAP

(fast recovery after photo-bleaching) techniquew17x. Inthis case, a small quantity of fluorescent dye is intro-duced on the film surfaces. When the film reaches itsequilibrium thickness the dye from a certain spot ispulse-bleached. The recovery of the fluorescence signalis attributed to the lateral diffusion of the dye molecules.One obtains the surface diffusion coefficient of the dyeas function of the film phase composition. It was foundthat there is a correlation between the surface mobilityof the dye and the surface rheological properties ofprotein and mixed protein-surfactant filmsw17x.

1.2.2. Miniaturized Scheludko cellThe principle of this cell is almost identical to that

described above. Using laser ablation technique, theauthors have fabricated a cell in between two sinteredglass lamellae. This cell is 10 times smaller than theconventional one. It has been applied for studyingemulsion films stabilized by Tween 20(nonionic surfac-tant) and Bovine serum albumin(BSA). As expected(see Eq.(2)) the drainage is much faster in comparisonwith the analogous films formed in the Scheludko cell.The hydrodynamics of the film formation is different aswell, being ‘thinning’ in the case of conventional celland ‘spreading’ in the case of miniaturized one. How-ever, this configuration is inconvenient for thickness andcontact angle measurementsw18x.

1.2.3. Drop pressed against a large homophaseSome authors investigate the lifetime of the emulsion

films formed between a drop pressed against a largehomophase by buoyancyw19,20x. The overall depend-ence of the lifetime on the drop radius passes through ashallow and broad minimum. For small non-deformabledroplets no film is formed and the drainage velocity ofthe liquid layer between the drop and the interface canbe described by the Stokes equation, with appropriateaccount for the viscous resistance(Taylor–Stokes regi-me). For large deformable droplets, the film lifetimeincreases with drop radius because larger drops experi-ence larger buoyancy, i.e. one forms larger films whosethinning is slower(see Eq.(2)).

1.2.4. Liquid surface force apparatusIn this case, an oil droplet is attached on a capillary,

and this assembly is manipulated so that the dropletapproaches water–oil interface. At a distance, where thesurface forces get important, the droplet is deformedand oil–water–oil film is formed. It is possible to varythe pressure between the drop and the oil phase, i.e. thedisjoining pressure inside the film by moving the dropletup and down. The film thickness is determined indepen-dently, employing light interferometry. This configura-


tion is supposed to deliver information about the surfaceinteractions in a wide range of distances and pressures.Until now, the set up has found application for surfac-tant-stabilized systems onlyw21x.

1.2.5. Film trapping techniqueThis technique allows determination of the barrier of

coalescence, , of a micron-sized droplets and a bulkCRPCoil phase. An important feature of the technique is thepossibility to vary the drop radius as well as to measurereliably very low pressures. For a detailed analysis, thereader is addressed to the paper by Denkov et al.w22x.

1.2.6. Mysels cellThis set-up was originally proposed by Myselsw23x

and further developed by Bergeronw24x. The film isformed in a porous plate, which allows increasing themaximal attainable pressure up to thousands of Pa. Thiscell is considered in great detail in the next sectionwhere we describe our own modifications, necessary forits application to emulsion films.

1.2.7. Magnetic chaining technique (MCT)Magnetic chaining techniquew25,26x allows to

explore the single contacts between the emulsion drop-lets. It may be considered as a direct link between thesingle contact experiments and the macroscopic studies.The method(see next section) works with droplets ofcolloidal size(approx. 0.2mm in diameter), i.e. at truecapillary pressures and with liquid interfaces. A veryimportant feature of it, is the possibility to explore verylow levels of forces: down to approximately 10 Nyy6

m. Another advantage of the method is the ‘build-in’ensemble averaging over millions inter-droplet contacts.

1.3. Aim of this work

There exists relatively little experimental informationconcerning the surface forces acting in the emulsionfilms stabilized by proteins. We applied two complimen-tary techniques, i.e. MCT and a version of Mysels cellto measure the forces acting between protein-stabilizedemulsion droplets. We also exploit the MCT in a way,which permits to obtain quantitative description theflocculation(threshold flocculation force) in some casesof protein-stabilized emulsions.

2. Experimental part

2.1. Substances

The proteins—Bovine Serum Albumin, essentiallyfatty acid free(BSA), b-lactoglobulin (BLG) and b-casein were Sigma products(catalogue numbers A-7511, L-0130, B-6905 lots 102H93101, 91H7005 and25H9550, respectively). Tween 20 was purchased from

Fisher Scientific(ICI Surfactants product, enzymaticalgrade, lot 982322). Sodium dodecyl sulfate(SDS) waspurchased from Aldrich. All surface-active productswere used as received.The oil used for emulsion preparation in MCT exper-

iments was a ferrofluid, prepared by Ferrofluidics. It isa 10% vol.yvol. dispersion of ferromagnetic(Fe O )2 3

particles in octane. The grains, 10 nm in size, arestabilized against aggregation by 2% wt.yvol. oleic acid.It is proven that the particles remain dispersed in therange of magnetic fields applied in the experiment(0–500 G).Hexadecane used as an oil phase for Mysels cell

studies was of analytical grade. It was purchased fromMerck and was used as received.NaN used for prevention of bacterial growth as well3

as NaCl used for the adjustments of the ionic strengthwere Merck products. NaCl was baked for approximate-ly 5 h at 450 8C in order to remove any organiccontamination.All solutions were prepared with water purified by

Millipore Milli-Q unit (Resistance 18.2 MV cm ) andy1

filtered through 0.22mm Millipore cut-off filter prior touse. The protein solutions were stored at constant tem-perature of 238C for no longer than 8 h. When a longerstorage was required, we used solutions that contained0.02 gyl of NaN .3

2.2. Experimental techniques

2.2.1. Magnetic chaining techniqueThe set up is schematized on Fig. 1. This technique

exploits the properties of paramagnetic monodispersedroplets. The applied field induces a magnetic dipolewithin each ferrofluid droplet. The interaction betweenthe magnetic dipoles leads to formation of linear arraysof particles(chains), which are parallel to the externalmagnetic field. At very low droplet volume fractions(f-0.1 vol.%) the chains are only one droplet thickand the droplets in the chains remain well separated. Ifthe chains are illuminated by a white light source,parallel to them, the emulsion appears beautifully col-ored in the back-scattering direction. These colors orig-inate from Bragg diffraction and provide a straight-forward measure of the spacing between droplets withinthe chains. For perfectly aligned particles at a separationd, illuminated by incident white light parallel to thechains, the first order Bragg condition reduces to:

l0ds (3)2 n0

where n is the refractive index of the suspending0

medium (n s1.331 for aqueous solutions), and l is0 0

the wavelength of the light Bragg scattered at an angleof 1808. The wavelength corresponding to the maximum


Fig. 1. Schema of the set-up of magnetic chaining technique(not to scale).

of the Bragg peak provides a direct measurement of theaverage spacing between the drops with a precision ofapproximately 1.5 nm. Because the drops are non-deformable owing to their large capillary pressure(;1atm), it is possible to determine the interfacial separa-tion, hsdy2a, wherea is the droplet radius.The repulsive force,F , between the droplets mustr

exactly balance the attractive force between the dipolesinduced by the applied magnetic field. Since the dipolesare aligned parallel to the field, this force can becalculated exactly and is given by:

21.202 3mF s F d s y = (4)Ž .r m 42 pm d0

wherem is the magnetic permeability of free space and0

m is the induced magnetic moment of each drop. Theinduced magnetic moment is determined self-consistent-ly from the magnetic susceptibility of the ferrofluid, andthe presence of the neighboring droplets. Thus:

4 3m sm pa x H (5)0 S T3

where H is the total magnetic field acting on eachT

drop, andx is the susceptibility of a spherical droplet.S

The total applied field,H , is given by the sum of theT

external applied field,H , and the field from theext

induced magnetic moments in all the neighboring dropsin the chain. This can easily be calculated for an infinitechain, assuming point dipoles, giving:

2mH s H q2=1.202= (6)T ext 34pm d0

This technique allows one to measure interparticleforces as small as 2=10 N, corresponding to they13

minimum force required for forming chains.MCT permits to measure only repulsive interactions,

as all ‘thin liquid film’ techniques. In contrast to theMysels or Scheludko techniques, when using MCT, onehas no direct glance on the contact zone of interactingdroplets. However, MCT has two very important advan-tages. First, the technique allows an on-line averagingover the pair interactions(at a given distance) betweena very large number of droplets, which is not possiblewith the other techniques, aimed to model the singlecontact between two emulsion droplets. Second, there isa possibility to induce flocculation in a controllable way.The latter is possible for flocculation barriers of somemNym. We find this possibility interesting and worthexploring.

2.2.2. Mysels cellWe developed a version of the Mysels type cell,

which allows relatively convenient operations with both


Fig. 2. Operational principle of Mysels cell employed in this study. The photomicrograph shows an example of a BSA stabilized foam film withconsiderable surface aggregation at ca. 2400 Pa. The foam film was chosen only for the sake of better contrast.

foam, and emulsion filmsw27x. In the set-up proposedby us (Fig. 2), the film is formed in a circular porousplate by sucking liquid via side mounted capillary. Thecell is directly connected to the pressure transducer(Omega PC136–G01 or Omega PC136–G05, dependingon the studied pressure range). The reference pressureis the atmospheric one. This configuration provides theopportunity to investigateboth foam and emulsion films.When the construction shown in Fig. 2 is used, themeasured value for the pressure,P, should be correctedfor the hydrostatic pressure difference between the planeof the film and the level of the measuring membrane ofthe transducer. That difference was obtained by calibra-tion in the following way: the cell was filled up withthe appropriate aqueous phase to the point where theupper edge of the porous plate is exactly at the level ofthe aqueous phase. The measured pressure is:

PsDrg DhsDrg Dh qDrg Dh , (7)f *

whereDr is the density difference between water andair, and the heightsDh, Dh and are shown in Fig.Dhf *

2. The value can be measured directly with2Dh*sufficient precision; thus,Dh is determined from Eq.f

(7). Let us now consider the situation where a film isformed. If in the system of Fig. 2 the big vesselcontaining the cell is filled up with oil(aqueous emul-sion films), then the real capillary pressure,P , is givenc

by the following relation:

P syP qDrg Dh qDr g Dh (8)c m f 1 *

Here P is the measured pressure, andDr is them 1

density difference between oil and air. All other symbolsare the same as above. Note that the lower the pressurethe higher the relative contribution of the corrections.The cell is open to the atmosphere, which is veryconvenient from a practical point of view, especiallywhen studying emulsion films. For foam films the termwith Dr in Eq. (8) will vanish. In this case, 2 ml of1

the corresponding protein solution is placed in thebottom of the big vessel(total volume ca. 20 ml) andthe latter is covered by a glass slide. This is done inorder to saturate the vapor pressure inside the chamber,thus preventing the evaporation from the film phase.The whole construction is made out of glass, whichpermits an easy and reliable cleaning with respect tosurface-active material.The cell is attached on the table of an Axioplan Zeiss

Microscope. The film thickness is determined via stan-dard interferometry. Therefore, the film has to be strictlyhorizontal. The intensity of the reflected light is con-nected with the film thickness via the expression:

B El IyIminC Fhs kpqarcsin , (9)yD G2pn I yI0 max min

where I and I denote the maximal and minimalmax min

intensity of the reflected light, respectively(measuredby means of a photo-multiplier tube), ks0,1« is theorder of the interference maximum,l is the wavelengthof the incident light(546 nm) and n is the refractive0


index of the liquid forming the film. This formulaneglects the interfacial layers, giving, as in the case ofMCT, the so-called ‘equivalent water thickness’. This isimportant for the further comparison of the resultsobtained by both methods. We always consider theequivalent water thickness, because physical propertiesof the protein layers are strongly dependent on thesample history. The literature data for the thickness andrefractive index of the protein layers are not alwaysconsistent and the adoption of ‘sandwiched’ models,like in Ref. w24x, cannot be justified.The reliability of the set up is tested on foam films

stabilized with the non-ionic surfactant Tween 20. Thedisjoining pressure vs. distance isotherm is very welldescribed by the classical electric double layer interac-tion combined with oscillatory repulsionw27x. The latterarises from the exclusion volume effect of the surfactantmicelles.We have the possibility to evaluate disjoining pressure

vs. thickness isotherms,P(h) for emulsion films. How-ever, special care has to be taken when attaching thepressure transducer to the glass part. Entrapment of airbubbles causes severe errors in pressure determinationbecause the gases are compressible. There are someother sources of artifacts as well. First, proteins adsorbon the glass surfaces and may render the latter hydro-phobic. This is observed for BSA and sometimes forBLG. As a consequence, the film frequently detachesfrom the cell and ruptures at very low disjoining pres-sure. Another crucial point is the regularity of the filmsurface. Light scattering experiments show that there isno clustering in the bulk protein solutions of BSA andBLG. However, the protein-stabilized films exhibit inboth cases surface clusteringw10,27,28x. An example ispresent in Fig. 2, where we show a BSA stabilized film,containing aggregates. The fact that these aggregates are‘surface born’ has been recently confirmed by Brewsterangle microscopy experiments performed on interfaciallayers of BLG and whey protein isolate(WPI) w29x.The interferometric method for thickness determina-

tion is applicable only to perfectly plane-parallel films,therefore it is necessary to work on aggregate-free filmsonly. After a few films(say maximum 3) have formedand ruptured, the aggregation becomes considerable.Therefore, we paid special attention to work only withthe first one or two films, formed after loading the twophases into the cell. We consider the surface aggregationas a considerable issue we shall discuss this point below.A few Mysels type cells were used in the experiments.

They differ in the size of the pores, i.e. in the maximumattainable capillary pressure. For each system we co-plotted the data from different runs and fitted thesecommon plots.

2.2.3. AuxiliariesPreparation of ferrofluid-in-water emulsions for MCT.

The primary crude ferrofluid-in-water emulsion wasprepared according to the method of emulsification inviscoelastic media developed by Mason and Bibettew30x. As emulsifier we used SDS. This technique leadsto relatively narrow sized droplet distributions, but notsufficient for the applied force measurement technique.So, the emulsion was diluted with 1=CMC SDS solu-tion (CMCs0.008 M) and further refined following afractionation crystallization procedure similar to thatsuggested by Bibettew31x. At least 6–7 steps werenecessary to obtain an emulsion with sufficient degreeof monodispersity. The size of the droplets was deter-mined by dynamic light scattering measurement(per-formed by means of a Brookhaven correlator). We founda mean diameter of 178 nm and polydispersity ofapproximately 5%.This SDS-stabilized emulsion was further treated in

order to cover the interfaces with the chosen surface-active molecules. This was achieved applying the so-called washing procedure, which consists of concen-trating the emulsion by means of centrifugation, fol-lowed by replacing the supernatant with a new aqueousphase, which contains the desired surfactant. When theprocess is repeated a few times, only negligible tracesof the primary surfactant(SDS) are left at the dropletinterface. It is also necessary to ensureno change of thesize distribution and no effects like flocculation andcoalescence. Replacing the surfactant with a protein istricky, because the surfactants(especially the ionic ones)have a strong tendency to bind to the proteins, causingunfolding, formation of complexes, etc. For that reason,it was necessary to exchange the SDS with a non-ionicsurfactant before adding the protein. We chose Tween20, which is a common, widely studied non-ionic sur-factant with a low CMC. After the coverage of theemulsion droplets with Tween 20 is achieved, we con-tinue the washing process in the same way in order toadsorb BSA,b-casein or BLG on the interface.

Electrophoretic mobilities of the emulsions weremeasured using a Malvern Zetasizer II. The electro-phoretic mobility was transformed intoz-potentialaccording to the Smoluchowski equationw15x.

pH control. The pH values were measured by meansof a digital pH-meter, equipped with protein and surfac-tant resistant electrode.The proteins obey buffering properties. When dis-

solved in water, they generally maintain certain pHvalue (which may depend on the purification process),frequently referred to as ‘natural pH’. This property ofthe proteins was used in our experiments to control thepH. For each protein(b-casein, BSA, BLG) we verifiedthat the pH is constant within 0.2 units over the rangeof concentrations studied. No drift of the pH values of


Fig. 3. (a) Disjoining pressure vs. thickness isotherm(dots-experi-mental data, line -DLVO fit) for an emulsion film stabilized by 0.1%b-casein, ionic strength of 1 mM NaCl, oil phase-hexadecane.(b)Comparison between the data obtained from TLF, MCT and SFA.

the protein solutions was observed over the period ofstorage(8 h).We chose this way to maintain the pH and did not

use a conventional buffer solution because buffers con-tribute to the total salt content(i.e. the ionic strength)and this significantly narrows the available range ofionic strengths.

All glassware, including the cells for film formation,was soaked for 20 min in concentrated chromic–sulfuricacid, then thoroughly rinsed with abundant quantities ofMillipore purified water and finally dried at 508C in aclosed oven.

3. Results

3.1. Force–distance measurements

3.1.1. b-caseinEmploying the Mysels cell, we measured the disjoin-

ing pressure vs. thickness isotherms of thin liquid films(TLF) between hexadecane droplets stabilized by 0.1wt.% b-casein. The pH was 6.1, the ionic strength wasfixed by 0.001 M NaCl. In this case, we find a DLVObehavior. Fig. 3a shows the experimentally obtainedP(h) isotherm(dots) and the best DLVO-fit(Eq.(10)).The fit is done supposing constant surface potential,C ; the plane of charge is taken to coincide with thes

wateryoil interface. The value of the Hamaker constant,A , is 0.49=10 J. The Debye length,k, is calculatedy20

H

from the electrolyte concentration,C , (Eq. (11)):el

B EA eCH s2C FP sy q 64C kTtanh expykh (10)Ž .DLVO el3 D G6ph 4kT

y1 yk nm s 3.29 C molyl (11)Ž . Ž .el

The only free parameter is the surface potential, whichis found to bey27.8 mV. It agrees fairly well with thedata in the literaturew32x (y30 to 36 mV) for the z-potential ofb-casein covered particles. Since casein isreported in the literature to form micelle-like aggregatesof various sizes, it is important to estimate the magnitudeof the oscillatory structural forces. Taking the averagediameter of theb-casein aggregates(micelles) to be 26nm w33x and 250 kDaw12x for their molecular mass,one calculates exactlyw15x the contribution of theoscillatory disjoining pressure to the measured pressure.The calculations show that the volume fraction of themicelles is less than 2% and the oscillatory pressureexerted by them is of the order of magnitude of theexperimental uncertainty in the pressure determination.It is interesting to compare these data with the output

of MCT and surface force apparatus(SFA). Thisrequires a conversion of the disjoining pressure in energyper unit area. By analytical integration over the thicknessof the expression for the disjoining pressure(taking forC the value provided by the best fit) one obtains thes

corresponding energy per unit area,f(h), between twoinfinite planes:w15x

`

f h s P h dh (12)Ž . Ž .|h

Adopting the Derjaguin approximation one can findthe real force,F(h), acting between two spheres ofequal radii, which is exactly the quantity measured inthe MCT experiments:

F(h)yasp f(h) (13)

In Eq. (13), a is the radius of the spheres. For crossedcylinders of the radius of curvatureR one obtains:

F(h)yRs2p f(h) (14)

The comparison is plotted in Fig. 3b. The SFA datawere taken from the literaturew34x. They have beenobtained for hydrophobised mica surfaces, protein con-centration of 0.01 wt.%, ionic strength of 0.001 M, andpH 7. Evidently TLF and MCT lead to very similarresults, which permit us to state that under these condi-tions the electrostatic repulsion is the main force gov-erning the behavior of the studied system. Thecomparison with the SFA data demonstrates that the


Fig. 4. (a) Disjoining pressure vs. thickness isotherm for an emulsionfilm stabilized by 0.1% BSA, ionic strength of 1 mM NaCl, oil phase-hexadecane. The dots are the experimental data, dashed line is theDLVO contribution to the total disjoining pressure, and the solid lineis the best fit done supposing additivity of the DLVO and steric forces.(b) Force vs. distance profiles for ferrofluid emulsions stabilized withmixed BSA-Tween 20 adsorption layers. The total concentration ofthe Tween 20 is kept constant-5=CMC, pH 5.8.

force laws are only qualitatively similar. This is moreor less expected, since the protein layers formed in thetwo types of experiments are incompatible, due to thesubstantial differences in the conformation at the twotypes of surfaces. The difference between the values isapproximately 1 decade. This result strongly suggeststhat extrapolation of the data concerning the forcesbetween protein layers formed on a surface of a giventype to another surface cannot be always justified.

3.2. BSA

For the TLF experiments we worked with 0.1 wt.%BSA solutions. The ionic strength is 0.001 M NaCl, andpH of 5.7"0.1 (natural). The oil phase is again hex-adecane. The results from the Mysels-type cell areshown as points in Fig. 4a.z-potentials of hexadecanedroplets, covered by BSA, measured under similar pHand salt conditionsw35x provide values betweeny5 and0 mV. Hence, we usey10 mV for the surface potentialin order to estimate the maximal DLVO-contribution tothe total disjoining pressure(dashed line in Fig. 4a). Asin the case ofb-casein A s0.49=10 J and they20

H

Debye length is 9.6 nm(corresponding to 1 mM 1:1

electrolyte). Apparently, the measured repulsive pressureis longer-ranged compared to the one predicted by theDLVO model. This means that there is an additionalrepulsive interaction, which remains not captured by thetheory. It is established in the literaturew36,37x thatBSA layers exhibit strong and long ranged, steric repul-sion, so it is natural to expect such interactions to bepresent in our films. For numerous proteins it is knownthat after reaching the equilibrium interfacial tension(for a certain protein concentration) the adsorption,G,continues to increasew4,6x. The latter fact is attributedto the formation of extra surface protein layer(s) ofpartially unfolded protein molecules. To take intoaccount the formation of the second protein layer(s) onemay use the approach of Israelachvili and Wennerstrom¨w38x. The idea is to derive an expression for thedisjoining pressure exerted by ‘ordering’ of BSA entitiesjust under the dense interfacial protein layer. Supposingthe density,r(z), of the sub-surface layer(s) decreasesexponentially with the distance,z, from the interface,we can write:

r(z)sr(z ) exp(yhyl*) (15)0

wherel* is the characteristic size of the protein speciesthat constitute the second layer(s). We neglect thecorrelations between the moieties in the sub-surfacelayer and consider the case when two surfaces approacheach other. Imposing non-overlapping of the proteinsub-layers, one obtains the following expression for therepulsive pressure:w38x

UU UG kTyl hyl expyhylŽ .Ž . Ž .2

P h s , (16)Ž .repulsion UU1y 1qhyl expyhylŽ . Ž .

whereG is the adsorption in the second(sub-surface)2

protein layer only. For distances larger than the charac-teristic size of the protein species forming the sub-layer,Eq. (16) reduces to:

U U UP h sG kTyl hyl expyhyl (17)Ž . Ž .Ž . Ž .repulsion 2

Assuming additivity of the DLVO pressure(Eq.(10))and the repulsive steric pressure Eq.(17), we fit ourexperimental data using as free parametersG and l*2

(solid line in Fig. 4). The values obtained are 0.23 mgym and 10.7 nm, respectively. From a physical point of2

view these values are quite reasonable. An increase ofthe total adsorption(after the equilibrium value of thesurface tension is reached) of approximately 0.5 mgym is reported in the literaturew39x. This is exactly what2

G stands for in model. The value ofl* is consistent2

with the size of the BSA molecule(11.4=11.4=4.1nm), as well as with the radius of gyration of a BSAmolecule of;9 nm.


Fig. 5. Comparison between the data obtained from TLF, MCT andSFA for systems stabilized with BSA. See text for details.

We consider now the MCT experiments. In all ourattempts to wash the emulsions with ‘pure’ BSA wealways obtain flocculated samples. When all Tween 20is removed, the droplets flocculate and form largeclusters. Therefore, it was necessary to prevent theflocculation of the samples by adding traces of Tween20.We perform a set of measurements in which we varied

the Tween 20 and BSA concentrations over a widerange. At constant Tween 20 concentration, when theprotein content is increased from 0 to 0.4 wt.%, there isa progressive change with respect to classical double-layer repulsion. Typical results are shown in Fig. 4b,where the Tween 20 concentration is kept at 5=CMC.For pure Tween 20, the repulsion profile is fitted bystandard DLVO curve, yielding a surface potential ofy17 mV, which is in very good agreement with thez-potential of the dropletsw8x. The profiles in the presenceof the same quantity of BSA at surfactant content of20=CMC and 50=CMC are very similar and do notdepend on the Tween 20 concentration as wellw8x. Thissuggests that the presence of the protein dominates thebehavior and the properties of these systems.z-potentialmeasurements of these droplets gave values of 0–3 mV,meaning that the contribution of the electrostatics to thetotal repulsive force is negligible. This result undoubt-edly shows that the operative repulsion, which weregister, is due to specific(non-DLVO) interactions.Bearing in mind this remark we compare the data

from the Mysels cell experiments with those from MCTin Fig. 5. The disjoining pressure is transformed intoforce in the same manner as in the case ofb-casein andcompared with data from MCT and SFAw40x. The datafrom MCT and TLF show reasonably good agreement.The results from the SFA studies are only qualitativelysimilar to both MCT and TLF data. The reason idessentially the same as in the case ofb-casein-stabilizedfilms, i.e. the difference in the conformational state ofBSA on solid–fluid and on fluid–fluid interfaces.

3.3. Threshold flocculation force

During the preparation of the samples for MCT, weobserved a very strong dependence of the properties ofemulsions on the history of preparation. In some casesthe emulsions were irreversibly flocculated during thewashing steps. The flocculation is always observed aftercentrifugation, i.e. when the droplets are brought incontact. As already discussed, it was impossible toproduce non-flocculated droplets stabilized solely withBSA. For the cases of BLG andb-casein it is possibleto produce non-flocculated emulsions stabilized by theprotein only, however, these systems show the sametendency to flocculate when centrifuged at accelerationbigger than 150–200=g. We use this fact for determi-nation of the threshold flocculation force employing theMCT in the following way.The investigated sample was submitted to a magnetic

field. We registered the force and inter particle distance,as described in Section 2.2.1 and switch off the magneticfield. Then we observed the emulsion under microscope.The removal of the magnetic field is expected to leadto instantaneous decomposition of the formed chains.This is observed for ferrofluid oil in water emulsionsstabilized by low molecular weight surfactantsw25,26x.For the proteins studied here, we observe under micro-scope that above some critical force, the dropletsremained irreversibly chained even in the absence of amagnetic field. Fig. 6 shows the force profiles forferrofluid oil-in-water emulsions stabilized by 0.1 wt.%protein at various ionic strengths. When the dropletsurfaces were covered byb-casein, we observe standardDLVO behavior up to a force,F*, above which dropletsare held together in flexible chains that persist in time.Importantly,F* is lower than the force corresponding tothe maximum of the repulsive barrier. The dashed linesin Fig. 6a are the best DLVO fits with a detailed accountfor the exclusion volume effect ofb-casein micelles.The dependence ofF* on the ionic strength is shownin Fig. 6c. The decrease in the resistance against floc-culation when the ionic strength increases may be dueto the screening effect of salt. As far as we know, valuesof that kind are the first directly measured.We tried to measure also the forces between droplets,

covered by BLG. We were able to measure only lowforces, since the application of forces as small as 1 pNlead to irreversible flocculation of the emulsion droplets.In fact, the quality of the Bragg peaks in this case isvery low. The latter results in a big uncertainty in thedistance determination. A bad quality diffraction patternmay come either from non-perfect alignment of thedroplets in chains or from the large polydispersity. Weshould admit that some small flocs(containing two orthree droplets) are observed even after the first washingcycle. A few typical force profiles are shown in Fig. 6b,where the concentration of the protein in the continuous


Fig. 6. (a) Force vs. distance profiles for ferrofluid emulsions stabi-lized with 0.1 wt.%b-casein at pH 6.2 and at different ionic strength(points). The lines(where presented) are the best fits.(b) Force vs.distance profiles for ferrofluid emulsions stabilized with 0.1 wt.%BLG at pH 6.0 and at different ionic strengths.(c) Threshold floc-culation force for ferrofluid oil-in-water emulsions stabilized withb-casein and BLG as a function of the ionic strength. The lines areguides for the eye.

Fig. 7. Optical micrographs of a ferrofluid oil-in-water emulsion sta-bilized by 0.04% BLG. The bar corresponds to 10mm. (a) on fieldcorresponding to force of ca. 5 pN.(b) ca. 30 min after switching offthe magnetic field.(c) 24 h after switching off the magnetic field.

phase is 0.1 wt.%, and the ionic strength is varied from5=10 to 1=10 M. The pH of the samples isy5 y2

6.0"0.1. No force profile is really measured for ionicstrength of 1=10 . The emulsion flocculates irrevers-y2

ibly at the lowest applied force of ca. 0.5 pN. Oneanticipates that the electrolyte makes the force shorterranged,(as expected in the framework of the conven-tional double-layer repulsion). Because of both distanceuncertainty and narrow force range we do not make anyattempts to fit these experimental data. Evidently, thethreshold flocculation force, plotted in Fig. 6c refers tothe irreversible chaining and has to be considered as anupper limit.Fig. 6c suggests that the critical threshold flocculation

force (under equivalent conditions) is lower for BLG-stabilized emulsions. The effect is more pronounced forhigher ionic strength. These results are inline withexperiments on bulk quantities of micron sized emul-sions stabilized by BLG andb-casein. While the BLG-stabilized samples are almost always flocculated(tightflocs are observed), the flocculation in theb-casein case

(under similar experimental conditions) is transient, withtemporarily existing flocsw10x.Another interesting phenomenon is observed for BLG-

stabilized system. The droplets are so tightly joined thatthe ‘irreversible’ chains are linear rods that remainunchanged for a period as long as 48 h. The chainsformed byb-casein stabilized particles are rather flexibleand tend to decompose for the same period of time. Anexample is shown in Fig. 7, where we present opticalmicrographs of a BLG-stabilized emulsion. Applicationof a magnetic field corresponding to a force of 1 pNleads as expected to a formation of chains, which areoriented parallel to the field. The imperfections in thechains (arrows in Fig. 7a) are most probably smallclusters comprising several droplets. Such clusters arenot present in the original emulsion before the introduc-tion of BLG. They appear during the process of replace-ment of Tween 20 with BLG. The ‘grains’ in the chainsexplain the low quality of the Bragg peaks.


Fig. 8. Flocculation induced by the interaction between the adsorbedprotein layers. The data are the same as in Fig. 6a, theY-scale ischosen to show the repulsive profiles as obtained from the best fit.The thickness of the protein layers is exaggerated.

After removal of the field(Fig. 7b) the chains do notdecompose, and we observe rigid rods, which stay thesame even after 24 h(Fig. 7c). The existence of theserods suggests that the contacts between the individualdroplets, forming the chains are substantially strong andcannot be affected by the thermal motion(i.e. energyof the order of kT). The dark dots in Fig. 7b,c aresmaller flocs, comprising a few droplets. The imageslook somehow out of focus, which is due to the factthat the investigated objects are small. Both the rodsand the tiny flocs are close to the resolution limit of theoptical microscopy and are constantly moving due tothe Brownian motion.

4. Discussion

The measurements of the force–distance profiles,together with the threshold flocculation force mayexplain some of the discrepancies which are frequentlyobserved between the ‘single film’ studies and thebehavior of bulk emulsions. The output of the ‘singlefilm’ studies is always repulsive potentials: This is alsothe case in the present work. However, there are numer-ous experimental observations of irreversibly flocculatedmicron-sized dropletsw10x. Based on the experimentsdescribed above, we propose the following physicalpicture. When the droplets are apart from each other, ina way that the two protein layers are not in contact, theoperative laws are long-ranged repulsive forces of vari-ous origin. This is what we measure employing theMCT and the Mysels cell. Fig. 8 provides a graphicalguidance. This is the same plot as Fig. 6a, but theY-scale is chosen so, that the maximum repusion force isshown. When droplets get closer, the approaching pro-

tein layers start ‘touching’ each other. This allows someof the adsorbed entities to interact forming a local link.We emphasize that these arepoint-like contacts due tointeractions at molecular scale. We have termed theselinks ‘surface aggregates’ because in the thin liquid filmexperiment as well as in Brewster angle microscopystudies,w29x the biggest of them appear like lumps ofmaterial located on the surface. We stress again thatthose lumps appear on the interface as a result of rapidsurface distortion. They are not a product of bulkinteractionsw29x. AFM images ofb–casein and BLGlayers formed at water-tetradecane interface and furthertransferred on mica by Langmuir–Blodgett technique,reveal a network formed by the adsorbed speciesw41x.This is a further evidence for the interaction betweenthe proteins in the adsorbed state. The network formationis possible because the adsorbed proteins are unfolded(denaturated) and therefore segments of various typesare available for interaction. Evidently, a distortion of aprotein layer will generate more interaction sites. Thisexplains why we observe increasing number of aggre-gates each time a new film is formed in the Mysels cell.Provided that two protein-covered droplets are sufficient-ly close, there is no difference whether the interactingentities belong to the same or to different protein layers.This interaction of the adsorbed species is a local

spontaneous process; therefore it cannot be captured bythe classic mean-field theories(like DLVO for example).The latter consider the stabilizing layers as uniform.This assumption fails when two protein layers come ata distance, which allows spontaneous local interactions.Therefore, the value of the critical flocculation force,measured above, has to be considered as the forcenecessary to bring the droplets at the minimum distanceallowing for the formation of surface clusters betweenthe two protein layers. This is the reason whyF* islower than the maximum repulsive force, predicted bythe model.The flocculation is irreversible when the thermal

energy(somekT) becomes insufficient to disjoining theaggregates.

5. Conclusions

The experiments employing the magnetic chainingtechnique(MCT), as well as our variant of the Myselscell permitted to obtain data concerning the repulsivebranch(es) of the interactions between protein layersformed at liquid–liquid interfaces. For BSA-stabilizedsystems, a long-ranged repulsion is operative. It is notof an electrostatic origin, as both experiment and cal-culations proved. It most probably originates from theformation of multiple protein layers at the interface, aneffect that is well documented in the literature. Theinteractions betweenb-casein layers formed at thewateryhexadecane interface are governed by electrostatic


repulsion. Due to the relatively large final thickness ofapproximately 20 nm, the van der Waals component ofthe total disjoining pressure is negligible. The oscillatorycomponent of the disjoining pressure is also very smallfor the studied protein concentration of 0.1 wt.%. How-ever, our previous studiesw8x have revealed that theonset of the exclusion volume effect due to theb-caseinmicelles can be detected at similar concentrations.MCT and Mysels cell experiments are very delicate

in general, and we emphasize that in both cases theextracted information refers to the situation where theprotein-covered surfaces are approachedymanipulated ina quasi-static manner. The latter was found to be anecessary prerequisite to obtain layers free from surfaceaggregates.Our experiments reveal a substantial difference

between the pertinent force laws existing between pro-tein-stabilizedliquid droplets(measured both with MCTand Mysels cell) and mica surfaces covered by proteins(literature data from SFA experiments). This result canbe justified considering the difference of the proteinconformation on solid and liquid interfaces.We also employed the MCT for determining the

threshold flocculation force in the case ofb-casein andBLG stabilized emulsions. Importantly, the flocculationis due to the formation of local aggregates between theapproaching droplets, rather that the overcome of arepulsive barrier. Our results suggest that the criticalthreshold flocculation force(under equivalent condi-tions) is the lower for BLG. Furthermore, the flocscomprising BLG-covered droplets are more rigid thanthe flocs formed fromb-casein stabilized droplets.

Acknowledgments

This work was mainly done in Centre de RecherchePaul Pascal, Pessac, France and LCPE, Sofia. It wasfinancially supported by a Ph.D. scholarship from Labor-atoire Franco- Bulgare(TDD) and partially by KraftGeneral Foods. The stay in MPIP of TDD was possiblethrough a Marie Curie Fellowship of the EuropeanCommunity program ‘Human Potential’ under contractnumber HPMF-CT-2001-01188. The authors are indebt-ed to Dr Asen Hadjiiski for fruitful discussions as wellas to Ms Nikolina Vassileva for the assistance in someof thin films experiments.

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Surface forces in model oil-in-water emulsions stabilized by … · 2007-04-30 · phase, the surfactants lower the interfacial tension much more than the proteins (consider 13–15