Forces between Emulsion Droplets Stabilized with Tween 20 and

Langmuir 1999, 15, 8813-8821

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Forces between Emulsion Droplets Stabilized with Tween 20 and Proteins Tatiana D. Dimitrova*,† and Fernando Leal-Calderon‡ Laboratory of Thermodynamics and Physicochemical Hydrodynamics, Faculty of Chemistry, Sofia University, 1, James Bouchier Ave., 1164 Sofia, Bulgaria, and Centre de Recherche Paul Pascal, Av. Schweitzer, 33600 Pessac, France Received April 21, 1999. In Final Form: August 5, 1999 We measured forces between tiny emulsion droplets by using the magnetic chaining technique. Force versus distance profiles were obtained for emulsions stabilized by Tween 20, Tween 20/BSA (bovine serum albumin) mixtures, and β-casein. For emulsions stabilized with Tween 20 alone, we observed classical DLVO behavior. At very large Tween 20 concentration, the force profile is clearly deviating from the double-layer repulsion. An interpretation of the data is proposed combining classical DLVO with steric-like additional effective repulsion due to the micellar condensation on the droplet surface. The systems that contain both Tween 20 and BSA qualitatively show the same kind of behavior: the purely electrostatic repulsion is progressively transformed into a steric one in the presence of BSA. The emulsions stabilized by β-casein obey a different type of behavior: in that case the depletion attraction is found to be of great importance. The data are interpreted assuming additivity of standard DLVO forces and depletion attraction. The threshold flocculation force, as a function of the ionic strength, was measured for these systems.

I. Introduction A dispersion of a liquid in another, immiscible one, is often referred as emulsion. Emulsions are thermodynamically nonstable systems since their production requires formation of a large interfacial area, formed by the impact of mechanical energy. As a result, the stability of the emulsion systems can be achieved only kinetically, even though their shelf life could be as long as a few years if the stabilizing surfactant(s) is (are) properly chosen. In the present work, we study oil-in-water emulsion systems stabilized by proteins with great applicability in the industry. Actually most of the nutrition and cosmetic products delivered on the market are emulsions. In both cases, they are stabilized mainly by natural surface active products as proteins (treated or not with hydrolyzing enzymes), lipids, small amounts of nonionic surfactants such as Tweens, Spans, etc., and variety of combinations of all them. The complexity of the proteins themselves and the multicomponent composition of food systems definitely require the investigation of model systems in order to understand the main factors and mechanisms governing the properties and the stability of the emulsions. The model experiments could be divided in two typical groups. In the first one fall all the experiments on macroscopic quantities of batch emulsions.1-6 In that case one obtains different useful data for the overall emulsion behavior and evaluates the influence of different factors on droplet size growth, creaming, viscosity, flocculation, etc. The main drawback of this class of methods is the lack of straightforward quantitative theory that can be applied to describe the results. * Corresponding author. E-mail: [email protected]. † Sofia University. ‡ Centre de Recherche Paul Pascal. (1) Dickinson, E.; Golding M.; Povey, M. J. W. J. Colloid Interface Sci. 1997, 185, 515. (2) Dickinson, E.; Golding M. J. Colloid Interface Sci. 1997, 191, 166. (3) Chen J.; Dickinson E. J. Agric. Food Chem. 1998, 46, 91. (4) Agboola, S. O.; Singh, H.; Munro, P. A.; Dalgleish, D. G.; Singh, A. M. J. Agric. Food Chem. 1998, 46, 84. (5) Agboola, S. O.; Dalgleish, D. G J. Food Sci. 1998, 46, 84. (6) Horne, D. S. Curr. Opin. Colloid Interface Sci. 1996, 1 (6), 752.

In the second group are included the model experiments designed to mimic the interaction between two single drops (bubbles) or surfactant layers. These methods are mainly the surface force apparatus (SFA) and thin liquid film studies. Different studies7-11 are based on the use of the surface force apparatus or other techniques as bimorph surface force apparatus (MASIF)7 for a large variety of protein systems. The information obtained consists of isotherms of the force (or disjoining pressure) versus distance. It is shown that protein layers interact in a quite complicated way, and the conventional DLVO theory frequently fails in explaining the observed phenomena. In almost all cases a compression/decompression hysteresis in pressure versus distance curves is evidently observed,7 a fact that is an indirect proof of the existence of strong specific protein-protein interactions and/or cross-binding. The data extracted in this way are very important in many fields, but it is hard to establish a direct correlation between them and the properties of emulsions. The reason is mainly the totally different ways in which the proteins adsorb on solid and on liquid interfaces. It is believed that during the adsorption on a solid surface the loss of the secondary and tertiary structure is minimal,7,9,10 while proteins adsorbed on liquid (air-water or oil-water) interfaces are gradually unfolded.12 In emulsion systems the overall stability is governed by the properties of thin liquid film, which separates the droplets.13 In real emulsions the films are formed by (7) Claesson, P. M.; Blomberg, E.; Fro¨berg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57, 161. (8) Chowdhury, P. B.; Luckham, P. F. Colloids Surf. B. 1995, 4, 327. (9) Fitzpatrick, H.; Luckham, P. F.; Eriksen, S.; Hammond, K. Colloids Surf. 1992, 65, 43. (10) Gallinet, J. P.; Gauthier-Manuel, B. Colloids Surf. 1992, 68, 189. (11) Blomberg, E.; Claesson, P. M.; Tilton, R. D. J. Colloid Interface Sci. 1994, 166, 427. (12) Murray, B. S.; Færgemand, M.; Trotereau, M.; Ventura, A. In Food Emulsions and Foams; Dickinson, E., Rodrı´guez-Patino, J. M., Eds.; Royal Society of Chemistry: London, 1999; p 223. (13) Ivanov, I. B.; Dimitrov, D. S. In Thin Liquid Films; Ivanov, I. B., Ed.; Marcel Dekker: New York, 1988; Chapter 7 and references therein.

10.1021/la9904758 CCC: $18.00 © 1999 American Chemical Society Published on Web 11/17/1999

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Langmuir, Vol. 15, No. 26, 1999

pressing two drops one against the other under action of different forces, e.g., surface forces, gravity, Brownian motion, etc. A single emulsion type film can be formed by pressing an oil droplet against a large oil-water interface.14-16 Another central technique for mimicking the interaction between two single (protein-covered) droplets is the thin liquid film balance applied to emulsion films. The method, pioneered by Scheludko,17 consists of forming a thin film in a narrow capillary by sucking liquid from a biconcave meniscus. One can determine many important parameters such as lifetime, thickness, contact angle, energy of interaction, etc. Experiments in the conventional or miniaturized Scheludko-type cell18 were employed for examination of interfacial properties of proteins that differ in their structure, pI value (isoelectric point), etc. In the case of bovine serum albumin (BSA) strong sticking between the opposite protein layers and a well-pronounced aging effect were observed.19 In most experiments the same authors have observed also the formation of lumps and aggregates in the film and contact angle hysteresis, both indicating the importance of specific protein-protein forces. Clark and co-workers20 developed an experimental setup similar to that proposed by Scheludko.17 They combined a similar film formation technique with fluorescence recovery after photobleaching (FRAP). The method allows determining the surface diffusion coefficient in the protein or protein-surfactant mixed layers, which is useful for understanding the film stability. However both Clark and Scheludko techniques have one important disadvantage: the maximum attainable capillary pressure is very lowsabout 100 Pa. That problem could be overcome by employing the setup proposed by Mysels21 and further developed by Bergeron.22 In that case the film is formed in a hole, drilled in a porous glass plate, and the maximum attainable pressure is determined by the mean size of the pores. However in a greater number of cases where large protein films are investigated, film thickness measurements are impossible. For example, the protein aggregates19,23 in the film make the interface inhomogeneous. Hence the evaluation of the disjoining pressure isotherms is restricted because of the inapplicability of the interferometric method for thickness determination. In the case of BSA it is found19 that the aggregate formation in thin liquid films is a consequence of the aggregation on the surface, since no clustering in the bulk protein solution was observed. The magnetic chaining technique24-26 combines the advantages of the methods mentioned above, providing the possibility to study the phenomena in conditions very (14) Dickinson, E.; Murray, B. S.; Stainsby, G. J. Chem. Soc., Faraday Trans. 1988, 84, 871 (15) Basheva, E. S.; et al. submitted for publication in Langmuir. (16) Kralchevsky, P. A.; Danov, K. D.; Denkov, N. D. In Handbook of Surface and Colloid Chemistry; Birdi, K. S., Ed.; CRC Press: Boca Raton, FL, 1997, Chapter 11 and references therein. (17) Scheludko, A. Kolloid-Z. 1957, 155, 39. (18) Velev, O. D.; Constantinides, G. N.; Avraam, D. G.; Payatekes, A. S. Borwankar, R. P. J. Colloid Interface Sci. 1995, 175, 68. (19) Marinova, K. G.; Gurkov, T. D.; Velev, O. D.; Ivanov, I. B.; Campbell, B. Borwankar, R. P. Colloids Surf. 1997, 123-124, 155. (20) Clark, D. C.; Coke, M.; Mackie A. R.; Pinder, A. C.; Wilson, D. R. J. Colloid Interface Sci. 1990, 138, 207. (21) Mysels, K.; Jones, A. J. Phys. Chem. 1964, 68, 3441. (22) Bergeron, V. Ph.D. Thesis, University of California, Berkley, USA, 1993. (23) Gurkov, T. G.; Marinova, K. G.; Zdravkov, A. Z.; Oleksiak, C.; Campbell, B. Prog. Colloid Polym. Sci. 1998, 110, 263. (24) Leal-Calderon, F.; Stora, T.; Mondain-Monval, O.; Poulin P.; Bibette, J. Phys. Rev. Lett. 1994, 74, 2959. (25) Mondain- Monval, O.; Leal-Calderon F.; Bibette, J. J. Phys. II 1996, 6, 1313. (26) Espert, A.; Omarjee; P.; Bibette, J.; Leal-Calderon F.; MondainMonval, O. Macromolecules 1998, 31, 7023.

Dimitrova and Leal-Calderon

close to the those in practical systems. It works with droplets of colloidal size (about 0.2 µm in diameter), i.e., at true capillary pressures and with liquid interfaces. A very important feature of the method is the possibility to explore very low levels of forces: the force resolution is about 2 × 10-13 N, approximately a half decade below the force resolution in the SFA technique. This corresponds to a force normalized by radius of about 10-6 N/m. We applied the technique to emulsions stabilized with Tween 20, β-casein, and BSA/Tween 20 mixtures. In the present paper we report the experimental results and we propose some interpretations. II. Experimental Section Substances. Sodium dodecyl sulfate (SDS) was purchased from Aldrich. Tween 20, β-casein (C 6905), and NaN3 were Sigma products. BSA (fraction V, 96-100% protein on dry basis) and NaCl and HCl, which were used for pH adjustments, were Acros products. All substances were of the highest possible grade and were used as received. The oil used for emulsion preparation was ferrofluid, prepared by Ferrofluidics. It is a 10% (v/v) dispersion of ferromagnetic (Fe2O3) particles in octane. The grains, 10 nm in size, are stabilized against aggregation by 2% (w/v) oleic acid. It is proven that the particles remain dispersed in the range of magnetic fields applied in the experiment (0-500 G). Emulsion Preparation, Purification and Washing. The primary crude ferrofluid-in-water emulsion was prepared according to the method of emulsification in viscoelastic media developed by Mason et al.27 As emulsifier we used SDS. This technique leads to relatively narrow sized droplet distributions, but not sufficient for the applied force measurement technique. So the emulsion was diluted with SDS solution and further refined following a fractionation crystallization procedure similar to that suggested by Bibette.28 At least six to seven steps were necessary to obtain an emulsion with sufficient degree of monodispersity. This SDS-stabilized emulsion was further treated in order to cover the interfaces with the chosen surfactant. That was achieved by applying a washing procedure, which consists of concentrating the emulsion by means of centrifugation, followed by replacing the supernatant with a new aqueous phase which contains the desired surfactant. When the process is repeated a few times, only negligible traces of the primary surfactant (SDS) are left at the droplets interface. It is necessary to choose properly the experimental conditions in order to ensure no change of the size distribution and no effects as flocculation and coalescence. It is well-known that the ionic surfactants unfold protein molecules because of their specific adsorption on the protein molecule.29 The denaturation is almost negligible in the case of nonionic surfactants.30 For that reason it was necessary to cover the droplets with a nonionic surfactant before any attempt to add protein(s) was made. We chose Tween 20, which is a common ingredient in many dairy products such as ice creams, yogurts, etc. After the coverage of the emulsion droplets with Tween 20, we continued the washing process in the same way in an attempt to adsorb BSA or β-casein on the interface. All the solutions contained 0.02 g/L of NaN3 as a bactericide. They were prepared with water purified by a Millipore Milli-Q unit (Resistance 18.2 MΩ. cm-1) and filtered through a 0.22 µm Millipore cutoff filter prior to use. The protein solutions were stored at a constant temperature of 23 °C for no longer than 8 h. The size of the droplets was determined by dynamic light scattering measurement. We found mean diameter of 176 nm and polydispersity of about 5%. Dynamic light scattering measurements were performed by means of a Brookhaven correlator. (27) Mason, T. G.; Bibette, J. Phys. Rev. Lett. 1996, 77, 3481. (28) Bibette, J. J. Colloid Interface Sci. 1990, 147, 474. (29) (a) Turro, N. J.; Xue-Gong, L.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525. (b) Guo, X. H.; Zhao, N. M.; Chen, S. H.; Teixeira, J. Biopolymers 1990, 29, 335. (30) Dalgleish, D. G. Curr. Opin. Colloid Interface Sci. 1997, 2, 573.

Forces between Emulsion Droplets

Langmuir, Vol. 15, No. 26, 1999 8815 The repulsive force, Fr, between the droplets must exactly balance the attractive force between the dipoles induced by the applied magnetic field. Since the dipoles are aligned parallel to the field, this force can be calculated exactly and is given by24-26

Fr ) Fm(d) ) -

1.202 3m2 2πµ0 d4

(2)

where µ0 is the magnetic permeability of free space and m is the induced magnetic moment of each drop. The induced magnetic moment is determined self-consistently from the magnetic susceptibility of the ferrofluid, and the presence of the neighboring droplets. Thus

m ) µ0

4 πR3 χs HT 3

(3)

where HT is the total magnetic field acting on each drop and χS is the susceptibility of a spherical droplet. The total applied field, HT, is given by the sum of the external applied field, Hext, and the field from the induced magnetic moments in all the neighboring drops in the chain. This can easily be calculated for an infinite chain, assuming point dipoles, giving

HT ) Hext + 2 × 1202 Figure 1. Scheme of the experimental setup (not to scale). Electrophoretic mobilities of the emulsions were measured using a Malvern Zetasizer II. The electrophoretic mobility was transformed into ζ-potential according to the Smoluchowski equation.16 pH Control. The pH values were measured by means of a digital pH-meter, equipped with protein and a surfactantresistant electrode. The proteins obey buffering properties. When dissolved in water they generally maintain certain pH value (which may depend on the purification process), frequently referred as “natural pH”. This property of the proteins was used in our experiments to control the pH. For each protein (β-casein or BSA) we verified that the pH is constant within 0.2 units over the range of concentrations studied. No drift of the pH values of the protein solutions was observed over the period of storage (8 h). We chose this way to maintain the pH and did not use a conventional buffer solution because buffers contribute to the total salt content (i.e., the ionic strength) and this significantly narrows the available range of ionic strengths. Setup. The principle of the magnetic force measurement technique is described elsewhere.24-26 The scheme of the setup is presented in Figure 1. This technique exploits the properties of paramagnetic monodisperse droplets. The applied field induces a magnetic dipole within each ferrofluid droplet. The interaction between the magnetic dipoles leads to formation of linear arrays of particles (chains) which are parallel to the external magnetic field. At very low droplet volume fractions (φ < 0.1 vol %) the chains are only one droplet thick and the droplets in the chains remain well separated. If the chains are illuminated by a white light source, the emulsion appears beautifully colored in the backscattering direction. These colors originate from Bragg diffraction and provide a straightforward measure of the spacing between droplets within the chains. For perfectly aligned particles at a separation d, illuminated by incident white light parallel to the chains, the first-order Bragg condition reduces to

d ) λ0/2n

(1)

where n is the refractive index of the suspending medium (n ) 1.33 for water) and λ0 is the wavelength of the light Bragg scattered at an angle of 180°. The wavelength corresponding to the maximum of the Bragg peak provides a direct measurement of the average spacing between the drops with a precision of about 1.5 nm. Because the drops are non-deformable31 owing to their large capillary pressure (∼1 atm), it is possible to determine the interfacial separation, h ) d - 2R, where R is the droplet radius.

2m 4πµ0d3

(4)

This technique allows one to measure interparticle forces as small as 2 × 10-13 N, corresponding to the minimum force required for forming chains. All experiments were performed at room temperature (23 ( 1 °C).

III. Results and Discussion Tween 20 is the common name of the substance polyoxyethylene (20) sorbitan-monolaurate. It is a widely used nonionic surfactant, which has a very low critical micelle concentration (cmc)sabout 2 × 10-5 mol/L. The purity of the Tween 20 was checked by conductivity measurements. In all cases 3.08 × 10-4 mol/L of NaN3 was present. No increase in the conductivity of the surfactant solutions was found when the concentration of Tween 20 was varied from 0 to 650cmc. We can therefore conclude that the ionic impurities in the substance (if any) can be neglected. The absence of parasite charges permits the ionic strength of the surfactant solutions to be exactly calculated from the NaN3 content (see below). We examined the force isotherms at different concentrations of the surfactant and found a strong repulsion between the surfaces, which we believe to have an electrostatic origin at low Tween 20 concentrations. A set of curves is presented in Figure 2. In all cases we used 3.08 × 10-4 mol/L of NaN3 as an inorganic electrolyte to fix the ionic strength in the continuous phase. The measured pH value is 5.6 ( 0.1 in all cases. For the lowest concentration of Tween 20 in the aqueous phase (1cmc) we fitted the curve with a classical DLVO curve varying as a free parameter only the surface potential, Ψ0. The value used for the inverse Debye length, κ(nm-1) ) 3.92CNaN31/2 (mol/L), was calculated from the NaN3 concentration. The van der Waals attraction was calculated via the Hamaker formula for non-deformed spheres,32 taking AH ) 4.1 × 10-21 J. This value of the Hamaker constant corresponds to the case of octane droplets acting across aqueous media.33 Certainly the presence of 10 vol % of mineral particles in the oil leads to a slightly greater value of the effective Hamaker constant, in comparison with the value which (31) Ivanov, I. B. Pure Appl. Chem. 1978, 52, 1241. (32) Hamaker, H. C. Physics 1937, 4, 1058. (33) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992.

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Figure 2. Force versus distance profiles for ferrofluid emulsions, stabilized with Tween 20 at different surfactant concentrations (points). The ionic strength is 3 × 10-4 mol/L, pH ) 5.6. The line is the best DLVO fit for the lowest surfactant concentration (see the text for details).

Figure 3. Evolution of the force profiles for ferrofluid emulsions, stabilized with Tween 20 with the ionic strength and pH (points). The Tween 20 concentration in all cases is 25cmc. The lines are the best DLVO fits.

was used. However, at 9-10 nm (the smallest distance which is measured in our experiment) the contribution from the van der Waals attraction to the total force measured is calculated to be always smaller than 7% and further decreases at large distances. Hence the value for the Hamaker constant, quoted above, is reasonable as a first approximation. The expression used for the electrostatic force was34

[

Fel ) 4π0 Ψ02 R2

]

1 κ exp(-κh) (5) + h + 2R (h + 2R)2

Hereafter and 0 denote the dielectric permeability in the solution and under vacuum, respectively. Expression 5 is derived assuming linear superposition of single sphere potentials and it is generally valid for κR g 5 and κh g 2. When κR < 5 one should use the other expression for the force, derived adopting the Derjaguin approximation16,34

[

Fel ) 2π0 Ψ02 Rκ

]

exp(-κh) 1 + exp(-κh)

(6)

From the fit shown in Figure 2, we found Ψ0 ) -22 mV, which is in very good agreement with the experimentally measured value for the ζ-potential of droplets of -17 ( 3 mV. A value of -23 ( 3 mV was found for xylene droplets covered with Tween 20, under similar conditions.35 The iron oxide grains in the ferrofluid oil droplets are stabilized by 2% oleic acid, which is also present at the oil/water interface. Its presence could give rise to some additional charge on the droplet surface.25 To elucidate the role of the oleic acid, we performed two experiments maintaining the overall ionic strength at a constant value of 1.3 × 10-3 mol/L and the Tween 20 concentration at 25cmc. In the first case the pH was set to 3 by adding the appropriate amount of HCl. At this pH the ionization of the oleic acid at the interface is negligible.25 In the second case the ionic strength is fixed by 0.001 mol/L NaCl and 0.0003 mol/L NaN3. The pH was 5.6. The results are shown in Figure 3. Once again we used the calculated value for κ to fit the experimental data. We obtained different Ψ0 values for the two systems, -17.3 and -27.4 mV. This difference could be explained by the partial ionization of the oleic acid molecules at the interface, providing additional (34) Russel, W. B.; Saville, D. A.; Schowalter, W. R. Colloidal Dispersions; University Press: Cambridge, 1989. (35) Marinova, K. G.; Alargova, R. G.; Denkov, N. D.; Velev, O. D.; Petsev, D. N.; Ivanov, I. B.; Borwankar, R. Langmuir 1996, 12, 2045.

Figure 4. Force versus distance profiles for ferrofluid emulsions stabilized with Tween 20 at concentrations much above the cmc (points). The experimental conditions are the same as in Figure 2. The solid lines are the best fits with the additional repulsive force added. The dashed lines represent the classical DLVO interaction in each case.

charges. The charges produced by the ionized oleic acid at the interface contribute to the total surface potential at pH ) 5.6. As a result we observed stronger electrostatic repulsion. At higher surfactant concentration, one can see a wellpronounced deviation from the classical DLVO profile (see Figure 2 and Figure 4). This effect is evident for all Tween 20 concentrations above about 50cmc. The curves are roughly linear (in semilog plot) at large distances but clearly deviate from linearity at short distances. Let us consider the possibility of some depletion attraction occurring. The cmc of Tween 20 is very low; hence the value of 500cmc corresponds to about 1.1 wt %. For that concentration, taking the aggregation number of 80 we calculated the depletion potential16 at zero distance to be about 0.5kBT units (kB being the Boltzmann constant), which is not sufficient to cause depletion attraction and to modify the force profiles. We measured the hydrodynamic radii of our emulsion droplets at different concentrations of Tween 20 in the continuous phase. As shown in Figure 5. there is a continuous increase in the size in the interval of surfactant concentrations in the bulk solution between 1 and 100cmc. The ordinate in this graph corresponds to the ratio between the droplet size measured at high concentration and the size measured at 1cmc. The diameter of the Tween 20 micelles is 7.3-7.5 nm,36 and its variation over the range of concentrations studied falls in the experimental uncertainty. The mean diameter of our ferrofluid droplets at 1cmc is 176 nm. Hence for concentrations larger than 100cmc the effective size is increased by around two micellar diameters. Since the precision of the dynamic (36) Alargova, R. Private communication.


Langmuir, Vol. 15, No. 26, 1999 8817 Table 1. Fitting Parameters for the Highest Tween 20 Concentrationsa concn, cmc

ionic strength, mol/L

apparent Ψ0, mV

f0, pN

λ, nm

100 250 500

3.08 × 10-4 3.08 × 10-4 3.08 × 10-4

-17.5 (6) -16.1 (6) -14.3 (5)

361.3 140.7 32.1

3.70 4.12 4.37

a The values in parentheses correspond to the number of points used for the calculation of the apparent surface potential, Ψ0.

Figure 5. Relative increase of the mean droplet diameter (measured by dynamic light scattering) of the ferrofluid emulsion droplets, stabilized with Tween 20, as a function of the surfactant concentration. The experimental conditions are the same as in Figure 2.

light scattering (DLS) measurements is a few nanometers and the changes we measured were almost of the same order of magnitude, we took all precautions to ensure reliability of the results. Each point in Figure 5 is the average value of at least three measurements and corresponds to an independent experiment. The volume fraction of the oil droplets is the same in each case, so the differences could not be ascribed to some long-range interactions between the investigated species. Furthermore the value of the diameter is reproducible after three times dilution of the emulsion with the corresponding aqueous phase. An explanation for both the force profile evolution and the increase of the hydrodynamic radius at higher Tween 20 concentration may be found in ordering/adsorption of micelles at the surface of the droplets. The (most probably) loosely packed micellar layers introduce “roughness” at the surface, and thus modify the electrostatic repulsion at short distances. The adsorption of micelles on isolated interfaces is reported in the literature. For example X-ray reflectivity reveals condensation of one or two layers of AOT micelles away from an isolated air/water interface.37 Few different methods38 reveal direct adsorption of block copolymer micelles onto a solid substrate covered with molecules of the same type. Adopting the hypothesis for micellar condensation at the interface we fitted the observed profiles in the following way. At long distances, i.e., above 25 nm, we fitted the profiles for Tween 20 concentrations of 100, 250, and 500cmc supposing that the operative force is of DLVO type, where the electrostatic component was calculated through eq 5. The values for Ψ0 obtained were further used for the calculation of the total force over the whole distance range. At short distances, we assume additivity of DLVO forces and of an additional repulsion, which rises when the layers of adsorbed micelles are approached. This steric-like repulsion is empirically described by the formula

Fst ) f0 exp(-h/λ)

(7)

Here λ could be considered as a specific range parameter, responsible for the effective change of the droplet diameter as a result of the condensation of micelles onto the interfaces. The parameter f0 is the effective magnitude of the corresponding repulsion and reflects all the macroscopic consequences of micellar condensation. The calculations were performed using λ and f0 as free parameters. All parameters, relevant to the interpretation of the results (37) Schwartz, D. K.; Braslaw, A.; Ocko, B.; Pershan, P. S.; AlsNielsen, J.; Huang, J. S. Phys. Rev. A 1988, 38, 5817. (38) Meiners, J. C.; Quintel-Ritzi, A.; Mlynek, J.; Elbs, H.; Krausch, G. Macromolecules 1987, 30, 4945.

for the highest Tween 20 concentrations, are summarized in Table 1. It can be seen that by increasing the micellar concentration we get lower values of the effective surface potential. We believe that this is a consequence of the adsorption of uncharged micelles onto the interfaces, which screens the surface potential. The parameter λ has a value close to 4 nm, which is around one micellar radius. Figure 4 shows the force profiles at the highest Tween 20 concentrations and the corresponding fits. The dashed lines are the DLVO components of the total force acting between the droplets in each case. BSA. The pH values of the BSA solutions were 5.6 for the highest protein concentration (0.4 wt %) and 5.8 for the lower ones (0.04 and 0.1 wt %). In all our attempts to wash the emulsions with “pure” BSA, we always obtained flocculated samples. The washing procedure in these cases led to formation of flocs of droplets when almost all Tween 20 was removed. The observed effect could be explained in the light of the results describing sticking and adhesion of protein layers19 at short distances (e.g., the properties of thin emulsion films, stabilized only by proteins). When the emulsions are centrifuged (even at the lowest accelerations), droplets are pressed one against each other by forces which are large enough to cause deformation of the droplets and formation of thin films between them. In the case of BSA-stabilized emulsion films, it is proven that there is adhesion between the surfaces,19 which is observed in the range of pH between 3.5 and 6.5. Because of the adhesion, during the redispersion of the concentrated emulsion, the films, which are formed, cannot be disjoined and the emulsion remains irreversibly flocculated. Tween 20 and BSA. To obtain nonflocculated emulsions, we switched to mixtures of Tween 20 and BSA. This emulsifier mixture raises many questions connected with the adsorption and interactions between Tween 20 and the protein.39 The adsorption and interfacial properties of the protein and Tween 20, separately and mixed, have been extensively studied in recent years employing a large variety of direct and indirect techniques such as surface/ interfacial tension measurements,40 Langmuir balances,12,41 etc. All studies reveal the complexity of the phenomena of protein adsorption at liquid interfaces and the tendency of molecular weight species to fluidize the mixed adsorption layers.42 It is important to note that in our case the protein is not supposed to be gradually unfolded by the surfactant because Tween 20 is a nonionic surfactant, so the specific, electrostatically driven binding of individual surfactant molecules to the protein macromolecule is minimal. The presence of Tween 20 in the BSA solutions seems not to influence the pH. For each given concentration of BSA the variation of the surfactant content from 0 to (39) Lucassen-Reynders, E. Colloids Surf. 1994, 91, 79. (40) Lucassen-Reynders, E.; Benjamins, J. In Food Emulsions and Foams, Dickinson, E., Rodrı´guez-Patino, J. M., Eds.; Royal Society of Chemistry: London, 1999; p 195. (41) Murray, B. Colloids Surf. 1997, 125, 73. (42) Wilde, P. J.; Rodrı´guez-Nin˜o, R.; Clark, D. C.; Rodrı´guez-Patino, J. M. Langmuir 1997, 13, 7151.

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250cmc did not lead to change of the pH greater than 0.08 units. We found a pH value of 5.6 ( 0.1 for all BSA/ Tween20 solutions, containing 0.4 wt % protein. When the BSA concentration was 0.04 or 0.1 wt %, the pH was measured to be 5.8 ( 0.1 at all Tween 20 concentrations. The ionic strength was always 3.08 × 10-4 mol/L, fixed by NaN3. We performed a set of measurements in which we varied the Tween 20 and BSA concentrations over a wide range. The measurements in all cases showed the same evolution of the force profile. At constant Tween 20 concentration, when the protein concentration is increased from 0 to 0.4 wt %, there is a progressive deviation with respect to classical double-layer repulsion. Typical results are shown in parts a and b of Figure 6, where the Tween 20 concentration is kept constant at 5cmc and 50cmc, respectively. In both cases, the resulting profiles in the presence of the same quantity of BSA are very similar and do not depend strongly on the Tween 20 concentration. ζ-potential measurements of the mixed systems gave values of about 2-3 mV, which suggests that there is a negligible contribution from the electrostatic repulsion. For that reason, we fitted the experimental points for mixed protein-surfactant systems with a combination of van der Waals attraction and power-law steric repulsion, supposing again additivity. It is known that the extended (protein) layers modify the van der Waals attraction, especially at short distances.33 For that reason for fitting these experiments we used for the Hamaker constant, AH, a value of 1.21 × 10-20 J, which is determined43 for BSA layers, acting across aqueous media. The expression used for the steric force is:

FS ) f0st(h/hmin)-R

Figure 6. Force versus distance profiles for ferrofluid emulsions stabilized with mixed BSA-Tween 20 adsorption layers. The total concentration of the Tween 20 is kept constant: 5cmc (a) and 50cmc (b). The ionic strength is 3 × 10-4 mol/L, pH ) 5.8. The solid lines are the best fits (see the text for details). Table 2. Fitting Parameters for the Mixed Layers of BSA and Tween 20 Tween 20 concentration (cmc)

(8)

where R is a parameter that accounts for the range of the interaction. The value hmin is the minimum distance measured in each case and serves as a scaling factor. We empirically adopted this expression, because it is the one which provides the best description of the experimental data (the lines in Figure 6). The fits were obtained using both f0st and R as free parameters. The force profiles for the corresponding emulsions stabilized only with Tween 20 (fitted with DLVO theory, as described in previous section) are shown for comparison. In Table 2 are reported the values of the fitting parameters f0st and R for different BSA concentrations. We should note that f0st and R are effective parameters characterizing the mixed adsorbed layers. The obtained f0st values are quite similar, and the main differences come from the R factor which determines the range of the force. The change of its value with the increase of BSA content can be attributed to some changes, in the adsorption and/or structural parameters of the protein-surfactant mixed layer present at the interface. It was proven29 that a low molecular weight surfactant and a globular protein interact in the bulk solution forming a complex. The formation of this complex is driven by electrostatic and and/or hydrophobic interactions. Unfortunately the lack of detailed knowledge, concerning the surface/interfacial properties of protein-surfactant complexes, makes any further interpretation of our numerical results rather difficult. From Figure 6 we can conclude that the force profiles do not vary much with Tween 20 concentration at the same BSA content. The deviations are in the frame of the experimental error, which suggest that the presence of (43) Roth, C. M.; Neal, B. L.; Lenhoff, A. M. Biophys. J. 1996, 70, 997.

25a

5 st,

f0 pN

R

st,

f0 pN

50 R

st,

f0 pN

25 R

st,

f0 pN

R

0.04 wt % BSA 3.61 2.23 3.76 1.73 3.07 2.50 4.46 2.17 0.10 wt % BSA 3.74 2.36 3.21 1.92 3.46 2.44 3.79 2.45 0.40 wt % BSA 3.79 8.97 3.43 3.76 4.89 4.68 a

Data not shown.

the protein dominates the behavior and the properties of the systems studied. Our force profiles are qualitatively identical to those reported in the literature. Indeed long-range steric repulsion between BSA covered mica sheets has been observed.9 It was found that at pH ) 5.5 the force (measured by means of SFA) deviates from linearity (in semilog plot) at a separation of about 50 nm, indicating the presence of long-range steric repulsion. The increased stability against coalescence of BSA-stabilized oil-in-water emulsions is also attributed to steric repulsion between the BSA adsorption layers.44 Another interesting observation is the absence of any dependence of the force profiles on the history of preparation. This is quite unusual in the majority of systems containing BSA.10,19 To achieve the desired BSA/Tween 20 content, one could proceed in two ways. The first one is to increase the BSA content, starting from zero, keeping the concentration of Tween 20 constant. The second route is to start from the highest BSA/Tween 20 ratio (at a given surfactant content) and to wash the emulsion with the appropriate aqueous phase, until the desired BSA/Tween 20 ratio is reached. The same (in the frame of the experimental uncertainty) force profiles were obtained (44) Narsimhan, G. Colloids Surf. 1992, 62, 41.


Langmuir, Vol. 15, No. 26, 1999 8819

Figure 7. Force versus distance profiles for ferrofluid emulsions stabilized with β-casein at different concentrations (points). The ionic strength is 3 × 10-4 mol/L, pH ) 6.2. The lines are the best fits (see the text for details). The dashed line is the DLVO force calculated for the same ionic strength and surface potential of -37 mV.

for the equivalent emulsions prepared following the two procedures. We could explain this independence on the preparation history with some specific, maybe even reversible, adsorption and/or reconfiguration of the protein-surfactant layer (or complex). β-Casein. Caseins are common milk proteins widely used as emulsifiers due to their excellent stabilizing properties. The caseins are disordered proteins, which (within some limits) can be treated as specific random coil polymers. Here we focus our attention on β-casein, which represents almost 33-35 wt % of the total casein content in cow’s milk. This protein is an amphiphilic molecule that could be divided into hydrophilic and hydrophobic parts.45 Its surface properties have been studied extensively in the recent years both experimentally45-49 and theoretically.50 Caseins are reported in the literature to form “micelles” and submicelles of various sizes. An important property of β-casein is its ability to form extended toward the aqueous phase adsorption layers at an oil-water interface. Their structure has been studied in the literature.48-50 It was found that the β-casein layers are constituted by two sublayers. The first (inner) sublayer is dense, approximately 1 nm thick, and the volume fraction of the protein in it is 85-90%. The second (outer) sublayer is between 4 and 7 nm thick (depending on the pH and ionic strength). The volume fraction of the protein in it is 10-15%. The total thickness of adsorbed β-casein layers, obtained from dynamic light scattering,51 is 5-10 nm. We investigated the force profiles between β-casein covered droplets, at protein concentrations in the continuous phase equal to 0.1, 0.2, 0.33, and 0.5 wt %. The pH of the solutions measured was 6.2 ( 0.1 in all cases. The ionic strength was fixed by 3.08 × 10-4 mol/L of NaN3. When a higher ionic strength was investigated it was adjusted by adding the appropriate amount of NaCl during the last washing of the emulsion. (45) Velev, O. D.; Campbell, B. E.; Borwankar, R. P. Langmuir 1998, 14, 4122. (46) Kozco, K.; Nikolov, A. D.; Wasan, D. T.; Borwankar, R. P. Gonsalves, A. J. Colloid Interface Sci. 1996, 178, 694. (47) Horne, D. S.; Leaver, J. Brooksbank, O. V. Prog. Colloid Polym. Sci. 1994, 97, 271. (48) Nylander, T.; Wahlgren, M. N. Langmuir 1997, 13, 6219. (49) (a) Atkinson, P. J.; Dickinson, E.; Horne, D. S.; Richardson, R. M. J. Chem. Soc., Faraday Trans. 1995, 91 (17), 2847. (b) Dickinson, E.; Pinfield, V. J.; Horne, D. S.; Leermakers, F. A. M. J. Chem. Soc., Faraday Trans. 1997, 93 (3), 1785. (50) Leermakers, F. A. M.; Atkinson, P. A.; Dickinson, E.; Horne, D. S. J. Colloid Interface Sci. 1996, 178, 681. (51) Fang, Y.; Dalgleish, D. G. J. Colloid Interface Sci. 1993, 156, 329.

Figure 8. Sketch of the depletion effect of the β-casein aggregates. The specific distances included in the model are also shown.

In this case it was possible to wash the emulsions with the protein alone, preserving them from flocculation, most probably because of the relatively high interfacial potential (see below). The obtained force profiles represent the interaction between two β-casein layers, adsorbed at hydrophobic interfaces. None of the measured profiles could be interpreted by means of a DLVO model. Instead, we clearly observe that the long-range part of the interaction deviates from linearity (in a semilog plot), which suggests the existence of an attractive interaction. The higher the concentration, the stronger the deviation from the classical DLVO isotherm. This evolution25 strongly suggests the occurrence of depletion attraction due to the presence of β-casein micelles. The effect of the β-casein concentration on the total force is shown in Figure 7. The lines in the figure represent the fit of the data assuming additivity of the depletion attraction and DLVO forces. The depletion attraction was calculated through the following expression25

[ {RR++rh/2 + δ} ]

Fd ) -P0π(R + r + δ)2 1 -

2

(9)

where P0 is the osmotic pressure exerted by β-casein micelles, R is the droplet radius, r is the radius of the depleting species, and h is the interdroplet distance (Figure 8). Here δ is an extra exclusion distance52 that accounts for the electrostatic and steric repulsion between β-casein micelles and droplet surface. The range of the electrostatic repulsion is set by the Debye length, which is about 17 nm at ionic strength at 3.08 × 10-4 mol/L of NaN3 and about 10 nm at salt concentration of 10-3 mol/L. The range of the steric repulsion corresponds to the most extended (toward the aqueous phase) protein tails.45,48 Since the range of the electrostatic repulsion in our case is always longer or equal to the range of the steric repulsion, following refs 25 and 52 we choose δ to coincide with the Debye length. For calculating P0 we adopted the perfect gas approximation. That assumption is reasonable since (52) Richetti, P.; Ke´kicheff, P. Phys. Rev. Lett. 1992, 68, 1951.

8820



Table 3. Fitting Parameters for Emulsions Stabilized with β-Casein β-casein concn, wt %

ionic strength, mol/L

Ψ0, mV

size of the depleting species, nm

0.10 0.10 0.20 0.33a 0.50

3 × 10-4 1.3 × 10-3 3 × 10-4 3 × 10-4 3 × 10-4

-26.3 -43.5 -33.9 -33.1 -48.1

32.2 29.2 36.1 24.0 36.3

a

Data not shown in Figure 7.

the volume fraction of the depleting species is low enough. The surface potential, Ψ0, and the mean size r of the depleting species were used as adjustable parameters. The plane of charge is assumed to be situated at the droplet interface. The inverse Debye length, κ, was calculated from the total salt content. The values obtained for the adjustable parameters are summarized in Table 3. The calculated surface potential, Ψ0, is almost constant and is consistent with the values obtained from ζ-potential measurements that gave -37 ( 2 mV. Claesson and coworkers7 report Ψ0 of -48.3 mV in the case of β-casein layers adsorbed on mica surfaces. Furthermore the sizes (diameters) obtained from the fit are also almost the same, as expected, since the size of micelles is found not to vary in the range of protein concentrations between 0.1 and 1 wt %, as pointed by Leclerc et al.53 The same authors have found also that β-casein micelles are 26 ( 1 nm in diameter, loosely packed, and relatively monodisperse, which is in excellent agreement with our numerical data. Taking a mean radius of 15 nm, and molecular weight1,2 of 2.5 × 105 Da one calculates the corresponding depletion potential at zero distance to be about 2kBT units for the highest concentration of β-casein, which is rather sufficient to produce attraction.1,2 The exclusion volume effect of some caseinate micelles has been observed and reported in the literature,46 where it is found that liquid films stabilized with sodium-caseinate at concentrations 0.1-4 wt % undergo stepwise thinning. The height of the stratification steps was 20-25 nm. In the same study the average size of caseinate submicelles, measured by dynamic light scattering, is reported to be about 25 nm, which is also in very good correlation with our data. Our interpretation is also consistent with the microscopic observation of flocculation in emulsions stabilized with sodium-caseinate, performed by Dickinson et al.1 They microscopically observed depletion flocculation induced by micellar and/ or submicellar protein aggregates/micelles at free protein concentration in the aqueous phase equal (or higher) to about 0.7 wt %, which agree fairly well with the concentrations at which we detect depletion attraction. Furthermore our technique allows attaining very low levels of forces and we are able to detect the depletion attraction below the threshold concentration1,54 necessary to induce flocculation. This is why we report attraction at concentrations slightly lower than those at which depletion flocculation in the bulk emulsions is detected. The depletion effect is well pronounced at all ionic strengths (Figure 9). The increase of the salt content leads to suppression of the electrostatic repulsion, which leads to larger effective importance of the depletion attraction. The β-casein concentration in this set of experiments was kept 0.1 wt %. The data are fitted as described above. The profile corresponding to ionic strength of 0.01 mol/L NaCl consists of only few points, because at forces higher than 1 × 10-12 N the flocculation of the sample was considerable. (53) Leclerc, E.; Calmettes, P. Phys. Rev. Lett. 1997, 78, 150. (54) Bibette, J.; Roux, D.; Pouligny, B. J Phys. II 1992, 2, 401.

Figure 9. Force versus distance profiles for ferrofluid emulsions stabilized with 0.1 wt % β-casein at pH ) 6.2 and at different ionic strength (points). The lines (where presented) are the best fits.

Figure 10. The threshold flocculation force, F*, for ferrofluid emulsions stabilized with 0.1 wt % β-casein as a function of the ionic strength (left axis). On the right axis F* is normalized by the droplet radius. The experimental conditions are the same as in Figure 9.

Hence no attempts to fit these experimental points were made. At every salt concentration we were able to estimate the threshold force, F*, which induces irreversible flocculation. Indeed above this threshold force, we observed under microscope that the droplets remained irreversibly chained even in the absence of magnetic field. The dependence of F* on the ionic strength is shown in Figure 10. The decrease in the resistance against flocculation is due to the decreased electrostatic repulsion at higher salt concentration. As far as we know, values of that kind are the first directly measured. IV. Conclusion For the first time the magnetic chaining technique was applied to food type emulsions, and force vs distance profiles for liquid interfaces covered by proteins were obtained. The results show interesting aspects in the interaction between the emulsion droplets stabilized by proteins and/or the nonionic surfactant Tween 20, all of them intensively used in the food industry as emulsifiers. We first investigated the interactions between droplets covered with Tween20 and found that the presence of large excess of surfactant modifies the repulsion most probably because micelles condense at the oil/water interface. We studied two proteinssbovine serum albumin (BSA) and β-casein. They represent two important classes of proteinssthe globular and disordered ones, respectively. The presence of protein at the interfaces strongly modifies the force-distance isotherms, in all cases giving rise of specific non-DLVO forces, which are believed to be steric repulsion in the case of BSA and depletion attraction in the case of β-casein. The present work demonstrates the advantages and the applicability of the magnetic chaining technique in


probing the interactions that are involved in complex systems. It could be extended also to inverted food emulsions (water-in-oil), which, up to now, have not been explored by means of any available force measurement technique. Finally, we hope that our results will provide some guidance in understanding the complicated behavior of protein-stabilized emulsions.

Langmuir, Vol. 15, No. 26, 1999 8821

Acknowledgment. This work is financially supported by Laboratoire Franco-Bulgare. The authors are indebted to Professor Ivan Ivanov and Dr. Theodor Gurkov for fruitful discussions. LA9904758

Forces between Emulsion Droplets Stabilized with Tween 20 and

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