Potencial Zeta 2

Colloids and Surfaces B: Biointerfaces 25 (2002) 55–67

Zeta potential and droplet size of n-tetradecane/ethanol(protein) emulsions

Agnieszka Ewa Wiacek, Emil Chibowski *Department of Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Sklodowska Uni�ersity, Lublin 20031, Poland

Received 17 April 2001; received in revised form 25 July 2001; accepted 15 October 2001

Abstract

Zeta potential, average diameter and multimodal size distribution were studied for n-tetradecane emulsions inaqueous solution of ethanol (0.5 and 1.0 M) in which bovine serum albumin (BSA), �-lactalbumin or �-casein (1, 2,or 5 mg/100 ml) was also dissolved. The emulsion pH was natural (7.3) or regulated to 4 or 11. The parameters weredetermined as a function of time, i.e. after 5, 15, 30, 60, 120 min, 1, and 2 days, and 1 week, since the emulsionpreparation. The emulsions were prepared by dissolving 0.1 ml of the n-alkane in a proper amount of ethanol andthen water or protein solution was added to obtain 100 ml of the emulsion in which total concentration of ethanolwas 0.5 or 1.0 M. Next, the emulsion was sonicated for 15 min and the measuring polyacrylic cells of the apparatuswere filled with the emulsion. The isoelectric point (i.e.p.) of the droplets in the presence of investigated proteins (2mg/100 ml) and in 1 M ethanol occurred at pH 4.9, 4.7 and 2.2, for BSA, �-casein and �-lactalbumin, respectively.In pH range 5.5–10, the zeta potentials of freshly prepared emulsions in 1 M ethanol were negative and relativelylarge, from −45 to −60 mV. In �-casein presence, the n-alkane droplets were larger and negative zeta potentialshigher than in the presence of two other investigated proteins. However, in the presence of each of the investigatedproteins the droplet size increased slightly relative to that in ethanol solution alone. Nevertheless, the emulsions wererelatively stable. In 0.5 M ethanol, the protein presence stabilized the emulsions. On time scale, the changes ofnegative zeta potential did not correlate in a straight way with changes in the droplet size (the emulsion stability).Experiments showed that without ethanol presence in the emulsion, �-casein alone can be used as an emulsifieralready at its concentration of 1 mg/100 ml for 0.1 ml of n-tetradecane content both at natural and alkalineenvironment. However, no stable emulsion could be obtained using BSA or �-lactalbumin. Multimodal sizedistribution analysis showed that the droplet sizes in the studied emulsions could be grouped in one or twocomparable populations only. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Emulsions; Ethanol; Protein; Size distribution; Effective diameter; Zeta potential

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1. Introduction

Application of natural emulsifiers, like proteins,in food products, pharmaceutics, and other emul-sion systems is of a great interest [1]. In many

* Corresponding author. Tel.: +48-81-537-5651; fax: +48-81-533-3348.

E-mail address: [email protected] (E. Chibowski).

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A.E. Wiacek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 55–6756

naturally occurring systems, proteins emulsify theoily substances thus facilitate their nutrition. Sta-bility of emulsions largely depends on dimensionof the emulsion droplets, which in turn depends onthe conditions of the emulsion preparation.Macromolecules, such as proteins, form an ad-sorbed film on the surface of emulsion droplet,thus creating an energy barrier against coales-cence. The protein molecules, when adsorbed, canunfold and reorient their amino-acid groups, sothat the hydrophobic groups may align with theoil molecules and the hydrophilic groups maydirect toward the aqueous phase. Thus, at theinterface a visco-elastic or gel-like layer can beformed [2,3].

It is obvious now that stability/instability ofemulsion is controlled by properties of the layeradsorbed to the surface of oil droplets [1]. Theunfolding protein process depends upon theprotein molecule structure [2], but the process isnot fully known yet [1]. Such knowledge could behelpful for better understanding of the stabilizingmechanism at the interface by the adsorbingprotein. Surely, structure and ‘strength’ of theadsorbed protein film are of a crucial importancein preventing coalescence of the oil droplets, thatis, breaking of the emulsion structure. In general,the adsorbed film structure will depend on theconcentration, composition, and kind of theemulsifier. The adsorbed layer, especially ofprotein (or a polymer) plays also a very importantrole in aggregation and deposition processes of adispersed solid phase. In some systems, smallamounts of the adsorbed protein can promoteflocculation by a bridging mechanism [2]. On theother hand, proteins at a larger adsorbed amountcan produce a greatly enhanced stability by aneffect known as steric stabilization [2,3].

In practical emulsions systems the droplets aresub-, or about micrometer size. The smaller thedroplet size the more stable emulsion is [1]. As itwas mentioned above, the proteins usually changetheir conformation state after adsorption at theoil/water interface and the adsorption is strong [2].Other component present in emulsion, for examplealcohol, can weaken the adsorbed protein layer bya competitive adsorption. The competingmolecules usually do not form a strong adsorbed

layer themselves, but they adsorb alongside theprotein molecules, thus weakening the gel-likestructure [1–3]. The resulting adsorbed layerforms a network structure with fractures of an-other component [2,3]. The structure of adsorbedprotein layer can thus be broken down completely,which drastically reduced stability of the interface.In general, an emulsifier changes (decreases) theoil/water interfacial tension, as well as the surfacecharge of the droplet, what should appear in thechanges of zeta potential [1]. These two parame-ters, i.e. interfacial tension and zeta potential,from viewpoint of the DLVO theory, are mostimportant for description of dispersed system sta-bility [4]. However, recently published papers [5–7] show that the stability can be described betterby so-called ‘extended DLVO theory’, where, inaddition to the attraction Lifshitz-van der Waalsand repulsion electrostatic forces, the hydration(acid–base) and/or steric (long-chain molecules)interactions are also taken into account.

The aim of this paper was to investigate stabiliz-ing and/or emulsifying properties of some proteinsfor oil-in-water emulsion. For this purpose zetapotential, effective diameter and multimodal sizedistribution of n-tetradecane droplets were investi-gated by dynamic light scattering technique as afunction of time. The oil phase was emulsified byethanol (0.5 or 1.0 M) and stabilized (or destabi-lized) by BSA, �-lactalbumin or �-casein presence.Also, the effect of emulsion pH on the aboveparameters was investigated. The zeta potentialwas determined subsequently after the droplet sizedetermination for the same sample of the emul-sion, which was placed in measuring cell of Zeta-Pals/BI-MAS instrument [8,9]. It seemed usinteresting to learn whether on time scale changesin zeta potential and effective diameter of the samesample of emulsion correlate each other. Since,electrostatic repulsion is determined by zeta poten-tial [10], therefore, one can expect that the dropletsshould be more stable if the zeta potential ishigher.

We would also like to study on a macroscopiclevel impact of structural features of the proteinmolecules on the emulsion stability. Such knowl-edge may be helpful for applications of a widerrange of protein for some specific systems, forexample food or cosmetic products. Obviously,

A.E. Wiacek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 55–67 57

for full characterization of proteins behavior asemulsifier/stabilizer agents a multidisciplinary ap-proach, like molecular interactions (biochemicalanalysis and proteins characterization), interfacialbehavior (spectroscopy, microscopy, tensiometry),and bulk properties (dispersion formation, stabil-ity and rheology), studies are required [1–3,7,8,10–12].

2. Experimental

The emulsions were prepared in the 100 ml-cal-ibrated flasks. The components were added asfollows. First desired portion of ethanol (p.a.from POCh Gliwice, Poland) was introduced toobtain its final concentration of 0.5 or 1.0 M.Then 0.1 ml of n-tetradecane (p.a. from Fluka)was added, and then protein solution (1, 2 or 5 mlfrom a stock solution 0.1 g/100 ml) and deionizedwater was added to obtain 100 ml of the emul-sion. Next, the flasks were placed for 15 min in anultrasonic bath (50 W) to homogenize the emul-sions. Having thus prepared emulsion, appropri-ate number of the polyacrylic cells used in theZetaPals/BI-MAS apparatus were filled with theemulsion. These cells were next left without anyshaking or mixing before the droplet size and zeta

potential determination. The proteins used werefrom Sigma Chemical Co., and particularly theywere: bovine serum albumin (BSA, fraction V,min. 90% proteins), �-lactalbumin (from bovinemilk, lyophilized, min. 85%) and �-casein (frombovine milk, lyophilized, min 90%). Water usedfor the emulsion preparation was from MiliQ-Plussystem. The emulsion droplet sizes were deter-mined as a function of time, after 5, 15, 30, 60,120 min, and 1, 2 and 7 days, since the emulsionpreparation. The duration of droplet size determi-nation in particular sample was about 5 min.Then, zeta potential measurement followed forthe same sample using ZetaPals/BI-MAS appara-tus [8,9]. Since, �a products (Debye-Huckelparameter and the droplet radius) were small forthe emulsion droplets, the zeta potentials werecalculated from Huckel equation. All the experi-ments were carried out at 20 °C.

3. Results

The changes in zeta potential of n-tetradecanedroplets in the emulsions are presented in Fig. 1.Since �a product was small for the droplets sus-pended in the electrolyte-free liquid phase, e.g.8.93, 0.23, and 18.2 for pH 4, 7.3, and 11, respec-

Fig. 1. Zeta potentials of n-tetradecane/ethanol (1 M) protein (2 mg/100 ml) emulsions vs. pH. The pH of the i.e.p. for nativeproteins are shown by arrows: 1–4.8 for BSA, 2–4.5 for �-casein, 3–5.1 for �-lactalbumin.


tively, therefore Huckel equation was applied forzeta potential calculations instead of Smolu-chowski one. On the other hand, Oshima andKondo [13] suggested that Smoluchowski equa-tion is not suitable for calculation of zeta poten-tials for biological particles (cells), because, itleads to overestimation of the surface (zeta) po-tentials due to electrophoretic fluid flow inside,for example, adsorbed layer of protein. They [13]derived an approximate mobility expression forparticles with surface layers in which fixed chargeswere uniformly distributed and a planar doublelayer could be assumed, i.e. particle radius wasmuch higher than the double layer thickness, 1/�.The expression consists of two terms: (i) aweighted average of the surface potential (and/orthe Donnan potential) and (ii) resulting from themembrane-fixed charges, which is not subjected toshielding effects at high concentration of the elec-trolyte. At high electrolyte concentrations, whereowing to the shielding effects the potentials arevery low, the first term diminishes so that themobility is determined mainly by the second term.This means that a particle with negligible surface(zeta) potential can exhibit non-zero mobility, incontrast to the prediction of classical Smolu-chowski theory. From their derivation [13] it re-sults that in presence of a structured layer aroundthe particle its electrophoretic mobility is not sen-sitive to the position of the slipping plane andhence it is difficult to define the zeta potential.Finally, the authors [13] concluded that at highelectrolyte concentration both planar and spheri-cal colloid surfaces behave similarly. Our mea-surements have been carried out at low electrolyteconcentrations in the emulsion systems, where �avalue was small. Therefore, Huckel equation [10]was applied as an approximation for zeta poten-tial calculations from the measured mobility val-ues of tetradecane droplets. These emulsionscontained 0.1 ml of the dispersed tetradecane in 1M ethanol and 2 mg of the investigated protein.Nevertheless, of the interpretation problem ofelectrophoretic mobility in terms of zeta potential,the pH of the isoelectric point (i.e.p.) of thedroplets can be set correctly. Thus, the i.e.p.occurs at pH 4.9, 4.7 and 2.2, for tetradecanedroplets with adsorbed BSA, �-casein or �-lactal-

bumin, respectively, and it clearly depended uponthe type of the adsorbed protein. Moreover, ex-cept for �-lactalbumin, the droplet pHi.e.p. value isclose to that of appropriate native protein [14],which is: 4.8, 4.5 and 5.1, for BSA, �-casein and�-lactalbumin, respectively [11]. The zeta potentialchanges for BSA are complementary to elec-trophoretic mobility results obtained by van derMei et al. [14] for hexadecane droplets in 10 mMpotassium phosphate after adsorption of BSA orHSA.

In alkaline region, the zeta potentials dependonly slightly on the emulsion pH (Fig. 1), whatindicates that in pH range 7–10 the negativecharge of freshly formed droplets is practicallyconstant as a result of total dissociation of�COOH groups. In the acidic region (pH 3–6)dissociation of both amine and carboxyl groupsdetermine the surface charge and the potential.On the other hand, the drastic shift in pHi.e.p.

(about 3 pH units) in the case of adsorbed �-lac-talbumin suggests that during its adsorption someconformation changes have occurred.

The pH effect on the emulsion particle size andzeta potential in the presence of �-casein is shownin Fig. 2a and b, respectively, and standard devia-tions of ten readings are shown in Table 1. At pH4 zeta potential of the droplets is positive, butdecreasing during the emulsion lifetime. The rela-tively high concentration of OH− ions (pH 11)practically does not cause any increase in thenegative zeta potential of freshly prepared emul-sion in comparison to that at natural pH (7.3),but the ions clearly stabilize the zeta potential(Fig. 2b). At natural pH, similarly as at pH 4, thezeta potential also has decreased during 1 week,from approximately −82 (fresh emulsion) to −30 mV (Fig. 2b). The hydroxyl ions may interactwith the protein molecules as well as influence thecarboxyl groups �COOH dissociation [15]. Stabil-ity of such emulsions is often higher than expectedon the basis of zeta potential and the ionicstrength, and it is likely that steric stabilizationplays some role here [2]. Relatively high andstable zeta potential at pH 11 (Fig. 2b) probablycauses that the emulsion is stable during 1 weekwithout any shaking of the sample (Fig. 2a).Moreover, at this pH from the very beginning the


Fig. 2. Effective diameters (a) and zeta potentials (b) of n-tetradecane (0.1 ml)/�-casein (1 mg) vs. pH.

Table 1Effective diameters, zeta potentials and their standard errors of n-tetradecane/�-casein (1 mg) emulsion

Age of emulsion pH 7.3 (nat.)pH 4 pH 11

Effective Zeta potential Effective Zeta potentialZeta potential Effectivediameterdiameterdiameter

31.6�1.35 1105.2�66.2 −79.3�3.4 351.6�15.3 −70.8�10.05 min 471.7�65.835.5�2.7 853.3�14.5 −83.1�1.5570.7�75.6 302.3�37.015 min −83.5�2.8

30 min 613.0�59.7 4.0�5.4 827.0�36.0 −77.5�0.3 306.6�23.5 −76.0�1.527.4�1.5 753.1�39.7 −67.8�2.8314.5�2.8 320.6�30.41 h −67.5�3.734.9�1.6 476.9�22.3 −53.8�1.52 h 304.2�32.4271.7�10.9 −62.5�2.121.0�1.0 341.4�5.8 −31.2�4.2273.5�1.8 323.4�4.11 day −66.9�1.9

2 days 328.3�7.5 17.2�1.9 301.2�4.0 −58.2�0.9 306.3�3.5 −69.9�2.47.5�1.3 332.6�1.2 −32.7�1.51530.8�79.1 294.5�6.11 week −60.3�1.5


droplet size was similar to that at pH 7.3 and 4when partially the separation process had takenplace. At natural (7.3) and acidic (4) pH, theeffective diameter and zeta potential have changedmarkedly during 1 week. For this emulsion, astraightforward correlation between the zeta poten-tial and its stability occurred, especially in thealkaline environment (pH 11). Hence, it may beconcluded that in this case magnitude of zetapotential is a principal factor for the emulsionstability [12,16]. It should be stressed that deter-mined by dynamic light scattering technique de-creasing effective diameter of the droplets with thetime (Fig. 2a) obviously had not resulted from aspontaneous process of splitting of the largerdroplets, but contrary, it resulted from coalescence

of those droplets and their flotation to the emulsionsurface (phases separation). In results a populationof smaller droplets had remained in the bulkemulsion. It may also happen, like at pH 4 (Fig. 2a),that the coalesced droplets are still present in thebulk emulsion for a period of time (1 week oldemulsion). Here a drastic increase in the averagediameter is observed, from 300 nm (2 days oldemulsion) to 1500 nm (1 week old emulsion). Tobetter visualize these processes in Fig. 3a–c areshown multimodal size distributions of the dropletsfor the emulsion with �-casein at alkaline pH 11,for 5 min, 2 day, and 1 week old emulsion,respectively. The effective diameter of the dropletsin 5 min- and 2 day old emulsion is similar eachother, 330.5 and 306.4 nm. The polydispersity andthe sample quality (which informs about fittingprocedure of the correlation functions in a 10-Uarbitrary scale) parameters are similar too, 0.227,0.222 and 9.5, 9.4, respectively. After 1 week, forthe same emulsion the effective diameter is stillsimilar (288.7 nm), polydispersity amounts to0.149, and the sample quality is even better (9.8)than in former two cases, but in this emulsion twocomparable populations of the droplets coexists,one of which is slightly smaller (that of smallerdroplets, Fig. 3c). In 5 min and 2 day old emulsionpractically one population of the droplets is onlypresent (Fig. 3a and b).

The effective diameters and zeta potentials of theemulsions at natural pH and in the presence oftested proteins are shown in Figs. 4–6, and inTables 2–4, where the standard deviations (S.D.)are given. For comparison, in these figures there arealso shown the parameters for emulsions preparedin ethanol solution alone. In general, effectivediameters of the droplets are lower in the proteinspresence, and they are in the range of 250–350 nm.These results show that optimal amount of theproteins to stabilize the emulsions is 5 mg/100 ml,because, effective diameters of such droplets are themost stable during 1 week time of the experiment.The zeta potentials of these emulsions (Fig. 4b Fig.5b Fig. 6b) are rather small and fluctuate between−30 and 50 mV (the most comparable zeta poten-tials appear in up to 1-day-old emulsions). Since,the emulsions were relatively stable, it points thatsteric stabilization, as well as hydrogen bondingprobably play here an important role [2,3].

Fig. 3. Multimodal size distribution of n-tetradecane droplets(0.1 ml/100 ml) in �-casein (1 mg) solution at pH 11 for 5 min,2 days, and 1-week-old emulsion, respectively.


Fig. 4. Effective diameters (a) and zeta potentials (b) of n-tetradecane (0.1 ml)/ethanol (0.5 M) BSA emulsions at natural pH, fordifferent concentrations of the protein and the age of emulsion.

According to Haynes and Norde [17] adsorp-tion of globular protein to apolar surface mustcause removal or neutralization of the electricalcharge between the molecule and the surface. Thismay be achieved by formation of pair of ions(which case seems to be hardly possible for oilsurface), protonation/deprotonation of the ioniz-able residual groups, and co-adsorption of smallions in the contact layer. Lateral interactions be-tween the adsorbed molecules may then be attrac-tive or repulsive, depending on the kind andmagnitude of electric charge of the residues. Thesecomplicated processes involving protein adsorp-tion are also reflected in some way in the zetapotential changes (shown in Figs. 1 and 2b Fig.4b Fig. 5b Fig. 6b) of the oil droplets as afunction of time and pH of the continuous phase,and in multimodal size distribution, which isshown in Fig. 7a–c for 2-day-old emulsion with

�-casein as a function of pH 4, 7.3, and 11.Although, in 1-week-old emulsion at pH 4 (Fig.2a) big droplets appeared (1500 nm), in 2-day-oldemulsion the effective diameter was only about300 nm, and from the multimodal size distribu-tion (Fig. 7a) it can be seen that practically therewas present only one population of the droplets(with some traces of two larger). At natural (7.3)and alkaline (11) pH some small population thelarger droplets can bee seen in Fig. 7b and c,respectively. However, while at pH 4 and 7.3, theeffective diameter had decreased on the time scale,so at pH 11 it was practically constant (Fig. 2a).

4. Discussion

Considering stabilization of the emulsion byprotein adsorption, it should be kept in mind that


conformational processes occurring at the oil/al-cohol solution interface are slow ones [12,16]. Theobserved changes in the effective diameter, andespecially in the zeta potentials, which occurredafter 1 and 2 days, and even up to 1 week (Figs.4–6), may just result from such conformationalchanges in the structure of the adsorbed proteinmolecules and the layer in general. Therefore, itmay happen that two emulsions prepared in simi-lar conditions may be characterized by differentzeta potential. According to Dickinson and Mat-sumura [18] molecular mechanisms contributingto the free energy of adsorption are complicatedand may be following: dehydration of the hydro-phobic regions at the surface, protein unfolding atthe surface, charge redistribution due to overlap-ping of the electric fields of protein and surface,

change in pK values of side chains on adsorption,difference in ionic hydration between bulk andsurface, and restructuring of van der Waals inter-actions. However, finally the principal drivingforce for protein adsorption is removal of itshydrophobic aminoacid side-chains from the po-lar environment, i.e. bulk aqueous phase [18].Dilution of the emulsion by adding of the contin-uous phase (water) usually does not cause desorp-tion of protein (irreversible adsorption). However,there are always small areas among the adsorbedprotein train segments accessible for small surfac-tant molecules [18,19]. For example, adsorbed�-lactalbumin occupies only about 1/3 of the sur-face area and although, protein adsorption isconsidered to be irreversible, small surfactantmolecules, like ethanol, may desorb the molecules

Fig. 5. Effective diameters (a) and zeta potentials (b) of n-tetradecane (0.1 ml)/ethanol (0.5 M) �-casein emulsions at natural pH,for different concentrations of the protein and age of the emulsion.


Fig. 6. Effective diameter (a) and zeta potential (b) of n-tetradecane (0.1 ml)/ethanol (0.5 M) �-lactalbumin emulsion at natural pH,for different concentrations of the protein and the age of emulsion.

Table 2Effective diameters, zeta potentials and their standard errors of n-tetradecane/ethanol (0.5 M), BSA emulsion

Age of emulsion 2 mg0 mg 5 mg

Effective Zeta potential Effective Zeta potentialZeta potential Effectivediameterdiameterdiameter

−36.0�2.1 782.5�105.8 −52.5�0.6 302.1�3.8 −32.1�1.55 min 404.0�5.1−39.4�0.6 380.1�27.2 −26.2�1.2391.0�5.5 315.0�4.515 min −36.1�3.3

30 min 389.0�7.7 −36.1�1.5 285.6�4.4 −36.0�0.9 312.6�6.0 −21.9�1.8−45.0�0.4 274.6�4.1 −36.3�1.0415.3�25.1 309.6�5.41 h −36.9�1.8−36.0�0.9 271.1�7.3 −30.9�1.62 h 327.6�0.9381.1�5.2 −31.9�1.6−49.3�0.6 272.2�2.1 −38.4�0.4345.8�8.1 308.5�2.61 day −46.2�0.7

2 days 340.5�15.6 −46.8�1.6 281.5�6.7 −41.4�1.2 302.0�3.2 −48.1�1.2−39.1�1.6 273.6�2.9 −39.7�0.3351.9�4.1 310.1�2.71 week −39.1�0.6


[1]. Ethanol may also interact as a denaturatingagent already at 20 °C [2]. However, maximumconcentration of ethanol used in this study was onlyapproximately 5 wt.% (1 M solution).

Obviously, temperature, pH, and ionic strengthmay affect the adsorption [1,12,20]. In the case ofglobular protein (e.g. bovine serum albumin, BSA)the adsorbed molecules may possess some transi-tion forms between native and denaturated, andamong them the so-called ‘molten globule’, whichcan also exists in the solution phase [17,18]. Thepolar and apolar residues are nearly evenly dis-tributed on the globular protein compact surface.In denaturation process, from the free energychanges as a function of temperature it results thatthe whole globular protein molecule can unfold ifdisruption of an essential part of its molecule took

place [12]. The maximum protein adsorption usu-ally occurs at pH close to its i.e.p. [1].

The electrophoretic mobility of n-alkanedroplets with adsorbed protein, among others, werestudied by van der Mei et al. [14]. The authorsconcluded that comparable pH value of the i.e.p.for protein coated tetradecane droplets and thosefor native proteins confirm that at low proteinconcentrations the net charge addition upon theiradsorption determines effects of the final elec-trophoretic behavior of the protein–hexadecanecomplexes. Basing van der Mei et al.’s [14] resultsand those obtained in this paper it can be concludedthat in the adsorbed state there are more dissoci-ated residual �COOH groups and/or less dissoci-ated amine groups, or less number of these lattergroups is exposed toward the water phase [12]. It

Table 3Effective diameter, zeta potentials and their standard errors of n-tetradecane/ethanol (0.5 M), �-casein emulsion

0 mgAge of emulsion 2 mg 5 mg

EffectiveZeta potential Zeta potential Zeta potentialEffective Effectivediameter diameter diameter

5 min 314.7�5.3404.0�5.1 −39.4�0.7−36.0�2.1 466.1�14.2 −29.8�2.1340.6�8.2 −36.7�1.3 305.6�7.9 −37.3�0.9391.0�5.515 min −39.4�0.6

30 min 334.4�6.4389.0�7.7 −35.2�0.1−36.1�1.5 333.1�5.5 −43.9�1.01 h −28.9�0.6321.7�7.1−28.2�0.6304.8�6.8−45.0�0.4415.3�25.1

322.9�3.5−37.6�1.3319.2�7.7 −23.4�0.1−36.0�0.9381.1�5.22 h1 day 316.6�3.2345.8�8.1 −33.1�0.4−49.3�0.6 319.3�3.1 −45.3�0.7

340.5�15.6 −46.8�1.6 315.5�4.72 days −19.5�2.7 341.2�6.9 −14.5�3.61 week 351.9�4.1 −39.1�1.6 314.1�3.8 −28.0�0.6 478.7�7.6 −10.6�1.9

Table 4Effective diameter, zeta potentials and their standard errors of n-tetradecane/ethanol (0.5 M), �-lactalbumin emulsion

0 mgAge of emulsion 2 mg 5 mg

Effective Zeta potential Effective Zeta potential Zeta potentialEffectivediameter diameterdiameter

404.0�5.1 −36.0�2.15 min 337.2�7.4 −59.4�1.3 251.2�2.3 −39.3�1.2391.0�5.5 −39.4�0.615 min 343.0�6.2 −39.1�1.2 251.6�1.6 −31.9�3.0389.0�7.7 −36.1�1.5 338.0�4.230 min −45.0�1.8 244.8�4.8 −28.9�0.7

−32.7�1.9415.3�25.1 243.8�4.51 h −43.6�1.8320.9�1.9−45.0�0.4−31.8�1.6 238.7�2.0 −29.5�1.5346.7�6.2−36.0�0.9381.1�5.22 h

1 day 244.0�1.4345.8�8.1 −49.3�0.6 327.3�3.0 −40.3�1.5 −33.3�0.1−46.8�1.6340.5�15.6 −13.9�1.52 days 253.6�4.7−36.3�3.1323.8�5.0

351.9�4.1 −39.1�1.6 333.8�3.5 −40.5�2.1 −19.9�1.31 week 265.1�4.0


Fig. 7. Multimodal size distribution of n-tetradecane droplets(0.1 ml/100 ml) in �-casein (1 mg) solution for 2 day-oldemulsion at pH 4, natural 7.3, and 11, respectively.

some authors suggest a typically electrostatic mech-anism of protein adsorption [18].

The results obtained in this paper show that inemulsion without ethanol presence, globularprotein with ‘hard’ molecules are in general worsestabilizers than �-casein, which has elastic structure[11]. Small amount of �-casein (0.1 mg) producedrelatively stable emulsion, especially in alkalineenvironment (see Fig. 2). Analogous emulsionswith BSA or �-lactalbumin without ethanol (notpresented) were unstable. However, if 0.5 M etha-nol was present as the emulsifier, all investigatedproteins showed comparable behavior. In suchsystems, the differences in protein molecule struc-ture probably are not so important, because thealcohol may refold them [1,12].

Comparison of the measured changes in effectivediameters and zeta potentials shows that in generalthere is not straightforward relationship betweenvariation of these two parameters, i.e. decreasingstability with decreasing zeta potential for. Thispoints to essential role of steric stabilization and/orhydrogen bonding interactions between polargroups of the adsorbed ethanol (and water) andprotein molecules from the bulk phase [2,3,11]. Itcan be speculated that more effective stabilizers arethe proteins whose molecule segments stretch intothe aqueous phase upon their adsorption [1]. Since,such stretching segments should be polar andhydrated, their overlapping would cause somedehydration and hence an increase in the freeenergy, which of course is not a favorable process.Also, electrostatic repulsion between such chargedsegments should take place [1]. As protein adsorp-tion is accompanied not only by changes in theelectrostatic properties of the oil surface, but alsoby the surface free energy changes [14], the ad-sorbed protein molecules may keep apart thedroplets at a distance at which van der Waalsattraction has already decayed, or where thedroplets form weak aggregates, which can be easilybroken by a shear force [1]. However, the mostimportant factor determining degree of steric stabi-lization is thickness of the adsorbed layer in relationto the particle (droplet) size [2], and it has beenknown for a time that emulsion stability is relatedto strength of the adsorbed protein layer [1–3].Hence, if the ratio of oil phase increases in the

should be kept in mind that because of preparationprocedure of the emulsions, used in this paper, theprotein molecules probably compete with alreadyadsorbed ethanol molecules, which can essentiallyaffect their conformation, as well as dissociation ofthe polar groups upon adsorption. Of course, itdepends strongly on the concentrations of proteinand alcohol (and the concentration ratio too) andmolecular weight of the protein [12]. One can alsoconsider a role of hydrogen bonding formationbetween the protein molecule residuals and �OHgroups of the adsorbed ethanol molecules, whichare stronger than electrostatic repulsion. Such re-pulsion would occur between rather small numberof negatively charged groups (e.g. OH− ions adher-ing to ethanol molecules) on the emulsion surfaceand the protein molecules [12]. On the other hand,


protein emulsion there may occur stability lossof such emulsion. However, it should be remindthat the studied emulsions were diluted. Themaximum distance between the droplets wasusually several times higher than the droplet di-ameter, which resulted from the effective diame-ter and the total content of n-tetradecane in theemulsion. Unfortunately, much more concen-trated emulsions cannot be investigated by usingthe dynamic light scattering technique. Never-theless, it seems to us that results obtained inthis paper also give some insight into propertiesof emulsified systems in which the proteins arepresent.

5. Summary

The effective diameter of emulsion may resultfrom one, two or more populations of thedroplets present [8,9], and in the studied emul-sions the droplets could be grouped in only oneor two populations.

The most stable and reproducible emulsionswere obtained when the protein content was 2or 5 mg/0.1 ml of n-tetradecane in 100 ml of0.5 M ethanol. The results obtained for emul-sions of n-tetradecane prepared in the proteinssolutions alone (without ethanol) showed that�-casein was a good emulsifier already at itsconcentration (1 mg/100 ml). This probably re-sulted from a flexible structure of its molecules,while proteins having compact globularmolecules, like BSA, usually show worse emulsi-fying properties [17].

The measured zeta potentials of the emulsiondroplets were moderate and at natural pH(which was close to neutral one) the potentialswere negative. By decreasing the emulsion pH itwas possible to reverse the zeta potential signfrom negative to positive.

The i.e.p. occurred at a pH value dependedon the type of protein present. However, exceptfor �-lactalbumin, pHi.e.p. of the emulsiondroplets was comparable to, or slightly lowerthan pHi.e.p. of the appropriate native protein.In some emulsions, despite decreasing the zetapotential on time scale the emulsion was still

stable. It points that some other interactions op-erated at the interface, like steric stabilizationand acid–base interactions (hydrogen bonding),which are electron donors and acceptors in na-ture [5,12]. Therefore, for complete descriptionof energy balance, evaluation of the electron-donor and electron-acceptor interactions seemsto be necessary. Such approach was tested ear-lier for a simpler model emulsion withoutprotein presence [21,22]. The calculationsshowed that the hydrogen-bonding interactionsbetween water and the alcohol dipoles predomi-nated over the attractive London dispersion andrepulsive electrostatic interactions [22–24].

It seems that ethanol concentration and itspolar interactions (Lewis acid–base) play a sig-nificant role in protein adsorption, restructuringits molecule and in consequence also the zetapotential of the n-alkane droplet [21]. Zeta po-tential of n-alkane droplet in the alcohol solu-tion alone may be ascribed to adsorbed,immobilized and oriented dipoles of the alcohol.The mechanism of the zeta potential formationprobably relies on an ‘attachment’ of ions fromthe bulk phase to the first structured and immo-bilized layer of water (alcohol) dipoles, whichactually corresponds to the concept of preferen-tial (competitive) ‘solubility’ of the ions in thevicinal water layer [12].

However, time-depended electrokinetic behav-ior of the oil droplets in the studied emulsionsshows that in such complex systems the mecha-nism of zeta potential formation probably con-sists of several competing processes. They maylargely depend on the alcohol and protein con-centration, emulsion temperature, pH (ionicstrength), as well as on the procedure of emul-sion preparation. Hence, the electrokinetic be-havior and stability of this type of emulsionsneeds further investigations.

Acknowledgements

The authors very much appreciate financialsupport for these investigations from the StateCommittee for Scientific Researches in Warsawunder the project 2 T09A 099 16.


References

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