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

www.elsevier.com/locate/colsurfb

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 Uni6ersity, 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 in
aqueous solution of ethanol (0.5 and 1.0 M) in which bovine serum albumin (BSA), a-lactalbumin or b-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 were
determined as a function of time, i.e. after 5, 15, 30, 60, 120 min, 1, and 2 days, and 1 week, since the emulsion
preparation. The emulsions were prepared by dissolving 0.1 ml of the n-alkane in a proper amount of ethanol and
then water or protein solution was added to obtain 100 ml of the emulsion in which total concentration of ethanol
was 0.5 or 1.0 M. Next, the emulsion was sonicated for 15 min and the measuring polyacrylic cells of the apparatus
were filled with the emulsion. The isoelectric point (i.e.p.) of the droplets in the presence of investigated proteins (2
mg/100 ml) and in 1 M ethanol occurred at pH 4.9, 4.7 and 2.2, for BSA, b-casein and a-lactalbumin, respectively.
In pH range 5.510, the zeta potentials of freshly prepared emulsions in 1 M ethanol were negative and relatively
large, from 45 to 60 mV. In b-casein presence, the n-alkane droplets were larger and negative zeta potentials
higher than in the presence of two other investigated proteins. However, in the presence of each of the investigated
proteins the droplet size increased slightly relative to that in ethanol solution alone. Nevertheless, the emulsions were
relatively stable. In 0.5 M ethanol, the protein presence stabilized the emulsions. On time scale, the changes of
negative 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, b-casein alone can be used as an emulsifier
already at its concentration of 1 mg/100 ml for 0.1 ml of n-tetradecane content both at natural and alkaline
environment. However, no stable emulsion could be obtained using BSA or a-lactalbumin. Multimodal size
distribution analysis showed that the droplet sizes in the studied emulsions could be grouped in one or two
comparable populations only. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Emulsions; Ethanol; Protein; Size distribution; Effective diameter; Zeta potential

1. Introduction
* Corresponding author. Tel.: + 48-81-537-5651; fax: + 4881-533-3348.
E-mail address: emil@hermes.umcs.lublin.pl (E. Chibowski).

Application of natural emulsifiers, like proteins,

in food products, pharmaceutics, and other emulsion systems is of a great interest [1]. In many

0927-7765/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 7 - 7 7 6 5 ( 0 1 ) 0 0 3 0 4 - 6

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A.E. Wiacek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

naturally occurring systems, proteins emulsify the

oily substances thus facilitate their nutrition. Stability of emulsions largely depends on dimension
of the emulsion droplets, which in turn depends on
the conditions of the emulsion preparation.
Macromolecules, such as proteins, form an adsorbed film on the surface of emulsion droplet,
thus creating an energy barrier against coalescence. The protein molecules, when adsorbed, can
unfold and reorient their amino-acid groups, so
that the hydrophobic groups may align with the
oil molecules and the hydrophilic groups may
direct toward the aqueous phase. Thus, at the
interface a visco-elastic or gel-like layer can be
formed [2,3].
It is obvious now that stability/instability of
emulsion is controlled by properties of the layer
adsorbed to the surface of oil droplets [1]. The
unfolding protein process depends upon the
protein molecule structure [2], but the process is
not fully known yet [1]. Such knowledge could be
helpful for better understanding of the stabilizing
mechanism at the interface by the adsorbing
protein. Surely, structure and strength of the
adsorbed protein film are of a crucial importance
in preventing coalescence of the oil droplets, that
is, breaking of the emulsion structure. In general,
the adsorbed film structure will depend on the
concentration, composition, and kind of the
emulsifier. The adsorbed layer, especially of
protein (or a polymer) plays also a very important
role in aggregation and deposition processes of a
dispersed solid phase. In some systems, small
amounts of the adsorbed protein can promote
flocculation by a bridging mechanism [2]. On the
other hand, proteins at a larger adsorbed amount
can produce a greatly enhanced stability by an
effect known as steric stabilization [2,3].
In practical emulsions systems the droplets are
sub-, or about micrometer size. The smaller the
droplet size the more stable emulsion is [1]. As it
was mentioned above, the proteins usually change
their conformation state after adsorption at the
oil/water interface and the adsorption is strong [2].
Other component present in emulsion, for example
alcohol, can weaken the adsorbed protein layer by
a competitive adsorption. The competing
molecules usually do not form a strong adsorbed

layer themselves, but they adsorb alongside the

protein molecules, thus weakening the gel-like
structure [1 3]. The resulting adsorbed layer
forms a network structure with fractures of another component [2,3]. The structure of adsorbed
protein layer can thus be broken down completely,
which drastically reduced stability of the interface.
In general, an emulsifier changes (decreases) the
oil/water interfacial tension, as well as the surface
charge of the droplet, what should appear in the
changes of zeta potential [1]. These two parameters, i.e. interfacial tension and zeta potential,
from viewpoint of the DLVO theory, are most
important for description of dispersed system stability [4]. However, recently published papers [5
7] show that the stability can be described better
by so-called extended DLVO theory, where, in
addition to the attraction Lifshitz-van der Waals
and 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 stabilizing and/or emulsifying properties of some proteins
for oil-in-water emulsion. For this purpose zeta
potential, effective diameter and multimodal size
distribution of n-tetradecane droplets were investigated by dynamic light scattering technique as a
function of time. The oil phase was emulsified by
ethanol (0.5 or 1.0 M) and stabilized (or destabilized) by BSA, a-lactalbumin or b-casein presence.
Also, the effect of emulsion pH on the above
parameters was investigated. The zeta potential
was determined subsequently after the droplet size
determination for the same sample of the emulsion, which was placed in measuring cell of ZetaPals/BI-MAS instrument [8,9]. It seemed us
interesting to learn whether on time scale changes
in zeta potential and effective diameter of the same
sample of emulsion correlate each other. Since,
electrostatic repulsion is determined by zeta potential [10], therefore, one can expect that the droplets
should be more stable if the zeta potential is
higher.
We would also like to study on a macroscopic
level impact of structural features of the protein
molecules on the emulsion stability. Such knowledge may be helpful for applications of a wider
range of protein for some specific systems, for
example food or cosmetic products. Obviously,

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

for full characterization of proteins behavior as

emulsifier/stabilizer agents a multidisciplinary approach, like molecular interactions (biochemical
analysis and proteins characterization), interfacial
behavior (spectroscopy, microscopy, tensiometry),
and bulk properties (dispersion formation, stability and rheology), studies are required [1
3,7,8,1012].

2. Experimental
The emulsions were prepared in the 100 ml-calibrated flasks. The components were added as
follows. First desired portion of ethanol (p.a.
from POCh Gliwice, Poland) was introduced to
obtain 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 ml
from a stock solution 0.1 g/100 ml) and deionized
water was added to obtain 100 ml of the emulsion. Next, the flasks were placed for 15 min in an
ultrasonic bath (50 W) to homogenize the emulsions. Having thus prepared emulsion, appropriate number of the polyacrylic cells used in the
ZetaPals/BI-MAS apparatus were filled with the
emulsion. These cells were next left without any
shaking or mixing before the droplet size and zeta

57

potential determination. The proteins used were

from Sigma Chemical Co., and particularly they
were: bovine serum albumin (BSA, fraction V,
min. 90% proteins), a-lactalbumin (from bovine
milk, lyophilized, min. 85%) and b-casein (from
bovine milk, lyophilized, min 90%). Water used
for the emulsion preparation was from MiliQ-Plus
system. The emulsion droplet sizes were determined as a function of time, after 5, 15, 30, 60,
120 min, and 1, 2 and 7 days, since the emulsion
preparation. The duration of droplet size determination in particular sample was about 5 min.
Then, zeta potential measurement followed for
the same sample using ZetaPals/BI-MAS apparatus [8,9]. Since, ka products (Debye-Hu ckel
parameter and the droplet radius) were small for
the emulsion droplets, the zeta potentials were
calculated from Hu ckel equation. All the experiments were carried out at 20 C.

3. Results
The changes in zeta potential of n-tetradecane
droplets in the emulsions are presented in Fig. 1.
Since ka product was small for the droplets suspended 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 native
proteins are shown by arrows: 1 4.8 for BSA, 2 4.5 for b-casein, 3 5.1 for a-lactalbumin.

58

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

tively, therefore Hu ckel equation was applied for

zeta potential calculations instead of Smoluchowski one. On the other hand, Oshima and
Kondo [13] suggested that Smoluchowski equation is not suitable for calculation of zeta potentials for biological particles (cells), because, it
leads to overestimation of the surface (zeta) potentials due to electrophoretic fluid flow inside,
for example, adsorbed layer of protein. They [13]
derived an approximate mobility expression for
particles with surface layers in which fixed charges
were uniformly distributed and a planar double
layer could be assumed, i.e. particle radius was
much higher than the double layer thickness, 1/k.
The expression consists of two terms: (i) a
weighted average of the surface potential (and/or
the Donnan potential) and (ii) resulting from the
membrane-fixed charges, which is not subjected to
shielding effects at high concentration of the electrolyte. At high electrolyte concentrations, where
owing to the shielding effects the potentials are
very low, the first term diminishes so that the
mobility is determined mainly by the second term.
This means that a particle with negligible surface
(zeta) potential can exhibit non-zero mobility, in
contrast to the prediction of classical Smoluchowski theory. From their derivation [13] it results that in presence of a structured layer around
the particle its electrophoretic mobility is not sensitive to the position of the slipping plane and
hence it is difficult to define the zeta potential.
Finally, the authors [13] concluded that at high
electrolyte concentration both planar and spherical colloid surfaces behave similarly. Our measurements have been carried out at low electrolyte
concentrations in the emulsion systems, where ka
value was small. Therefore, Hu ckel equation [10]
was applied as an approximation for zeta potential calculations from the measured mobility values of tetradecane droplets. These emulsions
contained 0.1 ml of the dispersed tetradecane in 1
M ethanol and 2 mg of the investigated protein.
Nevertheless, of the interpretation problem of
electrophoretic mobility in terms of zeta potential,
the pH of the isoelectric point (i.e.p.) of the
droplets can be set correctly. Thus, the i.e.p.
occurs at pH 4.9, 4.7 and 2.2, for tetradecane
droplets with adsorbed BSA, b-casein or a-lactal-

bumin, respectively, and it clearly depended upon

the type of the adsorbed protein. Moreover, except for a-lactalbumin, the droplet pHi.e.p. value is
close to that of appropriate native protein [14],
which is: 4.8, 4.5 and 5.1, for BSA, b-casein and
a-lactalbumin, respectively [11]. The zeta potential
changes for BSA are complementary to electrophoretic mobility results obtained by van der
Mei et al. [14] for hexadecane droplets in 10 mM
potassium phosphate after adsorption of BSA or
HSA.
In alkaline region, the zeta potentials depend
only slightly on the emulsion pH (Fig. 1), what
indicates that in pH range 710 the negative
charge of freshly formed droplets is practically
constant as a result of total dissociation of
COOH groups. In the acidic region (pH 36)
dissociation of both amine and carboxyl groups
determine 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 a-lactalbumin suggests that during its adsorption some
conformation changes have occurred.
The pH effect on the emulsion particle size and
zeta potential in the presence of b-casein is shown
in Fig. 2a and b, respectively, and standard deviations of ten readings are shown in Table 1. At pH
4 zeta potential of the droplets is positive, but
decreasing during the emulsion lifetime. The relatively high concentration of OH ions (pH 11)
practically does not cause any increase in the
negative zeta potential of freshly prepared emulsion 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, the
zeta potential also has decreased during 1 week,
from approximately 82 (fresh emulsion) to
30 mV (Fig. 2b). The hydroxyl ions may interact
with the protein molecules as well as influence the
carboxyl groups COOH dissociation [15]. Stability of such emulsions is often higher than expected
on the basis of zeta potential and the ionic
strength, and it is likely that steric stabilization
plays some role here [2]. Relatively high and
stable zeta potential at pH 11 (Fig. 2b) probably
causes that the emulsion is stable during 1 week
without any shaking of the sample (Fig. 2a).
Moreover, at this pH from the very beginning the

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

59

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

Table 1
Effective diameters, zeta potentials and their standard errors of n-tetradecane/b-casein (1 mg) emulsion
Age of emulsion pH 4

5 min
15 min
30 min
1h
2h
1 day
2 days
1 week

pH 7.3 (nat.)

pH 11

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

471.79 65.8
570.7 9 75.6
613.0 9 59.7
314.5 9 2.8
271.7 9 10.9
273.5 9 1.8
328.3 9 7.5
1530.89 79.1

31.69 1.35
35.59 2.7
4.0 9 5.4
27.49 1.5
34.99 1.6
21.09 1.0
17.2 9 1.9
7.59 1.3

1105.2 9 66.2
853.3 9 14.5
827.0 9 36.0
753.1 939.7
476.9 922.3
341.4 9 5.8
301.2 9 4.0
332.6 91.2

79.39 3.4
83.19 1.5
77.59 0.3
67.892.8
53.891.5
31.29 4.2
58.29 0.9
32.791.5

351.6 915.3
302.3 937.0
306.6 923.5
320.6 930.4
304.2 932.4
323.4 94.1
306.3 93.5
294.5 96.1

70.8910.0
83.59 2.8
76.091.5
67.59 3.7
62.59 2.1
66.99 1.9
69.992.4
60.39 1.5

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A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

droplet size was similar to that at pH 7.3 and 4

when partially the separation process had taken
place. At natural (7.3) and acidic (4) pH, the
effective diameter and zeta potential have changed
markedly during 1 week. For this emulsion, a
straightforward correlation between the zeta potential and its stability occurred, especially in the
alkaline environment (pH 11). Hence, it may be
concluded that in this case magnitude of zeta
potential is a principal factor for the emulsion
stability [12,16]. It should be stressed that determined by dynamic light scattering technique decreasing effective diameter of the droplets with the
time (Fig. 2a) obviously had not resulted from a
spontaneous process of splitting of the larger
droplets, but contrary, it resulted from coalescence

Fig. 3. Multimodal size distribution of n-tetradecane droplets

(0.1 ml/100 ml) in b-casein (1 mg) solution at pH 11 for 5 min,
2 days, and 1-week-old emulsion, respectively.

of those droplets and their flotation to the emulsion

surface (phases separation). In results a population
of smaller droplets had remained in the bulk
emulsion. It may also happen, like at pH 4 (Fig. 2a),
that the coalesced droplets are still present in the
bulk emulsion for a period of time (1 week old
emulsion). Here a drastic increase in the average
diameter is observed, from 300 nm (2 days old
emulsion) to 1500 nm (1 week old emulsion). To
better visualize these processes in Fig. 3a c are
shown multimodal size distributions of the droplets
for the emulsion with b-casein at alkaline pH 11,
for 5 min, 2 day, and 1 week old emulsion,
respectively. The effective diameter of the droplets
in 5 min- and 2 day old emulsion is similar each
other, 330.5 and 306.4 nm. The polydispersity and
the sample quality (which informs about fitting
procedure of the correlation functions in a 10-U
arbitrary scale) parameters are similar too, 0.227,
0.222 and 9.5, 9.4, respectively. After 1 week, for
the same emulsion the effective diameter is still
similar (288.7 nm), polydispersity amounts to
0.149, and the sample quality is even better (9.8)
than in former two cases, but in this emulsion two
comparable populations of the droplets coexists,
one of which is slightly smaller (that of smaller
droplets, Fig. 3c). In 5 min and 2 day old emulsion
practically one population of the droplets is only
present (Fig. 3a and b).
The effective diameters and zeta potentials of the
emulsions at natural pH and in the presence of
tested proteins are shown in Figs. 4 6, and in
Tables 24, where the standard deviations (S.D.)
are given. For comparison, in these figures there are
also shown the parameters for emulsions prepared
in ethanol solution alone. In general, effective
diameters of the droplets are lower in the proteins
presence, and they are in the range of 250350 nm.
These results show that optimal amount of the
proteins to stabilize the emulsions is 5 mg/100 ml,
because, effective diameters of such droplets are the
most 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 potentials appear in up to 1-day-old emulsions). Since,
the emulsions were relatively stable, it points that
steric stabilization, as well as hydrogen bonding
probably play here an important role [2,3].

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

61

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

According to Haynes and Norde [17] adsorption of globular protein to apolar surface must
cause removal or neutralization of the electrical
charge between the molecule and the surface. This
may be achieved by formation of pair of ions
(which case seems to be hardly possible for oil
surface), protonation/deprotonation of the ionizable residual groups, and co-adsorption of small
ions in the contact layer. Lateral interactions between the adsorbed molecules may then be attractive or repulsive, depending on the kind and
magnitude of electric charge of the residues. These
complicated processes involving protein adsorption are also reflected in some way in the zeta
potential changes (shown in Figs. 1 and 2b Fig.
4b Fig. 5b Fig. 6b) of the oil droplets as a
function of time and pH of the continuous phase,
and in multimodal size distribution, which is
shown in Fig. 7a c for 2-day-old emulsion with

b-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-old
emulsion the effective diameter was only about
300 nm, and from the multimodal size distribution (Fig. 7a) it can be seen that practically there
was present only one population of the droplets
(with some traces of two larger). At natural (7.3)
and alkaline (11) pH some small population the
larger droplets can bee seen in Fig. 7b and c,
respectively. However, while at pH 4 and 7.3, the
effective diameter had decreased on the time scale,
so at pH 11 it was practically constant (Fig. 2a).

4. Discussion
Considering stabilization of the emulsion by
protein adsorption, it should be kept in mind that

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A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

conformational processes occurring at the oil/alcohol solution interface are slow ones [12,16]. The
observed changes in the effective diameter, and
especially in the zeta potentials, which occurred
after 1 and 2 days, and even up to 1 week (Figs.
46), may just result from such conformational
changes in the structure of the adsorbed protein
molecules and the layer in general. Therefore, it
may happen that two emulsions prepared in similar conditions may be characterized by different
zeta potential. According to Dickinson and Matsumura [18] molecular mechanisms contributing
to the free energy of adsorption are complicated
and may be following: dehydration of the hydrophobic regions at the surface, protein unfolding at
the surface, charge redistribution due to overlapping of the electric fields of protein and surface,

change in pK values of side chains on adsorption,

difference in ionic hydration between bulk and
surface, and restructuring of van der Waals interactions. However, finally the principal driving
force for protein adsorption is removal of its
hydrophobic aminoacid side-chains from the polar environment, i.e. bulk aqueous phase [18].
Dilution of the emulsion by adding of the continuous phase (water) usually does not cause desorption of protein (irreversible adsorption). However,
there are always small areas among the adsorbed
protein train segments accessible for small surfactant molecules [18,19]. For example, adsorbed
a-lactalbumin occupies only about 1/3 of the surface area and although, protein adsorption is
considered to be irreversible, small surfactant
molecules, 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) b-casein emulsions at natural pH,
for different concentrations of the protein and age of the emulsion.

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

63

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

Table 2
Effective diameters, zeta potentials and their standard errors of n-tetradecane/ethanol (0.5 M), BSA emulsion
Age of emulsion 0 mg

5 min
15 min
30 min
1h
2h
1 day
2 days
1 week

2 mg

5 mg

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

404.095.1
391.0 95.5
389.0 97.7
415.3 925.1
381.1 95.2
345.8 98.1
340.5 915.6
351.9 94.1

36.09 2.1
39.49 0.6
36.19 1.5
45.09 0.4
36.09 0.9
49.39 0.6
46.89 1.6
39.19 1.6

782.5 9105.8
380.1 927.2
285.6 94.4
274.6 9 4.1
271.1 9 7.3
272.2 92.1
281.5 96.7
273.6 9 2.9

52.59 0.6
26.29 1.2
36.09 0.9
36.391.0
30.991.6
38.49 0.4
41.49 1.2
39.790.3

302.1 9 3.8
315.0 94.5
312.6 9 6.0
309.6 95.4
327.6 90.9
308.5 92.6
302.0 93.2
310.1 92.7

32.191.5
36.19 3.3
21.991.8
36.99 1.8
31.99 1.6
46.29 0.7
48.191.2
39.19 0.6

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A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

[1]. Ethanol may also interact as a denaturating

agent already at 20 C [2]. However, maximum
concentration of ethanol used in this study was only
approximately 5 wt.% (1 M solution).
Obviously, temperature, pH, and ionic strength
may affect the adsorption [1,12,20]. In the case of
globular protein (e.g. bovine serum albumin, BSA)
the adsorbed molecules may possess some transition forms between native and denaturated, and
among them the so-called molten globule, which
can also exists in the solution phase [17,18]. The
polar and apolar residues are nearly evenly distributed on the globular protein compact surface.
In denaturation process, from the free energy
changes as a function of temperature it results that
the whole globular protein molecule can unfold if
disruption of an essential part of its molecule took

place [12]. The maximum protein adsorption usually occurs at pH close to its i.e.p. [1].
The electrophoretic mobility of n-alkane
droplets with adsorbed protein, among others, were
studied by van der Mei et al. [14]. The authors
concluded that comparable pH value of the i.e.p.
for protein coated tetradecane droplets and those
for native proteins confirm that at low protein
concentrations the net charge addition upon their
adsorption determines effects of the final electrophoretic behavior of the proteinhexadecane
complexes. Basing van der Mei et al.s [14] results
and those obtained in this paper it can be concluded
that in the adsorbed state there are more dissociated residual COOH groups and/or less dissociated amine groups, or less number of these latter
groups is exposed toward the water phase [12]. It

Table 3
Effective diameter, zeta potentials and their standard errors of n-tetradecane/ethanol (0.5 M), b-casein emulsion
Age of emulsion 0 mg

5 min
15 min
30 min
1h
2h
1 day
2 days
1 week

2 mg

5 mg

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

404.09 5.1
391.09 5.5
389.09 7.7
415.3 9 25.1
381.1 9 5.2
345.89 8.1
340.59 15.6
351.9 9 4.1

36.09 2.1
39.49 0.6
36.19 1.5
45.09 0.4
36.09 0.9
49.39 0.6
46.89 1.6
39.19 1.6

466.1 914.2
340.698.2
333.1 95.5
304.896.8
319.29 7.7
319.3 9 3.1
315.5 9 4.7
314.1 93.8

29.892.1
36.791.3
43.991.0
28.290.6
37.691.3
45.390.7
19.592.7
28.090.6

314.7 95.3
305.6 97.9
334.4 96.4
321.7 97.1
322.9 93.5
316.6 93.2
341.2 96.9
478.7 97.6

39.490.7
37.390.9
35.290.1
28.990.6
23.490.1
33.190.4
14.593.6
10.691.9

Table 4
Effective diameter, zeta potentials and their standard errors of n-tetradecane/ethanol (0.5 M), a-lactalbumin emulsion
Age of emulsion 0 mg

5 min
15 min
30 min
1h
2h
1 day
2 days
1 week

2 mg

5 mg

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

Effective
diameter

Zeta potential

404.09 5.1
391.09 5.5
389.09 7.7
415.39 25.1
381.1 9 5.2
345.89 8.1
340.5 9 15.6
351.99 4.1

36.09 2.1
39.49 0.6
36.19 1.5
45.09 0.4
36.09 0.9
49.39 0.6
46.89 1.6
39.19 1.6

337.29 7.4
343.0 96.2
338.0 94.2
320.991.9
346.7 96.2
327.3 9 3.0
323.895.0
333.8 93.5

59.491.3
39.191.2
45.091.8
43.691.8
31.891.6
40.391.5
36.393.1
40.592.1

251.2 92.3
251.6 91.6
244.8 94.8
243.8 94.5
238.7 92.0
244.0 91.4
253.6 94.7
265.1 94.0

39.39 1.2
31.99 3.0
28.99 0.7
32.79 1.9
29.59 1.5
33.39 0.1
13.99 1.5
19.99 1.3

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

Fig. 7. Multimodal size distribution of n-tetradecane droplets

(0.1 ml/100 ml) in b-casein (1 mg) solution for 2 day-old
emulsion at pH 4, natural 7.3, and 11, respectively.

should be kept in mind that because of preparation

procedure of the emulsions, used in this paper, the
protein molecules probably compete with already
adsorbed ethanol molecules, which can essentially
affect their conformation, as well as dissociation of
the polar groups upon adsorption. Of course, it
depends strongly on the concentrations of protein
and alcohol (and the concentration ratio too) and
molecular weight of the protein [12]. One can also
consider a role of hydrogen bonding formation
between the protein molecule residuals and OH
groups of the adsorbed ethanol molecules, which
are stronger than electrostatic repulsion. Such repulsion would occur between rather small number
of negatively charged groups (e.g. OH ions adhering to ethanol molecules) on the emulsion surface
and the protein molecules [12]. On the other hand,

65

some authors suggest a typically electrostatic mechanism of protein adsorption [18].

The results obtained in this paper show that in
emulsion without ethanol presence, globular
protein with hard molecules are in general worse
stabilizers than b-casein, which has elastic structure
[11]. Small amount of b-casein (0.1 mg) produced
relatively stable emulsion, especially in alkaline
environment (see Fig. 2). Analogous emulsions
with BSA or a-lactalbumin without ethanol (not
presented) were unstable. However, if 0.5 M ethanol was present as the emulsifier, all investigated
proteins showed comparable behavior. In such
systems, the differences in protein molecule structure probably are not so important, because the
alcohol may refold them [1,12].
Comparison of the measured changes in effective
diameters and zeta potentials shows that in general
there is not straightforward relationship between
variation of these two parameters, i.e. decreasing
stability with decreasing zeta potential for. This
points to essential role of steric stabilization and/or
hydrogen bonding interactions between polar
groups of the adsorbed ethanol (and water) and
protein molecules from the bulk phase [2,3,11]. It
can be speculated that more effective stabilizers are
the proteins whose molecule segments stretch into
the aqueous phase upon their adsorption [1]. Since,
such stretching segments should be polar and
hydrated, their overlapping would cause some
dehydration and hence an increase in the free
energy, which of course is not a favorable process.
Also, electrostatic repulsion between such charged
segments should take place [1]. As protein adsorption is accompanied not only by changes in the
electrostatic properties of the oil surface, but also
by the surface free energy changes [14], the adsorbed protein molecules may keep apart the
droplets at a distance at which van der Waals
attraction has already decayed, or where the
droplets form weak aggregates, which can be easily
broken by a shear force [1]. However, the most
important factor determining degree of steric stabilization is thickness of the adsorbed layer in relation
to the particle (droplet) size [2], and it has been
known for a time that emulsion stability is related
to strength of the adsorbed protein layer [1 3].
Hence, if the ratio of oil phase increases in the

66

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

protein emulsion there may occur stability loss

of such emulsion. However, it should be remind
that the studied emulsions were diluted. The
maximum distance between the droplets was
usually several times higher than the droplet diameter, which resulted from the effective diameter and the total content of n-tetradecane in the
emulsion. Unfortunately, much more concentrated emulsions cannot be investigated by using
the dynamic light scattering technique. Nevertheless, it seems to us that results obtained in
this paper also give some insight into properties
of emulsified systems in which the proteins are
present.

5. Summary
The effective diameter of emulsion may result
from one, two or more populations of the
droplets present [8,9], and in the studied emulsions the droplets could be grouped in only one
or two populations.
The most stable and reproducible emulsions
were obtained when the protein content was 2
or 5 mg/0.1 ml of n-tetradecane in 100 ml of
0.5 M ethanol. The results obtained for emulsions of n-tetradecane prepared in the proteins
solutions alone (without ethanol) showed that
b-casein was a good emulsifier already at its
concentration (1 mg/100 ml). This probably resulted from a flexible structure of its molecules,
while proteins having compact globular
molecules, like BSA, usually show worse emulsifying properties [17].
The measured zeta potentials of the emulsion
droplets were moderate and at natural pH
(which was close to neutral one) the potentials
were negative. By decreasing the emulsion pH it
was possible to reverse the zeta potential sign
from negative to positive.
The i.e.p. occurred at a pH value depended
on the type of protein present. However, except
for a-lactalbumin, pHi.e.p. of the emulsion
droplets was comparable to, or slightly lower
than pHi.e.p. of the appropriate native protein.
In some emulsions, despite decreasing the zeta
potential on time scale the emulsion was still

stable. It points that some other interactions operated at the interface, like steric stabilization
and acidbase interactions (hydrogen bonding),
which are electron donors and acceptors in nature [5,12]. Therefore, for complete description
of energy balance, evaluation of the electrondonor and electron-acceptor interactions seems
to be necessary. Such approach was tested earlier for a simpler model emulsion without
protein presence [21,22]. The calculations
showed that the hydrogen-bonding interactions
between water and the alcohol dipoles predominated over the attractive London dispersion and
repulsive electrostatic interactions [22 24].
It seems that ethanol concentration and its
polar interactions (Lewis acidbase) play a significant role in protein adsorption, restructuring
its molecule and in consequence also the zeta
potential of the n-alkane droplet [21]. Zeta potential of n-alkane droplet in the alcohol solution alone may be ascribed to adsorbed,
immobilized and oriented dipoles of the alcohol.
The mechanism of the zeta potential formation
probably relies on an attachment of ions from
the bulk phase to the first structured and immobilized layer of water (alcohol) dipoles, which
actually corresponds to the concept of preferential (competitive) solubility of the ions in the
vicinal water layer [12].
However, time-depended electrokinetic behavior of the oil droplets in the studied emulsions
shows that in such complex systems the mechanism of zeta potential formation probably consists of several competing processes. They may
largely depend on the alcohol and protein concentration, emulsion temperature, pH (ionic
strength), as well as on the procedure of emulsion preparation. Hence, the electrokinetic behavior and stability of this type of emulsions
needs further investigations.

Acknowledgements
The authors very much appreciate financial
support for these investigations from the State
Committee for Scientific Researches in Warsaw
under the project 2 T09A 099 16.

A.E. Wia cek, E. Chibowski / Colloids and Surfaces B: Biointerfaces 25 (2002) 5567

References
[1] P. Wlastra, in: P. Becher (Ed.), Encyclopedia of Emulsion
Technology, vol. 4, Marcel Dekker, New York, 1996.
[2] D. Song, D. Forciniti, J. Coll. Int. Sci. 221 (2000) 25.
[3] P.C. Sontum, C. Christiansen, J. Pharm. Biomed. Anal.
16 (1997) 295.
[4] B.V. Derjaguin, N.V. Churajev, Coll. Surf. A 41 (1989)
223.
[5] C.J. van Oss, M.K. Chaudhury, R.I. Good, Chem. Rev.
88 (1988) 927.
[6] C.J. van Oss, R.I. Good, M.K. Chaudhury, Langmuir 4
(1988) 884.
[7] Y.J. Rabinovich, A.A. Baran, Coll. Surf. A 59 (1991) 47.
[8] E.F. Grabowski, J.D. Morrison, in: B. Dahneke (Ed.),
Particle Size Distributions from Analysis of Quasi-Elastic
Light Scattering Data, Wiley, New York, 1983.
[9] Manual Instruction for BI-FOQELS, Brookhaven Instr.,
Holtsville, New York 1995.
[10] R.J. Hunter, Zeta Potential in Colloid Science, Academic
Press, London/San Francisco, 1981.
[11] M.H. Baron, J. Coll. Int. Sci. 221 (2000) 273.

67

[12] E. Chibowski, A.E. Wia cek, Electrokinetics of n-alkane

O/W emulsions, in: A.V. Delgado (Ed.), Interfacial Electrokinetics and Electrophoresis, Marcel Dekker, New
York, 2001 Ch. 32, pp. 893 931.
[13] H. Ohshima, T. Kondo, Biophys. Chem. 39 (1991) 191.
[14] H.C. van der Mei, S. Meijer, H.J. Busscher, J. Coll. Int.
Sci. 205 (1998) 185.
[15] A.E. Wia cek, E. Chibowski, Coll. Surf. B 17 (2000)
175 190.
[16] J. Israelachvili, H. Wennerstro m, Nature 379 (1996) 219.
[17] C.A. Haynes, W. Norde, J. Coll. Int. Sci. 169 (1995) 313.
[18] E. Dickinson, Y. Matsumura, Coll. Surf. B 3 (1994) 1.
[19] A. Dussaud, G.B. Han, L. Ter Minassian-Saraga, M.
Vignes-Adler, J. Coll. Int. Sci. 167 (1994) 247.
[20] K. Kato, S. Sano, Y. Ikada, Coll. Surf. B 4 (1995) 221.
[21] B. Jan czuk, T. Biaopiotrowicz, W. Wo jcik, Coll. Surf. A
36 (1989) 391.
[22] A.E. Wia cek, E. Chibowski, Coll. Surf. B 14 (1999) 19.
[23] S. Servagent-Noinville, M. Revault, H. Quiquampoix,
M.-H. Baron, J. Coll. Int. Sci. 221 (2000) 273.
[24] C.J. van Oss, Interfacial Forces in Aqueous Media,
Marcel Dekker, New York, 1994.