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