Langmuir 1996, 12, 5419-5424
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Interaction Forces in Foam Films Stabilized with Lysophosphatidylethanolamine in the Presence of Na+ and Ca2+ R. Cohen,*,† D. Exerowa,† and T. Yamanaka‡ Institute of Physical Chemistry, Bulgarian Academy of Sciences, Sofia 1040, Bulgaria, and Faculty of Education, Chiba University, Chiba 263, Japan Received March 22, 1996. In Final Form: June 17, 1996X The interaction forces in microscopic foam films stabilized with the soluble zwitterionic phospholipid lauroyl lysophosphatidylethanolamine have been studied by measuring the thickness/electrolyte concentration and disjoining pressure/thickness isotherms in the presence of Na+ and Ca2+ in solution. Electrostatic long-range repulsive interactions leading to formation of thick silver-colored films are found to be operative at low NaCl concentrations. The determined diffuse electric layer potentials (φo) for the two studied surfactant concentrations (Cs) are low, 35 and 20 mV, the lower value corresponding to the higher Cs. These potential values are considered negative in conformity with the previously advanced assumption of the important role of OH- ions for formation of negative charge at the film interfaces. The obtained results agree well with previous results obtained for foam films formed from nonionic surfactant and phospholipid solutions showing that in the case of 1-1 valent electrolyte the film behavior is determined by the zwitterionic character of the phospholipid head group and not by its structure. The results obtained at low CaCl2 concentrations show lower φo-values due to Ca2+ ions binding at the initially negative film interfaces. The increased positive charging leads to a charge reversal at the film interfaces at concentrations higher than 0.02 mol dm-3 CaCl2 causing a Newton to common black film transition. Further increase in CaCl2 concentration leads to decrease in film thickness until again Newton films are obtained. The comparison of these results with previous data obtained for foam films stabilized with lysophosphatidylcholine shows the importance of the phospholipid head group structure for the Ca2+ binding which determines the electrostatic interactions in foam films stabilized with neutral phospholipids with CaCl2 added.
Introduction The interaction between biological cells, which is of major importance for many medical and biotechnological processes, has attracted the interest of many investigators. Many studies, using different techniques, have been carried out on model neutral bilayer systems, as is wellknown the cell membrane is mainly constituted of zwitterionic phospholipids. Important information on the effect of divalent ions on interbilayer interactions in such systems has been obtained, for instance, by electrophoretic, NMR, and X-ray studies1-3 and the osmotic stress method.4-6 The direct measurement of the forces acting between phospholipid bilayers7 deposited on mica has also shown the effect of different ions on long-range interaction forces. During recent years a number of investigations performed with free liquid (foam) films stabilized with phospholipids have demonstrated possibilities for the study of both long-range and short-range interaction forces, e.g., refs 8-16. Recent reviews17,18 also show the * To whom correspondence may be addressed. E-mail, cohen@ ipchp.ipc.acad.bg. † Bulgarian Academy of Sciences. ‡ Chiba University. X Abstract published in Advance ACS Abstracts, October 1, 1996. (1) Inoko, Y.; Yamaguchi, T.; Furuya, K.; Mitsui, T. Biochim. Biophys. Acta 1978, 413, 24. (2) Oshima, H.; Inoko, Y.; Mitsui, T. J. Colloid Interface Sci. 1982, 86, 57. (3) McLaughlin, A.; Grathwohl, C.; McLaughlin, S. Biochim. Biophys. Acta 1978, 513, 338. (4) Lis, L. J.; Parsegian, V. A.; Rand, R. P. Biochemistry 1981, 20, 1761. (5) Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P. Biochemistry 1981, 20, 1771. (6) Rand, R. P. Annu. Rev. Biophys. Bioeng. 1981, 10, 277. (7) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608. (8) Yamanaka, T.; Hayashi, M.; Matuura, R. J. Colloid Interface Sci. 1982, 88, 458.
S0743-7463(96)00280-6 CCC: $12.00
possibilities that such amphiphile bilayers offer in the field of model biological membrane studies. Surface forces in foam films have been investigated by different methods using both the microscopic film model9-14 (radius ∼0.01 cm) and the macroscopic one8,15,16 (with an area ∼1 cm2). Foam films stabilized with biological surfactants that form micellar solutions in water, such as the lysophospholipids, are particularly good for such studies. Lysophospholipids attract the interest of investigators also because of the role they play in the functioning of biological membranes. Useful information on interfacial and biological properties of lysophospholipids is given in the extensive studies of Stafford et al.19,20 Studies of surface forces in microscopic foam films stabilized with dimyristoylphosphatidylcholine11 and palmitoyl lysophosphatidylcholine (lyso PC)12 have shown that at low NaCl concentrations in the solution long-range electrostatic repulsive forces are operative. The study21 corroborates the previously made assumption for films stabilized with nonionic surfactants22,23 that the charging (9) Exerowa, D.; Lalchev, Z.; Marinov, B.; Ognianov, K. Langmuir 1986, 2, 664. (10) Exerowa, D.; Lalchev, Z. Langmuir 1986, 2, 668. (11) Cohen, R.; Koynova, R.; Tenchov, B.; Exerowa, D. Eur. Biophys. J. 1991, 20, 203. (12) Cohen, R.; Exerowa, D.; Kolarov, T.; Yamanaka, T.; Muller, V. M. Colloids Surf. 1992, 65, 201. (13) Exerowa, D.; Nikolova, A. Langmuir 1992, 8, 3102. (14) Nikolova, A.; Exerowa, D.; Lalchev, Zdr.; Tsonev, L. Eur. Biophys. J. 1994, 23, 145. (15) Yamanaka, T.; Tano, T.; Kamegaya, O.; Exerowa, D.; Cohen, R. D. Langmuir 1994, 10, 1871. (16) Yamanaka, T.; Tano, T.; Tozaki, K.; Hayashi, H. Chem. Lett. 1994, 1143. (17) Exerowa, D.; Kashchiev, D.; Platikanov, D. Adv. Colloid Interface Sci. 1992, 40, 201. (18) Exerowa, D.; Kashchiev, D.; Platikanov, D.; Toshev, B. Adv. Colloid Interface Sci. 1994, 49, 303. (19) Stafford, E. R.; Fanni, T.; Dennis, E. A. Biochemistry 1989, 28, 5113. (20) Stafford, E. R.; Dennis, E. A. Colloids Surf. 1988, 30, 47. (21) Cohen, R.; Exerowa, D. Colloids Surf. 1994, 85, 271.
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behavior of these films is most probably due to preferential adsorption of OH- at the solution/air interface. In the case of added CaCl2, Ca2+ binding to the zwitterionic phospholipid head groups has been found,8,12,15,16 leading to positive charge formation at the film interfaces. These experimental results are in good agreement with the studies of interbilayer interactions of multilamellar zwitterionic phospholipid systems in the presence of Ca2+ ions, e.g., refs 1-6. Although the mechanism of Ca2+ binding by zwitterionic phospholipds is not completely clarified, it seems that it should depend on the structure and properties of the polar head groups. To get more of an insight of the effect of the hydrophilic head group on surface forces, it is important to investigate foam films stabilized with phospholipids with different head groups, for instance, phosphatidylethanolamines, where we can expect that greater intermolecular hydrogen bonding and dipole-dipole attractions24 would lead to different film properties. As is wellknown, the microscopic films can be formed even at minimum surfactant concentrations13,14,17,18,25 which allows investigations also of the effect of phospholipid concentration on interaction forces in the foam films. The aim of this work was to study long-range electrostatic interaction forces of microscopic foam films stabilized with lysophosphatidylethanolamine in the presence of monovalent and divalent ions. For that purpose the equilibrium thicknesses of the obtained foam films have been measured at different conditions (electrolyte concentration, external pressure) and the results have been interpreted according to the DLVO theoretical concepts. The comparison with previous studies of the interaction behavior and related quantities of lyso PC foam films allows more of an insight of the effect of phospholipid head groups on foam film interactions. Experimental Section Materials and Methods. Lauroyl lysophosphatidylethanolamine (lyso PE), purchased from Avanti Polar Lipids, was used in the experiments. Its purity was tested by thin-layer chromatography, which did not show any contaminations. To control the eventual presence of ionic surface-active impurities, the conductivity of the lyso PE solutions was measured. It was found to be equal to the conductivity of about 10-6 Ω-1 cm-1 of the thrice-distilled water used for the preparation of the solutions . The critical micelle concentration (cmc) of 3.3 × 10-4 mol dm-3 of lyso PE has not been found to significantly depend on the electrolyte concentration up to about 2 M NaCl.17,18 NaCl (Suprapur) and CaCl2‚2H2O (p.a.) were obtained from Merck. NaCl was roasted at 500 °C for 4 h to remove surfaceactive contaminations. CaCl2‚2H2O was used as obtained. All experiments were carried out at a temperature of 32 °C, which is well above the temperature of 28 °C under which the solutions become cloudy. The measured pH values of the solutions were of 5.9 ( 0.2. The measurements were performed using the well-known microinterferometric experimental technique17,25,26-28 as shown in previous microscopic film studies, e.g., refs 9-14, 17, 18, and 25. All films in our experiments were formed with the same radius, 0.01 cm. The capillary pressure, Pσ, in these films was determined as Pσ ) 2σ/R, where σ is the surface tension and R is the radius of the glass capillary where the microscopic horizontal film forms in the center of a double-concave drop. The surface tension measurements were carried out by the Wilhelmy method with a Pt plate, using a “Kru¨ss” tensiometer. (22) Exerowa, D. Kolloid-Z. 1969, 232, 703. (23) Manev, E. D.; Pugh, R. J. Langmuir 1991, 7, 2253. (24) Pascher, I.; Sundel, S.; Hauser, H. J. Mol. Biol. 1981, 153, 807. (25) Scheludko, A. Adv. Colloid Interface Sci. 1967, 1, 391. (26) Exerowa, D.; Zacharieva, M.; Cohen, R.; Platikanov, D. Colloid Polym. Sci. 1979, 257, 1089. (27) Scheludko, A.; Exerowa, D. Kolloid-Z. 1959, 165, 148. (28) Scheludko, A.; Exerowa, D. Kolloid Z. 1960, 168, 24.
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Figure 1. Equilibrium thickness h of microscopic foam films stabilized with lyso PE as a function of NaCl concentration (Cel): b, Cs ) 2.5 × 10-4 mol dm-3; O, Cs ) 5 × 10-4 mol dm-3. The direct measurements of the disjoining pressure isotherms were performed by means of the specially designed “porous-plate technique”,29,30 which has repeatedly been used in microscopic foam film studies. The external pressure that balances the pressure in the film was applied utilizing the special membrane pump30 and measured with an accuracy of (5 N m-2. The processing of the interferometrically obtained photometric data yields the so-called equivalent film thickness, which is found considering the film as homogeneous with an index of refraction equal to the refraction coefficient of the solution from which the film is obtained, in our case 1.33. The accuracy of the so determined microscopic thin liquid film thicknesses is (0.2 nm. To consider the film structure model, assumptions were made which will be discussed later in the paper. Experimental Results. Figure 1 shows the equilibrium thicknesses of foam films stabilized with lyso PE as a function of NaCl concentration for two phospholipid concentrations (Cs). At Cs ) 2.5 × 10-4 mol dm-3 (full points) at low electrolyte concentrations (Cel) thick films were formed that gradually decreased in thickness with increase in Cel until the so-called critical electrolyte concentration Cel,cr ) 1.5 × 10-2 mol dm-3 was reached where a transition to black film formation occurred. Further increase in Cel did not lead to any change of the thickness of 6.4 ( 0.2 nm of these films which indicated that Newton black films were obtained. The curve obtained at Cs ) 5 × 10-4 mol dm-3 (circles in Figure 1) follows a similar course but it is worth noting that in the region of thick film formation it lies entirely below the curve obtained at Cs ) 2.5 × 10-4 mol dm-3. A zone of fluctuating film thicknesses was observed at this Cs between Cel ) 1.5 × 10-3 mol dm-3 and Cel,cr ) 8 × 10-3 mol dm-3 where occasionally Newton films were formed. The figure shows the Newton film thicknesses after 5 × 10-3 mol dm-3 where such films were more often observed. The experimental points represent the mean film thickness values obtained after three to five separate experiments performed with freshly prepared solutions. Eight to ten microscopic films were measured in each experiment. In the region of the thicker (silver-colored) films an experimental scatter of about (5 nm was found. The Newton film thicknesses were all obtained within a region of (0.2 nm, equal to the accuracy of the interferometric method. Figure 2 shows the obtained equilibrium thicknesses of lyso PE foam films as a function of CaCl2 concentration. The experiments were carried out at the same Cs as with NaCl added. In this case thick films were obtained at the low Cs (full points in Figure 2) only in the narrow Cel interval between 0.7 × 10-4 and 2.5 × 10-4 mol dm-3 CaCl2. At 3 × 10-4 mol dm-3 Newton films were formed that did not change their thickness of 6.4 ( 0.2 nm until Cel ) 1.5 × 10-2 mol dm-3. Above this concentration a dramatic increase in film thicknesses was observed leading to a Newton black to common black film transition. The thickness of the common black films at Cel ) 2 × 10-2 mol dm-3 reached 12.9 nm. Further increase of the Ca2+ ions concentration led to a slow decrease in film thicknesses. Only in some experiments, (29) Exerowa, D.; Scheludko, A. C. R. Acad. Bulg. Sci. 1971, 24, 47. (30) Exerowa, D.; Kolarov, T.; Khristov, Khr. Colloids Surf. 1987, 22, 171.
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Figure 2. Equilibrium thickness h of microscopic foam films stabilized with lyso PE as a function of CaCl2 concentration (Cel): b, Cs ) 2.5 × 10-4 mol dm-3; O, Cs ) 5 × 10-4 mol dm-3.
Figure 4. Π(h) isotherm, measured at Cs ) 2.5 × 10-4 mol dm-3 with 2 × 10-4 mol dm-3 CaCl2 added: lower line, constant potential 16 mV; upper line, constant charge density 0.7 mC m-2. The symbols show the experimental results; solid lines are theoretical predictions. until a transition to Newton film formation occurs at (1.8-2.6) × 103 N m-2. The Newton film thickness of about 6 nm did not change with further increase of the applied pressure. No film rupture was observed in the studied pressure range. The course of the obtained isotherm with CaCl2 added, shown in Figure 4, is very similar to that in Figure 3. The transition to Newton film formation occurred in this case at a pressure of (1.2 -1.5) × 103 N m-2.
Discussion
Figure 3. Π(h) isotherm, measured at Cs ) 2.5 × 10-4 mol dm-3 with 5 × 10-4 mol dm-3 NaCl added: lower line, constant potential 32 mV; upper line, constant charge density 1.75 mC m-2. The symbols show the experimental results; solid lines are theoretical predictions. Newton black films were also obtained in the zone of 0.05-0.1 mol dm-3 CaCl2. The Newton film thickness was reached above Cel ) 0.3 mol dm-3. At Cs ) 5 × 10-4 mol dm-3 lyso PE only Newton (bilayer) films were formed in the low Cel region (circles in Figure 2). When the CaCl2 concentration reached 1.5 × 10-2 mol dm-3, again a Newton to common black film transition occurred leading to formation of 13.8 nm thick films. After this initially observed small difference, the film thicknesses measured at both Cs were practically equal within the accuracy of the method. In the experiments with CaCl2 added, an experimental scatter of about (3 nm was observed only in the region of formation of the thicker silver-colored films at the low Cs. In all other experiments the experimental film thickness values were obtained within the accuracy of the microinterferometric technique. Important information on foam film interactions is obtained from the directly measured disjoining pressure isotherm. The experiments were carried out at Cs ) 2.5 × 10-4 mol dm-3 lyso PE. The results obtained with 5 × 10-4 mol dm-3 NaCl added and 2 × 10-4 mol dm-3 CaCl2 added are shown in Figures 3 and 4, respectively. As seen in Figure 3, the increase in the applied pressure leads to a gradual decrease of the equilibrium thicknesses of the initially obtained silver-colored film to about 16 nm
Lyso PE Films with NaCl Added. As shown above, in the case of NaCl added at low Cel, thick equilibrium films are obtained which can be considered evidence of the action of long-range repulsive forces. Such an effect has been found before in films stabilized with nonionic surfactants22,26 and phospholipids12 where it has been shown that the decrease in film thicknesses with increase in electrolyte concentration indicates the electrostatic origin of these forces. In an attempt to better understand the origin of the surface charge and potential leading to electrostatic repulsive forces in nonionic surfactant films, detailed studies of the effect of pH on the equilibrium film thickness and diffuse electric layer potential have been carried out.22,23,32 It has been demonstrated that before the so-called “pH isoelectric” (pH*) where φo f 0, the φo(h) curve is very steep, i.e., small changes of the pH cause dramatic decrease of the potential. These results lead to the conclusion that electrostatic repulsion in such systems is most probably caused by specific adsorption of OHions at the solution/air interface.22,32 This assumption has been corroborated by the experiments with lyso PC foam films.21 These microscopic foam film studies are in good agreement with electrokinetic studies of microbubbles produced in nonionic surfactant solutions where the OH- ions excess leads to negative zeta potentials at the solution/air interface at neutral pH.33,34 Of course, there exists also the possibility of an eventual adsorption at the (31) Kolarov, T.; Cohen, R.; Exerowa, D. Colloids Surf. 1989, 42, 49. (32) Exerowa, D.; Zacharieva, M. In Research in Surface Forces; Plenum Press: New York, 1972; p 234. (33) Huddleston, R. W.; Smith, A. L. In Foams; Akers, R. J., Ed.; Academic Press: London, 1976; p 163.
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film interfaces of the HCO3- present in the solution. It seems unlikely, however, that if the HCO3- ions are involved in the φo formation, the φo change would follow the same law with the pH decrease. It is possible, however, that an eventual adsorption of HCO3- can have an effect on the value of pH* and on the electric interactions in the film, respectively. The zwitterionic nature of the studied phospholipid allows a comparison of the present results with these previous studies of microscopic foam films. So, we can consider that in the case of lyso PE foam films the formation of thick films at low Cel is caused by electrostatic long-range interactions as well. The thicknesses of these silver-colored films are in the area where the film stability is mainly determined by the action of long-range electrostatic repulsion and van der Waals attraction. In this case we can use the classical DLVO theory to determine the potential of the diffuse electric layer φo at the film interfaces as shown before, e.g., refs 12, 22, 26, and 31. So, Π ) Πel + Πvw, where Πel is the electrostatic and Πvw is the van der Waals component of the disjoining pressure Π. At equilibrium Π ) Pσ. From the experimentally obtained surface tension data we found for Cs ) 2.5 × 10-4 mol dm-3 lyso PE a capillary pressure Pσ of 32.5 N m-2 and for Cs ) 5 × 10-4 mol dm-3 lyso PE Pσ ) 32 N m-2. The addition of electrolytes in the studied Cel range did not affect these values. Πvw was computed according to the equations35 derived using the complete Lifshitz equations for three-layer systems36,37 with dielectric permeability assessed as in refs 38 and 39. The film was considered as a three-layer structure,40,41 consisting of an aqueous core surrounded by two hydrophobic surface layers with a thickness of 0.9 nm and an index of refraction 1.422 (bulk properties of dodecane were used). So we obtained the aqueous core thickness h2 ) h - 2.3 nm. Since the thickness of the films we study is much higher than the thickness of the adsorbed layers, we can assume that the distance between the planes of formation of the φo-potential equals the aqueous core thickness. We followed the computational procedure, based on the algorithm42 described in more detail previously.43 The obtained mean results for the φo vs Cel dependence are shown in Figure 5. As is seen, at Cs ) 0.25 mol dm-3 (closed circles) the φo-potential varies around a value of about 35 mV. The scatter of the results for φo is (4 mV. These values are close to previously obtained results for films stabilized with nonionic surfactants at comparatively low concentrations of the solutions.22,26,31 It is seen in Figure 1 that the increase in the lyso PE concentration leads to the formation of thinner films. A pronounced effect of surfactant concentration on the thicknesses of the obtained films has been noticed before in the case of films stabilized with nonionic surface-active agents at concentrations in and above the cmc region.24 This effect has been considered as a result of a decrease of the φo-potential leading to a decrease of the electrostatic (34) Yoon, R.-H.; Yordan J. L. J. Colloid Interface Sci. 1986, 113, 430. (35) Donners, W. A. B.; Rijnbout, J. B.; Vrij, A. J. Colloid Interface Sci. 1977, 60, 540. (36) Dzyaloshinski, I. E.; Lifshitz, E. M.; Pitayevskii, L. P. Adv. Phys. 1961, 10, 165. (37) Ninham, B. W.; Parsegian, V. A. J. Chem. Phys. 1970, 52, 4578. (38) Parsegian, V. A.; Ninham, B. W. Nature (London) 1969, 224, 1197. (39) Gingell, D.; Parsegian, V. A. J. Colloid Interface Sci. 1973, 44, 456. (40) Duyvis, E. M. Thesis, State University, Utrecht, 1962, p 19. (41) Frankel, S. P.; Mysels, K. J. J. Appl. Phys. 1966, 37, 3725. (42) Chan, D. I.; Pashley, R. M.; White, L. R. J. Colloid Interface Sci. 1980, 77, 283. (43) Kolarov, T.; Exerowa, D.; Balinov, B.; Martinov, G. A. Kolloidn. Zh. 1986, 48, 1076.
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Figure 5. Diffuse electric layer potential φo as a function of NaCl concentration, determined from the experimental results in Figure 1: b, Cs ) 2.5 × 10-4 mol dm-3; O, Cs ) 5 × 10-4 mol dm-3.
repulsion in the film. As the present study is carried out at Cs in the cmc region as well, the obtained results seem to be in conformity with this suggestion. Using the DLVO theory equations as above shown, we obtain for Cs) 5 × 10-4 mol dm-3 φo values between 20 and 12 mV with a tendency to decrease with increase in Cel. These lower φo values compared to the φo values obtained at Cs ) 2.5 × 10-4 mol dm-3 determine a lower barrier in the Π(h) isotherm leading to a silver to Newton film transition at a lower Cel. The results obtained for φo are consistent with the results found before for microscopic films obtained from lyso PC solutions at a Cs that is well above the cmc, with NaCl added.12 The barrier transition to Newton films manifests itself in the results of the direct measurement of the disjoining pressure isotherm (Figure 3). Increase in applied pressure leads to decrease in film thicknesses to about 15 nm where black spot formation begins and a Newton film is formed. The pressure where this transition occurs is close to the previously measured pressure of transition to Newton films obtained from nonionic surfactant solutions.31 It has also been demonstrated in ref 31 that the complete Poisson-Boltzmann and Lifschitz equations give a good description of the film interactions and related quantities in the case of films stabilized with nonionics. The low φo-potential values found for the zwitterionic lyso PE films justify such a comparison between experimental results and theoretical predictions. We used the computational procedure31,41,42 involving the solution of the complete Poisson-Boltzmann and Lifschitz equations for Πel and Πvw. As is seen in Figure 3, the experimental results follow a course between the limiting cases of constant potential (lower curve) and constant charge (upper curve) which is within the framework of the DLVO extremes of constant potential of 32 mV and the respective constant charge of 1.75 mC m-2. These φo and σo values are consistent with the results in Figure 5 within the limits of the experimental scatter. The validity of the DLVO theory for the studied phospholipid films justifies the assumption that longrange electrostatic repulsion is operative in the thick films. It is important to assess how φo and σo change when the pressure in the lyso PE films with NaCl added increases. The obtained results for φo and σo as a function of h determined for each experimentally obtained point, are shown in Figure 6. When the film thickness decreases, φo increases from 28 to 43 mV while the charge density of 1.28 ( 0.1 mC m-2 remains practically constant. These results show that in the studied case, double-layer repulsion under the conditions of constant charge is operative. This result is in conformity with the previous results31 for nonionic surfactant microscopic films. If we extrapolate the experimental data in Figure 6 to h ) ∞ as shown in refs 31 and 43, we can obtain the diffuse electric layer potential φo,∞ for an infinitely thick film,
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Figure 6. Diffuse electric layer potential φo (b) and charge density σo (O) as a function of equilibrium thickness h of lyso PE films with 5 × 10-4 mol dm-3 NaCl added, determined from the experimental results in Figure 3. Table 1. Diffuse Electric Layer Potential φo as a Function of CaCl2 Concentration, Determined from the Experimental Results in Figure 2 103Cel (mol dm-3)
potential (mV)
103Cel (mol dm-3)
potential (mV)
0.07 0.1
21 17
0.2 0.25
14 12
where the film interfaces do not influence each other. So we obtain that φo,∞ ) 27 mV. Lyso PE Films with CaCl2 Added. As is seen in Figure 2 at Cs ) 2.5 × 10-4 mol dm-3 silver-colored films are obtained only in a very narrow concentration interval. In this case the concentration Cel,cr at which a transition to Newton films is observed is much lower than in the case of NaCl added showing that compared to the monovalent Na+ ions the divalent Ca2+ ions lead to lower electrostatic disjoining pressure and the barrier of the Π(h) isotherm is passed over at very low Cel. As a basic parameter that characterizes the electrostatic forces, the φo-potential can be determined in the case of a nonsymmetrical 2:1 valent electrolyte as shown in ref 12 using the same three-layer model as in the case of lyso PE films with Na+ ions added. The results are shown in Table 1. It is seen that the obtained φo-potential values are very low, reaching 12 mV at the last Cel where a thick film is obtained. The barrier transition of the silver-colored films to Newton films where short-range interactions are operative is demonstrated in the Π(h) curve shown in Figure 4. In conformity with the lower potential here the transition to Newton films occurs at lower pressures compared to the measurements performed with NaCl added. The comparison between the experimentally obtained data and the theoretical predictions was carried out using the computational procedure12 that involves the numerical solution of the complete Poisson-Boltzmann equations for the case of a nonsymmetrical 2:1 valent electrolyte. The lower curve represents the limiting case of a constant potential of 16 mV and the upper one, the case of constant charge, equal to 0.7 mC m-2. As is seen, in this case the experimental values fit very well the curve determined at the assumption of constant charge. The potential and the charge determined for each experimentally obtained point, plotted in Figure 7, show that in this case again interactions under the conditions of constant charge are operative. The charge and potential decrease caused by the binding of the positive Ca2+ can be considered additional evidence
Figure 7. Diffuse electric layer potential φo (b) and charge density σo (O) as a function of equilibrium thickness h of lyso PE films with 2 × 10-4 mol dm-3 CaCl2 added, determined from the experimental results in Figure 4.
supporting the assumption that in the case of neutral interacting solution/air interfaces OH- uptake causes the build-up of negative charge leading to thick film formation in the low Cel range. The effect of increasing Cs leads to obtaining only Newton films at Cs ) 5 × 10-4 mol dm-3 lyso PE in the case of CaCl2 added. The addition of Ca2+ at this Cs causes a lower φo-potential and a decrease in the barrier of the Π(h) isotherm. So only Newton films are obtained even at very low CaCl2 concentrations. Similar conclusions have already been made in the study12 where due to the high lyso PC concentration only Newton films are formed in the presence of CaCl2 at concentrations less than 1 mM. The course of the film thickness vs CaCl2 concentration curve (Figure 2) seems particularly intriguing above Cel ) 0.01 mol dm-3 where the Newton to common black film transition occurs. The obtained results show that increase in Cel leads to increased Ca2+ binding by the phospholipid head groups. The charge reversal gives rise to electrostatic repulsive forces and causes a dramatic increase in film thickness. This effect is similar to the one observed in the previous lyso PC foam film studies. Here, however, this Newton to common black film transition occurs at a Cel, which is an order of magnitude higher than in the case of phosphatidylcholine. The authors7 have found that Ca2+ ions bind more strongly in the case of DPPC than in the case of DPPE. So, the higher Cel where the ion binding manifests itself in the case of lyso PE foam films may be connected with the lower Ca2+ binding by the lyso PE polar head groups in comparison with phosphatidylcholine. This can be due to the hydrogen bonds and the dipole-dipole interactions between the lyso PE molecules in the adsorbed layer.24 The positive charging in the low Cel region is sufficient only to neutralize the negative film interfaces due to the OH- ions uptake. Only above Cel ) 0.02 mol dm-3 does the charge at the film interfaces become sufficiently high to give rise to electrostatic repulsion. After the abrupt increase in film thickness the small differences in Ca2+ binding at both Cs (Figure 2) are probably connected with the surface concentration of the lyso PE molecules. The film thickness gradually decreases with the further increase in Cel until the Newton black film thickness is reached again. The course of this dependence is determined by the combined action of increased charge at the film interfaces due to the Ca2+ binding and the increased electrolyte concentration of the solution. So, in the case of CaCl2 added long-range interactions are found both in the low and in the high Cel range. Different ionic effects determine however the mechanism
5424 Langmuir, Vol. 12, No. 22, 1996
of their formation. In the low Cel range the film behavior seems similar to that found for microscopic films stabilized with nonionic surfactants, whereas in the high Cel range specific Ca2+ binding leads to charge reversal at the film air/solution interfaces and transition from short-range to long-range interactions. Concluding Remarks The present study shows that the experimental isotherms (thickness/electrolyte concentration) and disjoining pressure/thickness are a good demonstration of the existence of electrostatic repulsive forces between the two interacting air/solution interfaces and allow detection of ionic effects in the case of foam films stabilized with lyso PE. The obtained results are in conformity with previous studies of microscopic foam films stabilized with other neutral phospholipids: lyso PC,12,21 and dimyristoylphosphatidylcholine.11 The formation of thick lyso PE foam films with NaCl added provides additional evidence of the existence of electrostatic interactions in the low Cel range. The properties of the obtained films in the case of 1:1 valent electrolyte added are similar to those observed previously in experiments with films stabilized with nonionic surfactants and phospholipids. This shows the essential role of the zwitterionic character of the phospholipid head group for the electrostatic interactions in the film determined most probably by OH- uptake at the solution/air interface. It seems that in this case the structure of the head group has not any significant effect on the film properties.
Cohen et al.
In the case when the positive Ca2+ bind to the phospholipid head group, they reduce the initially negative surface charge and lead to weaker repulsive electrostatic interactions than in the case with NaCl added. The important role of the phospholipid head group exhibits itself at higher Ca2+ concentrations where the positive surface charge becomes sufficient to give rise to long-range electrostatic repulsion, causing a Newton to common black film transition. In the case of lyso PE it seems that the Ca2+ ions bind less to the phospholipid head groups compared to the results found for foam films stabilized with phosphatidylcholine.8,12,15 The results obtained in the studies of the interaction forces in phospholipid foam films show that these foam films can be useful in investigations of the interactions between model lipid membranes. The obtained effect of charge reversal of the film interfaces with Ca2+ ions added offers also the good possibility to study experimentally and theoretically electrostatic repulsive forces in the case of an initially nonionic substance. Acknowledgment. The authors are grateful to Dr. T. Kolarov, Dr. B. Balinov, and the late Dr. V. Muller for the supplying of the computer programs for the determination of the theoretical double-layer interactions. This work is financially supported by a grant of the Bulgarian Ministry of Education and Science. LA960280P
