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Langmuir 2005, 21, 2804-2810
Formation of a Liquid Crystalline Phase from Phosphatidylcholine at the Oil-Aqueous Interface Jan-Willem Benjamins, Krister Thuresson, and Tommy Nylander* Department of Physical Chemistry 1, Lund University, P.O. Box 124, S-221 00 Lund, Sweden Received April 27, 2004. In Final Form: December 18, 2004 Adsorption of phospholipid (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) and formation of a surface phase at the oil-water interface has been followed by using ellipsometry. The properties of the interfacial phase were found to depend strongly on whether phospholipid was added to the oil phase or to the aqueous phase as liposomal structures. In the latter case a monolayer formed, while if the phospholipid was supplied from the oil phase a lamellar phase appeared at the interface. The effect on the stabilizing surface phase of a surface-active protein (β-lactoglobulin) was also investigated. The observations are important for understanding stabilizing properties of surface-active compounds commonly used to stabilize emulsions. In addition it has been demonstrated that ellipsometry can be used to study the initial process when a two-phase system consisting of a water and an oil phase is transformed into a three phase system or eventually to a one-phase system.
Introduction Many foodstuffs, such as dairy products, consist of oilin-water emulsions. These emulsions have to be stabilized by suitable stabilizing agents or mixtures thereof. Generally, it can be stated that surfactants or lipids that have the optimal hydrophilic and hydrophobic balance to make them good emulsifiers also form lamellar liquid crystalline phases.1 A commonly used example of such an emulsifier is lecithin, where the main constituent is phosphatidylcholine (PC). Often, stability of emulsions is further improved by adding also a second stabilizing compound, such as proteins or (semi)synthetic surface-active polymers. In fact, it has been shown that certain oil/water/PC emulsions are not stable unless a stabilizing polymer is added.2 The complex mechanisms that render emulsions stable are further demonstrated by better stability being obtained by adding lecithin to the oil phase than by adding it to the aqueous phase. It is also known that interactions with proteins and, consequently, the emulsion stability are dependent on the purity of the lecithin.3 Thus, apart from the ratio between the total amount of emulsifier present and the surface area of the dispersed phase, stability is also determined by competitive adsorption of different surface-active components and, subsequently, the composition of the interfacial layer. A limited number of fundamental studies on interfacial properties of oil/water/ phospholipid/protein systems have been published.4-6 Heertje et al. found, using confocal scanning light microscopy, that sodium caseinate, adsorbed at an oil-water * To whom correspondence should be addressed. Fax: +46 46 2224413. Phone: +46 46 2228158. E-mail: tommy.nylander@ fkem1.lu.se. (1) Bergenståhl, B. A.; Claesson, P. M. In Food Emulsions, 3rd ed.; Larsson, K., Friberg, S. E., Eds.; Marcel Dekker: New York, 1997; pp 57-109. (2) Karlberg, M.; Thuresson, K.; Lindman, B. Colloids Surf., A 2004, submitted for publication. (3) Yamamoto, Y.; Araki, M. Biosci. Biotechnol. Biochem. 1997, 61, 1791. (4) Heertje, I.; van Aalst, H.; Blonk, J. C. G.; Don, A.; Nederlof, J.; Lucassen-Reynders, E. H. Lebensm.-Wiss. Technol. 1996, 29, 217. (5) Bylaite, E.; Nylander, T.; Venskutonis, R.; Jo¨nsson, B. Colloids Surf., B 2001, 20, 327. (6) Tanaka, M.; Saito, H.; Arimoto, I.; Nakano, M.; Handa, T. Langmuir 2003, 19, 5192.
interface, is displaced by DPPE.4 Bylaite et al. found that β-lactoglobulin adsorbed at an olive oil/water interface was to a large extent (but not completely) displaced by PC.5 To gain more insight on the stabilizing layer between polar and nonpolar regions in an emulsion we have used ellipsometry to study the behavior of PC, 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC), at oil-water interfaces when DOPC was added to the oil phase, as well as when DOPC was dispersed in the aqueous phase. In this way the structure and kinetics of interfacial layer formation can be obtained. The aim is to explore the possibilities to use ellipsometry to study the initial process when a two-phase system consisting of a water and an oil phase is transformed into a three-phase system or eventually to a one-phase system. Additional information on interfacial layer structure was obtained by small-angle X-ray diffraction (SAXD). Ellipsometry is a well-established, nondestructive optical method to characterize ultrathin films.7 Until recently, only a handful of studies have been published that concerned the liquid-liquid interface.4,8-12 Ellipsometry measurements at liquid-air and liquid-liquid interfaces require special arrangements, which, for instance, ensure a well-defined angle of incidence.4,8,11 Another difficulty with measurements at the liquid-air and liquid-liquid interfaces is the lack of contrast between a formed transparent layer and a substrate that does not absorb light at the used wavelength. However, efforts undertaken at this department13-15 have resulted in a methodology (7) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland Publication: Amsterdam, 1979. (8) Nylander, T.; Hamraoui, A.; Paulsson, M. Int. J. Food Sci. Technol. 1999, 34, 573. (9) Russev, S. C.; Arguirov, T. V.; Gurkov, T. D. Colloids Surf., B 2000, 19, 89. (10) De Hoog, E. H. A.; Lekkerkerker, H. N. W.; Schulz, J.; Findenegg, G. H. J. Phys. Chem. B 1999, 103, 10657. (11) Hutchison, J.; Klenerman, D.; Manning-Benson, S.; Bain, C. Langmuir 1999, 15, 7530. (12) Harke, M.; Teppner, R.; Schulz, O.; Orendi, H.; Motschmann, H. Rev. Sci. Instrum. 1997, 68, 3130. (13) Benjamins, J. W.; Jo¨nsson, B.; Thuresson, K.; Nylander, T. Langmuir 2002, 18, 6437. (14) Kapilashrami, A.; Malmsten, M.; Eskilsson, K.; Benjamins, J. W.; Nylander, T. Colloids Surf., A 2003, 225, 181.
10.1021/la048957t CCC: $30.25 © 2005 American Chemical Society Published on Web 03/02/2005
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Langmuir, Vol. 21, No. 7, 2005 2805 is described by two parameters, ∆ and Ψ. These angles are related to the reflectivity coefficients, Rp and Rs, parallel and perpendicular to the plane of incidence, respectively, F being the ellipticity coefficient.
Rp/Rs ) F ) tan Ψei∆ ) tan Ψ (cos ∆ + i sin ∆)
Figure 1. Reflection of light, incident through the oil phase, at a thin film with a thickness d1 and a refractive index n1, on an aqueous phase. The upper (oil) and lower (water) phases have a refractive indices of n2 and n0, respectively.
which enables us to perform ellipsometry measurements at liquid-liquid interfaces and to evaluate their results both qualitatively and quantitatively.
The presence of a film at the interface will affect the reflectivity coefficients. A schematic drawing of light reflected at a film at the oil-water interface is given in Figure 1, where we also define some of the parameters used in this work. In principle the thickness, d1, and the refractive index, n1, of a film present at the surface can be determined from ∆ and Ψ.7 From these parameters the adsorbed amount (Γ), expressed as a function of n1 and d1 (eq 2), can be determined according to the formula derived by Cuypers et al.,16 based on the Lorenz-Lorenz equation.
(15) Benjamins, J. W.; Thuresson, K.; Nylander, T. Langmuir 2005, 21, 149-159.
0.3d1(n12 - n02)
Γ)
Materials and Methods Materials. The triglyceride oil that was used in these experiments was Miglyol 812N (batch 921226), provided by Hu¨ls AG, and was used without further purification. This is a synthetic caprylic/capric triglyceride and can, therefore, be expected to be free of impurities. Its refractive index (at λ ) 5320 Å) is 1.454 ( 0.001. Water was deionized and passed through a Milli-Q water purification system (Millipore Corp., Bedford, MA). Deuterated water (D2O, 99.8% D) was purchased from Dr. Glaser AG, Basel, Switzerland. We found that this water did not have the same purity as the Milli-Q water. The water was, therefore, filtered using a Sartorius Minisart syringe filter, with a pore size of 0.2 µm, and then degassed under vacuum. DOPC (lot no. 181PC-161), was purchased from Avanti PolarLipids, Inc., Alabaster, AL, and was used as received. β-Lactoglobulin was provided by Domo Food Ingredients (Beilen, The Netherlands, batch no. Rgs. 367102g). The protein was purified by dialysis at 4 °C using an 8000 molecular-weightcutoff dialysis membrane (Spectra/Por). First, 5 g of β-lactoglobuline was dissolved in 100 mL of Milli-Q water containing 0.03 wt % sodium azide. Dialysis was then performed during 6 h against Milli-Q water to remove salt, then for another 6 h against a 50 mM NaCl solution to replace any remaining ions with Na+ and Cl-, and finally 6 + 6 h against Milli-Q water to remove as much of the salt as possible. The resulting β-lactoglobulin solution was then freeze-dried and stored at -18 °C until used. Methods. Sample Preparation. Two sets of stock solutions of both DOPC and β-lactoglobulin were prepared. In one set, the phosphate buffer solution (PBS, pH ) 6.7, n ) 1.3348) in which the compounds were dispersed or dissolved was made using Milli-Q water, while another set was prepared in which the PBS was made using D2O (n ) 1.3299). All stock solutions had a concentration of 5 wt %. The DOPC solutions were then placed in an ultrasonic bath (Liarre Starsonic 90) for 30 s, to form a vesicular dispersion. The stock solutions were diluted 10 times (0.5 wt %) by injecting it in the aqueous phase (PBS or PBSD2O) in the measuring cell. To perform measurements in which the DOPC adsorbs from the oil phase, three DOPC-in-oil solutions were prepared, with concentrations of 0.05, 0.1, and 0.5 wt %, respectively. Ellipsometry. The ellipsometry measurements were performed using an Optrel Multiskop ellipsometer (as described in detail in ref 12), which was fitted with light guides to enable measurements at oil-water interfaces. These light guides are immersed in the top liquid phase to avoid reflections at the liquid-air interface. The modifications are extensively described in ref 13. A specially designed measuring cell was used to obtain a planar interface. This cell consists of a stainless steel rim, wetted by the aqueous phase, covered by a Teflon ring, which is wetted by the oil phase.13 Ellipsometry is based on measuring a change in polarization of a light beam that is reflected at the sample surface. This change
(1)
(2)
(n12 + 2)[r(n02 + 2) - v(n02 - 1)]
In this equation, n0 is the refractive index of the bulk solution, r is the specific refractivity of the adsorbed molecules, and v their partial specific volume. The values of r and v for β-lactoglobulin are r ) 0.249 and v ) 0.749 mL/g17 and for DOPC are r ) 0.274 and v ) 0.980 mL/g.16 One problem with measuring very thin, transparent layers on a substrate that does not absorb light at the used wavelength is that only ∆ changes when a layer is formed, while Ψ remains virtually constant.4 This means that the two desired parameters, the thickness of the adsorbed layer (d1) and its refractive index (n1), are impossible to calculate from the only affected variable, ∆. This means that we require an additional parameter. As shown by Antippa et al. consecutive measurements where either n2 or n0 is changed will allow determination of both of the unknown parameters.27 In their approach they suggested measurement at different wavelengths, which changes the refractive index of the media. Here we have performed each measurement twice: once with PBS as the aqueous phase and once with PBS-D2O as the aqueous phase because the refractive index of H2O is sufficiently different to that of D2O. Our earlier work has shown that, by doing this, it is possible to evaluate the data without having to make assumptions about the adsorbed layer’s nature and without the need for data from other techniques.15 The analysis rests on the assumption that d1 and Γ are the same in D2O and H2O and by combining the analysis of data for H2O with the corresponding ones for D2O both d1 and Γ can be obtained through an iterative procedure. If the layer does not contain any solvent, D2O or H2O, we can use the iterative procedure for determining n1. For a thin film (orders of magnitude thinner than the wavelength of the light) we can use an approximative expression, which relates the optical properties of the film to the change in ∆, δ∆ (in radians):27
[
)]
( ) (
d1 n|2 + n22 n02
n22 1 + n02 2 R0 R0n⊥
)Q
(3)
where
[
()
Q ) δ∆
]
1 - (n22/n0)2 tan2 φ λ (n02 - n22) 4π n2 sin φ tan φ
and the anisotropy, R0, is expressed as
R0 )
( )(
n⊥2 n|2 - n02 n|2 n⊥2 - n02
)
Here, λ is the wavelength of light, φ is the angle of incidence, and (16) Kop, J. M. M.; Cuypers, P. A.; Lindhout, T.; Hemker, H. C.; Hermens, W. Th. J. Biol. Chem. 1984, 259, 13993. (17) Nylander, T.; Wahlgren, N. M. J. Colloid Interface Sci. 1994, 162, 151.
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n2 and n0 are the refractive indices of the top (in our case oil) and the bottom (in our case H2O or D2O) phase. The refractive indices for the film are denoted n| and n⊥ for the components perpendicular and parallel to the interface, respectively. We can now express n| as
n|2 )
(n22/R0) + n02 + (Q/d1) + 2 2 [(n2 /R0) + n02 + (Q/d1)]2 n22 n02 4 R0
{
}
1/2
(4)
Equation 4 was used for evaluating data for the layer formed from dispersions of DOPC in the aqueous phase, using the iterative procedure described above and further discussed in the discussion section. For an isotropic layer, n| ) n⊥) n1. This was the case when DOPC was dispersed in the oil phase as further discussed below. As the formed liquid crystalline layer was fully swollen as expected for the equilibrium system and confirmed by the SAXD data of a sample collected from the interface, we actually know the composition of the layer and, hence, its refractive index. We, therefore, only need the δ∆ value to determine the thickness of the layer according to eq 3 with n| ) n⊥) n1, and the amount of DOPC in the layer can then be obtained from eq 2. The ellipsometry measurements were performed at an angle of incidence (φ) of 50° at λ ) 532 nm. This angle was chosen because it is relatively close to the Brewster angle (42.6°). At that angle, the formation of a layer would cause the changes in ∆ to be the largest. However, the intensity of the reflected light is at a minimum at the Brewster angle, increasing the risk for inaccuracies in the detector. The value of ∆ was determined with an accuracy of (0.02°. Brewster Angle Microscopy (BAM). BAM was used to image an interfacial layer by transforming the lateral inhomogeneity of the interface into a lateral inhomogeneity of the state of polarization of the reflected beam.18 This produces a black and white image of a small area of the interface, which can be interpreted in terms of interfacial structure with micrometer lateral resolution. The Optrel Multiskop ellipsometer can be easily converted into a Brewster angle microscope by replacing the light detector with a charge-coupled device camera connected to a personal computer and by attaching an objective lens to the detector arm. This procedure is described in detail in ref 12. To obtain maximum contrast, the angle of incidence was set to 42.6° during the imaging. The BAM image of the surface layer as a function of adsorption time was followed on a video monitor and recorded using a S-VHS video recorder. At several points, digital pictures were stored using a frame-grabber card and a personal computer. Small-Angle X-ray Scattering (SAXS). When a sufficiently thick interfacial layer was formed during the course of an ellipsometry measurement, it was transferred from the measuring cell into a small vial. This was done by first removing the top oil phase and subsequently pipetting a sufficient amount of the layer’s material into the vial. SAXS measurements were performed on a Kratky compact small-angle system equipped with a position-sensitive detector (OED 50M, Mbraun, Graz, Austria) containing 1024 channels, each with a width of 53.6 µm. Cu KR radiation of wavelength 1.542 Å was provided by a Seifert ID300 X-ray generator operating at 50 kV and 40 mA, and the sample-to-detector distance was 277 mm. To minimize scattering from air and increase the signal-to-noise ratio, the volume between the sample and the detector was under vacuum. During the measurement, the samples were placed in a capillary, and the temperature (25 °C) was controlled to within 0.1 °C by using a Peltier element.
Results The results below will be presented in terms of changes in the ellipsometry angle ∆, δ∆, for three different cases: (18) Reiter, R.; Motschmann, H.; Orendi, H.; Nemetz, A.; Knoll, W. Langmuir 1992, 8, 1784.
Figure 2. Change of δ∆ (degrees) as a function of time for a system of oil on PBS containing 0.5 wt % DOPC when the PBS is prepared from H2O and the corresponding data when the buffer is prepared from D2O. No further changes were observed in H2O within 210 min (data not shown).
1. DOPC added to the aqueous phase: Here we found that only one or a few monolayers of DOPC are formed at the oil-aqueous interface. 2. DOPC dispersed in the oil phase: Here we found that a microscopic layer of a lamellar liquid crystalline phase of DOPC is formed at the oil-aqueous interface. The layer thickness depends on the concentration of DOPC, and at sufficiently low DOPC concentration the amount adsorbed is similar to the one recorded when the lipid is added to the aqueous phase. 3. The effect of adding β-lactoglobulin on the formed layer of DOPC: The presence of the protein has little effect on the thickness of the DOPC layer. These findings, which can be related to the differences in kinetics, are presented in detail below, where the data for the amount of DOPC in the formed layer are analyzed as described in the methods section. The results will be thoroughly analyzed in the discussion section. DOPC Added to the Aqueous Phase. The results from the ellipsometry measurements of the adsorbed layer when DOPC is added to the aqueous phase are shown in Figure 2 for H2O and D2O, respectively. In the figures the change in the ∆ angle (δ∆) is plotted as a function of time. The measured parameters immediately reach a plateau value after addition of DOPC to the aqueous phase. A larger value of δ∆ is recorded in D2O than in H2O. This clearly follows from eq 3, because n0 ) 1.3348 in H2O and n0 ) 1.3298 in D2O with n2 ) 1.454. DOPC Added to the Oil Phase. When DOPC is added to the oil phase the equilibrium process takes a much longer time, and changes in ∆ are much larger (Figure 3). It can also be seen that the δ∆ values vary strongly, although they do not seem to be scattered but rather increase/decrease over time (“nonrandom scatter”). It is also interesting to note that the variatons in the beginning and the end of the adsorption process are less than at intermediate time. The plausible reasons for the nonrandom scatter will be discussed thoroughly in the discussion section. The tendency to “nonrandom scatter” of δ∆ decreases with the initial concentration of DOPC in the oil phase (data not shown). It is noteworthy that, at a DOPC concentration of 0.05 wt % in the oil phase, the observed value of δ∆ of 1.0 is the same as when the lipid is dispersed in the aqueous phase (Figure 4). As a result of the large spread in the ∆ values for DOPC adsorbing from the oil phase, it has not been possible to evaluate that data and
Liquid Crystalline Phase from PC at Oil/Aqueous
Figure 3. Change of δ∆ (degrees) as a function of time for a system of 0.5 wt % DOPC in oil on PBS when the PBS is prepared from H2O and the corresponding data when the buffer is prepared from D2O.
Langmuir, Vol. 21, No. 7, 2005 2807
Figure 6. Change of δ∆ (degrees) as a function of time for a system containing 0.5 wt % DOPC in oil on protein-free PBS and PBS containing 0.5 wt % β-lactoglobulin.
aqueous interface. When β-lactoglobulin is added to a DOPC layer formed from an aqueous dispersion of DOPC, an initial increase in δ∆ is observed. After a few minutes δ∆ decreases and seems to approach the values recorded for a DOPC layer formed from an aqueous dispersion. Addition of a mixture of DOPC and β-lactoglobulin leads to an initial sharp increase followed by a decrease in δ∆. In both H2O and D2O buffers the values approach those of DOPC layers formed from aqueous dispersions. Figure 6 shows the formation of a DOPC layer from the oil phase at the interface toward an aqueous buffer solution with and without 0.5 wt % β-lactoglobulin. Both systems show a large increase in δ∆ as well as the nonrandom scatter discussed above. With the available data we cannot conclude that there is a significant effect of the presence of β-lactoglobulin. Figure 4. Estimated values of the change of δ∆, recorded 300 min after adding DOPC in the oil phase as a function of the DOPC concentration. The aqueous phase consisted of PBS.
Figure 5. Change of δ∆ (degrees) as a function of time for the sequential and competitive adsorption of DOPC and β-lactoglobulin. The filled squares show the adsorption of β-lactoglobulin to the DOPC layer formed from an aqueous dispersion of DOPC in PBS-H2O. The open squares show the competitive adsorption from PBS-H2O containing 0.5 wt % DOPC and 0.5 wt % β-lactoglobulin, while the open circles show the corresponding data from PBS-D2O.
obtain clear-cut numbers on adsorbed amount and layer thickness. This will be further discussed below. Effects of β-Lactoglobulin. Figure 5 shows the sequential and competitive adsorption of DOPC and β-lactoglobulin from the aqueous dispersion to the oil-
Discussion Formation of the Interfacial Layer. The most striking result of the present study is that the change in the ellipsometry angle δ∆, which is proportional to the adsorbed amount,14,15 is different depending on whether the DOPC is added to the oil phase or the aqueous phase. When DOPC is added to the oil phase, the δ∆ values are much larger than when DOPC is added as a liposomal dispersion to the aqueous phase. Qualitatively this can be explained by assuming the formation of a lamellar phase at the interface, as commonly observed for PC-based phospholipids in excess of water.1,19 The results from the SAXD experiments of a sample collected from the oilaqueous interface with DOPC added to the oil (as shown in Figure 7) seem to confirm the interpretation. These measurements show a repeating distance of 66.1 Å, which can be compared with 50 Å for DOPC-alkane-water systems,20 and both 64 Å21 and 62.1 Å22 for a swollen DOPC-water lamellar structure. Here it is also important to remember that the formed liquid crystalline layer when DOPC is added to the oil is in thermodynamic equilibrium. The formed lamellar phase is in excess of water (and oil), and, therefore, the structure of the extracted middle phase will not be affected. SAXD measures the interlayer spacings in this phase on the nanometer scale. (19) Denizot, B. A.; Tchoreloff, P. C.; Proust, J. E.; Puisieux, F.; Lindenbaum, A.; Dehan, M. J. Colloid Interface Sci. 1991, 143, 120. (20) Sjo¨lund, M.; Rilfors, L.; Lindblom, G. Biochemistry 1989, 28, 1323. (21) Khakhar, J.; Nylander, T.; Khan, A. Unpublished results. (22) Costigan, S. C.; Booth, P. J.; Templer, R. H. Biochim. Biophys. Acta 2000, 41, 1468.
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Figure 7. SAXD spectra for two interfacial layers. The repeat distance in the lamellar phase is not affected by the presence of β-lactoglobulin.
On the basis of the small changes in δ∆ and the fact that the plateau value is reached almost instantaneously, the adsorption of DOPC from a liposomal dispersion onto the oil-water interface can be interpreted as the adsorption of a monolayer of DOPC. When we have added DOPC to the aqueous phase, no changes were observed during the first few minutes until over 3 h. However, when DOPC is added to the oil phase a dramatic increase occurs already with in the first hour. After 3 h, δ∆ is about factor of 10 larger than when DOPC was added to the aqueous phase. In fact at this stage the presence of the film was visible at the oil-aqueous interface. Here it is important to note that when DOPC is added to the oil phase, the system approaches the thermodynamic equilibrium much more rapidly. An additional experiment was performed where the oil containing increasing amounts of DOPC was equilibrated for 1 month with an equal amount of water. The presence of a lamellar phase, as apparent from inspecting the sample between crossed polarizers, could be observed at the interface above a DOPC concentration of about 0.015 wt %. This is at a lower concentration than where we observed a dramatic increase in the ellipsometer reading δ∆ (Figure 4). However, these data were recorded after only 300 min. These results show that it takes considerable time to reach equilibrium even though the process is much faster than when DOPC is added as vesicles to the aqueous phase. The difference in behavior between DOPC added to the oil phase and DOPC added to the aqueous phase can be explained as follows. The liposomal dispersion is more or less stable on the relevant time scale (hours). As the phospholipids are transferred or adsorbed to the oil-water interface the surface will, as monolayer coverage is approached, gain surface characteristics similar to the dispersed liposome. Once the monolayer covering of the oil-water interface is reached there will, therefore, be no driving force for liposomes to interact with the adsorbed layer. Because adsorption will be limited to a monolayer, formation of multilamellar structures will not occur on the investigated time scale. DOPC has a relatively low solubility in triglyceride oils. In fact we are even at the lowest concentration used in the present study, 0.05 wt %, expected to be close to the solubility limit of the phospholipids. In the presence of small amounts of water the PC can form a reversed micellar, L2 phase in organic solvents.23 This is expected to be the case at the higher concentrations of DOPC that we have used. In a previous study we compared the (23) Schurtenberger, P.; Scartazzini, R.; Magid, L. J.; Leser, M. E.; Luisi, P. L. J. Phys. Chem. 1990, 94, 3695.
Benjamins et al.
adsorption of soybean PC from caraway oil (containing limonene and carvone in a 1:1 ratio) and olive oil (mainly triolein).4 We can conclude that there are marked differences in the formation of soybean PC layers at the caraway- and olive oil-aqueous interfaces. The soybean PC has a much higher solubility in the caraway oil than in the olive oil. The lower solubility of soybean PC in the olive oil was suggested to drive the lipid to the aqueous interface, where it eventually can form of a liquid crystalline phase. The present studies are in line with these earlier observations. The nonrandom scatter in the δ∆ values, in the cases where DOPC was added to the oil, might be attributed to inhomogeneities or patches in the interfacial layer that fluctuate on the time scale of the measurements. It is also possible that the layer consists of anisotropic domains that are all misaligned compared to the horizontal plane. If these domains in the interfacial layer are free to move, due to thermal convection in the bulk phases, the light would at each measurement be reflected from a part of the layer that has slightly different optical properties than the preceding. This would account for the apparent periodicity in the spread of the δ∆ values. To further investigate this phenomenon, the experiment was repeated and studied using a Brewster angle microscope, where the process of DOPC adsorbing at the oilwater interface was followed in real time. Three images of the different stages of the adsorption process are shown in Figure 8. The first image is taken during the initial stages of the adsorption process, with relatively small values of δ∆ (see Figure 3). No particular features are observed, and the images are relatively stable with time. This corresponds to the homogeneous surface, and, consequently, virtually no nonrandom scatter in δ∆ is observed (see Figure 3). After about 40 min of adsorption, a markedly different image was recorded. Here, the formation of large domains can be observed and the images changed rapidly with time. This would explain the large variation in δ∆ values observed at intermediate adsorption times. Once the layer thickness has reached a certain level, the nonrandom scatter is reduced. This is also reflected in the BAM images taken after 300 min. Here again fewer features are observed and the images are more stable with time. This indicated a more rigid layer. Finally, we note that both ellipsometry and BAM monitor an area of about 1 mm2. However, what is more important is that both techniques give a snapshot of the events. This means that even if ellipsometry captures the average over a certain area this value will change with time as the domains are moving. This is obvious from the BAM images where the domains move with a speed that is about the same as the time it takes to record one set of the ellipsometry parameters ∆ and Ψ. Because the changes occur during the time of measurements, the recorded values tend to drift in a certain direction rather than scatter randomly. If the movements of the domains were faster the recorded data would represent the average value. Effect of β-Lactoglobulin on the Interfacial Layer. When both DOPC and β-lactoglobulin are added to the aqueous phase (Figure 5), we initially see a high adsorption, which gradually decreases. This indicates a sequential adsorption of the components at the oil-aqueous interface. Another possibility is that a lipid protein complex formed in the solution is adsorbed and gradually disintegrates at the interface. Unfortunately the available data does not allow us to separate between the two mechanisms. Upon adding β-lactoglobulin to a DOPC layer formed from the aqueous dispersion, the adsorption increases instantly,
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Langmuir, Vol. 21, No. 7, 2005 2809
Figure 8. Sequence of images of the formation of the DOPC layer obtained using BAM. The first image was recorded a few minutes after DOPC addition (0.5 wt %), the second after 40 min, and the last after about 300 min. The polarizer angle was 122°, and the analyzer angle was -31.5°. The displayed area is 700 × 500 µm2.
suggesting the adsorption of protein at the interfacial DOPC layer. The gradual decrease of the adsorption suggests that the protein is incorporated in the DOPC layer, thus, displacing part of the DOPC from the interface. A similar interaction between DOPC and β-lactoglobulin has been observed using microtitration calorimetry and surface tension measurements.24 Apparently, the hydrophobic domains of the β-lactoglobulin molecule can interact with the hydrophobic regions of the DOPC layer. The present study relates to a recent study by Waninge et al.25 concerning the interaction between β-lactoglobulin and β-casein and milk membrane lipids at the oil-aqueous interface in emulsions. They found that the membrane lipid emulsified emulsions were dominated by the membrane lipids even after equilibrium with protein solutions. Protein displacement was not observed for β-lactoglobulin with time; in contrast, the displacement effects were observed for the emulsions with β-casein, when both membrane lipids and β-casein were included during the emulsification. The addition of β-lactoglobulin has also little effect on the formation and the formed DOPC layer when the DOPC is dispersed in the oil phase. This adsorbed layer still is a slowly evolving lamellar phase, as can be seen in Figure 7. Khakhar et al. observed that the addition of β-lactoglobulin to a lamellar DOPC-water system led to a decrease in the interlamellar spacing.21 However, a similar behavior is not observed for the present case, which is in agreement with the findings by Waninge et al., who did not observe any significant change in the interlamellar spacing in multilamellar DOPC/DOPE vesicles when β-lactoglobulin was added.26 Our results are in agreement with a recent study that showed that the order of adding the protein and lipid component does matter; that is, if β-lactoglobulin is added to a milk phospholipid stabilized emulsion very little amount of protein is present at the oil-aqueous interface.25 However, if the emulsification is carried out in the presence of both β-lactoglobulin and phospholipids a mixed layer is formed at the interface. This indicates that this process is controlled by kinetics rather than thermodynamics. In principle one would expect that phospholipids would displace the protein at the interface as they generally give a lower interfacial tension. However, because a protein molecule can form a larger number of contact points with the interface as well as with other protein molecules, it is more difficult for the phospholipids to displace the protein. (24) Benjamins, J. Personal communication. (25) Waninge, R.; Walstra, P.; Bastiaans, J.; Nieuwenhuijser, H.; Nylander, T.; Paulsson, M.; Bergenståhl, B. J. Agric. Food Chem. 2005, 53, 716. (26) Waninge, R.; Nylander, T.; Paulsson, M.; Bergenståhl, B. Colloids Surf., B 2003, 31, 257. (27) Antippa, A. F.; Leblanc, R. M.; Ducharme, D. J. Opt. Soc. Am. A 1986, 3, 1794.
Quantification of the Interfacial Layer. The adsorption of DOPC from a liposomal dispersion onto the oil-water interface can be interpreted as the adsorption of a monolayer DOPC. However, it was not possible to evaluate the data assuming an isotropic film, neither for a mixture of DOPC and aqueous solvent, for a mixture of DOPC and oil, nor pure DOPC. The difference in δ∆ obtained in D2O and H2O, 1.55 and 1.22, respectively, is simply to large. However, if we assume that a monolayer of 20 Å is formed (about half the bilayer thickness of DOPC),4,16 we can fit the data if we assume that the layer is slightly anisotropic. Using the procedure described in our earlier work15 and eq 4, we obtain n1,| ) 1.545 ad n1,⊥ ) 1.541. These are quite reasonable values for a compact, nonhydrated monolayer for DOPC. The effect of β-lactoglobulin is modest, and in connection with Figure 5 it was concluded that the values of δ∆ approach those obtained without β-lactoglobulin. This means that also the layer thickness and the adsorbed amount are expected to be similar.14,15 Because we do not know the composition of the formed layer it is difficult to estimate the “plateau” value of the adsorbed amount in the mixed DOPC and β-lactoglobulin. Obviously a very thick layer is formed when DOPC is added to the oil phase, and eq 4 is, therefore, no longer applicable. From our SAXD data we know that the layer formed is a fully swollen lamellar phase. This means that we know the composition of the layer and, hence, the refractive index of the layer. Because the layer is fully swollen and the BAM images indicate that the layer is not ordered parallel to the interface, but rather in domains with rather random orientation, we can assume that the anisotropy effects are considerably smaller than for the DOPC monolayer formed from the aqueous dispersion. The anisotropy effect can, therefore, as a first approximation, be neglected. This means that we only need to measure one parameter and it, therefore, allows us to directly calculate the layer thickness and adsorbed amounts from one experiment, and the combined evaluation from experiments both in H2O and in D2O is not needed. The interlayer spacing, d () 66.1 Å), from the SAXD measurements can be used to give us the composition of the interfacial layer according to eq 5,22 which will enable us to get a value for the layer’s refractive index (n1).
1 d ) 2L Φlipid
(5)
Here, 2L is the bilayer thickness (34.9 Å, from ref 22) and Φlipid is the volume fraction of the lipid in the lamellar structure. The resulting value for Φlipid can then be used to obtain a weighted average of the refractive indices of PBS and DOPC. The resulting value for n1 is 1.395. Figure 9 gives the thickness of the adsorbed layer, calculated from the full evaluation of the data,7 assuming a constant
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Figure 9. Change of df as a function of time for a system of 0.5 wt % DOPC in oil on PBS when the PBS is prepared from H2O and the corresponding data when the buffer is prepared from D2O. The refractive index of the adsorbed film (n1) is assumed to be 1.395. Data are from Figure 3. Note that the adsorbed amounts can be calculated from eq 2, which for a constant refractive index of the film is directly proportional to the thickness, with factors of 0.0755 in water and 0.0744 in D2O.
refractive index and taking only the change in ∆ into account. The adsorbed amounts can be calculated from eq 2, which for a constant refractive index of the film is directly proportional do the thickness, with a factor of 0.0755 in water and 0.0744 in D2O. To obtain a better understanding of the relationships between the different parameters in an ellipsometry experiment and to have support in the interpretation of the much higher values for δ∆ when DOPC is added to the oil phase than as a liposomal dispersion to the aqueous phase, a model simulation was performed. In this simulation, ∆ was calculated for a model system with the same optical properties as the one studied during the actual experiments. The variables were the refractive index, n1, and the thickness, d1, of the intermediate phase. For all calculated points, the adsorbed amount (Γ) was calculated as well. The results of this simulation are shown in Figure 10. By entering the graph using the refractive index obtained from the SAXD data (n1 ) 1.395) these results indicate that the experimentally obtained values are definitely in the same range as that predicted by the simulations.
Benjamins et al.
Figure 10. Calculated values for ∆ as a function of the adsorbed layer’s refractive index (nf) at an oil (n2 ) 1.454)-aqueous (n0 ) 1.3348) interface for different adsorbed layer thicknesses: 1000, 1250, 1500, 1750, and 2000 Å. The calculations are for λ ) 5320 Å and φ ) 50°. The data for ∆ as a function of nf for adsorbed amounts (Γ) of 25, 50, 75, and 100 mg/m2, using eq 20 with r ) 0.274 and v ) 0.980 mL/g, are also inserted. The line corresponds to nf ) 1.395, calculated for the thick layer from the SAXD data.
Conclusions The properties of the interfacial phase were found to be very different depending on whether phospholipids were added to the oil phase or to the aqueous phase as liposomal structures. While a phospholipid monolayer is formed at the interface in the latter case a lamellar phase was observed in the former. The kinetics of the processes differ. Monolayer coverage from the liposomal dispersion is a rapid process, while the formation of the intermediate lamellar phase takes a much longer time. Furthermore, the present investigation has demonstrated that ellipsometry can be a useful tool to study the initial process when a system consisting of two phases, water and oil, is transformed into a three-phase system or eventually to a one-phase system. Acknowledgment. The financial support from The Swedish Research Council and Swedish Foundation for Strategic Research is gratefully acknowledged. LA048957T
