Is Brewster Angle Microscopy a Useful Technique ... - ACS Publications

Langmuir 1999, 15, 4777-4788

4777

Is Brewster Angle Microscopy a Useful Technique To Distinguish between Isotropic Domains in β-CaseinMonoolein Mixed Monolayers at the Air-Water Interface? Juan M. Rodrı´guez Patino,* Cecilio Carrera Sańchez, and Ma. Rosario Rodrı´guez Ninõ Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, Profesor Garcıá Gonza´ lez, s/nu´ m. 41012-Seville, Spain Received September 30, 1998. In Final Form: December 22, 1998

The surface pressure (π)-area (A) isotherms and Brewster angle microscopy (BAM) images of monooleinβ-casein mixed films spread on buffered water at pH 5 and 7 and at 20 °C were determined as a function of the mass fraction of monoolein in the mixture (X). The structural characteristics, miscibility, and morphology of monoolein-β-casein mixed films were very dependent on surface pressure and monolayer composition. The structure in monoolein-β-casein mixed monolayers was liquid-expanded-like, as for pure components. The monolayer structure was more expanded as the pH and the monoolein concentration in the mixture were increased. From the concentration and surface pressure dependence on excess area, elasticity, and collapse pressure it was deduced that monoolein and β-casein form a practically immiscible monolayer at the air-water interface. The BAM images and the evolution with the surface pressure of the relative reflectivity of BAM images give complementary information on the interactions and structural characteristics of monoolein-β-casein mixed monolayers, which corroborated the conclusions derived from the π-A isotherm. The morphology of monoolein, β-casein, and monoolein-β-casein domains at surface pressures lower than that for β-casein collapse cannot be observed by BAM due to the fact that pure components and mixed monolayers form isotropic domains at the air-water interface. However, the high relative reflectivity of β-casein domains after the collapse point leads to the conclusion that monoolein was unable to displace totally the protein from the mixed monolayer at the air-water interface, even at higher monoolein concentrations in the mixture and at higher surface pressures.

Introduction In recent years considerable progress has been made in the structural characterization of monolayers at the airwater interface, utilizing techniques such as fluorescence microscopy,1,2 Brewster angle microscopy,3,4 X-ray and neutron methods,5-8 ellipsometric measurements,9 infrared reflection-absorption spectroscopy,10 and so forth. With the development of Brewster angle microscopy (BAM),3,4 a sensitive and effective method is available for studying the organization in spread monolayers at the air-water interface at the microscopic level. BAM provides information on the morphology of amphiphilic monolayers,11-16 including the inner structure of condensed * To whom correspondence should be addressed. Telephone: +34 5 4557183. Fax: +34 5 4557134. E-mail: [email protected]. (1) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (2) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. (3) Heńon, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (4) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (5) Als-Nielsen, J.; Kjaer, K. K. In Phase Transition in soft and Condensed Matter; Riste, T., Sherrington, D., Eds.; NATO Ser. B, Vol. 211; Plenum Press: New York, 1989; pp 251-321. (6) Als-Nielsen, J.; Mo¨wald, H. In Handbook on Synchroton Radiation; Ebashi, S., Koch, M., Rubenstein, E, Eds.; Elsevier/North-Holland: Amsterdam, 1991; pp 1-53. (7) Kenn, R. M.; Bo¨hm, C.; Bibo, A. M.; Peterson, I. R.; Mo¨hwald, H. J. Phys. Chem. 1991, 95, 2092. (8) Paudler, M.; Ruths, J.; Riegler, H. Langmuir 1992, 8, 184. (9) Reiter, R.; Motschmann, H.; Orendi, H.; Nemetz, A.; Knoll, W. Langmuir 1992, 8, 1784. (10) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (11) Werkman, P. J.; Schouten, A. J.; Noordegraaf, M. A.; Kimkes, P.; Sudho¨lter, E. J. R. Langmuir 1998, 14, 157. (12) Gehlert, U.; Weidemann, G.; Vollhardt, D.; Brezesinski, G.; Wagner, R.; Mo¨wald, H. Langmuir 1998, 14, 2112.

domains and phase transitions in monolayers,17-20 the orientational order of the monolayer domains,19,21,22 deformation23-25 and relaxation phenomena23,26-28 in monolayer domains caused by compression-expansion cycles or by interfacial flow, and so forth. Recently, this technique has been applied to the analysis of the morphology in adsorption layers of one-29,30 and two-component systems.31,32 As the light intensity at each point in the BAM image depends on the local thickness and monolayer (13) Deschenaux, R.; Megert, S.; Zumbrunn, C.; Ketterer, J.; Steiger, R. Langmuir 1997, 13, 2363. (14) Cohen Stuart, M. A.; Wegh, R. A. J.; Jroon, J. M.; Sudho¨lter, E. J. R. Langmuir 1996, 12, 2863. (15) Vollhardt, D. Adv. Colloid Interface Sci. 1996, 64, 143. (16) Ho¨nig, D.; Overbeck, G. A.; Mo¨bius, D. Adv. Mater. 1993, 4, 419. (17) Vollhardt, D.; Melzer, V. J. Phys. Chem. B 1997, 101, 3370. (18) Melzer, V.; Vollhardt, D. Phys. Rev. Lett. 1996, 76, 3770. (19) Overbeck, G. A.; Ho¨nig, D. Mo¨bius, D. Thin Solid Films 1994, 242, 213. (20) Overbeck, G. A.; Mo¨bius, D. J. Phys. Chem. 1993, 97, 7999. (21) Brezesinskin, G.; Scalas, E.; Struth, B.; Mo¨wald, H.; Bringezu, G.; Gehlert, U.; Weidemann, G.; Vollhardt, D. J. Phys. Chem. 1995, 99, 8755. (22) Overberck, G. A.; Ho¨nig, D.; Wolthaus, L.; Gnade, M.; Mo¨bius, D. Thin Solid Films 1994, 242, 26. (23) Laüger, J.; Robertson, C. R.; Frank, C. W.; Fuller, G. G. Langmuir 1996, 12, 5630. (24) Friedenberg, M. C.; Fuller, G. G.; Frank, C. W.; Robertson, C. R. Langmuir 1996, 12, 1594. (25) Mann, E. K.; Heńon, S.; Langevin, D.; Meunier, J. Phys. Rev. E 1995, 51, 5708. (26) Gehlert, U.; Vollhardt, D. Langmuir 1997, 13, 277. (27) Lautz, C.; Fisher, Th. M.; Kildea, J. J. Chem. Phys. 1997, 106, 7448. (28) Reda, T.; Hermel, H.; Ho¨ltje, H.-D. Langmuir 1996, 12, 6452. (29) Henon, S.; Meunier, J., Thin Solid Films 210/211, 1992, 121. (30) Melzer, V.; Vollhardt, D. Phys. Rev. Lett. 1996, 76, 3770. (31) Sundaram, S.; Ferri, J. K.; Vollhardt, D.; Stebe, K. J. Langmuir 1998, 14, 1208.

10.1021/la981361j CCC: $18.00 © 1999 American Chemical Society Published on Web 06/05/1999

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optical properties, this technique can be used to determine the thickness of film regions, even when the optical properties of the film (refractive index) are unknown,33 if a model for the observed morphology is adopted.19,33-35 The application of BAM for the analysis of morphology and relative film thickness is of considerable practical importance in the field of food dispersions (emulsions and foams). In fact, in recent years extensive studies concerning the influence of emulsifiers (proteins and low-molecular weight surfactants) on the stability of food dispersions have been reported.36-40 These works showed that the stability of food dispersions depends on the amount of emulsifiers adsorbed and on the conformation adopted at the interface. As most food dispersions are stabilized by protein-lipid mixtures, the study of mixed emulsifier films is important because it leads to an understanding of the preferential structuring at the interface. The interactions and film characteristics (structure, rheology, miscibility, mobility, etc.) of protein-lipid mixed films can be studied by a combination of complementary techniquessincluding tensiometry,41,42 spread monolayers using a Langmuir or Wilhelmy film balance (unpublished results), dilational rheology,43,44 drainage and diffusion in thin liquid films,45 and more recently by BAM.32 This information may be used to improve the surface properties by means of an appropriate mixture of emulsifiers. From a practical point of view, a more complete understanding of these issues would facilitate the selection and use of mixed emulsifiers in order to improve the formulation of traditional products or facilitate the development of new formulations. This work is an extension of a previous study of proteinlipid mixed monolayers spread at the air-water interface.32 In that study we concluded that BAM makes it possible to distinguish between different patterns in mixed monolayers composed of a lipid (monopalmitin), with anisotropic crystalline-like domains in most regions of the phase diagram,46 and a protein (β-casein), with isotropic structure at the air-water interface.47 With these mixtures it is possible to determine the miscibility of or interactions in the mixed film at a microscopic level. In addition, the relative intensity-surface pressure dependence can be used not only to identify the existence of interactions between both emulsifiers at the interface but also to determine the relative thickness of the mixed film, (32) Rodrı´guez Patino, J. M.; Carrera Sańchez, C.; Rodrı´guez Ninõ, Ma. R. J. Agric. Food Chem., submitted. (33) de Mul, M. N. G.; Mann, J. A., Jr. Langmuir 1998, 14, 2455. (34) Overbeck, G. A.; Ho¨nig, D.; Wolthaus, L.; Gnade, M.; Mo¨bius, D. Thin Solid Films 1994, 242, 26. (35) Weidemann, G.; Gehlert, U.; Vollhardt, D. Langmuir 1995, 11, 864. (36) Dickinson, E. An Introduction to food Colloids; Oxford University Press: Oxford, 1992. (37) Sjo¨blom, J. Emulsions and Emulsion Stability; Marcel Dekker: New York, 1996. (38) Friberg, S. E., Larsson, K., Eds. Food Emulsions; Marcel Dekker: New York, 1997. (39) Hasenhuette, G. L., Hartel, R. W., Eds. Food Emulsions and their Applications; Chapman & Hall: New York, 1997. (40) Damodaran, S., Paraf, A., Eds. Food Proteins and their Applications; Marcel Dekker: New York, 1997. (41) Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M. J. Am. Oil Chem. Soc. 1998, 75, 1233. (42) Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M. J. Am. Oil Chem. Soc. 1998, 75, 1241. (43) Rodrı´guez Ninõ, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Langmuir 1998, 14, 2160. (44) Rodrı´guez Ninõ, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 1998, 46, 2177. (45) Wilde, P. J.; Rodrı´guez Ninõ, Ma. R.; Clark, D. C.; Rodrı´guez Patino, J. M. Langmuir 1997, 13, 7151. (46) Rodrı´guez Patino, J. M.; Carrera Sańchez, C.; Rodrı´guez Ninõ, Ma. R. Langmuir 1999, 15, 2484. (47) Rodrı´guez Patino, J. M.; Carrera Sańchez, C.; Rodrı´guez Ninõ, Ma. R. Food Hydrocolloids, in press.

Rodrı´guez Patino et al.

which is of considerable practical importance. The question that emerges from those studies is the following. Is it possible to determine the existence of interactions or miscibility in mixed monolayers by BAM if the two components have a homogeneous structure with isotropic domains, as is the case of monoolein46 and β-casein?47 The aim of the present work is to answer that question. Experimental Section Chemicals. Synthetic 1-mono(cis-9-octacenoyl)glycerol (monoolein) was supplied by Danisco Ingredients with over 95-98% purity. To form the surface film, monoglyceride was spread in the form of a solution, using hexane/ethanol (9:1, v/v) as a spreading solvent. Analytical grade hexane (Merck, 99%) and ethanol (Merck, >99.8%) were used without further purification. β-Casein (99% pure) was supplied and purified from bulk milk from the Hannah Research Institute, Ayr, Scotland. The sample was stored below 0 °C, and all work was done without further purification. Samples for interfacial characteristics of β-casein monolayers were prepared using Milli-Q ultrapure water and were buffered at pH 5 and 7. Analytical-grade acetic acid, sodium acetate, and Trizma for buffered solutions were used as supplied by Sigma (>95%) without further purification. The ionic strength was 0.05 M in all the experiments. The absence of active surface contaminants in the aqueous buffered solution was checked by surface tension measurements before sample preparation. No aqueous solutions with a surface tension other than that accepted in the literature (72-73 mN/m at 20 °C) were used. Methods. Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on a fully automated Langmuir-type film balance using a maximum area of 5.62 × 10-2 m2, as described elsewhere.48,49 The mean deviation was within (0.1 mN/m for surface pressure and (1.25 × 10-4 m2 per mg for area. The subphase temperature was controlled at 20 °C by water circulation from a thermostat, within an error range of (0.5 °C. The temperature was measured by a thermocouple located just below the air-water interface. Before each measurement, the film balance was calibrated at 20 °C. Mixtures of particular mass ratiossranging between 0 and 1, expressed as the mass fraction of monoolein in the mixture Xswere studied. To allow the quantitative adsorption of the protein on the interface, the monolayer was not under any surface pressure during the spreading process. Thus, the β-casein necessary to form the mixed film should be spread before the lipid. An aliquot ranging from 175 to 475 µL of aqueous solutions of β-casein (1.543 × 10-4 mg/µL) at pH 7 was spread on the interface by means of a micrometric syringe. To allow for spreading, adsorption, and rearrangements of the protein, 30 min was allowed to elapse before measurements were taken. The spreading method adopted in these experiments ensured the quantitative spreading of the protein on the interface as was discussed in a previous paper.50 Afterward, the lipid was spread at different points on the β-casein film. To allow for spreading and β-casein-monoolein interactions, 30 min was allowed to elapse before compression was performed. To ensure interactions and homogeneity, the mixed film was compressed near the collapse point of the mixture and then expanded immediately to avoid collapse. After 30 min at the maximum area, measurements of compression-expansion cycles were performed with 30 min of waiting time between each compression-expansion cycle. The compression rate was 6.2 × 10-2 nm2‚molecule-1‚min-1, which is the highest value for which isotherms have been found to be reproducible in preliminary experiments. Each isotherm was measured at least three times. The reproducibility of the surface pressure results was better than (0.5 mN/m. All isotherms were recorded continuously by a device connected to the film balance and then analyzed off-line. Brewster Angle Microscopy (BAM). A commercial Brewster angle microscope BAM2, manufactured by NFT (Go¨ttingen, (48) Rodrı´guez Patino, J. M.; Ruı´z, M.; de la Fuente, J. J. Colloid Interface Sci. 1992, 154, 146. (49) Rodrı´guez Patino, J. M.; Ruı´z, M.; de la Fuente, J. J. Colloid Interface Sci. 1993, 157, 343. (50) Rodrı´guez Ninõ, Ma. R.; Carrera, C. S.; Rodrı´guez Patino, J. M. Colloids Surf. B 1999, 12, 161.

Isotropic Domains in β-Casein-Monoolein Mixed Monolayers

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Germany) was used to study the morphology of the monolayer. The principles of BAM have been described in detail in the literature.3,4 Our design follows that of Overbeck, Ho¨ning, and Mo¨bius.51 Further characteristics of the device and operational conditions were described elsewhere.46,47 The Brewster angle microscope was positioned over the film balance on a specially designed frame structure which makes it possible to move the Brewster angle microscope easily along the length of the film balance. The location of the Brewster angle microscope along the film balance makes it possible to visualize any inhomogeneity in the overall film. Measurements of surface pressure, area, and gray level as a function of time were carried out simultaneously by means of a device connected between the film balance and the Brewster angle microscope. The frequency was fixed at one measurement every 5 s in order to reduce the noise in the gray level signal not related to the optical properties of the monolayer. These measurements were performed during continuous compression and expansion of the monolayer at a constant rate with different shutter speeds ranging from 1/50 to 1/500 s. To measure the relative thickness of the film, a camera calibration is necessary previously in order to determine the relationship between the gray level (GL) and the relative reflectivity (I), according to a procedure described previously.46,47 The theoretical intensity derived from Fresnel equations applied to the pure water surface, Rpw52 Brewster angle microscope gives a calibration to transform gray level into real intensities (eq 1)

Rpw )

n2 cos φ - xn2 - sin2 φ n2 cos φ + xn2 - sin2 φ

(1)

where n is the refractive index for water (n ) 1.334 at 680 nm) and φ is the incident light angle relative to the plane of incidence. The intensity at each point in the BAM image depends on the local thickness and film optical properties. These parameters can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light through the method based on the Fresnel reflection equations.52 At the Brewster angle

I ) |Rp|2 ) Cd2

(2)

where I is the relative reflectivity, C is a constant, d is the film thickness, and Rp is the p-component of the light. In previous papers the relationships between gray level (GL) and incident angle (φ) were determined, as well as the relative reflectivity dependence of the gray level as a function of the shutter speed.46,47

Results and Discussion Structural Characteristics of Mixed Films. Monolayer structural characteristics can be obtained from π-A isotherms. The π-A isotherms for monoolein and β-casein mixed monolayers at a mass fraction of monoolein in the mixture X ranging between 0 and 1, and at 20 °C, are shown in Figures 1 and 2, for monolayers spread on buffered water at pH 5 and 7, respectively. As expected,53 monoolein monolayers had a liquid-expanded structure under all experimental conditions (Figures 1A and 2A). This expanded structure for a monoolein monolayer is a consequence of weak molecular interactions because of the double bond of the hydrocarbon chain. However, the monoolein monolayer was pH dependent. The monoolein monolayer was more expanded at pH 7 than at pH 5, a phenomenon opposite to that observed for a monopalmitin monolayer with a liquid-condensed structure.46 The differences observed here for monoolein monolayers as a (51) Overbeck, G. A.; Ho¨nig, D.; Mo¨bius, D. Langmuir 1993, 9, 555. (52) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light, 1st ed.; North-Holland: Amsterdam, 1992. (53) Ruı´z, M.; Gonza´lez, I.; Rodrı´guez Patino, J. M. Ind. Eng. Chem. Res. 1998, 37, 936.

Figure 1. (A) Surface pressure-area isotherms (compression curve) for monoolein-β-casein mixed monolayers on buffered water at pH 5 and at 20 °C. Mass fraction of monoolein in the mixture (X): (4) 0; (s) 0.2; (- - -) 0.4; (‚‚‚) 0.6; (-‚-) 0.8; (O) 1. (B and C) Molecular area and excess area, respectively, for monoolein-β-casein mixed monolayers as a function of surface pressure (mN/m): (0) 5; (O) 10; (4) 15; (3) 20; and (]) 25.

function of pH should be attributed to the effect of different salts used for buffer solutions both on the distribution of water molecules around the monolayer head group and on the existence of interactions between monooleinmonoolein and monoolein-salt at the interface.53 The collapse of monoolein monolayers occurred at surface pressures close to the equilibrium surface pressure.54 This behavior is typical of spread lipids with an expanded structure which are liquids at the working tempera(54) Rodrı´guez Patino, J. M.; Martin, R. J. Colloid Interface Sci. 1994, 167, 150.

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Figure 2. (A) Surface pressure-area isotherms (compression curve) for monoolein-β-casein mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monoolein in the mixture (X): (4) 0; (s) 0.2; (- - -) 0.4; (‚‚‚) 0.6; (-‚-) 0.8; and (O) 1. (B and C) Molecular area and excess area, respectively, for monoolein-β-casein mixed monolayers as a function of surface pressure (mN/m): (0) 5; (O) 10; (4) 15; (3) 20; and (]) 25.

ture.55,56 The collapse pressure of monoolein monolayers was practically independent of the aqueous phase pH. β-Casein monolayers also had a liquid-expanded-like structure under all experimental conditions (Figures 1A and 2A). The π-A isotherm was displaced toward the surface pressure axis, and the monolayer structure was more condensed in an acidic subphase. Such behavior suggests that the amino acid residues of β-casein are more closely packed at the interface on acidic subphases.50 (55) Smith, R. D.; Berg, J. C. J. Colloid Interface Sci. 1980, 74, 273. (56) Ternes, R. L.; Berg, J. C. J. Colloid Interface Sci. 1984, 98, 471.


However, in contrast to the behavior of monoolein monolayers, from the π-A isotherm it appeared that critical surface pressures of about 14.4 and 10 mN/m for pH 5 and 7, respectively, existed, at which the film properties change significantly. In fact, as in a previous work50 with β-casein monolayers, we distinguish two different structures (structure 1 and 2) and the monolayer collapses at the highest surface pressure. In structure 1, β-casein molecules exist as trains with all amino acid segments located at the interface. As the surface pressure or the surface concentration exceeds that of the transition toward structure 2, a looping of the segments into the underlying aqueous solutions is produced. The acidic subphase caused an increase in the pressure of the transition between structures 1 and 2 (Figures 1A and 2A). Thus, the zone of existence of the β-casein monolayer with a more expanded structure (structure 1) was higher at pH 5 than that for a neutral subphase. The collapse of β-casein monolayers occurred at surface pressures close to the equilibrium surface pressure.50 Given that β-casein has an isoelectric point of about 4.9-5.2 in water, the condensation of the monolayer structure (Figures 1A and 2A) observed with β-casein monolayers on an acidic aqueous subphase must be attributed to a reduction in the repulsive interactions between negative amino acid residues due to the fact that at pH 5 the overall charge of β-casein molecules approaches zero. This phenomenon could produce the interfacial aggregation of β-casein, as was observed in the bulk phase.36,40 The monolayer structure in monoolein-β-casein mixed monolayers was liquid-expanded-like, as for pure monolayer components. Moreover, it can be seen that there was a monolayer expansion as both the pH and the monoolein content in the mixture were increased (Figures 1A and 2A). There was a marked transition, as for a pure β-casein monolayer, at surfaces pressures of about 14 and 10 mN/m for pH 5 and 7, respectively, which was more evident at the higher contents of β-casein in the mixture. At surface pressures higher than that for β-casein collapse, the π-A isotherms for mixed monolayers were parallel to that of pure monoolein. This result suggests that the composition of mixed monolayers was very dependent on the surface pressure. At surface pressures lower than that for β-casein collapse, a mixed monolayer of monoolein and β-casein could exist, β-casein molecules adopting different structures in the isotropic liquid-expanded monoolein monolayer, depending on the pH and the surface pressure, as described above. However, at surface pressures higher than that for β-casein collapse, the mixed monolayers were practically dominated by monoolein molecules. That is, at higher surface pressures, collapsed β-casein residues could be displaced from the interface by monoolein molecules. To acquire more information on the composition of monoolein-β-casein mixed monolayers, complementary data deduced from π-A isotherms are drawn in Figures 3 and 4 for pH 5 and 7, respectively. In Figures 3 and 4 we shown the hypothetical π-A isotherms for mixed monolayers calculated on the basis that only monoolein (Figures 3A and 4A) or only β-casein (Figures 3B and 4B) is present at the air-water interface. We can see that, on the basis that the mixed monolayers are dominated by monoolein (Figures 3A and 4A), the π-A isotherms for monoolein and monoolein-β-casein mixed monolayers at X ) 0.8 are practically the same, under all experimental conditions, especially on buffered water at pH 5. That is, at higher monoolein content in the mixture, the mixed monolayer behaves as a pure monoolein monolayer. In addition, the π-A isotherms at surface pressures higher


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Figure 3. Surface pressure-area isotherms (compression curve) for monoolein-β-casein mixed monolayers on buffered water at pH 5 and at 20 °C. Mass fraction of monoolein in the mixture (X): (O) 0; (s) 0.2; (- - -) 0.4; (‚‚‚) 0.6; (-.-) 0.8; and (4) 1. The molecular area was calculated on the basis that (A) only the monoolein and (B) only the β-casein was adsorbed on the interface.

Figure 4. Surface pressure-area isotherms (compression curve) for monoolein-β-casein mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monoolein in the mixture (X): (O) 0; (s) 0.2; (- - -) 0.4; (‚‚‚) 0.6; (-.-) 0.8; and (4) 1. The molecular area was calculated on the basis that (A) only the monoolein and (B) only the β-casein was adsorbed on the interface.

than that for β-casein collapse are practically coincident with the π-A isotherm for a pure monoolein monolayer (the mixed monolayer with lower monoolein concentration, at X ) 0.2, is an exception). That is, at surface pressures higher than that for β-casein collapse, mixed monolayers behaved practically as a pure monoolein monolayer. In contrast to the above data, π-A isotherms calculated on the basis that the mixed monolayers are dominated by β-casein (Figures 3B and 4B) are totally different from those for pure components under all experimental conditions. These results suggest that monoolein molecules are able to displace β-casein residues from the air-water interface, especially at surface pressures higher than that for β-casein collapse and at the higher concentrations of monoolein in the mixture, no matter what the surface pressure. Miscibility of Monoolein-β-Casein Mixed Monolayers. The interactions between monolayer components in the mixed monolayers can be studied from the π-A isotherms. In Figures 1B and 2B we show the mean area versus composition for monoolein-β-casein mixed monolayers at pH 5 and 7, respectively, at surface pressures lower than that for β-casein collapse. The dotted lines in Figures 1B and 2B correspond to an immiscible monolayer demonstrating ideal behavior of a mixed monolayer,

according to the additivity rule:57

A ) A1X1 + A2X2

(3)

where A is the molecular area at a given surface pressure for the mixed monolayer, A1 and A2 are the molecular areas of pure components (monoolein and β-casein, respectively), and X1 and X2 are the mass fractions of pure components in the mixed monolayer. From the fulfillment of the additivity rule (Figures 1B and 2B) we can deduce that, at surfaces pressures lower than that for β-casein collapse, monoolein and β-casein are nearly immiscible or that the mixed monolayer behaves as an ideal mixture with practically complete miscibility. The small deviations of the molecular area from the additivity rule indicate some degree of interaction between monolayer-forming components as a function of surface pressure and monolayer composition.

Aexc ) A - (A1X1 + A2X2)

(4)

The excess area (Aexc) versus monolayer composition (Figures 1C and 2C) makes possible the quantification of interactions between monolayer-forming components. The excess area was calculated by eq 4. For monoolein-β(57) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interface; Wiley: New York, 1966.

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Figure 5. Collapse pressure versus surface composition for monoolein-β-casein mixed monolayers on buffered water at pH (4) 5 and (O) 7. Temperature: 20 °C.

casein mixed monolayers at pH 5 (Figure 1C) the excess area was positive at every surface pressureswith the maximum Aexc values at the lower surface pressuress and for a mass fraction of monoolein in the mixture of 0.2. The positive values of Aexc are indicative that repulsive interactions between monolayer-forming components exist. Because an increase in surface pressure is followed by a decrease in the Aexc absolute value, the interactions in the mixed monolayer decrease with surface pressure. For monoolein-β-casein mixed monolayers at monoolein concentrations higher than 0.2 and at pH 5, and for all mixtures at pH 7, the Aexc values are practically zero. Thus, the existence of molecular interactions at the interface, either attractive or repulsive, between monoolein and β-casein molecules must be ruled out. Figure 5 shows the effect of monolayer composition on experimental collapse pressure (πc) for monoolein-βcasein mixed monolayers at pH 5 and 7, at 20 °C. It can be seen that the collapse pressure is similar to that for monoolein and does not depend on the monolayer composition. The practical independence of πc on monolayer composition also implies an immiscibility of the two components in the mixed monolayer at the highest surface pressure (under the collapse conditions). Elasticity of Monoolein-β-Casein Mixed Monolayers. The elasticity is defined as E ) -A(δπ/δA)T. The elasticity defined here is the elasticity at zero deformation rate. The monolayer elasticity is a measure of the monolayer resistance to a change in area and can be calculated directly from the slope of the π-A isotherm at any temperature. Thus, high elasticity values are associated with a monolayer which has a strong cohesive structure at the surface. Figure 6 shows the elasticity versus surface pressure for monoolein-β-casein mixed monolayers at 20 °C and at pH 7. For a pure monoolein monolayer, the elasticity increased with superficial density until the monolayer collapse. However, for pure a β-casein monolayer the elasticity increased with superficial density, passed through a maximum at a superficial density close to the transition between structures 1 and 2 (see arrow A in Figure 6), and then decreased to a plateau at the collapse point (see arrow B in Figure 6). The elasticity was similar for monoolein and β-casein pure components at superficial densities lower than that for the transition between structure 1 and 2 in a β-casein pure monolayer, at a surface pressure of 10.0 mN/m (Figure 2). In this region the elasticity of the mixed films was also the same as those for pure


Figure 6. Elasticity modulus versus surface density for monoolein-β-casein mixed monolayers on buffered water at pH 7 and at 20 °C, as a function of monoolein mass fraction in the mixture (X): (‚‚‚) 0; (0) 0.2; (O) 0.4; (4) 0.6; (3) 0.8; (s) 1. The arrows A and B indicate the transition between structures 1 and 2, and the collapse point for a β-casein pure component, respectively. The dashed line corresponds to a surface pressure of 25 mN/m, just after the β-casein collapse point.

components. However, important differences were observed at higher superficial densities as β-casein adopts structure 2. It can be seen that, for pure β-casein and mixed monolayers, the elasticity decreased with both the superficial density and the content of monoolein in the mixture sthe behavior of a mixed monolayer at X ) 0.2 is an exception, with an elasticity which was practically similar to that of pure β-casein. The discontinuity in the superficial density dependence of mixed monolayer elasticity was observed at a superficial density which corresponded to a surface pressure of 25 mN/msthe surface pressure of β-casein at the collapse point (Figure 2)swhich is indicated in Figure 6 by means of a discontinuous line. Thus, at surface pressures higher than 25 mN/m the elasticity of mixed monolayers increased with superficial density and with the content of monoolein in the mixture until the collapse point of the mixed monolayer was attained. In the last two regions, at surfaces pressures higher than 10.0 mN/m, no linear dependence was observed between elasticity and superficial density nor between elasticity and mixture composition at constant surface pressure. These results imply an immiscibility between the two components in the mixed monolayer. Brewster Angle Microscopy of Monoolein-βCasein Mixed Monolayers. From the discussion in previous sections we have concluded that monoolein and β-casein molecules are practically immiscible at the airwater interfacesespecially at pH 5 at mass fraction of monoolein in the mixture higher than 0.2, and at pH 7 at any compositionsand that the monolayer characteristics of the mixed monolayers are practically dominated by the presence of monoolein at the interfacesat mass fraction of monoolein in the mixture higher than 0.2 and at surface pressures higher than that for β-casein collapse. To further our knowledge of the monolayer composition and the existence of miscibility or interactions between the monolayer-forming components on one hand and to investigate the possibility of using BAM to analyze the behavior of mixed component monolayers with isotropic structures at the air-water interface on the other hand, we aimed to study the morphology and the relative thickness of monoolein-β-casein mixed monolayers at the air-water interface. In this study we selected two representative mixtures at low (X ) 0.4) and high (X ) 0.8) monoolein


Figure 7. Relative reflectivity as a function of surface pressure during the (A) compression and (B) expansion of (O) β-casein and (4) monoolein pure monolayers spread on buffered water at pH 7, and at a shutter speed of 1/50 s. The line fits for monoolein (continuous line) and β-casein (discontinuous line) pure monolayers are included in plots A and B. Temperature: 20 °C.

content in the mixed monolayers spread on buffered water at pH 7, as an example. Brewster Angle Microscopy of a Pure Component Monolayer. Figure 7 shows the relative reflectivity versus surface pressure for compression (Figure 7A) and expansion (Figure 7B) of monoolein and β-casein monolayers at 20 °C and at pH 7, for a shutter speed of 1/50 s. As discussed in a previous work,32 the shutter speed must be optimized in order to obtain a wide range in relative reflectivity, which makes it possible to determine significant differences between monoolein and β-casein with the maximum precision in measurements. With high shutter speed values the range for I measurements increases, which is of utility for the analysis of proteins, but with a loss in the sensitivity in the range of low π values, which is of utility for the analysis of lipids. From preliminary experiments (data not shown) it was deduced that a shutter speed of 1 /50 s is adequate for the analysis of monoolein-β-casein mixed monolayers. However, at the low shutter speed we used here, the camera was saturated with a thick protein film (image completely white), even before the protein collapse (Figure 7), but this low shutter speed was optimum for the analysis of a monoolein monolayer. The plots in Figure 7 will be used as reference for the analysis of mixed monolayers. The I-π plots reflect the surface equation of state for monoolein and β-casein spread monolayers. The I-π plot for monoolein was a continuous line which corresponds to a liquid-expanded structure with an isotropic morphology, as observed by BAM.46 During

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monolayer compression and expansion some holes were observed by BAM images with a higher frequency at the higher surface pressures.46 The absence of noise peaks in I-π plots is an indication that no anisotropic domains were formed over all the experimental conditions, as observed for monopalmitin monolayers.46 The I-π plots were practically the same for compression and expansion, indicating that the molecular organization of the monoolein monolayer after the collapse did not require any timesin agreement with the absence of any hysteresis in the π-A isotherm during the compression-expansion cycle (data not shown). The relative reflectivity versus surface pressure, I-π, plot for compression (Figure 7A) and expansion (Figure 7B) of β-casein monolayers at 20 °C and at pH 5 and 7 shows the typical sigmoidal shape of the π-A isotherms for proteins. It can be seen that β-casein monolayers have a liquid-expanded-like structure under these experimental conditions. Before the monolayer collapse, at a surface pressure of approximately 20 mN/m, a plateau was observed in I-π plots at a shutter speed of 1/50 s, due to the camera saturation by a thick β-casein monolayer, a phenomenon not observed for a monoolein monolayer. The same results were observed for β-casein monolayers at different shutter speeds47 from that used here. In contrast to that observed for a monoolein monolayer, the I-π plot was displaced toward the I axis during expansion (Figure 7B), indicating that the molecular reorganization of β-casein molecules at interface upon expansion required some time, as a consequence of its viscoelastic characteristics. The relative reflectivity increased with the monolayer compression and tended to a plateau at the collapse point. The absence of any defined structures reduced the noise peak in the I-π plot under both monolayer compression and expansion, a consequence of the monolayer isotropy as deduced from BAM images.47 It should be emphasized that β-casein monolayers have an I value higher than that of a monoolein monolayer at any surface pressure, indicating that the thickness of β-casein monolayers is significantly higher than that of a monoolein monolayer. In fact, the I value for β-casein at 20 mN/m is an order of magnitude higher than that of monoolein, which indicates that the thickness of a β-casein monolayer at the precollapse point is about three times higher than that of monoolein at the same surface pressure (20 mN/m). The differences observed in I-π plots for monoolein and β-casein, coupled with BAM images, will be used in the next section to analyze the behavior of mixed monolayers. For comparison purposes a continuous line was deduced by fitting the I-π data, derived from different measurements. The I versus π data for a monoolein monolayer can be fitted by a second-order polynomial, whereas a thirdorder polynomial fit the data for a β-casein monolayer better. Brewster Angle Microscopy of a Monoolein-β-Casein Mixed Monolayer at X ) 0.4. In Figure 8 we show the π-A isotherm for a compression-expansion cycle at pH 5 and 7 (Figure 8A) and the I-π plots for compression (Figure 8B) and expansion (Figure 8C) of a monooleinβ-casein mixed monolayer at 0.4 mass fraction of monoolein in the mixture, at 20 °C and at pH 7, as an example. In Figure 9 we show the BAM images for mixed monolayers at some representative surface pressures during the compression-expansion cycle. From the π-A isotherm (Figure 8A) it can be deduced that monoolein-β-casein mixed monolayers at surface pressures lower than that corresponding to β-casein collapse (at about 25 mN/m) were more condensed on an acidic subphase. However, as

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Figure 8. (A) Surface pressure-area isotherm (compression and expansion curves) for monoolein-β-casein mixed monolayers on buffered water at pH 5 (O, compression curve; 4, expansion curve) and pH 7 (s, compression curve, ‚‚‚, expansion curve). (B and C) Relative reflectivity as a function of surface pressure during the compression and expansion, respectively, for monoolein-β-casein mixed monolayers on buffered water at pH 7, and at a shutter speed of 1/50 s. The line fits for monoolein (continuous line) and β-casein (discontinuous line) pure monolayers are included in plots B and C. Monoolein mass fraction in the mixture: 0.4. Temperature: 20 °C.

for a pure monoolein monolayer, the pH had no significant influence on the π-A isotherm at surface pressures higher than that for β-casein collapse. This result strengthens the idea that at higher surface pressures the mixed monolayer is practically dominated by the presence of monoolein at the interface. The I-π plot gives complementary information about the behavior of the mixed monolayer especially at the lower (π < 10 mN/m) and higher (π > 25 mN/m) surface pressures. In fact, at surface pressures lower than 10


mN/m a pure β-casein monolayer adopts the most expanded structure (structure 1), the I-π for the mixed monolayer was essentially the same but a little lower than that for a pure β-casein monolayer. These results suggest that at these surface pressures the mixed monolayer is dominated by the presence of β-casein molecules at the interface. Unfortunately we are unable to deduce any difference from BAM images between pure components and the mixed monolayer because in this region both components and the mixed monolayer as well form an isotropic monolayer without any difference in the domain morphology. At surfaces pressures between 10 and 25 mN/m a sudden drop in I values was observed and the I-π plot was practically parallel to that for pure monoolein. In this region, BAM images (Figure 9A and B) demonstrated that monoolein and β-casein molecules adopt an isotropic structure in the mixed monolayer with some holes (Figure 9A) associated with the presence of monoolein at the interface and some white regions (Figure 9B) which correspond to the presence of a thicker β-casein monolayer domain. In fact, we could observe some peaks of higher intensity in the I-π plot (Figure 9B), when a domain of collapsed β-casein molecules passed through the spot where this measurement was performed. At surface pressures higher than 25 mN/m the I-π plots approached that for a pure monoolein monolayer, with a higher I value for the mixed monolayer, which corresponds to a higher monolayer thickness than that for pure monoolein. We attributed this fact to close packing of monoolein molecules and/or to the presence of β-casein molecules in a layer behind but close to that of monoolein. In this region, some noise peaks with a relative reflectivity similar to that for a collapsed β-casein monolayer were observed, which demonstrated that the monoolein monolayer was unable to displace completely the β-casein molecules from the interface. BAM images show that in this region the hole frequency increased (Figure 9C), which we associated with the presence of monoolein. However, some white spots were also observed (Figure 9D) due to the presence of collapsed β-casein molecules. We suggest that in this region a squeezing out phenomena was produced, with β-casein molecules being displaced from the interface by monoolein molecules. Finally, at the higher surface pressures, and especially at the collapse point of a pure monoolein monolayer, the noise peaks disappeared in the I-π plot and the relative reflectivity decreased to a value close to that for a pure monoolein monolayer (Figure 8B). This result suggests that in this region the mixed monolayer was dominated by the presence of monoolein at the interface, as deduced in previous sections. This conclusion is corroborated by BAM images (Figure 9E). However, BAM images (Figure 9F) also demonstrated for the first time that, even at the highest surface pressures, a monoolein monolayer is unable to displace completely β-casein molecules from the air-water interface. In fact, in this region some paths of collapsed β-casein residues (observed as a white path in the image) with holes of stacked monoolein domains were detected (Figure 9F) by the movement of the Brewster angle microscope along the film balance. The I-π plot during the monolayer expansion (Figure 8C) was similar to that of the monolayer compression, but the noise peaks decreased in frequency and in intensity. In fact, no I peaks with an intensity similar to that for pure β-casein were observed after repetitive measurements. BAM images showed that the morphology of the mixed monolayer was similar during compression and expansion (Figure 9G). However, the characteristic mor-


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Figure 9. Visualization of monoolein-β-casein mixed monolayers by Brewster angle microscopy at 20 °C and at pH 7. (A) π ) 15 mN/m; (B) π ) 15 mN/m; (C) π ) 30 mN/m (just after the β-casein collapse); (D) π ) 30 mN/m; (E) π ) 46.4 mN/m (at the monolayer collapse); (F) π ) 41.1 mN/m; (G) monolayer expansion at π ) 22 mN/m; (H) 2D foam after monolayer expansion at π ) 3 mN/m. Monoolein mass fraction in the mixture: 0.4. Temperature: 20 °C. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

phology of the monolayer during the expansion and after the breakup of the β-casein collapse structure was the formation of a 2D foam (Figure 9H), due probably to the penetration of β-casein molecules into the monoolein monolayer as the surface pressure decreased.

Brewster Angle Microscopy of a Monoolein-β-Casein Mixed Monolayer at X ) 0.8. In Figure 10 we show the π-A isotherm for a compression-expansion cycle (Figure 10A) and the I-π plots for compression (Figure 10B) and expansion (Figure 10C) of a monoolein-β-casein

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Figure 10. (A) Surface pressure-area isotherm (compression and expansion curves) for monoolein-β-casein mixed monolayers on buffered water at pH 5 (0, compression curve; 4, expansion curve) and pH 7 (s, compression curve; ‚‚‚, expansion curve). (B and C) Relative reflectivity as a function of surface pressure during the compression and expansion, respectively, for monoolein-β-casein mixed monolayers on buffered water at pH 7, and at a shutter speed of 1/50 s. The line fits for monoolein (continuous line) and β-casein (discontinuous line) pure monolayers are included in plots B and C. Monoolein mass fraction in the mixture: 0.8. Temperature: 20 °C.

mixed monolayer at 0.8 mass fraction of monoolein in the mixture, at 20 °C and at pH 7, as an example. In Figure 11 we show the BAM images for mixed monolayers at some representative surface pressures during the compression-expansion cycle. The behavior of monoolein-β-casein mixed monolayers at X ) 0.8 was different from that for lower concentrations of monoolein in the mixture (Figures 8 and 9), as deduced from π-A isotherms (Figure 10A), from I-π plots (Figures


10B and C), and BAM images (Figure 11). The compression-expansion isotherm was now more pH dependent, with a more condensed structure for an acidic aqueous subphase. During the compression and expansion the I-π plots followed the same trends as pure monoolein but with a higher I value for the mixed monolayer, especially during the monolayer compression. It should be noted that no peak with the relative intensity typical of a β-casein collapse monolayer was observed during the compression-expansion cycle. The BAM image at surface pressures lower than that for β-casein collapse (Figure 11A) shows the existence of an isotropic morphology. After the β-casein collapse the overall morphology of the mixed monolayer shows numerous holes (Figure 11B), associated with the presence of monoolein at the interface. At the collapse point of the mixed monolayer, some regions with more numerous and more closely packed small holes (Figure 11C) and other regions with fewer, less closely packed, bigger holes (Figure 11D) dominated the interfacial morphology. The overall morphology of the mixed monolayer was quite different from that for pure monoolein,46 but from the BAM image it is impossible to distinguish between domains of β-casein and monoolein at the interface. The paths of a collapsed β-casein monolayer were not observed at the highest surface pressures, as for a concentration of monoolein in the mixture of 0.4 (Figure 9F); instead some spots of collapsed β-casein (Figure 11E) with holes of stacked monoolein domains were detected in this region. This result suggests that even at the higher monoolein concentration some regions of collapse β-casein were present in the mixed monolayer. During the expansion, the morphology of the mixed monolayer was similar to that of the compression, with numerous holes at the higher surfaces pressures (Figure 11F) which decrease with the surface pressure (Figure 11G). Finally, the formation of a 2D foam at the lower surface pressure was observed (Figure 11H). This result suggests that, even at the higher surface pressures, with high content of monoolein in the mixture, some minor interaction between components in the mixed monolayer could exist (at a microscopic level) and, as a consequence of these reduced interactions, β-casein residues displaced by monoolein molecules toward the sublayer region during the compression return to the interface with the monolayer expansion. This hypothesis is in agreement with the 2D foam morphology at the lower surface pressures (Figure 11H) and with the reproducibility of the π-A isotherms after a waiting time at the maximum area (data not shown). Conclusions Structural characteristics and miscibility for monooleinβ-casein mixed monolayers spread on buffered water at pH 5 and 7 and at 20 °C have been determined. The structure of monoolein-β-casein mixed monolayers was liquid-expanded-like, as for pure components. The monolayer structure was more expanded as the pH and the monoolein concentration in the mixture were increased. The composition of the mixed monolayer was very dependent on the surface pressure. At surface pressures lower than that for β-casein collapse (π = 25 mN/m) a mixed monolayer formed by β-casein and monoolein could exist. However, at higher surfaces pressures (π > 25 mN/ m) the mixed monolayer is practically dominated by monoolein molecules. From the concentration and surface pressure dependence of excess area, elasticity, and collapse pressure, it was deduced that monoolein and β-casein forma practically immiscible monolayer at the air-water interface. The BAM images and the evolution with the


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Figure 11. Visualization of monoolein-β-casein mixed monolayers by Brewster angle microscopy at 20 °C and at pH 7. (A) π ) 20 mN/m; (B) π ) 30 mN/m (just after the β-casein collapse); (C) π ) 46 mN/m (at the monolayer collapse); (D) monolayer relaxation at π ) 40 mN/m; (E) monolayer relaxation at π ) 38 mN/m; (F) monolayer expansion at π ) 36 mN/m; (G) monolayer expansion at π ) 36 mN/m; (H) 2D foam after monolayer expansion at π ) 2 mN/m. Monoolein mass fraction in the mixture: 0.8. Temperature: 20 °C. The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

surface pressure of the relative reflectivity of BAM images give complementary information on the interactions and structural characteristics of monoolein-β-casein mixed monolayers, which corroborated the conclusions derived from the π-A isotherm. The morphology of monoolein,

β-casein, and monoolein-β-casein domains at surface pressures lower than that for β-casein collapse cannot be observed by BAM due to the fact that a pure component and mixed monolayers form isotropic domains at the air-water interface. However, the high relative reflect-

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ivity of β-casein domains after the collapse point allows us to conclude that monoolein was unable to displace totally the protein from the mixed monolayer at the air-water interface, even at the higher monoolein concentration in the mixture and at the higher surface pressures. In summary, Brewster angle microscopy is a useful technique to distinguish between isotropic domains in protein-lipid mixed monolayers at the air-water interface by means of the relative reflectivity.


Acknowledgment. The authors thank Prof. D. Horne for providing the β-casein sample and Danisco Ingredients for providing the monoolein sample. This research was supported by the European Community through Grant FAIR-CT96-1216 and by CICYT through Grant ALI971274-CE. LA981361J

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