Structural and Topographical Characteristics of Adsorbed WPI and

Langmuir 2004, 20, 4515-4522

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Structural and Topographical Characteristics of Adsorbed WPI and Monoglyceride Mixed Monolayers at the Air-Water Interface Juan M. Rodrı´guez Patino* and Marta Cejudo Fernańdez Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcıá Gonza´ lez 1, 41012-Seville, Spain Received November 21, 2003. In Final Form: March 15, 2004 In this work we have analyzed the structural and topographical characteristics of mixed monolayers formed by an adsorbed whey protein isolate (WPI) and a spread monoglyceride monolayer (monopalmitin or monoolein) on the previously adsorbed protein film. Measurements of the surface pressure (π)-area (A) isotherm were obtained at 20 °C and at pH 7 for protein-adsorbed films from water in a Wilhelmy-type film balance. Since the surface concentration (1/A) is actually unknown for the adsorbed monolayer, the values were derived by assuming that the A values for adsorbed and spread monolayers were equal at the collapse point of the mixed film. The π-A isotherm deduced for adsorbed WPI monolayer in this work is practically the same as that obtained directly by spreading. For WPI-monoglyceride mixed films, the π-A isotherms for adsorbed and spread monolayers at π higher than the equilibrium surface pressure of WPI are practically coincident, a phenomenon which may be attributed to the protein displacement by the monoglyceride from the interface. At lower surface pressures, WPI and monoglyceride coexist at the interface and the adsorbed and spread π-A isotherms (i.e., the monolayer structure of the mixed films) are different. Monopalmitin has a higher capacity than monoolein for the displacement of protein from the air-water interface. However, some degree of interactions exists between proteins and monoglycerides and these interactions are higher for adsorbed than for spread films. The topography of the monolayer corroborates these conclusions.

Introduction The stability and mechanical properties of dispersed food systems (emulsions and foams) depend on the way in which the constituent emulsifiers (lipids and proteins) adsorb and interact at fluid interfaces. The optimum use of emulsifiers depends on knowledge of their interfacial physicochemical characteristicsssuch as surface activity, structure, miscibility, superficial viscosity, etc.sand the kinetics of the film formation at fluid interfaces.1 In addition, in many emulsifier applications, mixtures of different emulsifiers (mainly polar lipids and proteins) often exhibit properties superior to those of the individual emulsifier alone due to synergistic interactions between emulsifier molecules. In previous works with mono- and diglycerides2,3 or mixtures of monoglycerides and milk proteins4 we have observed that when molecules of both emulsifiers are spread at the air-water interface, they are more expanded or packed more closely together than when either emulsifier is present alone, indicating some form of association. Interactions between molecules of emulsifiers could affect not only the film structure and topography but also dynamic phenomena in mixed films.4 Information about these phenomena would be very helpful in the prediction of optimized formulations for food foams and emulsions. So far, there are important experimental results available on structural characteristics of mixed emulsifiers spread at the air-water * To whom all correspondence should be addressed. Tel: +34 95 4556446. Fax: +34 95 4557134. E-mail: [email protected]. (1) Dickinson, E. An Introduction to Food Colloids; Oxford University Press: Oxford, England, 1992. (2) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M.; Carrera, C.; Cejudo, M.; Garcıá, J. M. Chem. Eng. Commun. 2003, 190, 15. (3) Horne, D.; Rodrı´guez Patino, J. M. In Biopolymers at Interfaces, 2nd ed.; Malmsten, M., Ed.; Marcel Dekker: New York, 2003; p 857. (4) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Curr. Opin. Colloid Interface Sci. 2003, 8, 387.

interface.4-8 However, as far as we know, there have been few studies of the structural characteristics of adsorbed films at the air-water interface,9,10 although in practice mixtures of these emulsifiers are usually used in order to reach an optimal effect in food formulations.5-7 In this contribution we are concerned with the analysis of structural characteristics of mixed monolayers formed by an adsorbed milk protein (whey protein isolate, WPI) and a spread monoglyceride monolayer (monopalmitin or monoolein). These experiments mimic the behavior of emulsifiers in food emulsions in which an oil-soluble lipid (monopalmitin or monoolein) interacts at the interface with a protein film previously adsorbed from the aqueous bulk phase. Monolayer technique has been used successfully for studying the properties of mixed emulsifiers spread at the air-water interface. Although these experiments have demonstrated the relevance of spread monolayers for fundamental studies,2-4 adsorbed monolayers are more interesting from a technological point of view. Experimental Section Chemicals. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN PA 90) and 1-monoglycerol (cis-9-octadecanoyl) (monoolein, RYLO MG 19) were supplied by Danisco Ingredients (Brabran, Denmark) with over 95-98% of purity. Whey protein isolate (WPI), a native protein with very high content of β-lactoglobulin (protein 92 ( 2%, β-lactoglobulin > (5) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsions and their Applications; Hasenhuette, G. L., Hartel, R. W., Eds.; Chapman and Hall: New York, 1997; p 95. (6) Nylander, T. In Proteins at Liquid interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998; p 365. (7) Wilde, P. J. Curr. Opin. Colloid Interface Sci. 2000, 5, 176. (8) Dickinson, E. Colloids Surf., B 2001, 20, 197. (9) Li, J. B.; Kra¨gel, J.; Makievski, A. V.; Fainermann, V. B.; Miller, R.; Mo¨hwald, H. Colloids Surf., A 1998, 142, 355. (10) Li, J. B.; Zhao, J.; Miller, R. Nahrung 1998, 42, 234.

10.1021/la036190j CCC: $27.50 © 2004 American Chemical Society Published on Web 04/30/2004

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95%, R-lactalbumin < 5%) obtained by fractionation, was supplied by Danisco Ingredients. Samples for interfacial characteristics of WPI adsorbed films were prepared using Milli-Q ultrapure water and were buffered at pH 7. To form the mixed surface film on a previously adsorbed β-lactoglobulin monolayer, 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. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). A commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl, Sigma, >99.5%) was used to achieve pH 7. Ionic strength was 0.05 M in all the experiments. Surface Film Balance. Measurements of surface pressure (π)-area (A) isotherms of WPI-monoglyceride mixed films at the air-water interface were performed on a fully automated Wilhelmy-type film balance as described previously.11-13 Before each measurement, the film balance was calibrated at 20 °C. For protein adsorbed films from water a protein solution at 1 × 10-6-1 × 10-5% (wt/wt) was left in the trough and time allowed for protein adsorption at the interface. These protein concentrations ware selected from previous data of the adsorption isotherm.14 At this protein concentration in solution the surface pressure at equilibrium is zero. In fact, after 24 h the surface pressure (π) at the maximum area of the trough was practically zero and then the monoglyceride was spread at different points on the WPI film. For pure adsorbed protein films the maximum protein concentration in the bulk phase should be selected to obtain a reasonable rate of adsorption at the interface but maintaining as zeroed the equilibrium surface pressure.18 On the other hand, for mixed films, at low protein concentrations in the aqueous phase we cannot observe the collapse point, especially for low monoglyceride concentrations. Thus, in these experiments we have selected optimum conditions in order to obtain the complete π-A isotherm of the mixed film, from the more expanded monolayer (at the higher areas) to the more condensed monolayer, at the collapse point (at the lower areas). Mixtures of particular mass ratiossexpressed as the mass fraction of monoglyceride in the mixture, Xswere studied. The compression rate was 3.3 cm‚min-1, which is the highest value for which isotherms were found to be reproducible in preliminary experiments. The π-A isotherm was measured at least five times. The reproducibility of the results was better than (0.5 mN/m for surface pressure and (0.05 m2/mg for area. Brewster Angle Microscopy (BAM). A commercial Brewster angle microscope (BAM), BAM2, manufactured by NFT (Go¨ttingen, Germany), was used to study the topography of the monolayer. The BAM was positioned over the film balance. Further characteristics of the device and operational conditions have been described elsewhere.15,16 The surface pressure measurements, area, and gray level as a function of time were carried out simultaneously by means of a device connected between the film balance and BAM. These measurements were performed during sinusoidal compression and expansion of the monolayer. To measure the relative reflectivity (I) of the film a previous camera calibration is necessary.15,16 The imaging conditions were adjusted to optimize both image quality and quantitative measurement of reflectivity. Thus, generally as the surface pressure or the protein content increased, the shutter speed was also increased. (11) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. Langmuir 2001, 17, 4003. (12) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Ind. Eng. Chem. Res. 2002, 41, 2652. (13) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. J. Agric. Food Chem. 2003, 51, 112. (14) Rodrı´guez Ninõ, M. R.; Carrera, C.; Cejudo, M.; Rodrı´guez Patino, J. M. J. Am. Oil Chem. Soc. 2001, 78, 873. (15) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Langmuir 1999, 15, 2484. (16) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R. Food Hydrocolloids 1999, 13, 401. (17) Murray, B. S.; Faergemand, M.; Trotereau, M.; Ventura, A. In Food Emulsions and Foams: Interfaces, Interactions and Stability; Dickinson, E., Rodrı´guez Patino, J. M., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1999; p 223. (18) Rodrı´guez Ninõ, M. R.; Rodrı´guez Patino, J. M. Ind. Eng. Chem. Res. 2002, 41, 1489.

Rodrı´guez Patino and Ferna´ ndez

Figure 1. (A) π-A isotherms for an adsorbed monolayer of WPI after successive compressions: (0) 11.5 h; (O) 24 h; (4) 26 h; (3) 28 h. (B) π-A isotherms for (s) adsorbed and (O) spread WPI monolayers. (C) π-A isotherms for adsorbed WPI monolayers. The aqueous subphase is at pH 7, and the temperature is 20 °C. The πe of WPI is indicated by means of an arrow.

Results and Discussion Structural and Topographical Characteristics of WPI Monolayer Adsorbed at the Air-Water Interface. Figure 1A shows the π-A isotherms for an adsorbed monolayer of WPI after successive compressions, formed from adsorption in solution at 1 × 10-6-1 × 10-5%, wt/wt. There was a difference in the π-A isotherms as a function of time after protein addition to the aqueous bulk phase. It can be seen that there was a shift of the π-A isotherms toward higher areas as the protein adsorption time increased. The π-A isotherm for a first compression at 30 min of adsorption time (data not shown) indicates that a low amount of protein was adsorbed at the interface because the surface pressure at the minimum area was much lower than the equilibrium surface pressure for WPI (πe = 30 mN/m).14 In addition, in Figure 1A we also observe that the maximum surface pressure also increased with the aging time. These data reveal that a long time interval of adsorption allows more WPI to adsorb at the surface, especially from low protein concentration in solution. At 24 h of adsorption time the π-A isotherms after successive compressions were practically coincident (Figure 1A). In addition, the π-A isotherms at long-term adsorption were reproducible after repeated experiments using new aliquots of the protein in solution, for different protein concentrations in the range 1 × 10-6-1 × 10-5%, wt/wt (Figure 1C). These results demonstrate that it is possible to measure reproducible π-A isotherms for adsorbed WPI

Adsorbed WPI and Monoglyceride Mixed Monolayers

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Figure 2. Visualization of WPI-monopalmitin mixed monolayers by Brewster angle microscopy at 20 °C: (a) pure WPI monolayer, pure monoolein monolayer, or WPI-monoolein mixed films at π < πc (WPI); (b) pure WPI monolayer, pure monoolein monolayer, or WPI-monoolein mixed films after the WPI collapse; (c) WPI at the collapse point; (d) WPI-monopalmitin mixed films at π < πc (WPI); (e) WPI-monopalmitin mixed films at π = πc (WPI); (f) WPI-monopalmitin mixed films at π > πc (WPI); (g) WPImonopalmitin mixed films at π > πc (WPI); (h) WPI-monopalmitin mixed films at π = πc (WPI-monopalmitin mixed film); (i) WPI-monoolein mixed films at π > πc (WPI). The horizontal direction of the image corresponds to 630 µm, and the vertical direction, to 470 µm.

monolayers from low protein concentration in the bulk phase. This is clear evidence that the π-A isotherms obtained after long adsorption time have a thermodynamic character. Since the surface concentration is actually unknown for the adsorbed monolayer, the values were derived by assuming (Figure 1B) that the A values for adsorbed and spread monolayers were equal at the collapse point of the mixed film.17,18 This assumption can be supported by the fact that, for protein films, the equilibrium spreading pressure (πe), the surface pressure at the plateau for a saturated WPI adsorbed film,14 and the collapse pressure for adsorbed (Figure 1A) and spread12,19,20 WPI monolayers are practically equal. The π-A isotherm deduced for adsorbed WPI monolayer is practically the same as that obtained directly by spreading (Figure 1B). Thus, the structures of the monolayers formed in the two different ways must be identical, at least for adsorption from low bulk protein concentrations. The same agreement between the adsorbed and spread isotherms was observed for other proteins.17,18,21,22 This good agreement between spread and adsorbed π-A isotherms also supports the relevance of adsorption studies by performing surface pressure measurements.21 The results of the π-A isotherms (Figure 1C) confirm that a WPI monolayer at the air-water interface adopt (19) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C.; Cejudo, M. Langmuir 2001, 17, 7545. (20) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, M. R.; Carrera, C. Ind. Eng. Chem. Res. 2002, 41, 3169. (21) Benjamins, J. Static and Dynamic Properties of Proteins Adsorbed at Liquid Interfaces. Ph.D. Dissertation, University of Wageningen, Wageningen, The Netherlands, 2000. (22) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 415.

a liquid expanded-like structure and the collapse phase. However, according to Graham and Phillips (1979b), WPI retains elements of the native structure, not fully unfolded at the interface. Thus, most amino acid residues in WPI adopt loop conformation at the air-water interface. But the loop conformation is more condensed at higher surface pressures and is displaced toward the bulk phase at the collapse point. The monolayer collapses at a surface pressure of about 30 mN/m (Figure 1), a value close to the surface pressure at the plateau for a saturated adsorbed film and the equilibrium surface pressure, which is indicated in Figure 1C by means of an arrow.14 The topography (Figure 2) and, especially, the relative reflectivity (Figure 3) as a function of surface pressure obtained with WPI monolayers clearly show the same structural characteristics as those deduced from the π-A isotherm (Figure 1C). The domains that residues of protein molecules adopt at the air-water interface appeared to be of uniform reflectivity (Figure 2a), suggesting homogeneity in thickness and film isotropy with dust causing the only visible features. The I-π dependence (Figure 3) is characteristic for this protein and is independent of the experimental conditions adopted (either for spread or adsorbed monolayers). The relative monolayer thickness increases with the surface pressure and is a maximum at the collapse, at the highest surface pressure. The increase in reflected light intensity with surface pressure, and especially at the monolayer collapse, suggests that an increase in the monolayer thickness from more expanded to more condensed structure, and a further increase at the monolayer collapse takes place. At the highest surface pressure I ) 2.5 × 10-6 au, which means that for adsorbed WPI the monolayer thickness is of the same order of magnitude as for milk23 and soy globulin24 spread mono-

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Figure 3. Relative reflectivity (arbitrary units) as a function of surface pressure during the (A) compression and (B) expansion for an adsorbed WPI monolayer on buffered water at pH 7 and at 20 °C. Shutter speed (s): (O) 1/50; (4) 1/250.

layers at the air-water interface. Minor regions with reflectivity peaks were observed, even at the beginning of the compression (at π = 0 mN/m). Even for a visible “homogeneous” interface (Figure 2a) the intensity peaks (Figure 3), which are an indication of the local heterogeneity of the interface at a microscopic level, are due to the existence of isolated interfacial regions with fold (Figure 2b) or aggregations (Figure 2c) of collapsed WPI formed at the higher surface pressures, which were present at the interface during the monolayer expansion, even at the lower surface pressures (Figure 3). At a microscopic level, the compression-expansion cycle was reversible due to the fact that the film adopted practically the same states (Figure 3). Structural and Topographical Characteristics of WPI-Monopalmitin Mixed Monolayers Adsorbed at the Air-Water Interface. Mixtures of particular mass ratiossranging between 0 and 0.6, expressed as the mass fraction of monoglyceride in the mixture, Xswere studied. The amount of spread monoglyceride was calculated on the basis of the mass of previously adsorbed WPI, which was calculated form the adsorbed π-A isotherm. Thus, as opposed to spread monolayers,19 for adsorbed monolayers the mixtures with mass fractions higher than X ) 0.6 saturate the interface, under the experimental conditions used in this work. The surface pressure as a function of the trough area for WPI + monopalmitin mixed films during a compression-expansion cycle is shown in Figure 4. As for pure WPI adsorbed film the actual π-A isotherm for WPI + monopalmitin mixed films was derived by assuming that the A values for adsorbed and spread monolayers were equal at the collapse point (Figure 4B,C). This assumption can be supported by the fact that the surface pressures (23) Rodrı´guez Patino, J. M.; Carrera, C.; Rodrı´guez Ninõ, M. R.; Cejudo, M. In Food Colloids: Fundamentals of Formulations, Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2001; p 22. (24) Carrera, C.; Rodrı´guez Ninõ, M. R.; Molina, S.; Anõn M. C.; Rodrı´guez Patino, J. M. Food Hydrocolloids 2004, 18, 335.

Figure 4. (A) Surface pressure-area isotherms (compressionexpansion curves) for adsorbed WPI-monopalmitin mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (s) 0; (---) 0.2; (4) 0.4; (- ‚ -) 0.5; (O) 0.6. (B) π-A isotherms for (s) adsorbed and (4) spread11 WPI-monopalmitin mixed monolayers at X ) 0.4. (C) π-A isotherms for (---) adsorbed and (O) spread19 WPImonopalmitin mixed monolayers at X ) 0.6. The πe of WPI is indicated by means of an arrow.

at the collapse point for adsorbed (Figure 4) and spread12,19 mixed films are practically equal compared that for the pure monoglyceride. This procedure and the comparison between adsorbed and spread π-A isotherms are illustrated in Figure 4B,C for two representative mass fractions of monopalmitin in the mixture. From the π-A isotherms for WPI + monopalmitin mixed films (Figure 5), it can be seen that there was a monolayer expansion as the monopalmitin concentration in the mixture was increased, especially at higher surface pressures. That is, the π-A isotherm is displaced toward higher A as the concentration of monopalmitin in the mixture increases. At surface pressures higher than that for WPI collapse, the π-A isotherm for mixed monolayers was parallel to that of monopalmitin. These data are in agreement with those deduced for spread β-lactoglobulinmonopalmitin25 and WPI + monopalmitin12,19 mixed films. For WPI + monopalmitin mixed films, the π-A isotherms for adsorbed and spread monolayers at surface pressures higher than the equilibrium surface pressure of WPI are practically coincident (Figure 4B,C), a phenomenon which may be attributed to protein displacement by the monoglyceride from the interface. At surface (25) Cornec, M.; Narsimhan, G. Langmuir 2000, 16, 1216.


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Figure 5. (A) Surface pressure-area isotherms (compression curves) for adsorbed WPI-monopalmitin mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (4) 0; (s) 0.2; (---) 0.4; (‚‚‚) 0.5; (+++) 0.6; (O) 1.0. (B) Area as a function of mass fraction of monopalmitin in the mixture (XMP) and surface pressure (mN/m): (3) 5; (4) 10; (O) 20; (0) 30. The πe of WPI is indicated by means of an arrow.

Figure 6. Surface pressure-area isotherms (compression curves) for adsorbed (A) WPI-monopalmitin and (B) WPImonoolein mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monoglyceride in the mixture (X): (4) 0; (s) 0.2; (---) 0.4; (‚‚‚) 0.5; (+++) 0.6; (O) 1.0. The molecular area was calculated on the basis that only the monoglyceride was adsorbed on the interface. The πe of WPI is indicated by means of an arrow.

pressures lower than the equilibrium surface pressure of WPI, both WPI and monopalmitin coexist at the interface and the adsorbed and spread π-A isotherms (i.e., the monolayer structures) are different (Figure 4B,C). These results suggest that at the higher surface pressures the arrangement of the monopalmitin hydrocarbon chain in mixed monolayers is practically the same in the entire WPI-monopalmitin ratio. In fact, we present in Figure 6 hypothetical π-A isotherms for mixed monolayers calculated on the basis that only monopalmitin is present at the air-water interface. It must be emphasized that, due to this assumption, in Figure 6 the area in the x axis is not the true area/unit mass of mixed film but the apparent area. For this reason the x axis in Figure 5A (for true area/unit mass of mixed film) is different from that in Figure 6 (for apparent area/unit mass of monoglyceride in the mixture). We can see that, supposing that the mixed monolayers are dominated by monopalmitin (Figure 6A), the π-A isotherms for monopalmitin and WPI-monopalmitin mixed monolayers at X > 0.2 and at surface pressures higher than that for WPI collapse are practically coincident. In contrast to the above data, π-A isotherms calculated on the basis that the mixed monolayers are dominated by WPI (data not shown) are totally different from those for pure components under all experimental conditions. The interactions between film forming components in mixed monolayers can be studied from the point of view of miscibility by means of the excess area, Aexc.26 For adsorbed WPI-monopalmitin mixed monolayers (Figure 5B), the excess area was positive at low concentrations of monopalmitin in the mixture (X = 0.2), especially at low surface pressures. These results and those deduced from π-A isotherms prove that adsorbed WPI and monopalm-

itin form a mixed monolayer at the air-water interface with few interactions between film forming components, at surface pressures lower than that for the WPI collapse (π ≈ 30 mN/m). The repulsive interactions in the mixed film decrease with the surface pressure, as the filmforming components adopt a similar liquidlike structure. However, at higher monopalmitin concentrations in the mixed film (X > 0.5) the excess area was negative, especially at low surface pressures, which proves the existence of attractive interactions between monopalmitin and WPI at the air-water interface, especially at the lower surface pressures. At the highest surface pressures, at the collapse point of the mixed film, immiscibility between monolayer forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monoglyceride monolayer (Figure 5). The fact that, upon expansion and further compression, the π-A isotherms were repetitive (data not shown) suggests that the protein reenters the mixed monolayer and supports the idea that the protein remains underneath the monoglyceride film either through hydrophobic interactions between protein and lipid or by local anchoring through the monoglyceride layer.9,19 However, for adsorbed WPI-monopalmitin mixed monolayers a first order-like phase transition was observed upon the monolayer expansion (Figure 4A) at surface pressures close to the equilibrium surface pressure of WPIswith a degenerated plateau in the π-A isotherm. Interestingly, this plateau is more evident and more extended at higher concentrations of monopalmitin in the mixture. This result suggests that the readsorption of previously displaced WPI has a kinetic character, which was not evident for spread mixed films.19 Moreover, the readsorption of previously displaced WPI is hindered as the concentration of monopalmitin in the mixture increases. The evolution with the surface pressure of BAM images (Figure 2) and relative reflectivity (Figure 7) gives

(26) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interface; Wiley: New York, 1966.

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Figure 7. Relative reflectivity (arbitrary units) as a function of surface pressure during the compression for adsorbed WPImonopalmitin mixed monolayers on buffered water at pH 7 and at 20 °C. Monopalmitin mass fraction in the mixture: (A) 0.3; (B) 0.5. The maximum reflectivity for monopalmitin (continuous line) and adsorbed WPI (discontinuous line) pure monolayers are included in plots A and B. Shutter speed (s): (O) 1/50; (4 and 3) 1/250.

Figure 8. (A) Surface pressure-area isotherms (compression curves) for adsorbed WPI-monoolein mixed monolayers on buffered water at pH 7 and at 20 °C. Mass fraction of monopalmitin in the mixture (X): (4) 0; (s) 0.2; (---) 0.4; (‚‚‚) 0.5; (+++) 0.6; (O) 1.0. (B) Area as a function of mass fraction of monoolein in the mixture (XMO) and surface pressure (mN/ m): (3) 5; (4) 10; (O) 20; (0) 30. The πe of WPI is indicated by means of an arrow.

complementary information, at a microscopic level, on the structural characteristics and interactions of adsorbed WPI-monopalmitin mixed monolayers, as deduced from π-A isotherms (Figure 5). Briefly, the results reported here suggest that, in WPI-monopalmitin mixed films, islands of protein and monoglyceride do exist at the airwater interface but the interactions between them depend on the surface pressure and the composition of the mixed film. At surface pressures lower than that for WPI collapse a mixed monolayer of monopalmitin and WPI may exist (Figure 2d) with small domains of monopalmitin (with a liquid condensed structure at π > 5 mN/m) uniformly distributed on the homogeneous WPI layer. The circular dark domains of liquid condensed monopalmitin were more numerous as the surface pressure increased (Figure 2e). At surface pressures higher than that for WPI collapse, the mixed monolayers were practically dominated by monopalmitin molecules. That is, at higher surface pressures, collapsed WPI residues (bright region) may be displaced from the interface by monopalmitin molecules. This characteristic squeezing out phenomenon was observed in BAM images (Figure 2f) with liquid-condensed domains of monopalmitin (dark circle) floating over a sublayer of collapsed residues of WPI (bright region). However, the topography of the mixed film also proves the existence of some degree of interactions between filmforming components with regions of collapsed monopalmitin (dark region) and those of interacting monopalmitin and WPI (small bright regions of collapsed WPI and monopalmitin) separated by a shear plane observed during the monolayer compression (Figure 2g). Another topographical characteristic of the adsorbed film (not observed in spread mixed film19) was the presence of short fractures in the monolayer at the collapse point of the mixed film (Figure 2h). These results have shown that over the overall range of existence of the mixed film the monolayer presents some heterogeneity due to the fact that domains of

monopalmitin and WPI residues were present during the monolayer compression-expansion cycle, giving I peaks of collapsed WPI with high relative film thickness (Figure 7). Interestingly, the reflectivity of the mixed film at higher surface pressures was higher than that for pure WPI or pure monopalmitin (Figure 7). These results strength the hypothesis that for adsorbed WPI + monopalmitin mixed films some degree of attractive interactions between filmforming components do exist at a microscopic level, giving a mixed film with greater thickness compared with those of pure components. Structural and Topographical Characteristics of WPI-Monoolein Mixed Monolayers Adsorbed at the Air-Water Interface. The structural characteristics of adsorbed WPI-monoolein mixed monolayers were essentially different from those of monopalmitin in the mixture, as deduced from π-A isotherms (Figure 8A) and excess area (Figure 8B). Briefly, as expected,19 WPImonoolein mixed films (Figure 8) at surface pressures lower than that for WPI collapse (π = 30 mN/m) adopt a liquidlike-expanded structure, as for pure components. There was a monolayer expansion due to the presence of monoolein in the mixture. At surface pressures higher than that for WPI collapse, the π-A isotherm for mixed monolayers was practically parallel to that of monoolein. At these experimental conditions, the π-A isotherms for WPI-monoolein mixed monolayers (Figure 8A) and the hypothetical π-A isotherms for mixed monolayers calculated on the basis that only monoolein was present at the air-water interface (Figure 6B), at X > 0.2, were practically coincident. From the point of view of miscibility (Figure 8B), the excess area was practically zero at low concentrations of monoolein in the mixture, but it fell to negative values at higher monoolein concentrations in the mixed film, especially at low surface pressures. These results prove the existence of attractive interactions between monoolein and WPI at the air-water interface,


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Figure 10. Displacement surface pressure of WPI adsorbed film by (∆) monopalmitin and (O) monoolein spread at the airwater interface as a function of mass fraction of monoglyceride in the mixture. The horizontal lines represent the equilibrium surface pressure of pure WPI (s, πe WPI), monoolein (- ‚ -, πe MO), and monopalmitin (---, πe MP) films at 20 °C and at pH 7.

Figure 9. Relative reflectivity (arbitrary units) as a function of surface pressure during the compression for adsorbed WPImonoolein mixed monolayers on buffered water at pH 7 and at 20 °C. Monoolein mass fraction in the mixture: (A) 0.25; (B) 0.5. The maximum reflectivity for monoolein (continuous line) and adsorbed WPI (discontinuous line) pure monolayers are included in plots A and B. Shutter speed (s): (O) 1/50; (4 and 3) 1/250.

especially at the lower surface pressures. At the highest surface pressures, at the collapse point of the mixed film, immiscibility between monolayer forming components is deduced due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monoolein monolayer (Figure 8A). BAM images for adsorbed WPI-monoolein mixed monolayers (Figure 2) were different from those described above for adsorbed WPI-monopalmitin mixed monolayers (Figure 2). In fact, at surface pressures lower than that for WPI collapse (π ≈ 30 mN/m), the topography of pure components and the mixed monolayer was practically identical because in this region both components and the mixed monolayer form an isotropic (homogeneous) monolayer without any difference in the domain topography (Figure 2a). At surface pressures near and after WPI collapse BAM images (Figure 2b) demonstrated that monoolein and WPI molecules adopted an isotropic structure in the mixed monolayer with some white regions (Figure 2b) which correspond to the presence of a thicker WPI collapsed monolayer. At the higher surface pressures, and especially at the collapse point, the topography of the mixed monolayer was dominated by the presence of small domains of collapsed WPI (bright regions) and monoolein (dark regions) at the interface (Figure 2i). These results and those for WPI + monopalmitin mixed films prove that at a microscopic level (Figure 2) (i) some degree of interactions between monoglycerides and WPI in adsorbed films did exist at the air-water interface and (ii) a monoglyceride monolayer spread on a previously adsorbed WPI film was unable to completely displace WPI molecules from the air-water interface, even at the highest surface pressures. The I-π plots for the adsorbed WPI-monoolein mixed monolayers are shown in Figure 9. From these results some differences can be deduced as compared to those recorded for adsorbed WPI-monopalmitin mixed mono-

layers (Figure 7). Briefly, at the lower concentration of monoolein in the mixture (Figure 9A) intensity of some spots was the higher as for pure WPI. At the higher concentration of monoolein in the mixture (Figure 9B) the I-π plots were practically the same as those for pure monoolein, over the overall range of surface pressures, which strengthens the conclusion that monoolein, with the lower relative thickness, predominates at the interface. The displacement of WPI by monoglyceride from the air-water interface depends on the protein-monoglyceride system. From the hypothetical isotherms shown in Figure 6, we define a displacement surface pressure (πd) as the minimum surface pressure above which the π-A isotherms for monoglyceride and monoglyceride-protein mixed monolayers are coincident. Thus, at low πd protein displacement by the monoglyceride is facilitated. In Figure 10 we show the πd as a function of the mixture composition for different WPI-monoglyceride mixed films. The WPI displacement is easier for monopalmitin than for monoolein. In fact, at X > 0.2 the πd value for WPImonopalmitin mixed films is lower than those for WPImonoolein mixed films, especially at higher monoglyceride concentrations in the mixture. WPI displacement by monopalmitin at X ) 0.6 is deduced even for surface pressures close to that of WPI collapse (at π = πe). However, for monoolein higher surface pressures are necessary for a quantitative displacement of WPI from the interface, in particular at lower monopalmitin concentrations in the mixture. In fact, for WPI-monoolein adsorbed mixed films at X ) 0.2 the value of πd is practically the surface pressure of monoolein at the collapse point (which coincides with its πe). That is, the lower surface activity of monoolein justifies the idea that this lipid has a lower capacity than monopalmitin for protein displacement.19 Finally, the capacity of a monoglyceride for WPI displacement from the air-water interface is easier for adsorbed (Figure 10) than for spread19 monolayers, especially for the WPImonopalmitin system. Conclusions From the results derived from these experiments it can be concluded that interactions, miscibility, and displacement of WPI by monoglycerides in adsorbed mixed monolayers at the air-water interface depend on the particular WPI-monoglyceride system. WPI-monoglyceride mixed monolayers form a practically immiscible monolayer at the air-water interface, with repulsive interactions between film-forming components, at low concentrations of monoglyceride in the mixture, and at

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surface pressures lower than that for the WPI collapse (π ≈ 30 mN/m). However, at higher monoglyceride concentrations attractive interactions between WPI and monoglycerides were observed, especially at the lower surface pressures. At higher surface pressures the collapsed WPI is displaced from the interface by monoglyceride (either monopalmitin or monoolein). The WPI displacement by monoglycerides is practically total at the highest surface pressure, at the collapse point of the mixed monolayer, especially for WPI-monoglyceride mass fractions higher than 0.2. The immiscibility between monolayer forming components is also deduced at the highest surface pressures, due to the fact that the collapse pressure of mixed monolayers is similar to that of pure monopalmitin or monoolein monolayer. From these studies emerges the idea that monopalmitin has a higher capacity than monoolein for the displacement of WPI from the airwater interface. The lower surface activity of monoolein justifies the idea that this lipid has a lower capacity than monopalmitin for protein displacement. The displacement of WPI from the air-water interface by monoglycerides has been observed recently for spread mixed monolayers using the same experimental techniques.19 Some differ-


ences between adsorbed and spread mixed films are (i) the interactions between film-forming components are higher for adsorbed than for spread mixed films, (ii) the adsorbed films are more segregated than spread films, and (iii) the collapsed WPI domains in adsorbed films are smaller than for spread films. All these phenomena are a consequence of the increased interactions between components at the interface in the former. (iv) The readsorption of previously displaced WPI is slower for adsorbed than for spread films. As for spread monolayers,19 the displacement of WPI by monoglyceride from adsorbed mixed monolayers follows successive stages according to an orogenic mechanism.27-29 Acknowledgment. This research was supported by the CICYT through Grant AGL2001-3843-C02-01. LA036190J (27) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. J. Colloid Interface Sci. 1999, 210, 157. (28) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Langmuir 2000, 16, 8176. (29) Gunning, A. P.; Mackie, A. R.; Wilde, P. J.; Morris, V. J. Langmuir 1999, 15, 4636.

Structural and Topographical Characteristics of Adsorbed WPI and

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