Structural-Dilatational Characteristics Relationships of Monoglyceride

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Structural-Dilatational Characteristics Relationships of Monoglyceride Monolayers at the Air-Water Interface Juan M. Rodrı´guez Patino,* Cecilio Carrera Sańchez, Ma Rosario Rodrı´guez Ninõ, and Marta Cejudo Fernańdez Departamento de Ingenierıá Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, C/. Prof. Garcıá Gonza´ lez, s/nu´ m. 41012 Sevilla, Spain Received December 11, 2000. In Final Form: April 3, 2001 We have studied the effect of monolayer structure on dilational characteristics (surface dilatational modulus and its elastic and viscous components) of monoglyceride monolayers (monoolein and monopalmitin) spread on the air-water interface, at 20 °C and at pH 5 and 7. The stress response to compressionexpansion sinusoidal deformation of the interface in a modified Wilhelmy-type trough with two oscillating barriers was measured as a function of deformation amplitude (within the range of 1-20% of the initial area), frequency (within the range of 1-300 mHz), and superficial density (within the range of 1-3.5 mg/m2). The same experimental device coupled with Brewster angle microscopy makes it possible to determine the structure and morphology of the monolayer. The monolayer structure and, especially, the conditions at which the monolayer collapses determine the viscoelastic behavior of the monolayer and the linear response of the stress to area deformation. The nonlinear viscoelastic behavior of the interface has been associated with the monoglyceride monolayer collapse. It was found that the dilatational modulus is not only determined by the interactions between spread molecules (which depend on the surface density) but that the structure of the spread monolayer also plays an important role.

Introduction The stability and mechanical properties of dispersed systems depend on the way in which the constituent particles and macromolecules interact.1-3 To stabilize food emulsions and foams, emulsifiers (lipids and proteins) must be placed at the interface so they can form a film around droplets or bubbles, respectively.1 The optimum use of emulsifiers depends on knowledge of their interfacial physicochemical characteristicsssuch as surface activity, structure, stability, superficial viscosity, etc.sand the kinetics of film formation at fluid-fluid interfaces.4-7 The lipids and proteins at the interface reduce the surface or interfacial tension between the phases and form a continuous film at the interface via complex intermolecular interactions which impart structural rigidity1,7 and, thus, stabilize1,3 and improve the formation of food emulsions and foams.8-10 As a result of systematic studies of model systems, the colloidal and intermolecular interactions themselves are now becoming reasonably well understood. The question that emerges is whether there is a direct link between interfacial characteristics and emulsion and foam formation and stability. Although some correlation has been * To whom the correspondence should be addressed. Phone: +34 95 4557183. Fax: +34 95 4557134. E-mail: [email protected]. (1) Halling, P. J. CRC Crit. Rev. Food Sci. Nutr. 1981, 15, 1. (2) Damodaran, S. Adv. Food Nutr. Res. 1990, 34, 1. (3) Dickinson, E. An Introduction to Food Colloids; Oxford University Press: Oxford, U.K., 1992. (4) Phillips, P. M. Food Technol. 1981, 35, 50. (5) Dickinson, E.; Stainsby, G. Colloids in Food; Applied Science: London, 1982. (6) Dickinson, E.; McClements, D. J. Advances in Food Colloids; Blackie: Glasgow, 1995. (7) Murray, B. S.; Dickinson, E. Food Sci. Technol. Inst. 1996, 2, 131. (8) Larsson, K. Lipids: Molecular Organization, Physical Functions and Technical Applications; The Oily Press: Dundee, Scotland, 1994. (9) Walstra, P.; Smulders, I. In Food Colloids: Proteins, Lipids and Polysaccharides; Dickinson, E., Bergensthal, B., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1997; p 367. (10) Walstra, P. Chem. Eng. Sci. 1993, 48, 333.

established between interfacial characteristics (especially surface rheological properties) of pure and mixed emulsifiers and capacity and stability of food emulsions and foams,11-15 the detailed relationships between surface rheology and colloidal stability is poorly understood.11 On the other hand, the development of intermolecular associations at the interface leads to alterations in surface properties that have measurable rheological consequences. That is, surface rheology is very sensitive to the structural characteristics of surfactants at interfaces.16-20 In previous studies from our laboratory, we have demonstrated that the formation, structural characteristics, stability, and morphology of food emulsifiers (monoglycerides, diglycerides, and proteins) depend on the interfacial and subphase composition as well as the environmental conditions.21,22 In this work, we comple(11) Dickinson, E. Colloids Surf. B 1999, 15, 161. (12) Murray, B. S. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, The Netherlands, 1998; p 179. (13) van Kelsbeek, H. K. A. I.; Prins, 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 91. (14) Bos, M.; Nylander, T.; Arnebrant, T.; Clark, D. C. In Food Emulsifiers and their Applications; Hasenhuettl, G. L., Hartel, R. W., Eds.; Chapman & Hall: New York, 1997; p 95. (15) Clark, D. C.; Wilde, P. J. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, The Netherlands, 1998; p 267. (16) Kajiyama, T.; Morotomi, N.; Uchida., M.; Oishi, Y. Chem. Lett. 1989, 1047. (17) Noskov, B. A.; Zudkova, T. U. J. Colloid Interface Sci. 1994, 170, 1. (18) Kizling, J.; Stenhius, P.; Ericsson, J. C.; Ljunggren, S. J. Colloid Interface Sci. 1995, 171, 162. (19) Ruı´z, D. M.; Gonza´lez, N. I.; Rodrı´guez Patino, J. M. Ind. Eng. Chem. Res. 1998, 37, 936. (20) Rodrı´guez Ninõ, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Ind. Eng. Chem. Res. 1996, 35, 4449. (21) Rodrı´guez Patino, J. M.; Rodrı´guez Ninõ, Ma. R. Colloids Surf. B 1999, 15, 235. (22) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Ninõ, Ma. R.; Cejudo, F. M. In Food Colloids 2000: Fundamental of Formulations; Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2001, p 22.

10.1021/la0017375 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/02/2001

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ment previous studies by investigating the surface dilatational characteristics of accepted emulsifiers in food formulations (monopalmitin and monoolein). We have carried out experiments with different devices (Wilhelmytype film balances, Brewster angle microscopy, and surface dilatational rheology) to characterize the monolayer under dynamic conditions and at equilibrium. Most of the surface dilatational measurements were made on surfactants in solutions, with the surface rheological characteristics being interpreted in terms of surfactant adsorption and desorption at the interface. However, experiments on insoluble surfactants films (i.e., spread at the air-water interface) are scarce.16-20 The results derived from this experiment show that there exists a close relationship between dilatational properties and monolayer structural characteristics as a function of amplitude and frequency of the area deformation and superficial density. Emulsifierbased foods such as traditional foods, low-fat products (especially without concentrated emulsions), instant foods, or alcohol-free and low-alcohol beverages are some examples of food systems in which the data obtained in this research are of practical interest.23-25 Materials and Methods Materials. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODANR PA 90) and 1-mono(cis-9-octadecanoyl)glycerol (monoolein, RYLO MG 19) were supplied by Danisco Ingredients with over 95-98% of 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. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). To adjust subphase pH, buffer solutions were used. An acetic acid/ sodium acetate aqueous solution (CH3COOH/CH3COONa) was used to achieve pH 5, and a commercial buffer solution called trizma [(CH2OH)3CNH2/(CH2OH)3CNH3Cl] was used to achieve pH 7. All of these products were supplied by Sigma (>99.5%). Ionic strength was 0.05 M in all of the experiments. Methods. Surface Film Balance. Measurements of the surface pressure (π) versus average area per molecule (A) were performed on a fully automated Wilhelmy-type film balance (KSV 3000, Finland) as described elsewhere.19 The monoglyceride solutions were spread on the subphase by means of a micrometric syringe at 20 °C. Aliquots of 250 µL (6.1 × 1016-7.63 × 1016 molecules) were spread in each experiment. The mean deviation was within ( 0.1 mN/m for surface pressure and ( 0.125 × 10-3 m2/mg for the area. The subphase temperature was controlled by water circulation from a thermostat, within an error range of ( 0.5 °C. The experiments were carried out at 20 °C. The compression rate was 3.3 cm/min. Each isotherm was measured at least four times. The reproducibility of the surface pressure results was better than ( 0.4 mN/m. Brewster Angle Microscope (BAM). A commercial Brewster angle microscope, BAM2, manufactured by NFT (Go¨ttingen, Germany) was used to study the morphology of the monolayer. The BAM was positioned over the film balance on a specially designed frame structure, which allows easy movement of the BAM along the length of the film balance. The location of BAM along the film balance makes it possible to visualize any inhomogeneity or structural change in the overall film. Further characteristics of the device and operational conditions have been described elsewhere.26,27 These measurements were performed during continuous compression and expansion of the monolayer. (23) Als, G.; Krog, N. In Proceedings of the World Conference on Oleochemicals into the 21st Century; Applewhite, T. E., Ed.; American Oil Chemists’ Society: Champaign, IL, 1991; p 67. (24) Krog, N. J.; Riison, T. H.; Larsson, K. In Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1985; Vol. 2, p 321. (25) Friberg, S. E., Larsson, K., Eds.; Food Emulsions; Marcel Dekker: New York, 1997. (26) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Ninõ, Ma. R. Langmuir 1999, 15, 2484.

Rodrı´guez Patino et al. Surface Dilatational Rheology. There are many experimental devices for measuring dilatational rheology.12,28-32 Convenient techniques for measuring surface dilatational rheology are derived from the longitudinal wave method.33 Trough methods are preferable for insoluble films at the air-water interface. In these methods, the area change applied to the film may be oscillatory, a step change, or a continuous expansion-compression. The mechanically generated longitudinal or capillary wave, such as that obtained for the movement of the barriers in a Langmuir trough containing the film, produces a response of surface tension which is monitored by a probe (i.e., a Wilhelmy plate) some distance away from the barrier.34,35 With trough methods, it has been found that the value of surface tension measured can depend on the position and orientation of the Wilhelmy plate relative to the moving barriers.36,37 The fact that the interfacial tension is a tensorial quantity has been considered by Wijmans and Dickinson in the simulation of the surface rheology of protein adsorbed layers.38 Recently, Petkov et al.39,40 make use of the uniaxial deformation created by the moving barrier in a trough for measuring the surface shear elasticity modulus together with the dilatational modulus of gellike protein layers. Thus, good experimental practice in a Langmuir trough requires that films always be compressed symmetrically relative to the position of measurement of surface tension. Particularly with films showing appreciable viscous behavior (such as that for proteins), the resultant expansion may not be uniform across the film.37,41 Another problem here is that the amplitude of the area variations generated by the barrier may be substantially damped when the area disturbance reaches the probe. However, the Wilhelmy plate can be situated anywhere on the surface if the deformation of the interface is uniform, as was observed with many surfactants adsorbed at the air-water interface.34,42,43 Alternative methods have been developed to avoid this problem, in which isotropic compression-expansion allows isotropic deformation in surface area.32,43,44 To obtain surface rheological parametersssuch as surface dilatational modulus, elastic and viscous components, and loss angle tangentsa modified Wilhelmy-type film balance (KSV 3000) was used (Figure 1). In this method, the surface is subjected to small periodic sinusoidal compressions and expansions by means of two oscillating barriers at a given frequency (ω) and amplitude (∆A/A) and the response of the surface pressure is monitored (π). Surface pressure was directly measured by means of two roughened platinum plates that can be situated anywhere on the surface between the two barriers. With this (27) Rodrı´guez Patino, J. M.; Carrera, S. C.; Rodrı´guez Ninõ, Ma. R. Food Hydrocolloids 1999, 13, 401. (28) Kretzschmar, G.; Miller, R. Adv. Colloid Interface Sci. 1991, 36, 65. (29) Prins, A. In New Physico-Chemical Techniques for the Characterization of Complex Food systems; Dickinson, E., Ed.; Blackie A & P: Glasgow, Scotland; 1995, p 214. (30) Dukhim, S. S.; Kretzschmar, G.; Miller, R. Dynamic of Adsorption at Liquid Interfaces. Theory, Experiment, Application; Elsevier: Amsterdam, The Netherlands, 1995. (31) Franses, E. I.; Basaran, O. A.; Chag, C. H. Curr. Opin. Colloid Interface Sci. 1996, 1, 296. (32) Murray, B. S.; Nelson, P. V. Langmuir 1996, 12, 5973. (33) Lucassen, J.; van den Tempel, M. Chem. Eng. Sci. 1972, 27, 1283. (34) Ting, L.; Wasan, D. T.; Miyano, K. J. Colloid Interface Sci. 1985, 107, 345. (35) Bonfillon, A.; Langevin, D. Langmuir 1993, 9, 2172. (36) Malcolm, B. R. J. Colloid Interface Sci. 1985, 104, 520. (37) Peng, J. B.; Barnes, G. T. Colloids Surf. A 1995, 102, 75. (38) Wijmans, C. M.; Dickinson, E. Langmuir 1998, 14, 7278. (39) Petkov, J. T.; Gurkov, T. D.; Campbell, B. E.; Borwankar, R. P. Langmuir 2000, 16, 3703. (40) Petrov, J. T.; Gurkov, T. D.; Campbell, B. E.; Borwankar, R. P. In Food Colloids 2000: Fundamentals of Formulations; Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2000. In press. (41) Bois, A. G.; Panaiotov, I. J. Colloid Interface Sci. 1995, 170, 25. (42) Lucassen, J.; van den Tempel, M. J. Collid Interface Sci. 1972, 41, 491. (43) Lucassen, J.; Giles, D. J. Chem. Soc., Faraday Trans. 1 1975, 71, 217. (44) Kokelaar, J. J.; Prins, A.; de Gee, M. J. Colloid Interface Sci. 1991, 146, 507.

Monoglyceride Monolayers at Air-Water Interfaces

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Figure 1. Schematic representation of the surface film balance adapted for monolayer compression (determination of π-A isotherm), visualization of monolayer morphology, and sinusoidal oscillation of the area showing typical correlation of the sinusoidal surface pressure response due to a sinusoidal deformation in the interfacial area. device, an isotropic dilatational deformation of the surface, without interference of shear, can be achieved, as is demonstrated by the results derived for monoglyceride spread monolayers at the air-water interface. In fact, the sinusoidal response in surface pressure due to sinusoidal area deformation was both the same in the two barriers and independent of the position of the barriers along the length of the film balance. In addition, the results obtained in this work for monoglyceride monolayers are essentially the same as those obtained with the ring trough method in which isotropic compression and expansion is ensured, because the surface area deformation is caused by moving a cylindrical glass ring vertically in the liquid surface.20 The surface dilatational modulus derived from the change in surface tension (dilatational stress), σ, (eq 1) resulting from a small change in surface area (dilatational strain), A, (eq 2) may be described by eq 3:33

σ ) σ0 sin(ωt + θ)

(1)

A ) A0 sin(ωt)

(2)

E)

dπ dσ )dA/A d ln A

(3)

where σ0 and A0 are the strain and stress amplitudes, respectively, θ is the phase angle between the stress and strain, t is the time, π ) σο - σ is the surface pressure, and σο is the surface tension in the absence of monoglyceride. The dilatational modulus is a complex quantity and is composed of real and imaginary parts (eq 4). The real part of the dilatational modulus or storage component is the dilatational elasticity, Ed ) |E| cos θ. The imaginary part of the dilatational modulus or loss component is the surface dilatational viscosity, Ev ) |E| sin θ. The ratio σ0/A0 is the absolute modulus |E|, a measure of the total unit material dilatational resistance to deformation (elastic + viscous). For a perfectly elastic material, the stress and strain are in phase (θ ) 0) and the imaginary term is zero. In the case of a perfectly viscous material, θ ) 90° and

Figure 2. Surface pressure-area isotherm (compression curve) for (A) monopalmitin and (B) monoolein monolayers spread at the air-water interface at pH 5 (discontinuous line) and pH 7 (continuous line). Temperature: 20 °C. the real part is zero. The loss angle tangent can be defined by eq 5. If the film is purely elastic, the loss angle tangent is zero.

E ) (σ0/A0)(cos θ + i sin θ) ) Ed + iEv

(4)

tan θ ) Ed/Ev

(5)

The application of the method for a typical experiment is shown in Figure 1, where the fit of experimental response in surface pressure due to a sinusoidal variation in area makes it possible to determine E and θ and then the surface dilatational and viscous components.

Results and Discussion Structure and Morphology of Spread Monoglycerides at the Air-Water Interface. Monopalmitin Monolayers. Results derived from π-A isotherms in the Wilhelmy-type trough are in good agreement with those obtained in the Langmuir-type trough with the same monoglyceride spread on similar subphases at pH 5 and 7.22,26 Briefly, different structures can be deduced for the monopalmitin monolayer as a function of surface pressure (Figure 2A). Liquid expanded phase (LE; at π < 5 mN/m), liquid-condensed (LC) structure (at 5 < π < 32 mN/m), solid (S) structure (at 32 < π < 53 mN/m), and finally collapse at a surface pressure of about 53.1 mN/m were observed. BAM images (Figure 3) reveal that monopalmitin monolayers present a rich polymorphism as a function of surface pressure. In fact, a homogeneous LE phase is present during compression at π < 5 mN/m (Figure 3A). The morphology of the monopalmitin monolayer at π > 5 mN/m (Figure 3B) shows circular LC domains from the homogeneous ambient phase with an LE structure. The LC domains grow in size, and the monolayer is covered with LC domains as the surface pressure is increased (Figure 3C). At the highest surface pressure, the LC domains are so closely packed that they occupy the entire

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Figure 3. Visualization of monopalmitin and monoolein monolayers by Brewster angle microscopy at 20 °C and at pH 5. (A) LC domains of the monopalmitin monolayer at 4 mN/m, (B) LC domains of the monopalmitin monolayer at 20 mN/m, (C) LC domains of the monopalmitin monolayer at 30 m/Nm, (D) solid morphology of the monopalmitin monolayer at 50 mN/m, (E) fracture in a collapsed monopalmitin monolayer, and (F) LE morphology of the monoolein monolayer at π < 45 mN/m. The horizontal direction of the image corresponds to 630 µm, and the vertical direction corresponds to 470 µm.

field of view and the contrast vanishes suddenly, a typical morphology of a solid structure (Figure 3D). Finally, at the collapse point, the presence of monolayer fractures can be observed (Figure 3E) in different zones by the movement of BAM along the length of the film balance. After expansion, the monolayer undergoes a break up of the collapsed structure and the LC and LE phases were detected with decreased surface pressures. Monoolein Monolayers. In contrast with the monopalmitin monolayer, the monoolein monolayer (Figure 2B) presents only the liquid expanded structure and collapse at the equilibrium surface pressure (πe ≈ 45.7 mN/m). BAM images corroborate that only the homogeneous LE phase is present during the compression and expansion of a monoolein monolayer (Figure 3F). From the observation with BAM along the film balance, no fractures were visualized after the monoolein collapse, which demonstrates that the collapse of monopalmitin and monoolein monolayers is quite different and these differences are associated with different structures of these lipids at the surface pressures corresponding to πe. Dilatational Characteristics of Monopalmitin Monolayers at the Air-Water Interface. Effect of Amplitude. The surface viscoelastic properties of monopalmitin monolayers spread on the air-water interface

as a function of deformation amplitude and at pH 7 (as an example) are shown in Figures 4 and 5, for 20 mN/m and at the collapse point, respectively. Monopalmitin monolayers at pH 5 behave in a similar way (data not shown). As can be seen, the surface dilatational modulus and the elastic component increased by varying the deformation between 0.1 and 3% and reached a maximum value at an amplitude of about 3%, whereas the viscous component and the loss angle tangent decreased to a minimum value in the same deformation amplitude range, no matter what the deformation frequency or surface pressure. This phenomenon may be associated with large errors in π measurements due to the fact that for lower amplitudes πamplitude is of the same order of magnitude than the reproducibility of the surface pressure measurements. However, we do not reject the fact that at lower surface deformation the wave is damped along the length of the trough. At deformation amplitudes higher than 3%, the amplitude effect on surface dilatational properties depends on both the amplitude and frequency of deformation. At a surface pressure of 20 mN/m and at a frequency of 20 mHz (Figure 4A), the surface dilatational properties did not depend on the deformation. That is, at these experimental conditions monopalmitin monolayers present


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Figure 4. Effect of amplitude in area deformation on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monopalmitin monolayers spread on aqueous solutions at pH 7 and at 20 °C. Surface pressure: 20 mN/m. Frequency (mHz): (A) 20, (B) 50, and (C) 100.

Figure 5. Effect of amplitude in area deformation on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monopalmitin monolayers spread on aqueous solutions at pH 7, at the collapse point, and at 20 °C. Frequency (mHz): (A) 20, (B) 50, and (C) 100.

linear viscoelastic behavior. However, at higher deformation frequencies (Figure 4 parts B and C), and especially for the monopalmitin monolayer at the collapse point (Figure 5), the surface dilatational modulus and its elastic component decreased with deformation amplitude. Moreover, at higher amplitudes than 3%, the dilatational viscous component and the loss angle tangent did not depend on the deformation amplitude. On the other hand, it can be seen that at every deformation amplitude (i) the values for the surface dilatational modulus are very similar to those for the dilatational elasticity and (ii) the dilatational viscosity and the loss angle tangent values are low and practically zero. As a consequence of this behavior, it can be established that the surface dilatational characteristics of monopalmitin monolayers are essentially elastic within the range of frequencies shown in Figures 4 and 5, as will be discussed in the next section. The deformation dependence on dilatational modulus may be related to the breaking of intermolecular interactions in monopalmitin monolayers. This breaking of bonds seems to be determined not only by the extent of deformation but by the rate of deformation as well, because the effect of deformation is higher at increasing frequencies. To check whether the level of deformation can produce any effect on the degree of intermolecular interactions and, as a consequence, on the monolayer structure, we present in Figure 6 the results from a compression-

expansion cycle for a monopalmitin monolayer at 20 mN/ m, at the collapse point, and at a deformation frequency of 50 mHz. Two representative deformation amplitudes have been selected (5% and 20%), which correspond to the maximum deformation studied and to that close to the value at which the modulus is maximum (Figures 4 and 5). At 20 mN/m and at a deformation amplitude of 5%, the liquid-condensed monolayer structure is constant during the compression-expansion cycle (Figure 6A). The results indicate that the breaking of intermolecular interactions, mainly between LC domains and, most probably, with a change in domain size during the compression-expansion cycle (according to BAM images, see Figure 3 parts B and C), is reversible. Thus, the level of deformation produces a linear response of the system. This finding supports the hypothesis that for small deformations the effect of the compression or expansion of the interface is directly linked to fluctuations in LC domain size in relation to the equilibrium value. The change between the monopalmitin monolayer structure with deformation is practically reversible and independent of the rate of deformation, which agrees with the reproducibility26 and with the absence of any hysteresis in π-A isotherms after continuous compression-expansion cycles up to 20 mN/m (data not shown). In addition, at these experimental conditions, the good fit between eq 1 and experimental

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Figure 6. Dynamic surface pressure response to a sinusoidal change in area at a frequency of 50 mHz for monopalmitin monolayers spread on aqueous solutions at pH 5 as a function of the amplitude of area deformation: (O) 5% and (∆) 20%. (A) Surface pressure: 20 mN/m. (B) Monolayer collapse point.

points describes the stress response during the compression-expansion cycle. At the same deformation amplitude (5%) and for the monopalmitin monolayer at the collapse point, a change between LC and S structures is produced during the compression-expansion cycle (Figure 6B). That is, a second-order phase transition takes place and LC domains merge toward a solid structure with the compression and then this structure relaxes back to LC domains with the monolayer expansion (see Figure 3 parts C and D). As for a surface pressure of 20 mN/m, the good fit between eq 1 and experimental points describes the stress response during the compression-expansion cycle for monopalmitin monolayer at the collapse point. The effect of high deformation on the monopalmitin monolayer structure is quite different (Figure 6). It can be seen that, at 20 mN/m and for a deformation amplitude of 20%, monopalmitin monolayers present a second orderphase transition between LC and S structures and then collapse and, finally, an “over-collapse” is produced during the compression-expansion cycle. As a consequence of these drastic structural transformations, the stress response during the LC to S transition (between 32.5 and 42.5 mN/m) and, especially, during the monolayer “overcollapse” (at π > 53 mN/m) losses the sinusoidal symmetry and the fits of experimental stress data to sinusoidal area deformation are worse than for lower deformations (Figure 6). It should be noticed that the stress response during the expansion shows a kink after the monolayer collapse. These findings support the hypothesis that for high deformations the effect of the compression or expansion of the interface is directly linked to irreversible breaking


Figure 7. Effect of frequency on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monopalmitin monolayers spread on aqueous solutions at pH 7 and at 20 °C. (A) Surface pressure: 20 mN/m. (B) Monolayer collapse point. Amplitude: 5%.

of intermolecular interactions within the time of the experiment, because the modulus decreases with increasing deformations. From a structural point of view, we attribute the nonlinear response of the system, with the modulus dependent on the deformation, to monolayer collapse. In fact, when a second-order phase transition takes place during the compression-expansion cycle, sinusoidal symmetry and good fits of experimental stress data according to eq 1 characterize the linear response of the system. However, as the monopalmitin monolayer collapses, crystalline-collapse nuclei and monolayer fracture (Figure 3E) may be the causes of the irreversibility of the system during the compression-expansion cycle. We do not reject the existence of other relaxation phenomena (such as monolayer molecular loss) within the time of the experiment.45 The effect of the rate of deformation on the nonlinear behavior of the system (Figures 4 and 5) supports this hypothesis, as will be discussed in the next section. Effect of Frequency. Changes in surface dilatational properties for the monopalmitin monolayer at pH 7, as a function of frequency of oscillation over a range of 1-300 mHz at two representative monolayer structural characteristics (at 20 mN/m and at the collapse point), are illustrated in Figure 7. We did not observe any influence of pH on surface rheological parameters (data not shown). It can be seen that (i) the dilatational modulus increased slightly with the frequency, especially at 20 mN/m, (ii) the dilatational modulus and its elastic component are essentially the same at frequencies lower than 50 mHz. However, significant differences between both rheological parameters were observed at frequencies higher than 50 mHz, mainly because of the decrease of the elastic (45) Carrera, S. C.; Rodrı´guez Ninõ, Ma. R.; Rodrı´guez Patino, J. M. Colloids Surf. B 1999, 12, 175.


component at increasing frequencies, and (iii) the value of the viscous component increased with the frequency and exceeded that of the elastic component at higher frequencies (ω > 200 mHz). From these results, it can be concluded that monopalmitin monolayers present rheological behavior in dilatational conditions that is essentially elastic at low frequencies (ω < 50) and viscoelastic at higher frequencies (ω > 50). As a consequence of the viscoelastic behavior, the loss tangent angle increased with frequency (Figure 7). This behavior was observed with the same monoglyceride under dilatational deformation in a ring trough20 and in a Langmuir film balance.19 These findings should be associated with the effect of the rate of deformation on the structure and stability of the monopalmitin monolayer. Relaxation of the surface pressure by diffusion is the most common relaxation mechanism in soluble surfactants and proteins.30,33,46-49 However, for insoluble polar lipids, other relaxation mechanism should be more operative, as a function of surface pressure and the time scale considered.45,50-52 In fact, the viscoelastic behavior observed for monopalmitin monolayers in the frequency range of 1-10 mHz should be associated with desorption of monolayer molecules toward the subphase at 20 mN/m (Figure 7A) and with desorption and/or monolayer collapse at the collapse point (Figure 7B). These relaxation processes, especially during the first step of dissolution toward the subsurface region or nucleation , require a time of the same order of magnitude as the scale of the oscillation, in our experiments between 16.7 and 1.7 min.45,50-52 At higher surface frequencies (50 < ω < 200 mHz), the viscoelastic behavior of the monopalmitin monolayer is more complex and should be associated with formation/destruction of LC domains coupled with LC-S phase transition and the formation/destruction of 3-D collapse structures (with the formation of crystalline nuclei and the existence of monolayer fractures). We have observed recently by means of BAM images (data not published) that the time required for these relaxation mechanisms is of the same order of magnitude as the time scale of the deformation. At higher frequencies (ω > 200 mHz), the above-mentioned relaxation phenomenon does not follow the sinusoidal deformation (data not shown). Thus, shear effects between monolayer domains and/or collapsed fragments and the trough wall or the Wilhelmy plate should have a role, as was pointed out recently by means of simulation38 and experimentally.39,40 Effect of Surface Density. The surface viscoelastic properties of monopalmitin monolayers spread on the airwater interface have been studied, as a function of superficial density at pH 5 and 7 (Figure 8). It can be seen that (i) the values for the surface dilatational modulus are very similar to those for the dilatational elasticity at every subphase pH and (ii) the dilatational viscosity and the loss angle tangent values are low and practically zero. As a consequence of this behavior, it can be established that the surface dilatational characteristics of monopalm(46) Chen, P.; Polikova, Z.; Susnar, S. S.; Pace-Asciak, C. R.; Demin, P. M.; Newman, A. W. Colloids Surf. A 1996, 114, 99. (47) Serrien, G.; Geeraerts, G.; Ghosh, L.; Joos, P. Colloids Surf. 1992, 68, 219. (48) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (49) van den Tempel, M.; Lucassen-Reynders, E. H. Adv. Colloid Interface Sci. 1983, 18, 281. (50) de la Fuente, J.; Rodrı´guez Patino, J. M. Langmuir 1994, 10, 2317. (51) de la Fuente, J.; Rodrı´guez Patino, J. M. Langmuir 1995, 11, 2090. (52) Carrera, S. C.; de la Fuente, J.; Rodrı´guez Patino, J. M. Colloids Surf. A 1998, 143, 477.

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Figure 8. Effect of superficial density on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monopalmitin monolayers spread on aqueous solutions at 20 °C and at (A) pH 7 and (B) pH 5. Amplitude: 5%.

itin monolayers are essentially elastic over the range of superficial monopalmitin densities studied. The values for the surface dilatational modulus depend on the monolayer structure, as was deduced directly from the π-A isotherm slope (data not shown). The more condensed the structure is (at higher superficial density), the higher the surface dilatational modulus of the monolayer becomes until collapse is reached. From this point, there is a decrease in E values with a higher superficial density of monopalmitin molecules, especially at pH 5 (Figure 8). The same dependence as that with superficial density was observed with the monolayer thickness.22,26 In fact, the relative thickness increases as the monolayer is compressed, passes through a maximum at surfaces densities close to collapse, and then decreases at the monopalmitin collapse point. That is, more condensed monolayer structures may lead to a increase in the forces of interactions between molecules at the interface, which is consistent with the observed increase in E. The decrease in the surface dilatational modulus at the higher surface densities, after the monolayer collapse, appears to be related to monolayer molecular loss due to the formation of 3D heterogeneous collapse structures and monolayer fracture at the highest surface pressures. These phenomena were visualized by means of BAM images (Figure 3E) and are consistent with relaxation phenomena observed with the same monoglyceride on the air-water interface,45 as discussed in the preceding section. However, the pH effect on the surface dilatational modulus is lower, and practically insignificant, as compared with the surface density effect. That is, on buffered aqueous solutions the effect of pH is of minor importance compared with that observed between water and buffered aqueous solutions at various pH values.19 Dilatational Characteristics of Monoolein Monolayers at the Air-Water Interface. Effect of the Amplitude. The surface viscoelastic properties of mono-

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Figure 9. Effect of amplitude in area deformation on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monoolein monolayers spread on aqueous solutions at pH 7 and at 20 °C. Surface pressure: 20 mN/m. Frequency (mHz): (A) 20, (B) 50, and (C) 100.

Figure 10. Effect of amplitude in area deformation on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monoolein monolayers spread on aqueous solutions at pH 7, at the collapse point, and at 20 °C. Frequency (mHz): (A) 20, (B) 50, and (C) 100.

olein monolayers spread on the air-water interface as a function the amplitude of area oscillation and at pH 7 (as an example) are shown in Figures 9 and 10, for 20 mN/m and at the collapse point, respectively. Monoolein monolayers on pH 5 behave in a similar way (data not shown). As for monopalmitin monolayers (Figures 4 and 5), the surface dilatational modulus and the elastic component increased by varying the deformation between 0.1 and 3% and led to a maximum value at an amplitude of about 3%, whereas the viscous component and the loss angle tangent decreased to a minimum value in the same deformation amplitude range, no matter what the deformation frequency or surface pressure. Thus, the same reasoning used for monopalmitin can be applied here. At deformation amplitudes higher than 3%, at a surface pressure of 20 mN/m, and at any frequency (Figure 9), the surface dilatational properties did not depend on the deformation, a behavior different than that observed with monopalmitin (Figure 5). That is, at these experimental conditions, monoolein monolayers present linear viscoelastic behavior. However, at the monoolein monolayer collapse the surface dilatational modulus and its elastic component decreases with the deformation amplitude (Figure 10). The deformation dependence on the dilatational modulus may be related to the monoolein monolayer structure during the compression-expansion cycle. To check this

assumption we present in Figure 11 the results from a compression-expansion cycle for a monoolein monolayer at 20 mN/m, at the collapse point, and at a deformation frequency of 50 mHz. Two representative deformation amplitudes have been selected (5% and 20%), which correspond to the maximum deformation studied and to that close to the value at which the modulus is maximum (Figures 9 and 10). At 20 mN/m and at deformation amplitudes of 5% and 20%, the liquid-expanded monolayer structure is constant during the compression-expansion cycle (Figure 11A). The results indicate that the breaking of intermolecular interactions is reversible and does not produce any change in the monolayer structure during the compressionexpansion cycle (Figures 2 and 3F). Thus, the level of deformation produces a linear response of the system. At the same deformation amplitude (5%) and for monoolein at the collapse point, the liquid-expanded monolayer structure was observed over the compressionexpansion cycle (Figure 11B). However, this structure coexists with the monolayer collapse at deformation amplitude of 20%. Moreover, at every experimental condition, a good fit between eq 1 and experimental points describes the stress response during the compressionexpansion cycle. The effect of high deformation on monopalmitin (Figure 6) and monoolein (Figure 11) monolayer structures is quite


Figure 11. Dynamic surface pressure response to a sinusoidal change in area at a frequency of 50 mHz for monoolein monolayers spread on aqueous solutions at pH 5 as a function of the amplitude of the area deformation: (O) 5% and (∆) 20%. (A) Surface pressure: 20 mN/m. (B) Monolayer collapse point.

different. However, the same nonlinear response was deduced at the collapse point. These findings strengthen the hypothesis that the nonlinear response of the system may be associated with monolayer collapse. However, some difference exists between monopalmitin and monoolein at the collapse point. In fact, (i) no “over-collapse” was observed with monoolein monolayer and (ii) the collapse of monopalmitin and monoolein monolayers are quite different, as discussed previously. These findings support the hypothesis that for high deformations the effect of deformation amplitude during compression or expansion of the interface is directly linked to irreversible breaking of intermolecular interactions, but this effect is more significant for monopalmitin than for monoolein. In fact, the impact of the deformation on the crystalline nucleus and fracture of collapsed monopalmitin is higher than that produced on the lenses of collapsed monoolein monolayer. We do not reject the existence of other relaxation phenomena (such as monolayer molecular loss) within the time of the experiment.45 In fact, a drift of the baseline was observed (data not shown) with the surface pressure decreasing over a time scale higher than that due to the period of oscillation. Finally, as for the monopalmitin monolayer, the values for the surface dilatational modulus for the monoolein monolayer were very similar to those for the dilatational elasticity and the dilatational viscosity and the loss angle tangent values are low and practically zero. As a consequence of this behavior, it can be established that the surface dilatational characteristics of monoolein monolayers within the range of frequencies between 20 and 100 mHz are essentially elastic.

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Figure 12. Effect of frequency on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monoolein monolayers spread on aqueous solutions at pH 7 and at 20 °C. (A) Surface pressure: 20 mN/m. (B) Monolayer collapse point. Amplitude: 5%.

Effect of Frequency. Changes in surface dilatational properties as a function of frequency of oscillation over a range of 1-300 mHz, at two representative monolayer structural characteristics, at 20 mN/m and at the collapse point, are shown in Figure 12. It can be seen that the frequency dependence on dilational properties of the monoolein monolayer is essentially the same as for monopalmitin monolayer Figure.7 Briefly, we did not observe any influence of pH on surface rheological parameters (data not shown). The dilatational modulus increased significantly with the frequency. The dilatational modulus and its elastic component are essentially the same at frequencies lower than 50 mHz. However, significant differences between these rheological parameters were observed at frequencies higher than 50 mHz, mainly because of the decrease of the elastic component at increasing frequencies. Finally, the value of viscous component increased with the frequency and exceeded that of the elastic component at the higher frequencies (ω > 200 mHz). From these results, it can be concluded that monoolein monolayers present rheological behavior in dilatational conditions, which is essentially elastic at low frequencies (ω < 50) and viscoelastic at higher frequencies (ω > 50). As a consequence of the viscoelastic behavior, the loss tangent angle increased with frequency (Figure 12). Thus, the same reasoning used for monopalmitin monolayer should be applied here. However, it should be noticed that the frequency dependence of the dilatational modulus in the regime 1 < ω < 20 mHz is higher for monoolein than for monopalmitin monolayers. This fact strengthens the hypothesis that monolayer molecule losses by desorption and/or collapse is the relaxation mechanisms operative in the time scale of the experiment, in agreement with previous data.52 Effect of Surface Density. The surface viscoelastic properties of monoolein monolayers spread on the air-

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Figure 13. Effect of superficial density on rheological parameters (O, E (mN/m); ∆, Ed (mN/m); ∇, Ev (mN/m); and (, tan θ) for monoolein monolayers spread on aqueous solutions at 20 °C and at (A) pH 7 and (B) pH 5. Amplitude: 5%.

water interface were studied, as a function of superficial density, at pH 5 and 7 (Figure 13). As a consequence of the surface dilatational properties (E, Ed, Ev, and tan θ), it can be established that the surface dilatational characteristics of monoolein monolayers are essentially elastic over the range of superficial monoolein densities studied. As for monopalmitin monolayers, the values for the surface dilatational modulus depend on the monolayer superficial density, as was deduced directly from the π-A isotherm slope (data not shown), a typical behavior of insoluble lipids. The more condensed the structure is (at higher superficial density), the higher the surface dilatational modulus the monolayer becomes until collapse is reached. From this point, there is a decrease in E values at higher superficial density of monoolein molecules. However, surface dilatational characteristics of monoolein spread monolayer (Figure 13) show important differences compared to monopalmitin monolayer (Figure 8). First, the magnitude of the maximum value of E is reduced by a factor of approximately 2.5. Second, the surface dilatational modulus decreased to a low value, which is practically zero, at the higher surface densities, after the monoolein monolayer collapse. Similar dependence of the surface density on surface dilatational properties has been observed with the monoolein monolayer in the ring trough, where an isotropic area deformation was allowed.20 Concluding Remarks A common trend of the surface density dependence of dilatational modulus for monopalmitin (Figure 8) and monoolein (Figure 13) monolayers is that E increased with increasing surface concentration up to the collapse point. This increase is a result of an increase in the interactions between the monolayer molecules (that is, of its structure), as deduced from π-A isotherms (Figure 2), BAM images


(Figure 3), and monolayer thickness.26 However, for the more condensed monolayer (monopalmitin), this increase is higher than that for the more expanded monoolein monolayer, at every deformation (Figures 4, 5, 9, and 10) and at every frequency (Figures 7 and 12). This indicates that the dilatational modulus is not only determined by the interactions between spread monoglyceride molecules (which depend on the surface pressure or surface density), but that the structure of the spread molecule also plays an important role. The conclusion that molecular structure is important to understand the dynamic interfacial behavior of adsorbed protein at fluid interfaces has been pointed out by Benjamins.53 Recently Wijmans and Dickinson,38 by means of a simulation of dilatational rheology of adsorbed protein monolayers, have demonstrated that at higher particle number density the amplitude of the stress response of the hard sphere system (consisting of unbounded particles only) is higher than that of the gelled system (consisting of extensively bounded particles). In this study, we have found the same behavior for monoglyceride spread monolayers. In fact, for the more aggregated monopalmitin molecules in LC domains (see Figure 3 parts D and E), the surface dilatational modulus is higher than that of monoolein molecules with LE structure, at the same surface density. The fact that monoolein and monopalmitin monolayers have a similar value of E (Figures 8 and 13) at a surface density corresponding to that at which both monoglycerides have the same LE structure (Figure 2) supports the hypothesis. Interestingly, at higher surface densities, the modulus reaches a maximum and decreases at further densities, after the monolayer collapse. At this point of inflection, we have observed the transition between linear and nonlinear viscoelastic behavior for monoolein and monopalmitin monolayers. The differences between superficial rheological characteristics of monopalmitin (Figure 8) and monoolein (Figure 12) spread films at surfaces densities above the monolayer collapse can be attributed to the differences in collapse behavior45 and film structure at equilibrium. Monopalmitin monolayers at equilibriumsπe are about 44.8 and 48.5 mN/m at pH 5 and 7, respectively45sexhibit a condensed structure (Figure 2A), and this structure favors the formation of liquid crystals at the interface “over collapse”.54 However, monoolein monolayers have a liquid-expanded structure (Figure 2B) at every surface density and collapse at the equilibrium surface pressures πe are about 45.2 and 46.1 mN/m at pH 5 and 7, respectively.45 Moreover, the collapse pressure is higher in monopalmitin (Figure 2A) than in monoolein (Figure 2B) monolayers. A relationship between chain packing structure and collapse pressure has been discussed in previous works with fatty acid,17 phospholipid,55 and monoglyceride56,57 monolayers. The lower collapse pressure in the monoolein film could be due to a reduction in the interactions between molecules in the film, which could explain the differences in E values between monoolein and monopalmitin monolayers at higher surface monoglyceride concentrations. That is, more condensed mono(53) Benjamins, J. Static and Dynamic Properties of Proteins Adsorbed at Liquid Interfaces, Ph.D. Thesis, Wageningen University, Wageningen, 2000. (54) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Iterface; Wiley: New York, 1996. (55) Dietrich, A.; Mo¨hwald, H.; Brezesinki, G. Makromol. Chem., Makromol. Symp. 1991, 46, 457. (56) Rodrı´guez Patino, J. M.; Ruı´z, D. M.; de la Fuente, F. J. J. Colloid Interface Sci. 1992, 154, 146. (57) Rodrı´guez Patino, J. M.; Ruı´z, D. M.; de la Fuente, F. J. J. Colloid Interface Sci. 1993, 157, 343.


layer structures may lead to an increase in the interaction forces between molecules at the interface, which is consistent with the observed increase in E. Reduced interactions between hydrocarbon chains could lead to the formation of lenses of monoolein molecules at the interface after collapse.45,54 This further reduction in molecular interactions could be the cause of the decrease in E at surface densities greater than that for the collapse point. However, the most important cause of the reduction of the surface dilatational modulus (and of all surface dilatational properties as well) to values close to zero is the fact that monoolein monolayer collapses (Figure 2B), as do all liquid surfactants at work temperature, at a surface pressure close to πe.58-60 Moreover, this surface pressure is practically constant during the monolayer

Langmuir, Vol. 17, No. 13, 2001 4013

“overcompression”. Thus, the sinusoidal oscillation in the area at the collapse point does not produce any variation in surface pressure, and as a consequence (see eq 3), the value of E approaches zero. Acknowledgment. This research was supported in part by EU through Grant FAIR-CT96-1216 and by DGICYT through Grant PB97-0734. LA0017375 (58) Smith, R. D.; Berg, J. C. J. Colloid Interface Sci. 1980, 74, 273. (59) Ternes, R. L.; Berg, J. C. J. Colloid Interface Sci. 1984, 98, 471. (60) Tomoaia-Cotisel, M.; Zsako, J.; Chifu, E.; Cadenhead, D. A. Langmuir 1990, 6, 191.

Structural-Dilatational Characteristics Relationships of Monoglyceride

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