Morphological and Structural Characteristics of Monoglyceride

The Journal of Physical Chemistry B 0 (proofing), .... Influence of the Saturation Chain and Head Group Charge of Phospholipids in ..... Colloids and ...
0 downloads 0 Views 212KB Size
Download PDF
2484

Langmuir 1999, 15, 2484-2492

Morphological and Structural Characteristics of Monoglyceride Monolayers at the Air-Water Interface Observed by Brewster Angle Microscopy Juan M. Rodrı´guez Patino,* Cecilio Carrera Sa´nchez, and Ma. Rosario Rodrı´guez Nin˜o Departamento de Ingenierı´a Quı´mica, Facultad de Quı´mica, Universidad de Sevilla, c/ Profesor Garcı´a Gonza´ lez, s/nu´ m, 41012-Seville, Spain Received July 27, 1998. In Final Form: December 21, 1998 In this paper we report the morphology of monolayer domains for some typical lipids used as food emulsifiers (monopalmitin, monoolein, and monolaurin). In addition, we propound the utility of BAM for quantitative characterization of the relative film thickness as a function of the lipid and surface density. The surface pressure (π)-area isotherms and the Brewster angle microscopy (BAM) images of monopalmitin, monoolein, and monolaurin monolayers spread on buffered water at pH 7 and at 20 °C indicate that the morphology and structural characteristics of these lipids are very dependent on the hydrocarbon chain length and the presence of a double bond in the hydrocarbon chain. With a camera calibration it is possible to determine the relationship between the gray level and the relative reflectivity. The relative reflectivity allows the determination of the relative thickness of the monolayer. The present studies show the utility of a master curve of relative reflectivity versus surface pressure that is characteristic for any lipid. The results of the relative thickness measurements show that the monolayer thickness increases with the surface pressure and is maximum at the collapse point. The monolayer thickness is higher for monopalmitin monolayers and lower for monoolein monolayers. The thickness of the monolaurin film is halfway between those for monopalmitin and monoolein monolayers. The higher monolayer thickness correlates with the higher long-range lipid-lipid interactions and with closer molecular packing at the air-water interface.

Introduction Monolayers at the air-water interface are interesting systems for studying two-dimensional structures of amphiphilic substances, depending on intermolecular interactions, including those between different components at the interface and between components at the interface and other components in the aqueous subphase. From a fundamental point of view, orientation phenomena and domain structure are of particular interest. In addition, insoluble monolayers at the air-water interface have a wide range of applications including optical devices, information storage, biological sensors, models for biological membranes and dispersed systems (emulsions and foams), and so forth. To develop the utility of traditional materials or to design new ones with appropriate properties for specific applications, a fundamental understanding is required of the interactions and structure that define their behavior at the air-water interface. Our interest is centered on the study of monolayer properties of food emulsifiers (low-molecular-weight surfactants and proteins) and their mixtures at fluidfluid interfaces. Previous studies, including analysis of π-A isotherms,1-3 relaxation phenomena,4-7 and rheo* To whom correspondence should be addressed. Telephone: +34 5 4557133. Fax: +34 5 4557134. E-mail: [email protected]. (1) Rodrı´guez Patino, J. M.; Ruı´z, M.; de la Fuente, J. J. Colloid Interface Science 1992, 154, 146. (2) Rodrı´guez Patino, J. M.; Ruı´z, M.; de la Fuente, J. J. Colloid Interface Sci. 1993, 157, 343. (3) Rodrı´guez Patino, J. M.; Rodrı´guez Nin˜o, Ma. R. Colloids Surf., A, in press. (4) de la Fuente, J.; Rodrı´guez Patino, J. M. Langmuir 1994, 10, 2317. (5) de la Fuente, J.; Rodrı´guez Patino, J. M. Langmuir 1995, 11, 2090. (6) Carrera, C.; de la Fuente, J.; Rodrı´guez Patino, J. M. Colloids Surf., A 1998, 143, 477.

logical properties8-10 of food emulsifiers (lipids and proteins) at the air-aqueous phase interface have demonstrated the importance of the interface and aqueous phase compositions on the interfacial interactions and characteristics (structure, stability, formation, rheology, compatibility, etc.) of these systems at the interface. The picture that emerges from these experiments is sometimes contradictory, especially when the behavior of mixed components at the interface is analyzed.6 The development of new techniques for microscopic observation and characterization of monolayers at the airwater interface, such as X-ray diffraction measurements,11,12 ellipsometric microscopy,13 polarized fluorescence microscopy,14,15 and, more recently, Brewster angle microscopy (BAM),16,17 has been used advantageously to clarify the structural characteristics of amphiphilic substances at fluid-fluid interfaces.18,19 Since its introduction,16,17 BAM has been used preferentially for the study of monolayers, due to the possibility of revealing domains (7) Carrera, C.; Rodrı´guez Nin˜o, Ma. R.; Rodrı´guez Patino, J. M. Colloids Surf. B 1998, 12, 175. (8) Rodrı´guez Nin˜o, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Ind. Eng. Chem. Res. 1996, 35, 4449. (9) Rodrı´guez Nin˜o, Ma. R.; Wilde, P. C.; Clark, D. C.; Husband, F. A.; Rodrı´guez Patino, J. M. J. Agric. Food Chem. 1997, 45, 3010; 1997, 45, 3016. (10) Rodrı´guez Nin˜o, Ma. R.; Wilde, P. J.; Clark, D. C.; Rodrı´guez Patino, J. M. Langmuir 1998, 14, 2160. (11) Kenn, R. M.; Bo¨hm, C.; Bibo, A. M.; Peterson, I. R.; Mo¨hwald, H. J. Phys. Chem. 1991, 95, 2092. (12) Paudler, M.; Ruths, J.; Riegler, H. Langmuir 1992, 8, 184. (13) Reiter, R.; Motschmann, H.; Orendi, H.; Nemetz, A.; Knoll, W. Langmuir 1992, 8, 1784. (14) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (15) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171. (16) He´non, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (17) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (18) Ruiz-Garcia, J.; Qiu, X.; Tsao, M.-W.; Marshall, G.; Knobler, C. M.; Overbeck, G. A.; Mo¨bius, D. J. Phys. Chem. 1993, 97, 6955. (19) Mo¨bius, D. Curr. Opin. Colloid Interface Sci. 1998, 3, 137.

10.1021/la980938u CCC: $18.00 © 1999 American Chemical Society Published on Web 03/05/1999

Monoglyceride Monolayers

and heterogeneities in thin films,20 without the use of any probe, as with fluorescence microscopy, that may disturb the local environment of the probe and thereby cause artifacts.21 The physical basis of BAM is the fact that p-polarized light is not reflected from an interface between two media with different refractive indices if incident at the Brewster angle. The formation of a thin film like a monolayer having a refractive index different from those of the two media changes the optical situation, and at constant angle light is now reflected that can be used for recording and imaging.19 On the other hand, quantitative characterization of thin films (refractive index and thickness) by ellipsometry requires more complicated analysis and rather bulky and expensive instruments.22 In this paper we report the morphology of monolayer domains for some typical lipids used as food emulsifiers (monopalmitin, monoolein, and monolaurin). In previous papers we have observed that these lipids present different structural polymorphism1,2 and relaxation phenomena7 at the air-water interface. In addition, we propound the utility of BAM for a quantitative characterization of the relative film thickness as a function of the lipid and surface density. The optimum use of emulsifiers depends on the knowledge of their interfacial physicochemical characteristics (such as surface activity, structure, stability, superficial viscosity, film thickness, etc.). The study of the characteristics of food emulsifier monolayers at the air-water interface as a food model presents several advantages. Relationships between the structural characteristics of the emulsifier monolayer at the air-water interface and dispersion stability, as well as between condensed film formation and emulsifier association in the bulk phase, have been established. Experimental Section Materials. Synthetic 1-monohexadecanoyl-rac-glycerol (monopalmitin, DIMODAN ML 90), 1-mono(cis-9-octadecenoyl)glycerol (monoolein, RYLO MG 1), and 1-monododecanoyl-rac-glycerol (monolaurin, DIMODAN ML 90) were 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. The water used as subphase was purified by means of a Millipore filtration device (Milli-Q). To adjust the subphase to pH 7, a commercial buffer solution called trizma ((CH2OH)3CNH2/(CH2OH)3CNH3Cl) supplied by Sigma (>99.5%) was used. The ionic strength was 0.05 M in all the experiments. Method. The monolayer structural characteristics were studied by measuring π-A isotherms on a computer-controlled Langmuir film balance, as described elsewhere.1,2,7 The monoglyceride solutions were spread on the subphase by means of a micrometric syringe at 20 °C. Aliquots of 250 µL (6.1 × 1016 to 7.63 × 1016 molecules) were spread in each experiment. The experiments were carried out at temperatures of 20 °C. The same precautions as in previous studies were taken to allow for the evaporation of the spreading solvent (a 15 min wait before beginning the isotherm recording) and for the choice of compression rate (0.062 nm2‚molecule-1‚min-1).1,2 Some experiments were repeated (at least twice). In these cases, the mean deviation was within (0.1 mN/m for surface pressure and (0.005 nm2/molecule for area. A commercial Brewster angle microscope (BAM2 plus, NFT, Go¨ttingen, Germany) was used to image monolayer structures and determine the relative film thickness. Our design follows that of Overbeck et al.23 Briefly, p-polarized light from a 690 nm, (20) Vollhardt, D. Adv. Colloid Interface Sci. 1996, 64, 143. (21) Lo¨sche, M.; Sackmann, E.; Mo¨wald, H. Ber. Bunsen-Ges. Phys. Chem. 1983, 87, 848. (22) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light, 1st ed.; North-Holland: Amsterdam, 1992. (23) Overbeck, G. A.; Ho¨nig, D.; Mo¨bius, D. Langmuir 1993, 9, 555.

Langmuir, Vol. 15, No. 7, 1999 2485 30 mW laser is reflected off the air-water interface at the Brewster angle. The angle of incidence of the laser beam was fixed to approximately 53.1°. The lateral resolution is 2 µm. The reflected beam passes through a focal lens, into an analyzer at a known angle of incident polarization, and finally to a CCD camera. Rotation of the analyzer allows the image contrast to be adjusted by varying the reflected polarization that is passing to the camera. The BAM images were digitized, filtered to reduce diffraction fringes caused by the coherent nature of the laser beam, and then processed to obtain the best quality images for subsequent analysis. The Brewster angle microscope was positioned over the film balance on a specially designed frame structure which allows easy movement of the Brewster angle microscope, 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. 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 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 a fixed shutter speed of 1/50 s. Relative Thickness Measurement. As an adaptation of ellipsometry, the light 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 by the method based on the Fresnel reflection equations.22 At the Brewster angle

I ) |Rp|2 ) C‚d2

(1)

where I is the relative reflectivity (defined as the ratio of the reflected intensity (Ir) and the incident intensity (I0), I ) Ir/I0), C is a constant, d is the film thickness, and Rp is the p-component of the light, given by eq 2.

Rp )

r01 + r12 × e-i2b 1 + r01 × r12 × e-i2b

(2)

with

b ) 2πn1 cos φ1‚(d/λ)

(3)

r01 )

n1 × cos φ0 - n0 × cos φ1 n1 × cos φ0 + n0 × cos φ1

(4)

r12 )

n2 × cos φ1 - n1 × cos φ2 n2 × cos φ1 + n1 × cos φ2

(5)

where λ is the wavelength of light, ni are the refractive indices of air (n0), the monolayer (n1), and water (n2), and φi are the incident light angles relative to the plane of incidence at the air (φ0), monolayer (φ1), and water (φ2). One of the major problems of thin film optics is that the optical properties (refractive index) are often unknown, and the film might even be anisotropic. If optical anisotropy is observed in the film (i.e. by using the analyzer), the film cannot be described by a single refractive index value. The dependence of R on d2 (eq 1) can be used to determine the relative thickness of film regions even if the optical properties of the film are unknown.24 Although this approach is sufficient for the work reported here, a further step to calculate values of the monolayer thickness based on a model for the observed morphology has been used recently.24-27 However, for our purpose (24) de Mul, M. N. G.; Mann, J. A., Jr. Langmuir 1998, 14, 2455. (25) Overbeck, G. A.; Ho¨nig, D.; Wolthaus, L.; Gnade, M.; Mo¨bius, D. Thin Solid Films 1994, 242, 26. (26) Overbeck, G. A.; Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1994, 242, 213.

2486 Langmuir, Vol. 15, No. 7, 1999

Rodrı´guez Patino et al.

Figure 1. Gray level response of the CCD camera versus incident light angle. Different data points are for different calibration measurements. The arrows indicate the incident light at the Brewster angle.

Figure 2. Theoretical relative reflectivity for a pure water surface as a function of the measured gray level at different incident light angles. Shutter speed 1/500 s.

the exact film thickness is not as interesting as the question of monolayer or multilayer formation depending on the experimental conditions. To measure the relative thickness of the film, a camera calibration is necessary in order to determine the relationship between the gray level (GL) and the relative reflectivity (I). For this purpose, the angle of incidence is modified slightly to deviate from the Brewster angle. This deviation causes an increase in the gray level signal from the pure water background. The signal is adjusted to the expected theoretical intensity derived from Fresnel equations applied to the pure water surface, Rpw, (eq 6). This procedure gives a calibration to transform gray level into real intensities.

Rpw )

n22 × cos φ2 - xn22 - sin2 φ2 n22 × cos φ2 + xn22 - sin2 φ2

(6)

Briefly, the instrument must be set to p-polarization, the analyzer must be removed, and the camera must be at maximum gain to ensure linear response of the camera. If the angle of incidence is set at the Brewster angle on the pure water surface, a dark area in the middle of the image can be observed. Next, the angle has to be changed in small steps to both sides of the Brewster angle, that is, changing the angle by a few tenths of a degree below and above the Brewster angle. The deviation from the Brewster angle must be as large as possible until a saturation in the middle of the image is observed (image completely white). The plot of gray level versus incident angle is a parabola with a minimum at the Brewster angle (Figure 1). Because of the inhomogeneous intensity profile of the laser, it is very important that all measurements during camera calibration and further image analysis of the film must be located on the same spot, just in the center of focus. Figure 2 shows the gray level measured as a function of the intensity derived from eq 6 at a shutter speed of 1/500 s. The linear fit (eq 7, linear regression 0.977) will be used to calculate the relative reflectivity (I) and then the relative film thickness by means of eq 1.

I ) -3.915 × 10-6 + (6.333 × 10-8)GL

(7)

Results and Discussion Morphological and Structural Characteristics of Monopalmitin Monolayers. Figure 3 shows the π-A isotherm during a compression-expansion cycle (Figure 3A), the time evolution of surface pressure and relative reflectivity during a compression-expansion cycle (Figure 3B), and the surface pressure dependence on the relative intensity during compression (Figure 3C) for a monopalmitin monolayer spread on buffered water at pH 7 and at 20 °C. (27) Weidemann, G.; Gehlert, U.; Vollhardt, D. Langmuir 1995, 11, 864.

Figure 3. π-A isotherm during a compression (0)-expansion (4) cycle (A), the time evolution of (O) surface pressure and (s) relative reflectivity during a compression-expansion cycle (B), and the surface pressure dependence on the relative intensity during compression (C) for a monopalmitin monolayer spread on buffered water at pH 7 and at 20 °C.

During compression and expansion of a monopalmitin monolayer, a hysteresis was observed in the π-A isotherm (Figure 3A). This hysteresis is not due to a relaxation process by monolayer molecular loss by desorption and/or collapse, as revealed in the π-A isotherm after repeated compression-expansion cycles. All compression and expansion curves, respectively, were coincident if a waiting time between each cycle was attained in order to allow organization/reorganization of the monolayer, as will be

Monoglyceride Monolayers

Langmuir, Vol. 15, No. 7, 1999 2487

Figure 4. Visualization of monopalmitin monolayers by Brewster angle microscopy at 20 °C and at pH 7: (A) LC domains at 5 mN/m and (B) 8 mN/m; (C) LC domains at 20 m/Nm without analyzer; (D) LC domains at 20 mN/m for a position p of the analyzer relative to the plane of incidence of 70°; (E) LC domains at 20 mN/m for a position p of the analyzer relative to the plane of incidence of 110°; (F) monolayer collapse; (G) monolayer collapse after 30 min of waiting time, without analyzer; (H) monolayer collapse after 30 min of waiting time, for a position p of the analyzer relative to the plane of incidence of 120°; (I) monolayer collapse after 30 min of waiting time, for a position p of the analyzer relative to the plane of incidence of 60°; (J) fracture in a collapsed monolayer; (K) 2D foam after expansion of the monolayer at 10 m/m; (L) expansion of the monolayer at the maximum area (π ≈ 0 mN/m). The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm.

discussed later with the aid of BAM images. From the π-A isotherm (Figure 3A), different structures can be deduced for monopalmitin monolayer as a function of surface density or surface pressure, such as a liquidexpanded phase (LE) (at π < 5 mN/m), a degenerate firstorder phase transition between liquid-expanded (LE) and liquid-condensed (LC) structures (at 5 < π < 30 mN/m), the liquid-condensed structure (at π > 30 N/m), and, finally, the collapse at a surface pressure of about 53.1 mN/m. However, monopalmitin exists as a metastable monolayer at surface pressures higher than 49 mN/m, the equilibrium surface pressure at 20 °C.7 Brewster angle microscopy allows direct visualization of changes in morphology and collapse of the monopalmitin

monolayer at the air-water interface. In Figure 4 we show the images of domain morphology for monopalmitin monolayers as a function of surface pressure. A monopalmitin monolayer at 5 mN/m (A) shows circular liquidcondensed domains from the homogeneous ambient phase with a liquid-expanded structure. The same condensed domains have also been observed by Brezesinski et al.28 These circular domains are the equilibrium shapes for the second and subsequent compression-expansion cycles. In fact, during the first compression of the monolayer, dentritic-shaped domains (B) were observed at the begin(28) Brezesinski, G.; Scalas, E.; Struth, B.; Mo¨hwald, H.; Bringezu, F.; Gehlert, U.; Weidemann, G.; Vollhardt, D. J. Phys. Chem. 1995, 99, 8758.

2488 Langmuir, Vol. 15, No. 7, 1999

Figure 5. π-A isotherm during a compression (0)-expansion (4) cycle (A), the time evolution of (O) surface pressure and (s) relative reflectivity during a compression-expansion cycle (B), and the surface pressure dependence on the relative intensity during compression (C) for a monoolein monolayer spread on buffered water at pH 7 and at 20 °C.

ning of the LE to LC transition. The dentritic shapes are due to a growth anisotropy, indicating that the incorporation of molecules in the condensed phase is favored in defined directions.29 In agreement with Gehlert and Vollhardt,29 the presence of circular domains after the first compression-expansion cycle could be the consequence of the absence of any significant supersaturation due to the slow compression rate. In addition, it is possible that, after the first compression-expansion cycle, nuclei of the LC phase exist even at waiting times higher than 60 min. The LC domains grow in size and the monolayer is covered with LC domains as the surface pressure is increased. Thus, at surface pressures lower than the equilibrium surface pressure πe (πe ≈ 49 mN/m), the monolayer is dominated by LC domains (C). Each domain does not have a uniform intensity, and this intensity changes with the analyzer angle (D and E). It can be seen that for different positions p of the analyzer relative to the plane of incidence, image D (p ) 70°) appears almost as an inverted image of E (at p ) 110°). The optical anisotropy, as visualized for different positions of the analyzer, is typical for LC structures due to crystallinelike domains being formed at the air-water interface.30 (29) Gehlert, U.; Vollhardt, D. Langmuir 1997, 13, 277. (30) Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1992, 210/211, 64.

Rodrı´guez Patino et al.

At the highest surface pressure, as the monolayer collapses, the LC domains are so closely packed that they occupy the entire field of view, the contrast vanishes suddenly (F), and the presence of monolayer fractures can be observed (J) in different zones by the movement of the Brewster angle microscope along the length of the film balance. Under these conditions, changes in relative reflectivity are observed by turning the analyzer angle (parts G, H, and I of Figure 4), suggesting that the hydrocarbon chains in the molecules have a vertical orientation, but with a different tilted angle and, probably, with an inhomogeneous thickness. The monolayer collapsed phase is also characterized by the absence of mobility in LC domains during compression, which correlates with the highest elasticity of the monolayer at the collapse point (data not published). Finally, after expansion, the monolayer undergoes a break up of the collapsed structure to a 2D foam structure (K). This 2D foam consists of collapsed plateau borders with foam cells containing an LC monolayer at higher surface pressures and finally with LC/LE structures at the maximum area, as the surface pressure approaches zero. Even under these conditions, a punctual collapse phase and LC domains can be observed (L), especially as the expansion finished. It must be emphasized that the reversible collapse mechanism as deduced from the repetitivity of the π-A isotherms after continuous compression-expansion cycles, with 60 min of waiting time between each cycle, is really a pseudoreversible collapse mechanism due to the fact that nuclei of LC domains on a submicroscopic scale (not observed with the resolution of BAM) could exist after this waiting time. This phenomenon could be the cause of the presence of circularshaped domains of LC phases during the recompression of the monolayer, as discussed previously. The evolution with the monolayer compression of the relative reflectivity (I) of the image gives complementary information about the morphology and structural characteristics of a monopalmitin monolayer during a compression-expansion cycle (Figure 3B). It can be seen that the relative reflectivity increases as the monolayer is compressed, passes through a maximum at the collapse (it must be noted that the plateau observed at the monolayer collapse is due to a saturation of the camera at the shutter speed used in this experiment), and then decreases with the expansion of the monolayer. In Figure 3B we observed some interesting features. At lower surface pressures, with an LE structure, the relative reflectivity shows little noise. The noise in the relative reflectivity increases in frequency during the compression of the monolayer with LC domains. Finally, the noise vanishes at higher surface pressures as the monolayer collapses and then increases during the expansion, as the monolayer recovers the LC structure. The noise peaks are not an artifact but are the consequence of the relative reflectivity when a circular domain with LC structure passes through the spot where this measurement is performed. It should be noted that in the region of LC structure the noise in the relative reflectivity is higher during the expansion than during the monolayer compression. This fact is a consequence of the relaxation phenomenon related to the organization/reorganization of the monolayer structure with the expansion, which could explain the hysteresis observed in the π-A isotherm (Figure 3A). Figure 3C gives the surface pressure dependence of the relative reflectivity for a monopalmitin monolayer spread on buffered water at pH 7. The I versus π curve could reflect the surface equation of state of the spread material at the air-water interface, and it is particularly sensitive

Monoglyceride Monolayers

Langmuir, Vol. 15, No. 7, 1999 2489

Figure 6. Visualization of monoolein monolayers by Brewster angle microscopy at 20 °C and at pH 7: (A) LE domains at 10 mN/m; (B) LE domains at 30 mN/m; (C) monolayer collapse; (D) 2D foam after expansion of the monolayer at the maximum area (π ≈ 0 mN/m). The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm.

for assessing phase structures in the monolayer. In addition, this master curve gives important and complementary information about the relative film thickness, as deduced from the application of eq 1. The data collected in Figure 3C clearly show the zone of existence of a LE phase, at lower surface pressures, with a minimum in relative reflectivity and, as a consequence, with lower monolayer thickness. The monolayer thickness increases suddenly as the LE to LC transition takes place, and finally reaches its maximum value with an LC structure. The data in Figure 3C show that a doubling in the thickness for the LC structure in relation to the LE one is produced because the relative reflectivity increases roughly four times. The fact that the relative reflectivity at the equilibrium surface pressure (49 mN/m) with a LC structure is practically the same as that at the collapse point is an indication that no multilayer formation takes place as monopalmitin collapses under dynamic conditions, that is, during the continuous compression of the monolayer at the higher surface pressures. Thus, it can be concluded that the collapse of a monopalmitin monolayer takes place after the fusion of LC condensed domains (see Figure 4F) with the formation of fractures in different zones along the monolayer (see Figure 4J). Morphological and Structural Characteristics of Monoolein Monolayers. Figure 5 shows the π-A isotherm during a compression-expansion cycle (Figure 5A), the time evolution of surface pressure and relative reflectivity during a compression-expansion cycle (Figure 5B), and the surface pressure dependence of the relative intensity during compression (Figure 5C) for a monoolein monolayer spread on buffered water at pH 7 and at 20 °C. In contrast with the monopalmitin monolayer (Figure 3), the monoolein monolayer (Figure 5) presents only the liquid-expanded structure and the collapse at the equilibrium surface pressure (πe ≈ 45.7 mN/m). In addition, the hysteresis observed in the π-A isotherm during a

compression and expansion cycle is lower for the monoolein monolayer. This hysteresis should be ascribed to organization/reorganization changes in the monolayer structure during the expansion, as will be discussed later by means of BAM images. In fact the repetitivity of the π-A isotherms after continuous compression-expansion cycles is an indication that no molecular loss takes place at the compression rate used in this experiment. BAM images (Figure 6) corroborate that only the homogeneous LE phase is present during the compression of a monoolein monolayer (A). Upon compression at 30 mN/m, LE domains were formed (B). These domains grew with the surface pressure, and their size was maximum at the monolayer collapse, but no change in the structure was observed (C). The absence of crystalline domains in the monoolein monolayer was confirmed by the uniform intensity and the independence of this intensity on the analyzer angle (data not shown). At any surface pressure the surface mobility of monoolein was higher than that for the monopalmitin monolayer. Even at the collapse point, the circular domains observed in monoolein monolayers are in motion. From the observation with BAM along the film balance, no fractures were visualized after the monoolein collapse. Clearly, the collapses of monopalmitin and monoolein monolayers are quite different and these differences are associated with different structures of these lipids at the surface pressures corresponding to πe. These qualitative conclusions agree quite well with the relaxation phenomena discussed in a previous paper7 and with the surface rheological characteristics of these lipids at the air-water interface.8 On the basis of these experiments, it was concluded that monoolein monolayers form lenses upon collapse.7,8 Finally, after the expansion, the monolayer undergoes breakup of the collapsed structure up to a 2D foam structure (D), which consists of a collapsed phase in the plateau borders with foam cells containing an LE monolayer. These structures are present

2490 Langmuir, Vol. 15, No. 7, 1999

during the expansion process, even as the surface pressure approaches zero. The evolution of the relative reflectivity for a monoolein monolayer with monolayer compression (Figure 5B) also shows important differences compared to the case of monopalmitin monolayers (Figure 3B). It can be seen that the relative reflectivity increases monotonically with monolayer compression, but in contrast to the case for monopalmitin monolayers, (i) no discontinuity was observed, which corroborates that during the monolayer compression a denser film is formed but without any change in its structure, (ii) the absence of defined structures reduces significantly the noise during monoolein monolayer compression, (iii) the higher noise during the monolayer expansion is associated with the presence of 2D foams of monoolein collapsed lenses, and (iv) the relative reflectivity during monoolein monolayer compression, and especially at the collapse point, is practically two times lower than that for monopalmitin. Finally, the surface pressure dependence of the relative reflectivity (Figure 5C), and thus the relative film thickness, shows that the thickness of monoolein monolayers increases monotonically with the surface pressure up to the monolayer collapse. Tentatively, the formation of lenses at the collapse point is the consequence of the fusion of domains with LE structure, but no formation of multilayers takes place because as the collapse is produced, at the equilibrium surface pressure with a LE structure, no discontinuity was observed in the relative film thickness (Figure 5C). As the relative reflectivity for a monopalmitin monolayer at πe is roughly two times higher than that for the monoolein monolayer, it can be concluded that the thickness at equilibrium of a monopalmitin film is four times higher than that for a monoolein film. Morphological and Structural Characteristics of Monolaurin Monolayers. Figure 7 shows the π-A isotherm during a compression-expansion cycle (Figure 7A), the time evolution of surface pressure and relative reflectivity during a compression-expansion cycle (Figure 7B), and the surface pressure dependence of the relative reflectivity during compression (Figure 7C) for a monolaurin monolayer spread on buffered water at pH 7 and at 20 °C. The hysteresis observed after a continuous compression-expansion cycle (Figure 7A) is higher than that for monopalmitin and monoolein monolayers. This hysteresis not only is due to organization/reorganization changes in the monolayer structure during the expansion but also must be associated with a process of monolayer molecular loss. In fact, the isothermal displacement toward the π-axis after continuous compression-expansion cycles at constant temperature (data not shown) is a consequence of a monolayer molecular loss, because the molecular area A is calculated by assuming that all the spread monolaurin molecules remain in the monolayer. The monolaurin molecular loss was quantified in a previous paper7 by means of a desorption mechanism including two steps: dissolution of monolayer molecules in the subsurface region, followed by the diffusion of the previously dissolved molecules into the bulk phase. The monolaurin molecular loss by desorption is an irreversible process due to the fact that the isotherm does not return to its original state on recompression after a waiting period of 24 h at the maximum area (π ) 0). In this regard, monolaurin spread monolayers behave in a different way than monopalmitin and monoolein monolayers, as discussed in previous sections. From the π-A isotherm and the surface dilatational modulus (E ) -A‚(δπ/δA), data not shown, a condensed structure can be deduced for a monolaurin monolayer at 20 °C. However,

Rodrı´guez Patino et al.

Figure 7. π-A isotherm during a compression (0)-expansion cycle (4) (A), the time evolution of (0) surface pressure and (s) relative reflectivity during a compression-expansion cycle (B), and the surface pressure dependence on the relative intensity during compression (C) for a monolaurin monolayer spread on buffered water at pH 7 and at 20 °C.

this condensed structure must be different for monopalmitin than for monolaurin because the interactions at the interface are lower for monolaurin monolayers. In fact, during the monolaurin compression it is impossible to achieve monolayer collapse at the minimum area or at the equilibrium surface pressure (πe ≈ 44 mN/m).7 BAM images (Figure 8) corroborated the existence of condensed domains (Figure 8A) even at low surface pressures. But these domains do not adopt circular crystalline-like shapes typical of liquid-condensed structures as for monopalmitin. The number of domains grows with the surface pressure (Figure 8B). In addition, the tilted angle of the molecules in these domains is the same as that deduced by the change in the analyzer angle (Figure 8C and D). In fact, at higher surface pressures no differences in the domain light intensity (as for monopalmitin, Figure 4) were observed by turning the analyzer angle, but a condensed structure with the same tilted angle can be deduced for these domains because the direction of the light reflected changed with the analyzer angle (see Figure 8C and D). The monolayer molecular loss is the cause of the differences observed between the series of Figure 8B-E corresponding to a compression of the monolayer at the same molecular area. Due to the existence of a relaxation phenomenon associated with monolayer molecular loss, the surface pressure and domain density decrease with

Monoglyceride Monolayers

Langmuir, Vol. 15, No. 7, 1999 2491

Figure 8. Visualization of monolaurin monolayers by Brewster angle microscopy at 20 °C and at pH 7: (A) condensed domains at 5 mN/m; (B) condensed domains at 18 mN/m without analyzer; (C) as in part B at 15 min of relaxation time (π ) 15 mN/m) for a position p of the analyzer relative to the plane of incidence of 70°; (D) as in part B at 15 min of relaxation time (π ) 15 mN/m) for a position p of the analyzer relative to the plane of incidence of 110°; (E) as in part B at 60 min of relaxation time (π ) 8 mN/m) without analyzer; (F) condensed domains at the minimum surface area (near the monolayer collapse); (G) as in part F after a waiting time of 60 min; (H) 2D foam after expansion of the monolayer at the maximum area (π ≈ 0 mN/m). The horizontal direction of the image corresponds to 630 µm, and the vertical direction to 470 µm.

the relaxation time from 20 mN/m (Figure 8B) to 15 mN/m (Figure 8C and D), and, finally, to 8 mN/m (Figure 8E). However, the domain size grows with the relaxation time due to a crystallization process. After the expansion, the

monolayer undergoes breakup of the condensed structure to a 2D foam structure (Figure 8H). This structure is present during the expansion process, even as the surface pressure approaches zero.

2492 Langmuir, Vol. 15, No. 7, 1999

The evolution with the monolayer compression of the relative reflectivity for the monolaurin monolayer (Figure 7B) also shows important differences with monopalmitin (Figure 3B) and monoolein (Figure 5B) monolayers. It can be seen that the relative reflectivity increases monotonically with the monolayer compression, but no discontinuity was observed, which corroborates that during the monolayer compression a condensed film is formed but without any change in its structure. The noise during monolaurin monolayer compression and expansion is practically the same due probably to the absence of collapse under the conditions specified previously. The surface pressure dependence of the relative reflectivity (Figure 7C), and then of the relative film thickness, shows that the thickness of monolaurin monolayers increases with the surface pressure, but without any discontinuity. The relative reflectivity for the monolaurin monolayer is of the same order of magnitude, but lower, as that for the monopalmitin monolayer, a consequence of the lower lipid-lipid interactions associated with the shorter hydrocarbon chain in the monolaurin monolayer. Conclusions The surface pressure-area isotherms and the Brewster angle microscopy images of monoglyceride (monopalmitin, monoolein, and monolaurin) monolayers spread on buffered water at pH 7 and at 20 °C indicate that the

Rodrı´guez Patino et al.

morphology and structural characteristics of these lipids are very dependent on the hydrocarbon chain length and the presence of a double bond in the hydrocarbon chain. With a camera calibration it is possible to determine the relationship between the gray level and the relative reflectivity. The relative reflectivity allows the determination of the relative thickness of the monolayer. The present studies show the utility of a master curve of relative reflectivity versus surface pressure that is characteristic for any lipid. The results of the relative thickness measurements show that the monolayer thickness increases with the surface pressure and is maximum at the collapse point. The monolayer thickness is higher for monopalmitin monolayers and minimum for monoolein monolayers. The thickness of the monolaurin film is halfway between those for monopalmitin and monoolein monolayers. The higher monolayer thickness correlates with the higher long-range lipid-lipid interactions and with closer molecular packing at the air-water interface. Acknowledgment. Drs. Ho¨nig and A. Pe´rez Izquierdo are thanked for their comments during this study. This research was supported by the European Community through Grant FAIR-CT96-1216 and by CICYT through Grant ALI97-1274-CE. The authors thank Danisco Ingredients for providing the monoglyceride samples. LA980938U