Interactions between Modified Mica Surfaces in Triglyceride Media

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Langmuir 1998, 14, 5546-5554

Interactions between Modified Mica Surfaces in Triglyceride Media Andra Dedinaite,*,† Per M. Claesson,† Bruce Campbell,‡ and Holger Mays§ Laboratory for Chemical Surface Science, Department of Chemistry, Physical Chemistry, Royal Institute of Technology, SE-100 44 Stockholm, Sweden, Institute for Surface Chemistry, P.O. Box 5607, SE-114 86, Stockholm, Sweden, Kraft Foods Technology Center, 801 Waukegan Road, Glenview, Illinois 60025, and Department of Physical Chemistry, Uppsala University, SE-751 21 Uppsala, Sweden Received February 26, 1998. In Final Form: June 17, 1998 Results obtained from surface force measurements using modified nonpolar mica surfaces immersed in triolein are presented. The force vs distance curves were determined for different water activities in the interaction medium. Two oscillations with a periodicity of 20 Å were observed in the force curve measured across anhydrous triolein. The force barriers appear at separations of 45-40 and 20-30 Å. It is suggested that triolein has no clear preferential orientation of the oleic acid chains outside a nonpolar surface. This is different from outside a polar mica surface where triolein adopts conformations with the three oleic acid residues directed toward the bulk. At high water contents the triolein molecules outside nonpolar surfaces suddenly change their orientation when a high compressive force is applied. The forces acting between mica surfaces were measured in triolein solutions containing phospholipids at different water activities. It was shown that the phospholipid self-assembled onto the mica surfaces and rendered them nonpolar. The forces between such surfaces in anhydrous triolein are similar to those observed between mica hydrophobized using the Langmuir-Blodgett technique. In addition, at high water activities a weak long-range repulsive force was observed. This force was interpreted as being due to weakly adsorbed phospholipid aggregates. We discuss the implications of the results for the stability and physical properties of colloidal particle dispersions in nonpolar media. Adsorption isotherms for the phospholipid from refined vegetable oil at a low water activity on mica and sucrose crystals are presented. They show that the phospholipid adsorbs in a monolayer on mica. On sucrose more than monolayer coverage is observed, which we interpret in terms of a phase separation of phospholipid into crevices and cracks.

Introduction Phospholipids of natural origin (mostly soybean lecithin) are commonly accepted additives in food and pharmaceutical industry. They are widely used as emulsifiers, as agents controlling rheological properties of dispersions1 and fat crystallization.2 In a series of previous studies attempts have been made to understand how lecithin controls the interactions between particle dispersions. The majority of the efforts were launched to elucidate the adsorption properties of lecithin and synthetic phospholipids with a well-defined chemical structure on wellcharacterized particles.3,4 It has long been realized that the adsorption properties of a certain substance on a surface depends not only on the properties of the surface and adsorbing material but also to a large extent on the properties of the solvent,4 in particular the interactions between solvent and surfaces and between solvent and adsorbing substances. The association behavior of phospholipids in nonpolar media (isooctane, cyclohexane, and toluene) has been investigated by Cirkel et al.5 They found clear evidence for formation of reversed spherical and † Royal Institute of Technology and Institute for Surface Chemistry. ‡ Kraft Foods Technology Center. § Uppsala University.

(1) Minifie, B. W. Chocolate, Cocoa and Confectionary: Science and Technology; AVI Publishing Company: Westport, CT, 1982. (2) Smith, P. R. The Molecular Basis for Crystal Habit Modification in Triglycerides; Smith, P. R., Ed.; University of Leeds: 1995. (3) Hey, M. J.; Mackie, A. C.; Mitchell, J. R. J. Colloid Interface Sci. 1986, 114, 286. (4) Tamamushi, B. In Adsorption from Solution; Ottewill, R. H., Rochester, C. H., Smith, A. L., Eds.; Academic Press: London, 1983; pp 79-86.

cylindrical aggregates. In isooctane at high phospholipid and water contents it was found that the reversed cylindrical micelles associated to form a network structure. The influence of the polarity of the dispersion medium on the adsorption of phospholipids onto silica surfaces has been mapped by Hey et al.3 Tamamushi investigated adsorption of egg lecithin on alumina and silica from hexane and water.4 These studies provided useful information about phospholipid adsorption. However, the investigated systems differed in many respects from typical food dispersions and gave only a limited insight into food colloid behavior. A system with close similarity to food colloids was investigated by Johansson et al.6 who studied adsorption of emulsifiers to various crystals (saccharose, fat) dispersed in vegetable oils by employing indirect methods. For instance, they measured the sediment volume of the dispersed particles, and from this they draw some conclusions about the character and the strength of the interparticle forces. A dramatic decrease in sediment volumes of sugar crystals was observed when lecithin was introduced to the system, indicating decreased particle-particle attraction.7 The link between the effect of phospholipids on bulk properties of colloidal dispersions and their surface chemical properties is still not sufficiently understood. The interferometric surface force technique employed in the present study provides a unique possibility to directly investigate interactions between surfaces of (5) Cirkel, P. A. Structure and Dielectric Properties of Lecithin Organogels; Cirkel, P. A., Ed.; Rijksuniversiteit te Leiden: Leiden, 1998. (6) Johansson, D.; Bergenståhl, B. A. J. Am. Oil Chem. Soc. 1992, 69, 705. (7) Johansson, D.; Bergenståhl, B. J. Am. Oil Chem. Soc. 1992, 69, 728.

S0743-7463(98)00237-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/26/1998

Mica in Triglyceride Media

different origin across various solutions and also to detect changes in interaction when the conditions are changed. The present work is the second article in a series where we are investigating the factors influencing interactions between solid surfaces in a triglyceride medium. In the first article we described interactions between polar mica surfaces across pure triolein, a vegetable oil representative, and the effect of humidity on these interactions.8 In this report we extend the investigation by employing modified nonpolar mica surfaces for modeling interactions of hydrophobic particles across anhydrous and watercontaining triolein. We also report some data showing the effect of a polar lipid, L-R-oleylpalmitoylphospatidylethanolamine (OPPE) on interactions between mica surfaces across triolein. It was found that OPPE molecules self-assembled in monolayers on the surface and thus modified their character. The interactions between OPPEcoated mica surfaces across triolein are similar to those between initially nonpolar surfaces in several respects. The information obtained by studying phospholipid adsorption on mica crystals and hydrophilic food particles (sucrose crystals) dispersed in vegetable oil are discussed in connection with data provided by surface force measurements. Experimental Section Surface Force Measurements. Interactions between mica (muscovite mica Reliance, New York) surfaces were measured using the interferometric surface force apparatus (SFA) using Mark II9 and Mark IV10 models. The detailed description of the technique is given previously;9,11 therefore, we shall mention just some essential features. Optically polished half cylindrical silica disks (radius of curvature ≈2 cm) with thin mica sheets silvered on one side were mounted in a crossed cylinder geometry inside the surface force apparatus. One of the disks was mounted on the force-measuring double cantilever spring. This spring design ensures that the surfaces do not roll over each other when the spring bends during the measurement of strong forces.12 When the gradient of the force, dFc/dD, exceeds the spring constant, the spring system becomes unstable. Under these conditions the surface separation changes suddenly and a “jump” to the next stable region of the force curve occurs. The force acting between the surfaces was determined by moving a motor to which one of the surfaces was mounted, by a known amount, and interferometrically measuring the resulting change in separation between the surfaces. Any difference between the motion of the motor and the actual change in separation when multiplied by the spring constant gives the difference between the force at the initial and final positions. The surface separation was determined using multiple beam interference fringes by first measuring the wavelengths of the interference bands when the surfaces were in contact and then analyzing the shift in wavelength when the surfaces were at a certain distance apart. In the present study we used two types of surfaces, namely, bare mica and dimethyldioctadecylammonium bromide (DDOA)-coated mica. It is important to note that the zero separation for bare mica surfaces was determined as mica-mica contact and for DDOAcoated mica surfaces as DDOA-DDOA contact. Results from the measurements are plotted as force, Fc, normalized by the undeformed geometric mean radius of the surfaces, R, as a function of surface separation, D. This quantity is related to the free energy of interaction per unit area, Gf, between two flat surfaces at the same separation13 (8) Claesson, P. M.; Dedinaite, A.; Bergenståhl, B.; Campbell, B.; Christenson, H. K. Langmuir 1997, 13, 1682. (9) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1 1978, 74, 975. (10) Parker, J. L.; Christenson, H. K.; Ninham, B. W. Rev. Sci. Instrum. 1989, 60, 3135. (11) Israelachvili, J. N. J. Colloid Interface Sci. 1973, 44, 259. (12) Christenson, H. K. J. Colloid Interface Sci. 1988, 121, 170. (13) Derjaguin, B. Kolloid-Z. 1934, 69, 155.

Langmuir, Vol. 14, No. 19, 1998 5547 Fc(D)/R ) 2πGf(D) ) 2π(γ(D) - γ(∞))

(1)

γ(D) and γ(∞) are the film tension at a separation D and at infinite separation, respectively. The quantity γ(∞) is equal to twice the solid liquid interfacial tension. This relation is valid provided R . D and provided the surfaces remain undeformed during the measurement. The latter condition is not fulfilled under high compressive forces, particularly when the force gradient is large.14 Thus, eq 1 is not applicable for the strongest forces encountered in this study. The surface force measurements were carried out with a droplet of about 50 µL (droplet of pure triolein (C57H104O6) or triolein containing 200 ppm of phospholipid) placed between the surfaces. The triolein was purchased from NU-Check-Prep, Inc., with purity greater than 99%. Prior to use the triolein preparation was centrifuged for ≈6 h at 300000g (L5-50 Ultracentrifuge, Beckman) in order to remove the particles that were present in the commercially available preparation. The phospholipid, L-Roleylpalmitoylphosphatidylethanolamine (OPPE), from soybean with purity greater than 99%, was purchased from Avanti Polar Lipids, Inc., and used as received. The surface interactions were investigated at different water activities (aw) at room temperature (≈20 °C). An environment with a desired water activity was created by introducing a beaker containing a saturated solution of a suitable salt or, alternatively, a powder of P2O5 into the measuring chamber. Prior to measurement, the drop of triolein was injected between the surfaces positioned in the SFA. Later, the measuring chamber was hermetically closed and equilibrated with the air of wanted water activity for at least 16 h. To obtain close to zero water activity the atmosphere in the measuring chamber and the sample were dried with P2O5 (Merck, p.a. grade). The aw ) 0.47 was obtained by equilibration with vapor of saturated KCNS (Fluka, puriss p.a.) solution. The aw ) 1.00 was obtained by equilibration with pure water vapor. The water used was pretreated with a Milli-RO 10 PLUS water purification system and further treated with a Milli-Q PLUS 185-unit. To acquire a nonpolar surface we deposited a monolayer of dimethyldioctadecylammonium bromide (DDOA) (Kodak) onto the mica surface employing the Langmuir-Blodgett (LB) technique. When the mica plate is moved through the DDOA monolayer spread on the water surface, DDOA molecules deposit on mica with the positively charged ammonium groups attached to the negatively charged surface and the hydrocarbon chains directed away from the surface. As spreading solution a 95/5 mixture of hexane (Merck, analytical grade) and ethanol (Kemetyl, spectrometric grade) containing 2.4 × 10-4 M DDOA was used. DDOA was deposited on muscovite mica at a constant surface film pressure of ≈20 mN/m. The temperature during deposition was 23 °C, and the surfaces were withdrawn from the water at a speed of 1 mm/min. The transfer ratio was 1.34 ( 0.08; i.e., the mean molecular area in the deposited LB film was ≈48 ( 3 Å2. This gives one DDOA molecule per negative charge site on the mica surface (the area per lattice charge on mica is 48 Å2.15 Surface Wetting. The wetting of DDOA-coated mica by triolein and by water in triolein was measured with a RameHart, Inc. (USA), goniometer (A-100). Turbidity. The turbidity of OPPE dispersion in triolein was measured employing a HACH ratio turbidimeter. Phospholipid Adsorption. The adsorption of OPPE onto sucrose crystals (BET surface area 0.88 m2/g) and mica particles (OMYA AB, Micro mica WI Sweden; (BET surface area 6.8 m2/g) was determined employing HPLC (high-pressure liquid chromatography) for quantitative analysis of phospholipids. All adsorption studies were performed with anhydrous dispersions. The drying procedure was accomplished by keeping melted oil, OPPE stock solution, and a thin layer of mica or sucrose crystals at 45 °C in a hermetically closed box together with P2O5 powder for a week prior to making the dispersions. The adsorption was performed in an anhydrous atmosphere. First, the particles were dispersed in refined vegetable oil (99.9% of triglycerides with (14) Attard, P.; Parker, J. L. Phys. Rev. A 1992, 46, 7959. (15) Claesson, P. M.; Herder, P. C.; Blom, C. E.; Ninham, B. W. J. Colloid Interface Sci. 1987, 118, 68.

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Table 1.

surface-liquid-medium

advancing contact angle, θA (deg)

receding contact angle, θR (deg)

DDOA-triolein-air DDOA-water-air DDOA-water-triolein

49 105 132

49 60 117

the main components being oleo-palmitostearin and oleodistearin1) by ultrasonicating for 4 min. The phospholipid was then added from a stirred stock solution containing 10 000 ppm OPPE in refined vegetable oil into the oil containing dispersed crystals. Adsorption was performed from solutions containing 200 and 2000 ppm of OPPE at 45 °C for 18 h. In each set of experiments the initial concentration of OPPE was kept constant and the amount of particles was varied. This procedure ensures that the amount of OPPE and its aggregation state is the same in all samples before adsorption. After the adsorption step was completed, the supernatant was separated from the particles by filtration through Teflon filters with a pore diameter of 1 µm (Gelman, Acrodisc CR PTFE, HPLC certified) and prepared for HPLC analysis for the phospholipid content as follows. One gram of OPPE solution in oil was dissolved in chloroform (Merck, for chromatography) to a total sample volume of 5 mL. Later, the sample was loaded onto a Sep-Pak cartridge (Sep-Pak Classic, Silica, Waters). Next, the cartridge was washed with 10 mL of 7% (v/v) petroleum spirit (BDH, GPR) in diethyl ether (Merck, GR dried) solution in order to remove compounds that may interfere (e.g., oil components containing double bonds in the hydrocarbon chains) with the OPPE signal. OPPE was eluted by applying 30 mL of methanol (BDH, for HPLC) onto the cartridge. Before the analysis, the OPPE solution in methanol was filtered through 0.2 µm Teflon filters (Gelman, AcrodiscCR PTFE, HPLC certified) and stored cold in sealed vials. To quantify the amount of OPPE in the samples 50 µL of the OPPE solution in methanol was loaded onto the HPLC column (Sperisorb S 5 W, 20 cm). As a mobile phase a mixture of acetonitrile, methanol, and phosphoric acid at the volume ratio 146/12/1 was used. The concentration of the OPPE was detected using a UV detector (Merck Hitachi L, 400.210 nm). The concentration of OPPE after adsorption was determined by integrating the HPLC peaks and comparing them with the ones obtained by analyzing solutions with known OPPE concentrations. A new calibration curve was determined for each set of experiments in order to take into account any change in sensitivity of the instrument. The adsorbed amounts of OPPE on mica and sucrose crystals were calculated from the difference in phospholipid concentration, ∆Cp, in the oil before and after adsorption (equilibrium concentration, Ceq ) and expressed as the surface excess Γex:

Γex )

∆CpV As

(2)

where V is the volume of the oil and As is the surface area of the crystals. The adsorption isotherms are reported as surface excess, Γex, as a function of equilibrium concentration, Ceq, of phospholipid. Differential interference contrast (DIC) microscopy images of mica and bright field microscopy images of sucrose particles were acquired using a ZEISS Axioplan MC 100 microscope coupled to a video camera.

Figure 1. Force normalized by radius as a function of surface separation between DDOA-coated mica surfaces (solid squares) and bare mica surfaces (open squares) interacting across anhydrous triolein.

Forces Across Triolein. The interactions between bare mica and between DDOA-coated mica surfaces across anhydrous triolein (aw ≈ 0) are shown in Figure 1. For bare mica two strong oscillations are observed. The first force barrier is located at a separation of 60-50 Å and the second one at a separation of 30-20 Å. The first force barrier is gradually overcome when applying a strong compressive force. The second force barrier is reached when this force is about 30 mN/m. The surfaces cannot be forced closer together than 20 Å even when very high forces are applied. The magnitude of the pull-off force measured when separating the surfaces is 14 ( 0.4 mN/ m, and the adhesive minimum is located at a distance of 30 Å. When two hydrocarbon surfaces, i.e., DDOA-coated mica surfaces, interact across anhydrous triolein, two pronounced force barriers of increasing strength occurring with a periodicity of about 20 Å can be identified in the force curve. The remarkable difference in periodicity of the structural force observed between the nonpolar DDOAcoated mica compared to bare mica implies that the triolein molecules pack very differently outside polar and nonpolar surfaces. The magnitude of the pull-off force measured upon separation of the surfaces at the position of the force barrier located at a distance interval D ) 45-40 Å was typically 0.8 mN/m. The magnitude of the pull-off force between DDOA-coated mica surfaces at the position of the force minimum at D ) 30-25 Å is considerably larger, typically 3 mN/m. We also measured the pull-off force between DDOA layers at D ) 0 in triolein. To accomplish this, the DDOA-coated surfaces were brought into contact in the anhydrous atmosphere and then a drop of anhydrous triolein was placed around the contact. Upon separation the normalized pull-off force, F/R, was measured, using a stiff spring, to be about 170 ( 10 mN/m. This value can be converted into an interfacial energy using the theory of Johnson, Kendall, and Roberts, the JKR theory.16

F/3πR ) γsl - γsls

(3)

Results Wetting. To study wetting of DDOA-coated mica surfaces by water in triolein and air, and by triolein in air, we employed contact angle measurements. The results obtained are presented in Table 1. The data in Table 1 show that triolein adsorbs more readily than water on nonpolar DDOA-coated mica surfaces.

This gives a value of F/3πR ≈ 18 ( 1 mN/m. For perfect surfaces the excess energy, γsls, associated with the contact between the surfaces is zero. The value of F/R and γsv γsvs for DDOA-coated mica was similarly determined to be about 350 and 37 mN/m, respectively. Hence, using (16) Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc. R. Soc. London 1971, A 324, 301.

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Figure 2. Force normalized by radius as a function of surface separation between DDOA-coated mica surfaces (open squares) and bare mica surfaces (solid circles) interacting across triolein saturated with water. The arrows pointing to the left show inward jumps occurring upon approach. Arrows pointing to the right show outward jumps occurring upon separation.

Young’s equation and assuming γsvs ) γsls we can obtain another estimate of γsl

γsl ) γsv - γlv cos θ

Figure 3. Force normalized by radius as a function of surface separation between mica surfaces interacting across anhydrous triolein containing 200 ppm of OPPE. The dashed line marks the thickness of an OPPE bilayer (one layer on each surface). Two clear force barriers are observed. The surfaces jump into the region of the inner force barrier from a distance of ≈100 Å (upper arrow). The lower arrows show outward jumps occurring during separation. Solid squares represent the force curves measured upon approach; open squares show the force curves measured upon separation.

(4)

where the triolein-vapor interfacial tension is about 32 mN/m and the contact angle 49°. This gives a value of γsl ) 16 mN/m, in good agreement with the one obtained from the measured pull-off force. The measured surface force profiles for bare mica and for DDOA-coated mica surfaces across triolein saturated with water (aw ≈ 1) are shown in Figure 2. The interaction between two bare mica surfaces was dominated by a strong long-range attraction due to capillary condensation of water in the gap between the surfaces that caused the surfaces to jump into contact form a distance of about 100 Å. Hence, the oscillating structural force was completely removed. The magnitude of the pull-off force measured upon separation was 350 ( 50 mN/m. When DDOA-coated mica surfaces interact across triolein saturated with water, the measured surface force profile was different both from that obtained for bare mica in triolein saturated with water and for DDOA-coated mica interacting across anhydrous oil. There existed two pronounced oscillations, with one force barrier located at a surface separation of 30-20 Å, i.e., at the same position as that of the inner force barrier in anhydrous triolein, and another one at a separation of 10 Å. Hence, the periodicity of the structural force between DDOA-coated surfaces was different in anhydrous and water-saturated triolein. The magnitude of the pull-off force at the position of 30-20 Å was 0.4 mN/m, and at the position of 10 Å it was 15 mN/m. In some experiments a third, weak force barrier was noted at separations of about 45 Å. Forces across OPPE Solutions in Triolein. The interactions of bare mica surfaces across anhydrous 200 ppm solutions of OPPE in triolein are presented in Figure 3. Two force barriers were observed in the surface force curve: a rather weak one, with a magnitude of about 0.8 mN/m, located in the region of D ) 100-120 Å, and a strong one, located in the distance interval D ) 70-85 Å. The pull-off force measured when separating the surfaces from the region of D ) 100-120 Å was about 0.5 mN/m and from the region of D ) 70-75 Å about 2 mN/m. This is comparable to the depth of the attractive minima

Figure 4. Force normalized by radius as a function of surface separation between mica surfaces interacting across triolein containing 200 ppm of OPPE at a water activity of 0.47. The dashed line marks the thickness of an OPPE bilayer. The arrow indicates an outward jump occurring upon separation. Solid squares represent the force curves measured upon approach; open squares show the force curves measured upon separation.

observed outside DDOA-coated mica surfaces where values of 0.8 and 3 mN/m were measured at the position of the outer and inner minima, respectively. The interactions changed when the water activity in the solution was increased to 0.47 (Figure 4). The location of the oscillations was shifted inward by ≈20 Å when compared to the anhydrous system (Figure 3). It was also found that the outer force barrier was not clearly expressed, but very weak, not exceeding 0.2 mN/m. The inner force barrier was in this case located at a separation of 53-65 Å. The repulsive force was not as steep as that in the anhydrous case. However, the surfaces could not be brought closer than to a separation of 53 Å. When the surfaces were pulled apart, they did not separate from the position of the inner force barrier by a sudden jump. Instead, they gradually moved outward, until the position of the weak outer oscillation was reached, and then the jump occurred. The magnitude of the pull-off force was close to 1 mN/m.

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Figure 5. Force normalized by radius as a function of surface separation between mica surfaces interacting across triolein containing 200 ppm of OPPE at a water activity of 1.00. The first measurement (squares) and the second measurement (circles) are shown. Forces measured upon approach and separation are presented by solid and open symbols, respectively. The arrow shows an outward jump occurring upon separation. The dashed line marks the thickness of an OPPE bilayer between the surfaces.

The forces measured across a 200 ppm solution of OPPE in triolein saturated with water were very different (Figure 5). During the first approach of the surfaces a rather strong, up to 0.8 mN/m, long-range repulsive force was measured until the surfaces came to a separation of ≈53 Å. At this point the repulsive branch of the force curve became very steep. It is interesting to notice the difference between the first and the consecutive approaches: once the mica surfaces have been brought into contact, separated, and the measurement was repeated, the long range repulsive branch was nearly completely removed but the strong steep repulsion was measured at exactly the same position as previously, at a separation of ≈53 Å. This behavior was reproducible whereas the range and magnitude of the force measured during the first approach were not. The measured pull-off force at this position was 0.8 mN/m. Note that the attractive branch of the force profile exhibited the same type of behavior as was described for the case of aw ) 0.47; i.e., first the surfaces gradually moved outward and then they ”jumped” apart. The repulsion detected at a distance of ≈53 Å could be overcome if a sufficiently strong compressive force was applied (Figure 6). In this case the mica surfaces were forced into a separation of ≈30 Å, which is some 3 Å larger than the thickness of one layer of anhydrous DPPE,17 which is very similar in size to OPPE. The measured pull-off force at D ) 30 Å was 5.7 mN/m. Adsorption. The adsorption of OPPE on mica and sucrose particles was determined as described in the Experimental Section. The mica particles were rather flat and smooth plates (Figure 7a), whereas the sucrose particles exhibited very uneven surfaces providing a host of fractures, crevices, and gaps (Figure 7b). The surface excess of OPPE as a function of total bulk OPPE concentration (i.e., OPPE present as dispersed molecules and in aggregates) in extensively dried (see methods section) refined vegetable oil is shown in Figure 8. Clearly, OPPE adsorbs on mica particles to form a monolayer. The adsorption of the phospholipid on sucrose particles is significantly different, and the amount of OPPE associated with the surface vastly exceeds that of a monolayer. We argue below that this is related to the uneven surface of the sucrose particles. We note that it is extremely difficult to remove trace amounts of water from the phospholipids (17) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608.

Dedinaite et al.

Figure 6. Force normalized by radius as a function of surface separation between mica surfaces interacting across triolein containing 200 ppm of OPPE at a water activity of 1.00. The force barrier is overcome by applying a compressive force of ≈2 mN/m; a jump occurs and the surfaces come to a separation of ≈30 Å. Solid squares represent the force curve measured upon approach; open squares show the force curve measured upon separation. The dashed line marks the thickness of an OPPE bilayer between the surfaces.

and the sugar particles, and thus we believe that despite the precautions taken some water likely remains in the sample. Hence, we do not regard this system to be anhydrous, but the water activity is low. Discussion Forces between DDOA-Coated Mica Surfaces. The surface force data for DDOA-coated mica interacting across triolein (Figure 1) show the existence of a structural force with two oscillations. The force barriers are located at separations of 45-40 and 30-20 Å; i.e., they appear with a periodicity of approximately 20 Å, which is significantly less than the dimensions of the triolein molecule along the oleic acid residues (the length along the oleic acid residue in extended conformation is calculated to be ≈27 Å8), indicating that, unlike outside polar surfaces, no clear preferential orientation of the triolein molecules can be envisaged from the force curve. The triolein molecules are not rigid; therefore, when compressive forces act on them, the conformation of the hydrocarbon chains can change. This is clearly expressed by the form of the repulsive branch in the force curve: It starts at a distance of ≈45 Å with a rather small slope, but when the separation is reduced to ≈40 Å, the slope of the force becomes steeper since it becomes more difficult for the molecules to adapt their conformation in response to the decreasing space between the surfaces. The properties of liquid molecules and the surface are the determining factors in inducing structural forces. Previously published surface force data8 for hydrophilic mica surfaces interacting across anhydrous triolein are compared with the surface force data for DDOA-coated mica interacting across anhydrous triolein in Figure 1. The magnitudes of the force barriers between hydrophobically modified mica surfaces are lower compared to those between hydrophilic mica surfaces. This is not surprising, considering the fact that the DDOA surface, consisting of hydrocarbon chains directed toward the solution, is less even than the atomically smooth bare mica basal plane. We note that the strength of the force barriers between DDOA-coated mica surfaces was strongly variable from experiment to experiment, and in some experiments the outer force barrier was not even observed. We suggest that this variation is due to a variable quality

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Langmuir, Vol. 14, No. 19, 1998 5551

Figure 8. Adsorption isotherms for OPPE from refined vegetable oil on mica particles (solid squares) and sucrose particles (open squares). The dashed lines show the limits of the surface excess, which corresponds to monolayer coverage of OPPE (limiting headgroup area in the range 40-60 Å2).

Figure 7. Optical microscopy pictures of (a) mica and (b) sucrose particles dispersed in triolein. The bars under the pictures correspond to 10 µm.

of the DDOA monolayers deposited on the mica surfaces. A less smooth surface is less efficient in inducing order in the liquid as also has been observed in previous studies using mica and hydrophobized mica surfaces in octamethylcyclotetrasiloxane.18 Another important factor for the different structural forces observed with bare mica and hydrophobically modified mica is that triolein molecules interact with nonpolar hydrocarbon surface and polar mica surfaces in different ways: At a bare mica surface the local dipolar interactions between ester groups of the glyceryl residue and the mica surface are important, and this leads to a strong preferential orientation at the mica surface. In contrast, at the DDOA-coated mica surface these interactions are insignificant. It is not expected that the van der Waals interaction between DDOA-coated mica and the various parts of the triolein molecule is very different, and therefore no significant preferential orientation occurs. Instead, the spatial confinement of the triolein molecules in a more random (18) Christenson, H. K. J. Phys. Chem. 1986, 90, 4.

conformation and orientation gives rise to the observed structural force. The presence of water in the triolein sample (aw ≈ 1) induced marked changes in the measured surface force profile (Figure 2). It is possible to identify three force barriers with a variable periodicity in the force curve. The two outermost force barriers were located at the same separations as in anhydrous triolein whereas the innermost one was only observed in the presence of water. It is to be noted though, that the outermost force barrier, located at a surface separation of about 45 Å, was very weak and not always observed. We suggest that the effect of water is due to its affect on the deposited layer. DDOA is an amphipilic molecule, consisting of two hydrocarbon chains and a positively charged ammonium headgroup that anchors it to the negatively charged mica surface. It was shown in the study of Eriksson et al.19 that despite a very good match between the number of negative sites on the mica basal plane and the number of deposited DDOA molecules (close to one DDOA molecule per one negative mica lattice charge), imperfections always exist in the DDOA layer, and thus a part of the mica surface is exposed. When water is present in the triolein, it will interact favorably with the polar part of the DDOA and the mica surface. Obviously, the ease at which the water molecules get access to the polar part of DDOA depends on the initial quality of the deposited layer. The presence of water makes the DDOA molecules more mobile and renders the DDOA monolayer less ordered. The amount of defects, apparently, varies from deposition to deposition, and as a consequence of this, we measure a surface force profile with two or three force barriers. Due to the reduced ordering in the DDOA monolayer when water is present, the triolein molecules become less ordered outside such a surface. Under the action of a compressive force the triolein molecules in the last layer are able to rearrange in such a way that their oleic acid residues become parallel to the surface. This is the reason for the innermost oscillation. In contrast, this rearrangement of the last triolein layer was not observed without the presence of water. Though the water influence was apparent, it was of much smaller degree and it did not remove the structural force as was the case when bare mica surfaces were interacting across triolein saturated with water. In the (19) Eriksson, L. G. T.; Claesson, P. M.; Ohnishi, S.; Hato, M. Thin Solid Films 1997, 300, 240.

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latter case water was preferentially adsorbed onto the mica surfaces, which resulted in a long-range attraction due to capillary condensation (Figure 2). For further discussion on this matter, see.8 Phospholipid Adsorption from Nonpolar Medium. Phospholipid adsorption onto solid polar surfaces from nonpolar media has been investigated in several studies using natural and synthetic phospholipids. However, the results reported are rather different and no consistent picture has emerged. Hey et al.3 investigated dipalmitoylDL-phosphatidylcholine (DPPC) adsorption onto silica particles from ethanol, methanol, and propanol and concluded that the binding affinity of DPPC increases when the polarity of the solvent decreases. No explanation for this observation was provided, but it seems reasonable that the effect is largely due to that the surface-solvent interactions become less favorable for less polar solvents. The limiting area at the adsorption plateau was found to be slightly larger than 100 Å2; i.e., a far from tightly packed monolayer was obtained. Similar results were obtained by Tamamushi for egg lecithin adsorption onto alumina from hexane, who reported a limiting area per adsorbed phospholipid of 140 Å2. On silica, however, the limiting area per molecule was only 30 Å2.4 For phosphatidylcholine adsorption from soybean oil on saccharose particles, multilayer adsorption or aggregate formation was reported.6 In our opinion, it is difficult to understand what factors could drive phospholipid multilayer adsorption from nonpolar media. Phospholipid adsorption from a nonpolar medium is likely driven by energetically favorable polar interactions between the phospholipid headgroup and the polar surface. Once a monolayer of phospholipids is formed on the surface, the surface becomes nonpolar and the polar interactions no longer play a role. For multilayer adsorption one thus has to look for other reasons than polar interactions between the phospholipid and the surface. One such reason could be that dipolar interactions between the phospholipid headgroups favor multilayer adsorption, particularly in the presence of water when liquid crystalline phases are formed. In that case multilayer adsorption ought to take place on every type of surface. However, our adsorption and surface force data show that this is not the case. Instead we found that on mica crystals OPPE adsorbed to give a monolayer, whereas onto sugar crystals the amount of adsorbed matter many times exceeded the amount corresponding to monolayer coverage. We view this excessive adsorbed amount as being due to phospholipid phase separation rather than due to multilayer adsorption. As revealed by the microscopy pictures (Figure 7), the sucrose particles have much rougher surfaces compared to mica crystals. Hence, the phospholipid phase (reversed micelles, reversed hexagonal or lamellar) can condensate in small crevices thus leaving the supernatant solution depleted from OPPE. The driving force for this process is a reduction in the area between the phospholipid phase and the oil. Phase separation of sparingly soluble solution components to microscopic gaps has been observed before20 and is well understood. OPPE exhibits low (below the order of 1 × 10-4M) solubility in triglyceride oil; therefore even very small amounts of OPPE present in the oil give a solution saturated with the phospholipid. The change in free energy (∆G) due to phase separation of OPPE from triolein in a crevice can be expressed as: (20) Christenson, H. K. J. Colloid Interface Sci. 1985, 104, 234.

Dedinaite et al.

∆G ) A1(γ12 - γ13) + A2γ23 +

V ∆g v

(5)

where ∆g is the change in free energy associated with bringing 1 mol of the condensing material from the bulk, where it has a low concentration, to the condensate where it has a larger concentration, γ12 is the interfacial tension between the surface and the condensing phase, γ13 is the interfacial tension between the surface and the bulk phase, γ23 is the interfacial tension between the bulk phase and the condensing phase, A1 is the surface area inside the crevice, A2 is the surface area between the condensing phase and the bulk phase, V is the volume of the condensate, and v is the molar volume of the condensing material. The free energy change can only be negative provided that the first term in the equation is sufficiently negative to compensate for the other two positive terms. It is wellknown that the addition of lecithin1 improves the flow properties of sugar dispersions in fat. Observations of particles by scanning electron microscopy revealed that added lecithin smoothens previously rough particle surfaces.21 This is fully consistent with the idea of phospholipid phase separation into crevices and gaps. By phase separation, the phospholipids fill existing cracks and crevices, thus making the particle surface smoother, which means that the particles can slide past each other easier. Forces Induced by the Presence of Phospholipids in Solution. As we mentioned above, OPPE has a very low molecular solubility in anhydrous triglyceride oil; therefore the mixtures of OPPE and triolein are turbid. By measurement of the turbidity of OPPE dispersions in triolein (at aw ≈ 0.4-0.6), it was observed that the solubility is below 80 ppm (1.02 × 10 -4 M), most likely even lower, but the accuracy of the method does not allow any more rigorous estimate. We measured the surface interactions across anhydrous 200 ppm OPPE dispersion, i.e., in the region where the OPPE concentration exceeds the solubility limit. We note that OPPE is hygroscopic and the turbidity of the solution decreases with increasing water activity. We interpret this observation as evidence for higher solubility and/or formation of smaller reversed aggregates at higher water activities. Preliminary light scattering experiments of OPPE solutions in triolein at high water activities show the presence of some small aggregates with a hydrodynamic radius of about 120 Å, presumably nonspherical reversed micelles, and larger aggregates with a mean hydrodynamic radius of about 5200 Å. The surface force profile for two mica surfaces interacting across anhydrous 200 ppm OPPE solution is shown in Figure 3. There are two oscillations identified in the surface force curve: one located at a surface separation of ≈100 Å and another one located at a surface separation of ≈70-75 Å. These results are consistent with a monolayer of OPPE adsorbed onto each mica surface and two layers of triolein molecules ordering themselves outside such a surface. The number and periodicity of the force barriers reminds one of those detected for DDOAcoated mica surfaces interacting across anhydrous triolein (Figure 1). This indicates that OPPE self-assembles to form monolayers onto mica surface thus making it rather nonpolar. This becomes even more clear if one subtracts the thickness of the OPPE layers from the measured distances and then compares the results with the forces observed between initially hydrophobic mica surfaces across triolein. Such a comparison is made in Figure 9. (21) Hoskin, J. M.; Dimick, P. S. J. Food Sci. 1980, 45, 1541.

Mica in Triglyceride Media

Figure 9. A comparison of the interactions between two DDOAcoated mica surfaces across anhydrous triolein (9) and two bare mica surfaces across anhydrous triolein containing 200 ppm of OPPE (0).

Indeed, the two force curves are rather similar, the main differences being that the outer oscillation is less pronounced and the inner one less steep for mica across the 200 ppm OPPE solution. The likely reason for this is that the self-assembled OPPE layer is less densely packed compared to the DDOA layer prepared by LangmuirBlodgett deposition. Our previous study8 revealed the significance of the interactions between the polar part of triolein and the polar mica surface leading to an adsorption of triolein with the ester groups toward the mica surface. The phospholipid, OPPE, possesses a strongly polar phosphatidylethanol amine group, and this group, being more polar than the triolein glyceryl residue, will drive the preferential adsorption of OPPE onto mica surfaces. Close to monolayer adsorption is confirmed by the adsorption isotherm of OPPE on mica particles from anhydrous refined vegetable oil (Figure 8). Despite this it is not certain that the hydrophobic monolayer is composed only of OPPE. Some triolein molecules may be incorporated into the adsorbed layer. As both the oleic acid residues of triolein and the hydrocarbon chains of OPPE are of a very similar length (18 C atoms in the oleic acid residue and 16 C atoms in the palmitic residue) it is impossible to distinguish between these two molecules by SFA measurements. When the water activity in the OPPE solution was increased to 0.47, some significant changes in the measured interaction profile were detected: the outer force barrier became undetectable and the inner one was shifted by some 10 Å to smaller separations (Figure 4). Also, it became less steep when compared to the same force barrier measured in anhydrous solution. By application of a compressive force of 9 mN/m, the surfaces were forced into OPPE-OPPE layer contact. It has been suggested that the binding of the polar part of phospholipids to polar surfaces in nonpolar media is mediated by water molecules. It is claimed that water helps to bind phospholipids stronger to sugar particles surfaces.1 This theory is partly supported and partly not confirmed by our experimental results using mica. It is clear that water is accumulated in the region between the polar part of the phospholipids and the polar surface. However, the presence of water reduces the order of the adsorbed layer as evidenced by the decreased strength of the structural force and by the fact that at very high water activities (aw ≈ 1) part of the phospholipid layer can be

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squeezed out by applying a compressive force of 2.2 mN/m (Figure 6), whereas at lower water activities even a force exceeding 10 mN/m was not able to affect the OPPE layers (Figure 4). Thus there is no doubt that the phospholipid polar part interacts with water, but this does not result in a stronger binding to the surface but rather in an increased lateral mobility. A similar conclusion can be drawn for triolein molecules adsorbing to mica at a water activity of 0.9.8 Another unexpected observation was that when the surfaces were separated in OPPE-triolein solutions, they moved apart by several nanometers before the position of the attractive minimum was reached. No similar effect was seen in pure triolein. The reason may be that OPPE phase separates in the gap between the surfaces when they are in contact. However, the refractive index difference between OPPE and triolein is too small to allow the direct observation of a separate phase between the surfaces, and the presence of such a phase between the surfaces should be regarded as a working hypothesis. A long range repulsion develops when the water activity is increased to close to 1 (Figure 5). We suggest that this repulsion is due to the presence of water containing OPPE aggregates, presumably, reversed micelles, that weakly attach to the surface. The formation of reversed micelles by phospholipids in nonpolar media has been observed previously by proton NMR.22 As is shown in Figure 5, the long range repulsion, which indicates that aggregates are attached to the surface, was measured just upon the first approach but not on consecutive approaches. Clearly, the aggregates are easily destroyed or pushed away from the surface, confirming their weak attachment. The finding that OPPE forms aggregates in nonpolar media when water is present has important consequences for the influence of trace amounts of water on the stability of colloidal dispersions. In our previous study we investigated in detail the effect of water on interactions between polar surfaces in triolein and showed that water removes the stabilizing structural force and gives rise to long-range attractive forces. In OPPE-triolein solutions the water effect is opposite. By the formation of small phospholipidwater aggregates that weakly adsorb to the surface and generate long-range repulsive forces, certain amounts of water present in a dispersion may stabilize it. Aggregate formation may account for another phenomenon observed in practice. Namely, it is known that not only the part of lecithin that is directly adsorbed on the particles contributes to a decrease in viscosity but also the “nonadsorbed” fraction. Hence, the lecithin molecules not directly attached to the particles play some part in decreasing the viscosity, but the mechanism of their action is not clear.1 Our experimental results give one clue: weak association between surfaces and lecithin aggregates induces repulsive forces and prevents the particles from sticking to each other. Conclusions Two molecular layers of triolein are ordered between DDOA-coated mica surfaces when no water is present. The triolein molecules do not adopt any clear conformation or orientation outside such surface. When water is present in the medium, it affects the DDOA layer and disturbs the packing in the deposited layer. Therefore the structural force is reduced compared to in anhydrous triolein. It is also observed that under the action of a strong compressive force the triolein molecules change their orientation with respect to the surface. These findings (22) Kumar, V. V.; Raghunathan, P. Chem. Phys. Lipids 1986, 41, 159.

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are different from the results obtained for bare mica interacting across anhydrous triolein, where it was concluded that mica surfaces induce an orientation of triolein with the oleic acid residues directed to the bulk and with the glyceryl residue adsorbed on the surfaces. The results are also different from those obtained for bare mica interacting across triolein saturated with water where the structural force was totally removed and instead a long-range attractive force due to capillary condensation was measured. OPPE self-assembles onto mica surface from OPPEtriolein mixtures and renders the surface rather nonpolar. The interactions of such surfaces across an anhydrous OPPE-triolein solution are similar to those of DDOAcoated mica across anhydrous triolein. When water is present, it interacts with the polar part of OPPE and the mica surface. This result in a reduced structural order of the OPPE layers, and the structural force is decreased.

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At high water contents reversed phospholipid aggregates weakly adsorb onto the surface giving rise to a long-range repulsive force. This may have a stabilizing effect on particle dispersions in the oil. We have noted that OPPE adsorbs on mica surface to give a monolayer, whereas adsorption on sucrose largely exceeds that of a monolayer. We suggest that it is due to phase separation of OPPE into cracks and crevices on the sucrose particles. Acknowledgment. We wish to thank Bjo¨rn Bergenståhl, Marianne Lindblom, and Paul Smith for inspiring discussions. Britt Hedlund and Nicklas Warne are thanked for performing quantitative analysis of phospholipids at Kraft Freia Marabou. Andra Dedinaite acknowledges financial support from Kraft Foods. LA980237X