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An AFM Study of Lipid Monolayers. 1. Pressure-Induced Phase Behavior of Single and Mixed Fatty Acids K. Ekelund,*,† E. Sparr,‡ J. Engblom,† H. Wennerstro¨m,‡ and S. Engstro¨m† Department of Food Technology, Chemical Center, Lund University, Lund, Sweden, and Department of Physical Chemistry 1, Chemical Center, Lund University, Lund, Sweden Received January 29, 1999. In Final Form: May 28, 1999 Monolayers of palmitic (C16:0) and lignoceric acid (C24:0) and their equimolar mixture were transferred to a hydrophilic mica substrate at various surface pressures and investigated by means of atomic force microscopy (AFM) in contact and lateral force modes. The first-order transition of lignoceric acid gives a plateau region, representing a liquid expanded to liquid condensed phase transition in the pressure-area isotherm. This was visualized by AFM as stripes of a condensed phase within the expanded phase, exhibiting a small height difference but a significant difference in friction. The corresponding phase transition of the palmitic acid was continuous, and no changes of the Langmuir-Blodgett films with respect to pressure were observed with AFM. Both the surface pressure-area isotherms and the direct observations of domains of irregular size and shape using the AFM showed that lignoceric and palmitic acid were immiscible. The height difference between the domains was 1.1 nm corresponding to the difference in hydrocarbon chain length of the two fatty acids.
Introduction Fatty acids are examples of water-insoluble amphiphilic compounds that self-assemble in aqueous solution and at water surfaces. Traditionally information on the organization of the monolayers has been obtained from surface pressure-area isotherms. During the past decade more sophisticated methods have been developed such as fluorescence microscopy,1 Brewster-angle microscopy (BAM),2 and synchrotron X-ray diffraction,3 which provide a much more detailed picture of the molecular organization in the monolayer. However, these methods show a limited lateral resolution of structures existing in the plane of the film. By transfer of the monolayer to a solid substrate, thereby creating a so-called Langmuir-Blodgett film, further techniques become available for the investigation of the monolayer. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR)4 have been used to investigate monolayer domain topography, but none of these techniques can provide direct information of heterogeneous domains as well as nanometer-scale structure and surface properties of heterogeneous multicomponent monolayers. Atomic force microscopy (AFM) is a surface imaging technique with angstrom-scale lateral and normal resolution that operates by measuring the forces acting between a probe and the sample. Biological membranes have complex organizations that are essential for their functionality. Phospholipids are the main constituents in many biological membranes, and many studies of their monolayers have been reported.5,6 We have a particular interest in the lipid bilayers existing between the corneocytes in the horny layer of the skin.7 * Corresponding author. † Department of Food Technology. ‡ Department of Physical Chemistry. (1) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171-195. (2) Wolthaus, L.; Schaper, A.; Mo¨bius, D. J. Phys. Chem. 1994, 98, 10809-10813. (3) Kenn, R. M.; Bo¨hm, C.; Bibo, A. M.; Peterson, I. R.; Mo¨hwald, H. J. Phys. Chem. 1991, 95, 2092-2097. (4) Kajiyama, T.; Oishi, Y.; Uchida, M.; Tanimoto, Y.; Kozuru, H. Langmuir 1992, 8, 1563-1569. (5) ten Grotenhuis, E.; Demel, R. A.; Ponec, M.; Boer, D. R.; van Miltenburg, J. C.; Bouwstra, J. A. Biophys. J. 1996, 71, 1489-1399. (6) Dufreˆne, Y. F.; Barger, W. R.; Green, J.-B. D.; Lee, G. U. Langmuir 1997, 13, 4779-4784.
Here phospholipids are rare, and the main classes of lipids are ceramides, cholesterol, and free fatty acids of different chain lengths and saturation.8,9 For a complex biological mixture it is hard to identify individual components from the topographic images. By the study of each component separately and then the formation of simplified models, more information of the complex system can be achieved. The aim of this work is to establish basic knowledge about lateral phase separation and the formation of domains on the nanometer scale by studying monolayers of two fatty acids of different chain lengths and their mixtures. To this end we perform AFM measurements on LangmuirBlodgett films transferred from the air-water interface at controlled surface pressures. Materials and Methods Palmitic (C15H31COOH) and lignoceric (C23H47COOH) acid (+99% purity) were purchased from Larodan Fine Chemicals (Malmo¨, Sweden). Monolayers were prepared on a LangmuirBlodgett trough type 611 from Nima Technology (Coventry, England). A 0.1 M acetate buffer, adjusted to pH 4.0 was used as subphase. The water was deionized, distilled, and filtered through a Millipore Q purification system (Millipore Corporation, Bedford, MA). Solutions of single fatty acids and their equimolar mixtures (1:1) were used in this study. Isotherms were reversible and reproducible. For deposition, sheets of freshly cleaved mica were immersed into the subphase. The lipids were dissolved (1 mg/mL) in chloroform and spread on the subphase. After the solvent was allowed to evaporate for 20 min, the monolayers were compressed at a speed of 20 cm2/min to the pressure of deposition. The monolayer was left standing at constant pressure for 20 min before it was transferred to the substrate at a dipping speed of 2 mm/min. All samples were prepared in a cleanroom at a constant temperature of 19 °C. The transfer ratios of the monolayers were close to unity. Constant force AFM and lateral force AFM (LFM)10 measurements were performed on a commercial Nanoscope IIIa (Digital Instruments, Santa Barbara, CA). Experiments were made in air at ambient temperature within 4 h from sample preparation. (7) Engblom, J.; Engstro¨m, S.; Jo¨nsson, B. J. Controlled Release 1998, 52, 271-280. (8) Wertz, P. W.; Swartzendruber, D. C.; Madison, K. C.; Downing, D. T. J. Invest. Dermatol. 1987, 89, 419-425. (9) Norle´n, L.; Nicander, I.; Lundh Rozell, R.; Ollmar, S.; Forslind, B. J. Invest. Dermatol. 1999, 112, 72-77. (10) Neubauer, G.; Cohen, S. R.; McClelland, G. M.; Horne, D.; Mate, C. M. Rev. Sci. Instrum. 1990, 61, 2296-2308.
10.1021/la990092+ CCC: $15.00 © 1999 American Chemical Society Published on Web 08/05/1999
AFM Study of Lipid Monolayers
Figure 1. Surface pressure-area isotherms for monolayers of lignoceric acid, palmitic acid, and their equimolar mixture. Deposition pressures used for the images shown are indicated in the isotherms (A-C). An E tube scanner with a 10 × 10 (x, y) × 2.5 (z) µm scan range was used for imaging. Microfabricated square pyramidal shaped tips of silicon nitride with a bending spring constant of 0.12 N/m (manufacture specified, Digital Instruments, Santa Barbara, CA) were used as received. The scan rate was 2 Hz, and the applied force was on the order of 1-10 nN. To eliminate imaging artifacts, the scan direction was varied to ensure the true image. Images were obtained from at least five macroscopically separated areas on each sample. All images were processed using procedures for plane-fit and flatten in Nanoscope IIIa software version 4.22 (Digital Instruments, Santa Barbara, CA) without any filtering. Dimensions of the domains were measured directly from the AFM height images, and thickness variations were estimated from section analysis of the topographic images.
Results and Discussion Surface Pressure-Area Isotherms. The pressurearea isotherms of the systems were carefully studied before depositing the monolayers on the mica support. The pressure-induced phase behavior was studied for single fatty acid monolayers and for mixed monolayers. Figure 1 illustrates the air-water pressure-area isotherms of lignoceric acid (C24:0), palmitic acid (C16:0), and the equimolar mixture at 19 °C. The isotherm of the lignoceric acid has a transition from liquid expanded to liquid condensed state at a surface pressure of approximately 8-10 mN/m and an area of 25-22 Å2/molecule; see B in Figure 1. This transition is extended and rather flat, and a large decrease in headgroup area takes place at equilibrium between the condensed and expanded phases at ideally constant surface pressure. Consequently, conformational order in the hydrocarbon chains must increase significantly. The transition occurs with a small variation in pressure and this slight deviation from the ideal behavior of a first-order phase transition11 can be taken as a sign that a true macroscopic phase separation is not realized in the sample. Nonequilibrium nanometer-sized domains or aggregates on the surface can result in nonhorizontal transitions in the monolayer isotherm.12,13 If one eliminates the possibility of an impurity effect, the (11) MacRitchie, F. Chemistry at Interfaces; Academic Press: San Diego, CA, 1990. (12) Israelachvili, J. Langmuir 1994, 10, 3774-3781. (13) Ipsen, J. H.; Mouritsen, O. G.; Zuckermann, M. J. J. Chem. Phys. 1989, 91, 1855-1865. (14) Bibo, A. M.; Peterson, I. R. Adv. Mater. 1990, 2, 309-311. (15) Overney, R. M.; Meyer, E.; Frommer, J.; Gu¨ntherodt, H.-J. Langmuir 1994, 10, 1281-1286.
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Figure 2. Topographic (top) and friction (bottom) AFM images (3 × 3 µm) of a transferred monolayer of lignoceric acid. The films were deposited on mica at a surface pressure corresponding to A (a) and B (b) in Figure 1. The difference between the phases is clearly seen in the friction images in (b). Z range: for the topographic images, 1.5 nm; for the friction images, 0.05 V.
most likely cause to the nonideal phase transition is the formation of small domains in the monolayer. The corresponding phase transition in the palmitic acid monolayer is continuous, with a small decrease in headgroup area, around 22 mN/m and 22 Å2/molecule; see Figure 1. Isotherms and headgroup areas of the fatty acids are in consistence with an earlier report.14 The mixed monolayer of lignoceric acid and palmitic acid gives a pressure-area isotherm where the phase transitions for both components are seen at unchanged surface pressures. This is an indication of immiscibility between the fatty acids, which is in agreement with previous studies of mixed fatty acids with several hydrocarbons difference in chain length.14 The nonhorizontal transition of lignoceric acid is even more pronounced in the mixed fatty acid isotherm, where the transition takes place under the same surface pressures (8-10 mN/m) but the reduction in headgroup area is smaller (25-23 Å2/ molecule). The transition from gaseous to liquid expanded state of the monolayers was clearly observed but not studied in detail. Single Fatty Acid Monolayers. Transferred monolayers of lignoceric acid are studied at the surface pressures A-C in Figure 1. Height and friction AFM images were simultaneously obtained, where the height images originate from normal forces and the friction images from lateral forces. Frictional measurements can give information on heterogeneities in samples that are not caused by height differences and are therefore a good complement to the topographic measurements. For the interpretation, absolute values of the friction measurements are not reliable, while relative values of friction within an image are more accurate.15 At pressure B in the isotherm in Figure 1, both the expanded and the condensed phase are present. Flat areas and some more crude areas with stripes are observed in the transferred monolayer at this surface pressure; see Figure 2b. The stripes have various orientations within the sample, and the distance between them is typically 150 nm. The height of the stripes is only about 0.1-0.2 nm and would be difficult to detect with methods other than AFM. In the friction image these stripes are much more pronounced showing 2-3 times higher friction relative to the flat areas within the same sample (Figure 2b). The orientation and size of these stripes are the same
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when (i) zooming in and out of an area examined and (ii) changing scan direction, strongly indicating that the stripes reflect a property of the system rather than one of the imaging processes. Lignoceric acid monolayers deposited at higher surface pressures, corresponding to a liquid condensed state of the monolayer, are flat with no visible stripes. The films seem quite robust, and no sign of film rupture by the tip can be seen. For low surface pressures, well below the phase transition pressure, the transferred monolayer shows stripes; see Figure 2a. However these stripes differ from those in the previously described sample in shape, and they exhibit much lower relative friction; compare Figure 2a,b. In the low-pressure sample, one can also see inhomogenities and a few holes or cracks. The depth of the holes are about 2 nm, and no internal structure can be observed. In the liquid expanded state of lignoceric acid, the headgroup area is relatively large which allows a less ordered hydrocarbon chain organization. This can explain the irregularities observed within the monolayer. The film also seems to be sensitive to the force applied by the tip on the sample. In transferred films of palmitic acid no stripes are observed at any surface pressure. Furthermore, palmitic acid monolayers are not as robust as the lignoceric acid monolayer and they seem to be affected by the tip. The condensed palmitic acid monolayer is more resistant to the tip than the expanded monolayer, and no signs of new defects caused by the tip are observed. No other significant differences between the expanded and condensed palmitic acid films are detected. At the first-order phase transition liquid expanded and liquid condensed phases coexist in the monolayer. This is demonstrated here as a two-dimensional phase separation in the transferred film, visualized as stripes in the lignoceric acid monolayer. The height images alone cannot prove that the small topographic irregularities in Figure 2b are due to the phase transition and are different from those in Figure 2a. By combination of height and friction images, a significant difference between the two samples can be shown, where the frictional fluctuations can be related to variations in the crystalline properties within the samples. From this it is concluded that the features observed in Figure 2b are coexistence of liquid expanded and liquid condensed lignoceric acid monolayers and not the same as the features in Figure 2a which lack this significant difference in friction. A corresponding phase transition is not observed for the palmitic acid monolayer, which is due to the continuous transition without a coexistence region. Inhomogenities within lignoceric acid monolayers have also been reported by Hosoi et al.16 but are not assumed to be the same as seen here. The internal inhomogeneous and triangular structures of lignoceric acid visualized by phase contrast microscopy are several magnitudes larger in size than the ones observed in our work. The structures reported by Hosoi et al. existed for all surface pressures. Mixed Monolayers. In all images of the equimolar mixture of lignoceric and palmitic acid, phase separation is observed with distinct domains of respective fatty acid; see Figure 3a. The relative height difference between the domains is ∼1.1 nm. For 8 carbon atoms in the all trans conformation of a normal alkane their estimated length is 8 × 0.127 ) 1.02 nm. A thinner continuous phase of (16) Hosoi, H.; Akiyama, H.; Hatta, E.; Ishii, T.; Mukasa, K. Jpn. J. Appl. Phys. 1997, 36, 6927-6931. (17) Koleske, D. D.; Barger, W. R.; Lee, G. U.; Colton, R. J. Mater. Res. Soc. Symp. Proc. 1997, 464, 377-383. (18) Widayati, S.; Dluhy, R. A. Microchim. Acta Suppl. 1997, 14, 683-685.
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Figure 3. (a) Topographic AFM image (3 × 3 µm) of a transferred monolayer of lignoceric acid-palmitic acid, molar ratio 1:1. Z range: 5 nm. (b) A cross section along the line indicated in (a).
palmitic acid (appears dark in the image) is embedding large domains of a thicker phase of lignoceric acid (appears bright in the image). The domains have an irregular shape with the boundaries consisting of nonuniformly connected straight stretches on a nanometer length scale. At increased surface pressure the interfaces become even less rounded. The size of the domains are about the same even at increased pressure. Small domains are observed within the lignoceric acid domains; see Figure 3. The relative height difference to the lignoceric acid domains is the same for these small domains as that for the continuous palmitic acid phase; see section graph in Figure 3b. From this it is concluded that the small domains consist of palmitic acid. These palmitic acid “lakes” within the lignoceric acid domains are almost circular with a diameter of ∼200 nm. Area ratios between the two different lipid phases correlate with the composition of the sample. The palmitic acid phase is less sensitive to the tip at increased surface pressure which is in consistence with the results for the pure palmitic acid monolayer. The height differences between lignoceric acid and palmitic acid domains was here measured to 1.1 nm, consistent with the difference of 8 methylene units. Koleske et al.17 reported almost twice the chain length difference for the same system measured with AFM. This deviation is claimed to be due to adhesion forces. On the other hand, Widayati et al. have reported an AFM study where the height differences in monolayers of mixed chain length fatty acids are in good agreement with the difference in methylene units.18 Small features can be observed within the lignoceric acid domains for samples prepared at pressures corresponding to the phase transition of lignoceric acid. These features are hard to detect in height images but are very distinct in friction images; see Figure 4. They do not show any regular size or shape, although they often resemble stripes. At pressures below the phase transition of lignoceric acid no signs of these features are observed. It is our interpretation that the small features in the lignoceric acid domains correspond to the stripes in the pure lignoceric acid monolayer. As a result of the large height difference between the two fatty acids, compared to the internal height difference in the lignoceric acid, the features in the lignoceric acid domains are less pronounced in the height image of the mixed samples compared to the single lignoceric acid samples. Despite this, the relative frictional differences within the lignoceric acid phase is
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Figure 5. Topographic AFM image (3 × 3 µm) of a transferred monolayer of palmitic acid-lignoceric acid, molar ratio 1:1. The film was relaxing at a surface pressure of 0 mN/m for 12 h before deposited on mica at a surface pressure of 22 mN/m. Z range: 5 nm.
Figure 4. Topographic (top) and friction (bottom) AFM images (3 × 3 µm) of a transferred monolayer of palmitic acid-lignoceric acid, molar ratio 1:1. Height difference between the phases was measured to 1.1 nm. The structures in the lignoceric acid phase are clearly seen in the friction image. The film was deposited on mica at a surface pressure corresponding to B in Figure 1. Z range: for the topographic image, 5 nm; for the friction image, 0.07 V.
of the same order of magnitude as the relative frictional differences between the two fatty acid phases. At pressures below and above the phase transition of lignoceric acid, no frictional differences within the lignoceric acid phase are detectable. This is true for both the lignoceric acid samples and the mixed samples, with exception for the very weak stripes in Figure 2a. One can therefore conclude that the frictional measurements in these samples visualize the coexistence of liquid condensed and liquid expanded phases in the lignoceric acid monolayer. Domain Shape. Equilibrium of the phase-separated domains would be expected to be of circular or at least regular shapes. Thus the shape of the domains in the mixed fatty acid monolayers exhibit a nonequilibrium state and incomplete phase separation. In all described experiments the monolayer was left for 20 min at a determined pressure before depositing. To investigate the equilibration for the domain formation more thoroughly, the mixed monolayer was left for 12 h at a constant pressure of 22 mN/m before deposition. AFM images of this sample showed domains of comparable size and shape as for the earlier experiments. Finally a spread monolayer was left at zero surface pressure for 12 h before compression to 22 mN/m and deposition onto mica. Figure 5 illustrates this monolayer where the domains of lignoceric acid are squared and have smooth borders to the palmitic acid and few “lakes” of palmitic acid within the lignoceric acid are seen. From these experiments it can be concluded that the phase separation in two dimensions is very slow. Perfect phase-separated structures were not even obtained after 12 h at zero surface pressure. The slow phase separation allows us to study monolayers transferred at nonzero surface pressures with reproducible results. Effects of Sample Preparation. Investigations of transferred Langmuir-Blodgett films generally aim to increase the understanding of the monolayer structure at the air-water interface. Even if there is a general belief (19) Schwartz, D. K.; Viswanathan, R.; Garnaes, J.; Zasadzinski, J. A. J. Am. Chem. Soc. 1993, 115, 7374-7380.
that the structure of a monolayer at the air-water interface resemble the structure of the monolayer transferred to a solid substrate,19 one has to be aware of factors that may influence the molecular arrangement during and after the deposition. Samples were therefore prepared with varying deposition dipping speed in the range of 2-10 mm/min, showing no visible differences. Some of the transferred samples were also reexamined after 1 and 2 days, and no significant changes could be noticed. To ensure that the routine for the monolayer preparation gives representative and reproducible results, the monolayer at the air-water interface was compressed and decompressed in isocycles six times before relaxing at constant pressure and then deposited onto mica. The compression speed was also varied in the range of 10200 cm2/min. No detectable differences were observed in these samples compared to films made according to the routine described in the Materials and Methods section, except that the lower palmitic acid phase was maybe slightly rougher after the isocycles. Varying the pH of the subphase resulted in a better film quality for monolayers prepared on a buffer of pH 4 than on one of pH 7. Since the fatty acids in the monolayer effectively titrate at a higher pH than in bulk, we assume that all lipids should be in an undissociated state at pH 4, resulting in a more homogeneous monolayer when no lipids self-assembles to form aqueous phases. Conclusion Domain formation in single and mixed LangmuirBlodgett films of free fatty acids has been studied by AFM. The method clearly reveals domains in the lignoceric acid monolayer at the liquid expanded to liquid condensed phase coexistence. These are particularly apparent using the friction mode of the AFM device. Similarly for the mixed palmitic lignoceric acid system separate domains of the two components are easily seen and they show a difference in thickness reflecting the difference in chain length of the two acids. The domains appear over a substantial variation in the procedure for preparing the monomolecular film, and we conclude that the domains are present also in the parent film at the air-water interface. The domains are most likely not equilibrium structures, but they form generically and their presence can explain the pressure variation in the coexistence region for liquid expanded and liquid condensed phases. The irregular shape of the domains in the mixed lignoceric acid-palmitic acid system shows that not only is the equilibration slow with respect to the formation of large, macroscopic domains but also the relaxation of the shape appears to be very slow under the experimental conditions. LA990092+
