Langmuir 1992,8, 197-200
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Evidence for First-Order Phase Transitions in Lipid and Fatty Acid Monolayers Y. F. Hifeda and G. W. Rayfield* Physics Department, University of Oregon, Eugene, Oregon 97403 Received July 18, 1991. In Final Form: September 3, 1991 Data are reported which show that the liquid-condensed-to-liquid-expanded(LC/LE) phase transitions of a pentadecanoic acid monolayer and a dipalmitoylphosphatidylcholine monolayer are first order. In particular we measured a zero slope of the T-A isotherm in the LC/LE transition region using a continuous compression method. The data were obtained by using a very careful experimental protocol. We also examined the state of equilibrium or stability of the monolayer system.
Introduction There are theories of critical behavior which are not solvable in three dimensions, but become solvable in two dimensi0ns.l Partly because of this there is considerable theoretical interest in the phase behavior of twodimensional systems.lP2 A particular two-dimensional system that has received much study is the molecular monolayer which consists of surfactant molecules spread on a water s ~ r f a c e .Some ~ ~ ~ theories and mathematical models developed to describe the monolayer system yield a first-order phase t r a n ~ i t i o n . ~The - ~ phase transition involves a change from a liquid-expanded state to aliquidcondensed state (LC/LE). A first-order phase transition requires a zero slope in the ?r-A isotherms in the transition region.' However, for a very long time, there was no experimental evidence for a region of zero slope in the ?r-A isotherms.3 This is strictly inconsistent with a firstorder phase transition. Experimental evidence for a kink in the ?r-A without a region of zero slope prompted efforts to devise models that yielded a higher order LC/LE phase transition."16 These higher order models are characterized by the lack of a coexistence of the liquid-expanded and liquidcondensed states. However, monolayer experiments, using fluorescence imaging17-19and electron microscopy,20have shown that lipid monolayers exhibit coexistence of two phases in the LC/LE transition region. This experimental evidence (1)Nagle, J. F. Ann. Rev. Phys. Chem. 1980,31,157. (2)Pink, D. A. Can. J. Biochem. Cell Biol. 1984,62, 760. (3)Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966; pp 15-188. (4)Adamson, A. W. Physical Chemistry of Surfaces; John Wiley and Sons: New York, 1976;pp 99-148. (5) Scott, H. L.; Cheng, W. H. Biophys. J. 1979,28,117. (6)O'Leary, T.J. Biophys. Chem. 1982,15,299. (7)Nagle, J. F. Faraday Discuss. Chem. SOC.1986,81,151. (8)Marcelja, S.Biochim. Biophys. Acta 1974,367, 165. (9)Bell, G. M.; Combs, L. L.; Dunne, L. J. Chem. Reu. 1981,81,15. (10)Baret, J. F.;Bois,A. G.;Dupin, J. J.; Firpo,J. L.J. Colloid Interface Sci. 1982,86,370. (11)Kaye, R. D.; Burley, D. M. J. Phys. A: Math. Nucl. Gen. 1974, 7,1303. (12)Langmuir, 1. J. Chem. Phys. 1933,1, 756. (13)Albrecht, 0.;Gruler, H.; Sackmann, E. J . Phys. (Paris) 1978,39, 301. (14)Gershfeld, N. L. J. Colloid Interface Sci. 1982,85,28. (15)Jalal, I. M.; Zografi, G. J. J . Colloid Interface Sci. 1979,68,196. (16)Fischer, A.; Sackmann, E. J . Phys. (Paris) 1984,45,517. (17)Peters, R.;Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983,80,7183. (18)McConnell, H.M.; Ta", L. K.; Weis, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984,81,3249. (19)Losche, M.; Mohwald, H. Eur. Biophys. J. 1984,11, 35. (20)Neuman, R. D.; Fereshtehkhou, S.; Ovalle, R. J.Colloid Interface Sci. 1984,101,309.
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makes the theories which describe the transition as hieher than first order inappr~priate.~ Georgallas and Pinkz1 proposed that the nonideal behavior of the ?r-A isotherms is due to impurities. Performing Monte Carlo simulations using a constructive model and assuming 4 % impurities in an otherwise homogeneous monolayer, they were able to produce theoretical u-A isotherms very similar to those observed experimentally, i.e., anonzero slope in the transition region. Pallas and PethicaZ2conducted monolayer studies using highly purified pentadeconoic acid. They were able, for the first time in the past 80 years, to obtain experimental data indicating a region of zero slope in T-A isotherms i.e., a first-order phase transition. They used a single shot method to measure the *-A isotherms. In this technique the area of the monolayer surface is held fixed and the surface area per molecule is determined by the amount of surfactant added to the surface. Surface pressure is measured after the monolayer has equilibrated. Each data point in the T-A isotherm is the result of a single delivery of surfactant to a clean substrate surface. When Pallas and Pethica used a discontinuous compression method instead of the single shot method to measure ?r-A isotherms, they obtained T-A isotherms with markedly nonzero slopes in the transition region, even with the highest purity samples. In the discontinuous compression method, the amount of surfactant on the surface is fixed and the surface area per molecule is varied by discrete displacements of the surface barrier. The surface pressure is recorded after it reaches a constant value following discontinuous compression. Pallas and Pethica suggest that the different results obtained using the discontinuous compression method were due to the extreme sensitivity of this method to impurities. Roland et al.23developed a model which indicated that nonequilibrium behavior also contributes to a nonzero slope in the LC/LE transition region. ?r-A isotherms obtained in the continuous compressionmethod are subject to nonequilibrium behavior particularly if rapid continuous compression is used to vary the surface area per molecule. It follows that very slow compression rates must be used to obtain nonzero slope in the LC/LE transition region. The application of thermodynamics or statistical mechanical theories to the monolayer system requires that it be stable. A criterion for establiihing monolayer stability is vital if meaningful results are to be obtained. We showed (21)Georgallas,A.; Pink, D. A. J.Colloid Interface Sci. 1982,89,107. (22)Pallas, N. R.;Pethica, B. A. Langmuir 1985. 1, 509. (23)Roland, C. M.;Zuckermann,M. J.;Georgallas. A. J. ('hew. Phys. 1987,86,5852.
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198 Langmuir, Vol. 8, No. 1, 1992 in previous that the equilibrium spreading pressure, a,, represents a good criterion for stable films. Monolayers at pressures above a, are thermodynamically metastable. In the present work we show that the continuous compression method can be used to obtain a-A isotherms with zero slope in the coexistence region of the LC/LE transition. The requirements are an ultraclean system, pure materials, slow compression rates, and a stable film (i.e., a < a,). Materials and Methods The Langmuir trough and control system were similar to that used in the previous The Teflon trough was 58 cm long, 13cm wide, and 1 cm deep. Temperature regulation of the trough was achieved by circulating constant-temperature water through metal tubing fitted into the outside surface of the bottom of the trough. A tight-fitting Plexiglas cover over the entire trough provided a seal against dust and foreign matter. A small window in the Plexiglas cover, which could be closed and sealed, provided access to the trough. An IBM-PC and Scientific Solutions Labmaster were used to control the stepping motor that moved the Teflon barrier. Under control from the IBM-PC, the Labmaster digitized the voltage signal from an electrobalance which indicated the surface pressure by use of a glass Wilhelmy plate. The surface pressure sensitivity was 0.040 dyn/cm and the Teflon surface barrier moved in 6.35 X cm increments. *-A isotherms were measured by the continuous compression method. The monolayer surface pressure, a, and area, A, were automatically stored on disk. The digitally stored data were easily corrected for experimental artifacts such as Wilhelmy plate buoyancy. Each measurement of a a-A isotherm was begun with a freshly cleaned apparatus. Several steps were involved in the cleaning procedure: First the Teflon trough and Teflon barrier were cleaned with a strong detergent followed by a thorough rinse with hot water. Then the trough and barrier were soaked in a cleaning solution for several hours (usually overnight). The cleaning solution was prepared just prior to ita use and consisted of 200 mL of concentrated hydrochloric acid, 80 mL of 30 % hydrogen peroxide, and 520 mL of triple-quartz-distilled water. Followingthe cleaning solution soak the trough and barrier were again rinsed with freshly prepared distilled water and used immediately. Glassware, including the glass Wilhelmy plate, was also cleaned immediately prior to use in a manner similar to the trough and barrier. Syringes were rinsed with ultrapure hexane before and after their use and then kept in a covered box. Only highly purified materials were used in the experiments. Glass-distilled hexanes of high purity were obtained from Burdick and Jackson Laboratories, Inc., Muskegon, MI. Other chemicals were obtained from standard sources. Pentadecanoic acid from Nu Chek Prep, Elysian, MN, was purified by multiple slow recrystallizations of material from hexane. A highly purified dipalmitoylphosphatidylcholine(DPPC) lipid from Avanti Biochemicals, Inc., was used without further purification. To start the experiment, the freshly cleaned trough was placed into position in the apparatus and immediatly covered with the Plexiglas cover. The barrier was then fastened to the driving mechanism. The water, upon which the monolayer was to be spread, was poured into the trough (24) Hifeda, Y.M.; Rayfield, G . W.J.Colloid Interface Sci. 1985,104, 209.
Hifeda and Rayfield until the water surface was higher than the brim of the trough. Freshly prepared, triple-quartz-distilled water with a pH near 7 was used. The Wilhelmy plate was suspended in position and arranged with its lower edge parallel to the water surface. I t was then lowered into the water and raised back into the air. This process was repeated 2 or 3 times to provide a last rinse of the plate in the water. Finally the temperature control system was turned on to regulate the temperature of the trough. The apparatus was left as described above for 2 to 3 h to reach thermal equilibrium and to allow the relative humidity in the box to reach its maximum value (near 100%1. During this time, airborne contamination, introduced before the box was sealed, was adsorbed onto the water surface. Before the monolayer film is formed, this contamination must be removed. This was accomplished by sweeping (compressing) the water surface with the barrier and removing absorbed contaminants with a clean capillary tube connected to a vacuum line. After lowering the Wilhelmy plate into contact with the water, the water surface was again checked for contamination by sweeping it with the barrier and measuring the surface pressure. If the surface was clean (Le., no change in surface pressure was recorded), the barrier was pulled back until it reached ita initial position (defining the initial surface area). The level of the liquid substrate was then adjusted to about 1 mm below the rim of the trough. Two techniques were used to form the insoluble monolayers, either solvent spread or crystal spread. In either case, material was added through the small window in the cover. The crystal spread method involved placing a small solid crystal of surfactant on the water surface. When surface pressure rose to some desired value, the crystal was removed from the water surface. In the solvent spread method, the surfactant was first dissolved to a desired concentration in a volatile organic solvent (usually hexane). A known amount of this solution was then placed on the water surface. Since the surface area of the water was known, the number of molecules per unit area of the monolayer was determined. The possibility of solvent Contamination is minimized by using a high surfactant concentration (about 10 mg/mL). Therefore only a very small volume of solution, usually 5 pL, was spread on the surface. This required an accurate delivery system. We used a 5-pL syringe (Hamilton Co., Reno, NV). After the monolayer was spread, the small access window was closed and the rest of the operation was controlled from the computer keyboard. A period of 30 min was allowed after the spreading to ensure complete solvent evaporation and to regain thermal and vapor equilibrium. Several hours were required to record a single a-A isotherm. A complete experiment took 1 day. In our experiments the equilibrium spreading pressure, r e , was taken as a criterion for monolayer film stability.24 We require a < a, for stable films. r e was measured by repeatedly adding a highly concentrated solution of fatty acid or lipid in organic solvent in small quantities to the liquid substrate surface. This process was continued until the surface pressure of the monolayer reached a maximum value. This maximum value of a was taken to be a,. The method gave quick, accurate, and reproducible values of a, which were in good agreement with the crystal spread method for measuring a,.24Moreover, the method could be used to measure aefor surfactants that do not spread from crystals at temperatures below their melting points such as DPPC.
Langmuir, Vol. 8, No. 1, 1992 199
Phase Transitions in Monolayers r
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Figure 1. Pentadecanoic acid t - A isotherm at 25 "C obtained with the continuouscompressionmethod comparedwith the data (points) of Pallas and Pethica22 using a one-shot method to measure the t - A isotherm. The surface pressure, measured at an area of 30.5 A2, is 7.4 dyn/cm and it is 7.3 dyn/cm at a surface area of 26.5 Az so that the slope in this region is less than 0.0286 (dyne/cm)/A2.
35
25
30 0 Area p e r M o l e c u l e (sq.A)
Figure 3. Pentadecanoic acid t - A isotherm at 25 "C (curve a) obtained using slow continuous compression compared with an isotherm at 23 "C (curve b) of the same fatty acid obtained by the discontinuous compression method.
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Figure 2. t - A isotherms for unpurified (Figure2a) and purified (Figure 2b) pentadecanoic acid are shown. The unpurified fatty acid was used as received from a commercial source without further purification.
Results The measured equilibrium spreading pressure, r e , for pentadecanoic acid was 21 dyn/cm at 25 "C. I t was found to vary linearly with temperature from 20 to 30 "C with apositive slope of 0.52 (dyn/cm)/"C. The measured equilibrium spreading pressure for DPPC was 42.6 dyn/cm at 25 "C with a positive slope of 0.15 (dyn/cm)/"C. In agreement with results reported p r e v i ~ u s l y we , ~ ~found that if the surface pressure is set to a value above ?re, it decays in less than an hour to re. If, however, the surface pressure of the monolayer is set to a value less than r e , it remains constant for periods in excess of 8 h. Thus we used a, as a criterion for monolayer film stability. Figure 1(solid line) shows a purified pentadecanoic acid r A isotherm measured at 25.0 "C. The monolayer was solvent spread onto a distilled water substrate and continuously compressed at a rate of 0.1 (A2/molecule)/ min. The surface pressure was kept below a, in order to maintain monolayer stability. The a-A isotherm data of Pallas and PethicaZ2for pentadecanoic acid taken with the single shot method is shown by individual data points. The figure shows the good agreement between our a-A isotherm data obtained with the continuous compression method and the single shot data of Pallas and Pethica. Curve a in Figure 2 shows a a-A isotherm taken at 24.0 "C for the commercial pentadecanoic acid used as received without further purification. Curve b in Figure 2 shows a a-A isotherm taken at the same conditions but with
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Figure 4. Family of DPPC *-A isotherms at five differenttemperatures is shown. Data were obtainedusing the slow continuous compression method. All data were taken with t less than A. to ensure a stable film.
purified pentadecanoic acid. The monolayers were spread using the solvent spread method. Continuous compression (0.1(A2/molecule)/min)was used to obtain the isotherms. The nonzero slope in curve b in the LC/LE transition region due to impurities is apparent. Curve a in Figure 3 shows a a-A isotherm for purified pentadecanoic acid at 25 "C obtained by the slow continuous compression method (0.1 (A2/molecule)/min). Curve b in Figure 3 shows a r A isotherm for the same purified fatty acid but at 23 "C using a discontinuous compression method. This latter method requires that the film is first compressed at a rapid rate (2 (A2/molecule)/ min) for a limited time (30 s). The compression is then stopped for 10 min. This is followed by another period of rapid compression. The surface pressure was continuously recorded both during the compression period and during the 10-min waiting period. During the waiting period the surface pressure slowly decreases to an equilibrium value. The locus of equilibrium values give an isotherm that is flat as one would expect since the average compression rate is the same for curves a and b. But during each rapid compression phase the isotherm is not flat. Thisshows that high compression rates generate a nonzero slope in the LC/LE transition region.25 Figure 4 shows a family of *-A isotherms for DPPC near the LC/LE transition. The isotherms were obtained (25) The deviation from the equilibriumvalue becomes greater at each waiting period as the LC phase is approached. This may be due to higher film stresses induced by the compression in this region of the phase transition. Longer waiting periods may be desirableto completely 'anneal" the film as the LC phase is approached.
200 Langmuir, Vol. 8, No. 1, 1992
by the slow continuous compression method (0.1 (A2/ molecule)/min). The surface pressure, a , was kept below the equilibrium spreading pressure, re,in order to maintain equilibrium. Below ae the surface pressure remains constant at fixed area for at least several hours. Discussion There is general agreement among investigators on the importance of monolayer stability in measurements of properties of monolayers, and there have been widespread studies of this important i s s ~ e . ' ~ , ~ However, ~ , ~ ' there is disagreement about defining a criterion for the stability of monolayers. Our previous results24are consistent with the notion that the equilibrium spreading pressure, a,, represents an upper limit on the surface pressure for stable films. We find stable monolayer films of DPPC providing a
c ae.
At very slow compression rates the continuous compression method produces a-A isotherms that show an ideal first-order LC/LE phase transition. This ends the speculation that the continuous compression method for measuring a-A isotherms fails to show a first-order transition in monolayer systems. The a-A isotherms for pentadecanoic acid (Figure 1) disagree with almost all of the past data in the literature. This is probably due to the use of rapid compression rates and/or the presence of impurities in the fatty acid monolayers. The impurities are due to contamination either in the chemical compounds or of the monolayer trough apparatus itself. Our data agree Very well with those of Pallas and Pethica.22 These investigators used the single shot method and carefully purified fatty acids to produce a-A isotherms with zero slope in the LC/LE transition region. However, they also report a nonzero slope in the transition region when the discontinuous compression method is used to measure the a-A isotherm. We agree with their conclusion that this is due to contamination of the monolayer trough apparatus. The compressionmethod is particularly sensitive to impurities. The continuous compression method has two disadvantages compared to the single shot method of Pallas and Pethica:22 First, in addition to compressing the molecules, any contaminants which may be present on the surface are also compressed. This is why an extra effort is needed to minimize contamination. Second, continuous compression or expansion can cause kinetic effects23as shown in Figure 3. This problem was minimized in our experiments by using a very slow compression rate of 0.1 (A2/molecule)/min. Advantages of the slow continuous compression method are as follows: (1)I t produces a complete isotherm with (26) Heikkila, R. E.; Kwong, C. N.; Cornwell, D. G. J.Lipid Res. 1970, 11, 190. (27) Horn,L. W.; Gershfeld, N. L. Biophys. J. 1977, 18, 301.
Hifeda and Rayfield
minimal scattering in the data. Scattering occurs in the single shot data due to the difficulty of spreading the same number of molecules for each data point. (2) The rounding of the isotherm at the beginning and the end of the phase transition can be studied in detail. It is difficult to use the single shot method for this task due to the scattering problem. (3) The continuous compression method is easier experimentally. A complete *-A isotherm can be measured in about l day compared to the several days of work required using the single shot method. Our studies of DPPC indicate that (1)DPPC exists in a stable state at or below the equilibrium spreading pressure, r e , and (2) a family of a-A isotherms for DPPC can be measured that show a first-order transition (Figure 4). Other experiments support the conclusion that the LC/LE phase transition in DPPC is first order. Electron microscopy studies of DPPCZ0and fluorescence microphotolysis of DPPC17-19indicate coexisting phases in the LC/LE transition region. There are two basic sources of uncertainties in attempting theoretical descriptions of complicated systems with phase transitions: model uncertainty and computational uncertainty.' Ideally one would like a realistic model that is rigorously solvable. Nagle28129 has studied one simplified model for the LC/LE phase transition in molecular monolayers that is rigorously solvable. The model gives a firstorder transition between a conformationally perfectly ordered solid phase and a chain-melted conformationally disordered liquid phase. However, the wedge-shaped coexistence region ends in an unusual critical point and with near vertical isotherms in the solid phase and no rounding at the end of the transition. We propose to examine the feasibility of testing this prediction with future experiments. Conclusions The main points of our study may be summarized: 1. Monolayers of both pentadecanoic acid and DPPC were found to be stable providing a < re. 2. The LC/LE transition region of monolayer a-A isotherms has zero slope. However in order to generate accurate isotherms great care must be taken to control contamination of the system and very slow compression rates must be used. 3. We suggest that most of the LC/LE phase transitions in monolayers are first order in nature. The apparent higher order phase transitions reported in the literature are the result of impure compounds, contaminants in the monolayer trough apparatus, or rapid compression rates. Acknowledgment. We are grateful to J. Long and J. Nagle for very helpful discussions. Registry No. HsC(CH2)&02H, 1002-84-2; DPPC,2644-64-6. (28) Nagle, J. F. J. Membr. Biol. 1976, 27, 233. (29) Nagle, J. F. J. Chem. Phys. 1975, 63, 1255.
