Langmuir 1995,11, 4803-4810
4803
Complete and Partial Miscibility in Binary Monolayers of Phosphatidylethanolamines with Different Lengths of Acyl Groups H.-D. Dorfler,” C. Koth, and W. Rettig TU Dresden, Fachrichtung Chemie, Lehrstuhl fur Kolloidchemie, Mommsenstrasse 13, 0-01062 Dresden, Germany Received April 6, 1995. I n Final Form: August 21, 1995@ A detailed miscibility analysis of the three systems of binary phospholipid monolayers (system dimyristoylphosphatidylethanolamineldipalmitoylphosphatidylethanolamine, system dipalmitoylphosphatidylethanolamine/distearoylphosphatidylethanolamine, and system dilauroylphosphatidylethanolamineldistearoylphosphatidylethanolamine)was carried out by application of a variety of spreading and surface techniques to produce mixed monolayers in different mixing states. Studies of the compression and spreading behavior of these binary systems and the application of the surface-phaserule form a basis for the determination of binary phase diagrams. From these phase diagrams of the binary monolayers we can analyze and compare the miscibility properties in the mixed films. We found complete or partial miscibility in the binary monolayers in dependence on the film state (liquid-expandedor condensed films) and the chain length differences between the mixing components.
Introduction In the last 30 years, the conceptions of structures and the properties of monolayers have become important, particularly with regard to the connection between structure and function of biological membranes.l-19 This concerns especially the phase and mixing behavior of monolayers. The understanding of the properties of bilayers in biomembranes, as a basis for their function, is closely connected with the knowledge of the phase and mixing behavior of these complex multicomponent systems. Therefore, as a first step, we shall discuss binary monolayers as a n oversimplified membrane model in order to develop a systematization for the phase and mixing behavior, preliminary to a more interface chemistry point of view.
* To whom correspondence should be addressed.
Abstract published in Advance ACS Abstracts, November 1, 1995. (1)Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966; Chapter 6. (2) Cadenhead, D. A. Recent Progress in Surface Science;Academic Press: New York, 1970; Vol. 3. (3) Phillips, M. C. Progress in Surface and Membrane Science; Academic Press: New York, 1972; Vol. 5. (4) Dorfler, H.-D. Biol. Rundschau 1973, 11, 1. (5)Chapman, D. Q.Rev. Biophys. 1975,8,185. (6) Huthier, H.; Galla, H. J.;Mohwald, H. 2.Naturforschen. 1978, 33c, 382. (7) Albrecht, 0.;Gruber, H.; Sackmann, E. J.Phys. (Paris)1978,39, 301. (8) Firpo, J. L.; Dupin, J. J.;Albinet, G.; Bois, A.; Casalta, L.; Baret, J. F. J. Chem. Phys. 1978, 68, 1369. (9) Albrecht, 0.;Gruber, H.; Sackmann, E. J.Colloid Interface Sci. 1981, 79, 319. (10)Suzuki, A.; Cadenhead, D. A. Chem. Phys. Lipids 1986,37,69. (11)Ringsdorf,H.; Schlarb,B.;Verzmev,J.Angew. Chem. 1988,100, 117. (12) Handa, T.; Nakagaki, M. Adu. Colloid Sci. 1992,38, 45. (13)Asgharian, B.; Cadenhead, D. A.; Tomoaia-Cotisel, M. Lungmuir 1993, 9, 228. (14) Yamauchi, H.; Takao, Y.; Abe, M.; Ogino, K. Langmuir 1993,9, 300. (15)Alsina, M. A.; Mestres, C.; Rabanal, F.; Busquets, M. A.; Reig, F. Lungmuir 1993,9, 1129. (16) Damodaran, K. V.; Merz, K. M., Jr. Langmuir 1993, 9, 1179. (17) Ahuja, R. C.; Caruso, P.-L.; Mobius, D.; Wildburg, G.; Ringsdorf, H.; Philp, D.; Preece, J. A.; Stoddart, J. F. Langmuir 1993, 9, 1534. (18)Mathauer, K.; Vahlenkamp, T.; Frank, C. W.; Wegner, G. Langmuir 1993, 9, 1582. (19) Hall, R. A.; Thistlethwaite, J.; Grieser, F. Langmuir 1993,9, 2128. @
~~
There are essential differences between the analysis of the mixing behavior in bulk and in monolayers. As a consequence of their predominantly two-dimensional character, the monolayers are not directly visible, in contrast to the corresponding bulk systems. So it is impossible to make distinctions between the “heterogeneity” and “homogeneity” of the layer’s structure-the criterion of complete miscibility-merely by visual inspection. Even electron microscopy and other optical methods give, in some cases, only restricted information on this point. l,zo Translation diffusion in binary phospholipid monolayers measured by fluorescence microphotolysis suggests that phase separation in mixed monolayers takes place.zl,zz Probably in the near future new experimental techniques as fluorescence spectroscopyz3-z8and the gracing incidence diffraction methodz9will be more applied to give further contributions to clear up the problems of miscibility in binary monolayers. Actually the pecularity of the miscibility tests of monolayers lies in the fact that the mixing state can be analyzed only in more indirect manner, using the phenomenological properties of the binary monomolecular systems by application of the surface-phase rule.30~31 This unavoidably leads to a concentration on the application of surface thermodynamic^^^-^^ and phenomenological ~
~~
(20) Fischer, A.; Sackmann, E. J.Colloid Interface Sci. 1986,112,l. (21) Peters, R.; Beck, K. Prm. Natl.Acad. Sci. U S A . 1983,80,7183. (22) Beck, K.; Peters, R. In Spectrosco y and the Dynamics of Molecular Biological Systems; Bayley, P. M., gale, R. E., Eds.; Academic Press: London, 1985. (23) Losche, M.; Sackmann, E.; Mbhwald, H. Ber. Bunsenges. Phys. Chem. 1983,87,848. (24) Ldsche, M.; Mohwald, H. Rev. Sci. Instrum. 1984, 55, 1968. (25) Miller, A.; Helm, C. A.; Mohwald, H. J.Phys. (Paris)1987,48, 693. (26) Losche, M.; Duwe, H.-P.; Mohwald, H. J. Colloid Interface Sci. 1988,126,432. (27) Rice, P. A.; McConnell, H. M. Proc. Natl.Acad.Sci. U S A . 1989, 86, 6445. (28) Beitinger, H.; Vogel, V.; Mobius, D.; Rahmann, H. Biochim. Biophys. Acta 1989, 984, 293. (29) Jacquemain, D.; Wolf, S. G.; Leveiller, F.; Deutsch, M.; Kjaer, K.; Als-Nielsen,J.;Lahav, M.; Leiserowitz, L. Angew. Chem. 1992,104, 134. (30) Crisp, D. J. Surface Chemistry (Supplement to Research) 23; Butterworths: London, 1949; p 17. (31) Defay, R. Thesis, Brussels, 1932. (32) Joos, P. J. Colloid Interface Sci. 1971, 35, 215. (33) Hall, D. G. J. Chem. SOC.,Faraday Trans. 2 1972, 68, 1439.
0743-746319512411-4803$09.00/0 0 1995 American Chemical Society
4804 Langmuir, Vol. 11, No. 12, 1995
methods46and considerations in the field of miscibility testing. The thermodynamic parameters required for such analyses are the surface pressure ~t as a function of the molecular area A (n/Aisotherm) and the dependence of the spreading pressure flon the concentration in the bulk phase (spreading diagram). The spreading pressure is the most important measurable quantity of the equilibrium between the bulk phase and the monolayer. Clearly, however, the lipids and phospholipids being studied must have a measurable amount of spreading pressure for this quantity to be determined. In addition, the purity of the lipids and the conditions for the equilibration play a n important role. For this reason the analysis ofthe mixing properties of monolayers is only feasible on the basis of well-founded and extensive experimental studies. In this paper we will give some examples of miscibility analysis of spreading and phase diagrams. The aim of this work is therefore to carry out an analysis of miscibility only in such phospholipid systems of phosphatidylethanolamines which give a measurable spreading pressure.46 The following essential questions will be answered: How do differences in the chain lengths of mixing components with the same head group influence their miscibility properties? Does the miscibility dependend on the film state of the monolayer? To answer these questions we shall discuss the miscibility properties of the following three binary monomolecular systems: dilauroylphosphatidylethanolaminel distearoylphosphatidylethanolamine, dimyristoylphos-
phatidylethanolamine/dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine/distearoylphosphatidylethanolamine. In connection with the application of the phase rule, the mixing properties and the phase diagrams of the equivalent binary bulk systems are of special i n t e r e ~ t . Therefore, ~ ~ , ~ ~ the mixing properties of these three binary systems were investigatedby measuring the concentration dependence of the equilibrium spreading pressures and by application of so-called mixed and separated spreading and long-time investigations to check the equilibrium and mixing state in the binary monolayers.
Experimental Section Phospholipids. The phospholipids 1,2-dilauroylphosphatidylethanolamine [di-(Cl2:O)-PEl,1,2-dimyristoylphosphatidylethanolamine [di-(C14:O)-PE],1,2-dipalmitoylphosphatidylethanolamine [di-(ClG:O)-PE],and 1,2-distearoylphosphatidylethanolamine [di-(C18:O)-PElwere products of Fluka, Switzerland. (34)Motomura, K. J.Colloid Interface Sci. 1974,48,307. (35)Motomura, K.; Sekita, K.; Matuura, R. J.Colloid Interface Sci. 1974,48,319. (36)Sekita, K.; Nakamura, M.; Motomura, K.; Matuura, R. Mem. Fac. Sci., Kyushu Univ., Ser. C 1976,10, 51. J. Colloid Interface Sci. 1977,62 ‘40)Motomura, K. Ad;. Colloid Interface sei. 1980,12, 1. (41)Rakshit, A.K.; Zografi, G. J.Colloid Interface Sci. 1981,80,474. (42)Pethica, B. A.;Glasser, M. L. J.Colloid Interface Sci. 1981,81, 41. (43)Gaines, G. L.,Jr. J. Colloid Interface Sci. 1982,85, 16. (44)Handa, T.; Ichihashi, C.; Nakagaki, M. Prog. ColloidPolym.Sci. 1985,71, 26. (451 Handa, T.; Tomita, K.; Nakagaki, M. Colloid Polym. Sci. 1987, 265, 250.
(46)Koth, C. Thesis (A),Martin-Luther-Universitat Halle-Wittenberg, Sektion Chemie 1985. (47)Miethe, P. Thesis (A),Martin-Luther-Universitat Halle-Wittenberg, Sektion Chemie 1986. (48)Dorfler, H.-D.; Brezesinski, G.; Miethe, P. Chem. Phys. Lipids 1988,48,254.
Dorfler et al. The degree of purity of these phospholipids was “per analyses” (pea.)and was checked by thin layer chromatography on silica gel (Merck, Darmstadt, FRG). SurfaceTechniquesfor Miscibility Tests. The application of the different spreading and surface techniques leads to complementaryresults concerningthe mixingbehavior in binary monolayers. Thus, the determination of the spreading pressure of the combined film in dependence on the concentration within the bulk phase is of central importance. After the attainment of thermodynamic equilibrium between the given mixed bulk phase and the mixed monolayerbeing formed, the concentration dependence of the spreading pressures yields essential information on the mixing behavior of the binary monolayer. However, the film spread in this manner can be used for additional analysis. These tests give further information on the state of miscibility in binary systems. This experimental procedure, however, supposes an observable spreading pressure of the film’s components. But this is not always the case, and so the abovementioned methods are not universally applicable. In addition to this spreading method, the technique of using other means for spreading in order to generate single or binary monolayers is a further vital tool for the critical examination of the miscibility of mixed monolayer^.^^ The maintenance of equilibrium conditions for the binary monolayer, however, is indeed the critical point of this experimental procedure. It is well-known that at the beginning,the auxiliary spreading solvent invariably produces a temperary mixing state which is not always identical with the equilibrium state of the binary system. The application of this spreading technique always requires longtime experiments to examine the attainment of equilibrium in the mixture. Further results may be obtained by correlating the different spreading methods to the shape of the compression isotherms. These different spreading techniques can be used for producing definite states of mixing or demixing in the binary monolayers. The results obtained by compressionexperiments on such mixed monolayers and produced by different spreading techniques are particularly suitable for a comparison between the miscibility analysis of the systems in combination with continuous or discontinuous compression techniques. In this way, a complementary, well-founded picture of the miscibility in binary lipid monolayerscan be built up from the overall experimental results. For analyzing the miscibility properties of applying various spreading and surface techniques for miscibility tests, the following techniques or methods have proved to be suitable: Mixed Spreading. Both components are dissolved in an auxiliary spreading solvent and mixed thoroughly. In this way their completemiscibility is maintained. For the phospholipids, we used CHC13 and CH30H ((v/v)85/15) as auxiliary spreading solvent. After the solution was spread on the water surface, the solvent evaporates. There remains a combined film,whose initial state corresponds to the complete miscibility of the two components. Long-time studies are then required to establish to what degree this state, brought about by the spreading solvent, represents the true equilibrium of the mixture. If the x/A isotherm does not alter during the long-time experiments, the two components are in fact completely miscible. If the curvature of the compression isotherm changes after a certain time, however, demixing processes occur in the binary system. In this case the components are either only partially miscible or completely immiscible. An exact distinction between partial miscibility and complete immiscibility cannot be based on this method alone. SeparatedSpreading. In this spreading technique, the two components are dissolved in separate volumes of the auxiliary spreading solvent. One of the solutions is first spread. When the solvent has evaporated-generally after about 5 min-the solution of the second component is added. After another 20 min, the solvent usually has evaporated. For complete evaporation, a waiting time of 30 min was applied. From the dependence of the trends of the compression isotherms on the time after complete solvent evaporation, conclusions can be drawn concerning the mixing or demixing processes in the binary monolayers. In our experience,it is irrelevant which of the components is spread first. Separated Spreading with a Barrier. The principle of this method is to apply both componentsin a completelydemixed
Miscibility in Binary Monolayers state, Le., in nonequilibrium, and to obtain results for the miscibility of the lipids from the mixing tendency, i.e., from the tendency to attain equilibrium. In this spreading technique, both components are dissolved separately in the auxiliary spreading solvent. The surface available for the formation of the monolayer is divided by a barrier into two equal areas. Within each of these areas a monolayer of one component solved in the spreading solvent is produced in the usual spreading technique. After the removal of the barrier a completely demixed film is obtained, whose demixing state can be characterized by the curvature of the compression isotherms. If a time-dependent compressionoccurs within this originally demixed binary monolayer, conclusionsmay be drawn from the trend ofthe compression isotherms concerning the miscibility of the components, Le., the mixing of the components or the maintenance of the demixed state, respectively. If, for instance, the curvature of the compression isotherm changes only slightly, completedemixing or partial mixing takes place. If, however, during repeated compressiodexpansion cycles a drastic alteration in the shape of the compressionisotherm is observed,the binary system tends to complete miscibility at equilibrium. Long-Time Investigations. Long-time tests for attaining the equilibrium state of the binary monolayer are indispensable in studies involving the various spreading techniques. These surface methods are characterized by leaving the mixed films undisturbed above the “lift-off area (generally 0.02 to 0.05 nm2 molecule-’) during waiting times between 8 min and 24 h. The system is then compressed continuously and the compression curve is recorded. This procedure is repeated cyclicallyuntil the attainment of equilibrium. Compression Techniques. The method and rate of compression are also ofimportance in the attainment of equilibrium. It is well-known that an overcompressionof the monolayer can be brought about by too high an excessive compression rate. In the case ofcontinuouscompressionat avariable compression rate, U k is used ( U k 0.05-0.20 nm2 molecule-*). Under these conditionsthe tested phospholipids showed no variations in their n/A isotherms. In the case of a discontinuous compression-starting from the “lift-off area Ao-a definite molecular area is adjusted. The dependence of the resulting film pressure on time is measured until it has reached a constant value. This constancy of the film pressure is taken as a criterion of the attainment of equilibrium. Several repetitions of this procedure, applied to a variety of definite areas, result in a plot of the so-called equilibrium isotherms which also represent the attainment of equilibrium in the binary system. CompositionAnalysisof the MixedFilmafter Spreading from Bulk Phase. It is well-knownthat the composition of the mixed monolayerformed by spreading from the mixed bulk phase is quite different from the composition of the bulk phase itself. Therefore a composition analysis of the mixed monolayer after spreading from bulk is necessary. The simplest way to determine the mole fraction ofthis mixed monolayeris to comparethe shape of the compression isotherms of the mixtures in the monolayer with those compression isotherms which we obtained by spreading a solution of the mixture with a defined composition. Consequently we have to measure supplementary the concentration dependence of the compression isotherms of defined mixed monolayersusing spreading solvents (see Mixed Spreading section). The composition analysis of the spread mixed films is carried out in the following way: The crystallites of the mixed bulk phase are put on the water surface. On the film-balance trough, the spreading of the monolayer occurs until the equilibrium state between bulk and monolayer is attained. Then the mixed monolayer is expanded by the movable barrier of the film balance after the removal of the rest of the bulk phase using a small glass slide. As already mentioned above, the compression isotherm of a mixture of unknown composition is then recorded. For the analysis of the composition of the so obtained spread mixture, we have to measure the compression isotherm of the pure components and also the concentration dependence of the compression isotherms of the monomolecular mixture over the whole concentration range. Then the comparison between the shape of the compression isotherm of the unknown composition produced by spreading of the mixed bulk phase and the compressionisotherm obtainedby spreadingof defined monolayer
-
Langmuir, Vol. 11, No. 12, 1995 4805
X
0.2 - x
04
0.6 b
08 G
c14, xc14 Figure 1. Concentration dependence of: the spreading pressures e of the binary system dimyristoylphosphatidylethanolaminddipalmitoylphosphatidylethanolamine at 295 K (-) experimental values; (- - -) calculated values (eq 1);( x ) nl values of the phase transition Me MC. The mole fraction x of the mixtures in the monolayer (a)and the bulk phase (b) is related to di-(C14:O)-PE.
-
mixtures using an auxiliary spreading solvent leads to the fixation of the composition in the monolayer by spreading from bulk. Measurements of the d A Isotherms and of the Spreading Pressures. Compression curves (nlA isotherms) of the pure components and of the mixtures were measured by an automatically registering film balance on %fold distilled water (pH = 7). All measurements were carried out at T = 295 K. The equilibrium spreading pressure was registered by the Wilhelmy plate method. The temporal turning-up of the equilibrium of the spreading pressure of the mixtures was examined by recording the et curves (t = time). The volume mixture was obtained by dissolving both components in benzene and vaporizing the solvents, using a rotational evaporator at T 315 K.
-
Results and Discussion System Dimyristoylphosphatidylethanolamine [di(C14:O)-PEl/Dipalmitoylphosphatidylethanolamine [di-(ClB:O)-PE]. ConcentrationDependence ofthe Spreading Pressures and Equilibrium Collapse Pressures. Figure 1illustrates the dependence of spreading pressures on the bulk phase composition at T = 295 K. As a consequence of the phase diagram of the bulk phase48both components are completely miscible in both the gel phase and the Laphase. Corresponding to the @/xb curves plotted in Figure 1,both components are also completely miscible in the monolayer. From these results it follows that at any spreading pressure @ a completely miscible bulk phase is at equilibrium with a completely mixed monolayer. Following a model proposed by J O O Sthis ~ ~ means complete miscibility in the bulk phase and in the monolayer. The spreading pressures are calculated with the aid of eq 1
with
Dorfler et al.
4806 Langmuir, Vol. 11, No. 12, 1995 X
b
(
a,k ---1 exp -)I;:
x2a
+
and presuming that w 1 = wp = w andxlb xpb = 1. When the parameters nlk, nzk,rile, w , xlb, and xpb of the single components are known, the concentration dependence of the spreading pressure can be calculated. The area w can be obtained from the corresponding nlA isotherms. The parameters ?tlk and npkstand for the surface pressures of the components (1, 2) a t the breakpoint K1 of the nlA isotherms. However, being based only on the knowledge of the spreading pressure Jte, statements concerning the composition of the coexisting monolayer phases xIacannot be made. It may be estimated qualitatively that the component with the gieater spreading pressure ought to be accumulated in the surface phase. As already demonstrated, the composition of the spread film is experimentally determinable by recording and comparing the nlA isotherms (see the section Composition Analysis of the Mixed Film after Spreading from Bulk Phase). The corresponding results are also shown in Figure 1. The differences between the measured values and the calculated ones are about An 2 mN m-l. In view of the steep gradients of the plots these deviations may be considered negligible. The equilibrium collapse pressures nkcanbe determined only in a n indirect manner, because the d A isotherms measured by continuous compression yield mostly only nonequilibrium collapse pressures on account of supercompression. Generally, they lie above the nkvalues. We therefore determined the film comopsition of this system (see the section Composition Analysis of the Mixed Film after Spreading from Bulk Phase). The concentration of the binary monolayer seems to differ only slightly from the concentration in the bulk phase. Figure 2 demonstrates a comparison between the collapse pressures calculated after Garrett3' and M ~ t o m u r aand ~ ~ the experimentally obtained values. The calculation was carried out by eqs 4, 5, and 6.
-
xib= xiu expG T
J:
ai
dn)
(4) (5)
(6) Garrett and Motomura proposed two different ways for calculating the composition xia (x = mole fraction, u = surface phase) of the binary monolayer. Garrett37starts from a n ideal miscibility behavior in the bulk phase (b) and in the surface (0)and thus eliminates the difficulties connected with the calculation of the activity coefficients. The dependence of xia and X? is expressed by eq 4. The integral of eq 4 can be calculated by determining the area, using for this purpose the nlA isotherm of one of the single components (i) with the limits of the corresponding spreading pressure Jte of the mixture. Equation 4 yields the value of xia, and consequently the composition of the spread binary monolayer. In his thermodynamic treatises, M ~ t o m u r apresumes ~~ a real miscibility behavior in the monolayer and a n ideal miscibility in the bulk phase, eq 5. When the gradient
L 0.2
0.4
0.6
0.8
xb, X E I C
Figure 2. Concentration dependence of measured equilibrium collapse pressures nk (0)and the corresponding calculated values after Garrett (-1 (eqs 4 and 6) and Motomura (- - - -1 (eqs 5 and 6) at 295 K. The mole fraction x of the mixtures in the monolayers (a)and the bulk phase (b)is related to di-(C14: 0)-PE. (d3f/dxzb)T and the average molecular area a, a t the spreading pressure $9have been determined by experiment, the composition ofthe monolayer can be calculated. The gradient (d d d x p b ) T is obtained from the concentration dependence of the spreading pressure. Thus the average areas of the components of the monolayer are determined from the nlA isotherms of the mixtures spread. To carry out the calculation, however, the average molecular areas of the single components must be assumed to be nearly independent of the composition of the monolayer. For this reason, the result so obtained must be regarded as only approximate. Considering the above-mentioned supercompression of the mixed films, it is evident that the determination of the equilibrium collapse pressures from the d A isotherms of the mixtures would yield no significant results. Therefore the nkvalues are determined indirectly. The results are also shown in Figure 2. The nklxbcurves calculated by eq 4 differ from those obtained from eq 5. The experimental data approximate to the values derived from Motomura's formula. Since in these binary systems the breakpoint K1 can be found only within a restricted concentration range, the concentration analysis of the spread film was carried out only for the case of two different bulk mixtures (compare Figure 3 ) . nlA Isotherms of the Mixed Spreading. Figure 4 gives a survey of the d A isotherms obtained by applying the method of mixed spreading. From the identity of the isotherms obtained by long-time investigations, we may conclude that the equilibrium in the mixture is already reached after 8 min. This can be taken as a further indicator of the complete miscibility of the components. The film state depends on the concentration. This fact can be expected because of the different film states formed by the components. At xu = 0.79, the phase transition Me MC has not been observed. Below this concentration, the composed films exist only in a condensed film state MC.
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Miscibility in Binary Monolayers
Langmuir, Vol. 11, No. 12, 1995 4807
t
I
-
AREA, A
[nmz molecule-']
Figure 3. nIA isotherms of the binary system dimyristoylphosphatidylethanolamine/dipalmitoylphosphatidylethanolamine spread from the bulk phase at 295 K. The estimated ~ 1 The 4 mole concentrations are (0)xbC14 = 0.1; ( x ) ~ ~ = 0.3. fraction xb of the mixtures in the bulk phase is related to di(C14:0)-PE.
I
. 0.L
-
, ,*.'
0.5
0.6
AREA, A
0.7
0.8
[nd.molecule-l]
Figure 5. Long-time tests. Time dependence of the n/A isotherms of the binary system dimyristoylphosphatidylethanolaminddipalmitoylphosphatidylethanolamineat 295 K mole fraction X'C14 = 0.50; applied spreading technique, separate spreading; waiting times (0)8 min, ( A ) 5 h, and ( x ) 24 h.
Q
4
0.5
t l
~
-
~
.
0.2 0.L 0.6 0.8
-
AREA, A
[nmz molecule-'1
Figure 4. Concentration dependence of the nIA isotherms of the binary system dimyristoylphosphatidylethanolamineldipalmitoylphosphatidylethanolamineat 295 K: applied spreading technique, mixed spreading. The mole fraction xu of the mixtures in the monolayers is related to di-(ClB:O)-PE: (0) 0.00;( x ) 0.48; (0)0.70; (01 0.79; (mi 0.85; (A) 0.92; (A)LOO.
nIA Isotherms of Separated Spreading and Alx" Diagram. Amixture ofcompositionx = 0.5 was subjected to long-time investigations under the conditions of the separated spreading method (see Figure 5). From the compression tests and the comparison with the nIA isotherms, it turns out t h a t the nlA isotherm breakpoint typical ofthe di-(C14:0)-PEisotherm is also observed when the sample is compressed 8 min aRer the separated spreading procedure. This indicates the formation of a monolayer in which the two components are almost completely separated in demixed regions, after the separated spreading. Prolongation of the waiting time between separated spreading and compressing makes the breakpoint disappear. This is a criterion of a timedependent mixing of the components and a n approach to equilibrium. The latter is characterized by the nlA isotherm of the mole fraction obtained by mixed spreading after 8 min. Within the surface pressure range n = 15-20 mN m-l,
Xb
ClL
Figure 6. MA? diagram of the binary system dimyristoylphosphatidylethanolamine/dipalmitoylphosphatidylethanolamine at 295 K. Film pressures n (mN m-l) were ( x ) 5, ( 0 )10, ( 0 ) 15, and (A) 20. The mole fraction x' of the mixtures in the monolayer is related to di-(C14:O)-PE.
the Alx" diagram in Figure 6 differs only slightly from a diagram resulting from additive behavior in this range of surface pressure. Here, the composed film exists in a condensed state. Assuming complete miscibility of the components, we may attribute the deviations to the intermolecular forces between the phosphatidylethanolamines in the mixed film (AA 0.02 nm2 molecule-l). Within the range of surface pressure n = 5-10 mN m-l and a t constant film pressure, the composed films form both of the concentration-dependent film states, MCand Me. This is demonstrated by the existence of a breakpoint in the Alx" curves and by obvious deviations from the additivity of the areas. Phase Diagram. The phase diagram obtained by application of the surface-phase rule30331$46 in Figure 7 gives a survey of the essential results from the tests on monolayers of mixtures of this binary system. There are three homogeneous phase regions: Me, MC,and S. Within these phase regions, the components form homogeneous mixed films. The curve of the equilibrium collapse
-
~
Dorfler et al.
4808 Langmuir, Vol. 11, No. 12, 1995
0.2
I 0.L
-
0.6
0.8
Xh8
Figure 8. Concentration dependence of the spreading pressures 5 of the binary system dipalmitoylphosphatidylethanolamine/distearoylphosphatidylethanolamine at 295 K (-) experimentalvalues:(- - -) calculatedvalues(eq 1).The mole fraction xu of the mixtures in the bulk phase is related t o di(C18:0)-PE.
-
0.2
0.4
08
0.6
xb, x&L
Figure 7. Phase diagram of the binary system dimyristoylphosphatidylethanolamine/dipalmitoylphosphatidylethanolamine at T = 295 K S, solid state of bulk; Me, liquid-expanded film state; MC,condensed film state. The mole fractionx ofthe mixtures (u = monolayer, b = bulk phase) is related to di(C14:O)-PE. pressures is calculated by eqs 5 and 6. The heterogeneous phase region of both film states Me MCis extended by the curve referring to the composition of the condensed film which is calculated by eqs 6 and 7. Motomura et al.35tried to calculate the composition of the mixture in the condensed film state. He assumed the average molecular areaAeto be concentration-independent a t a constant film pressure in the MC state of a binary monolayer. The compositionxi'rc ofthe condensed (c) film, coexisting with the film discussed, a t a film pressure nl, is then obtained by eq 7.
+
The areas A' and AC are the average molar areas of coexisting film states Me and MC. The area Ae can be obtained directly from the nlA isotherms of the mixtures. The gradient (dnlldxla*e)Tis obtained by plotting the nl values for the breakpoints K1 versus the film composition X Z ~ and , ~ AC;different extrapolation procedures are necessary. In Figure 7 we have drawn the results. The characteristic difference between the system di(C 12:0)-PE/di-(C14:O)-PEwhich was already discussed49 and the system now under discussion is the fact that the component di-(C14:0)-PEforms only the liquid-expanded film state Me. Consequently, the film state Me exists only in the phase diagram region 1.0 > xu =- 0.7. According to the criterion of miscibility, this indicates a partial miscibility of the components. (49)Rettig, W.; Koth, C.; Dortler, H.-D. Colloid Polym. Sci. 1984, 262, 747.
-AREA,
A
[nmz. molecule-']
Figure 9. z/A isotherms of diplamitoylphosphatidylethanolamine ( 0 )and distearoylphosphatidylethanolamine(A) at 295 K.
System Dipalmitoylphosphatidylethanolamjne [di(C16O)-PEl/Distearoylphosphatidylethanolamine [di-(C18&))-PElConcentration . Dependence ofthe Spreading Pressures. Since in this system the monolayers are only slightly different in their monolayer properties, the experiments must be regarded purely as an aid to further information. The spreading pressures of the pure components have nearly the same values. Figure 8 shows the resulting measurements of the e l x b curve. The curve has its maximum value at xb 0.4. The spreading pressures are calculated by eq 1,and result in a straight line between the values of the pure components. n / A Isotherms. Figure 9 compares the nIA isotherms of the pure components. They are nearly identical. Both components form condensed films MC. Therefore, the mixtures were not tested, because alterations in the concentration dependence of their isotherms are not expected and so the long-time investigations, generally used as a n additional tool in spreading technique, would yield no further useful results. Phase Diagram. By reason ofthe existence Qfcondensed film states in both components, the phase diagram (Figure 10) shows only two homogeneous miscibility states: MC,
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Miscibility in Binary Monolayers v E
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Langmuir, Vol. 11, No. 12, 1995 4809
S
e [ , ,
1
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0.2
, I
,
0.4
0.8
0.6
xb, x;8,
Figure 10. Phase diagram of the binary system dipalmitoylphosphatidylethanolamine/distearoylphos hatidylethanolamine at 295 K: S, solid state of bulk; MB, condensed film state. The mole fraction x of the mixtures (o.= monolayer, b = bulk phase) is related to di-(C18:O)-PE.
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-
AREA, A
[nmz.mo~ecu~e-']
Figure 12. Concentration dependence of the nIA isotherms of the binary system dilauroylphosphatidylethanolaminddistearoylphosphatidylethanolamineat 295 K applied spreading technique, mixed spreading. Mole fraction xu of the mixtures in the monolayer is related to di-(Cl2:O)-PE(0)0.00, ( x ) 0.18, (W) 0.25, ( 0 )0.50, (+) 0.88, and (0)1.00.
J
' I
0.2 0.4 0.6 0.8
-
Xk12
Figure 11. Concentration dependence of the spreading pressure 2 of the binary system dilauroylphosphatidylethanolaminddistearoylphosphatidylethanolamineat 295 K. The mole fraction xb of the mixtures in the bulk phase is related to di(C12:O)-PE.
S. The azeotropicpoint indicates considerable interactions between the components of the binary monolayer, which are completely miscible over the whole concentration range.
System Dilauroylphosphatidylethanolamine [ai(C12:O)-PEl/Distearoylphosphatidylethanolamine
[di-(Cl&O)-PE]. Concentration Dependence ofthe Spreading Pressures. In Figure 11,the concentration dependence of the spreading pressures resulting from experimental measurements is plotted. There are two distinct regions. In the concentration range up to xb 0.2 the 9 values increase markedly and then, in the neighboring region, they remain constant. In the gel phase between the composition xb 0.07 and xb = 0.17, the phase diagram of the bulk phase has a narrow mixing region, corresponding to the concentration range of increasing 9 1 x b values.48 On the other hand, the range of constant spreading pressure corresponds exactly to the miscibility gap in the gel phase. n l A Isotherms of the Mixed Spreading and Separated Spreading. Figure 12 shows the graph of the d A
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0.2
0.4
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0.6
0.8
NE12
Figure 13. Alx" diagram of the binary system dilauroylphosphatidylethanolaminddistearoylphosphatidylethanolam~eat 295 K. Film pressures x (mN m-l) were ( 0 )25, (0)0.30, ( x ) 0.35, and (A) 0.40. The mole fraction xu of the mixture in the monolayer is related to di-(Cla:O)-PE. isotherms obtained by tests on mixtures 8 min &r mixed spreading. Variations of concentration do not result in an alteration in the breakpoint position. The film pressure of n1 = 35 mN m-l, which is characteristic of di-(Cl2:O)PE, remains constant up to a concentration of xu 0.19. The breakpoint KIdisappears on further increasing the concentration. Our experiments also show that no change in the resulting nIA isotherm was obsemed, when the binary systems are produced by separated spreading and compressed after a waiting time of 8 min. This indicates the complete immiscibility of the two phosphatidylethanolamines within the concentration range tested in our experiments. Alxa Diagrams. Throughout the pressure range, the A/xU diagram of this system does not deviate from diagrams corresponding to a stronger additivity of the average molecular areas (Figure 13). This is an indicator of the immiscibility of the two phospholipids. Probably both
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4810 Langmuir, Vol. 11, No. 12, 1995
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Dorper et al.
xb, x E , 2
Figure 14. Phase diagram of the binary system dilauroylphosphatidylethanolamine/distearoylphosphatidylethanolamine at 295 K S, solid state ofbulk; Me, liquid-expanded film state; MC,condensed film state; the subscripts 1and 2 refer to component 1(di-(C18-0)-PE)and component 2 (di-(Cla:O)-PE), respectively. The mole fraction x of the mixtures (a = monolayer, b = bulk phase) is related to di-(Cl2:O)-PE. components coexist as separated clusters in a demixed state within the binary monolayer. Phase Diagram. The analysis of the mixing behavior of the binary systems by application of the surface-phase r ~ l e ~leads ~ , to ~ the ~ y phase ~ ~ diagrams. On the basis of the results of our measurements, we propose the phase diagram presented in Figure 14. In the following regions there is a mixing gap (MIC Mze; MIC MZC;MIC Sa; S1 S2). The mixing gap, established by calorimetric m e a s ~ r e m e n t slies , ~ ~between the solid phases SIand S2. In the homogeneous regions of the condensed film MC and the bulk phase SI,both components are partially miscible. On account of this, we find the heterogeneous two-phase region MC1 SI.At the spreading pressure n? = 46 mN m-l, there exists an equilibrium between three phases: SI, SZ,and MC1.
+
+
+
+
+
Conclusions Together with the results published al~-eadf~,~O and the results of Phillips3 on binary mixtures of phosphatidylcholines about the miscibility properties in monolayers of the binary systems di-(C12:O)-PE/di-(C14:0)-PE and di(C12:O)-PE/di-(Cl6:O)-PE we can draw the following conclusions: For the systems di-(C12:O)-PE/di-(C14:O)-PE, di-(C14: 0)-PE/di-(C16:0)-PE,and di-(C16:O)-PE/di-(C 18:0)-PE,in which the difference in the number of ACH2 units of the hydrocarbon chains is ACH2 = 4, the film states Me and MCdo not differ in their miscibility behavior remarkably. The components of the systems di-(C12:O)-PE/di-(C14:0)(50) Dorfler, H.-D.; Rettig, W.; Koth, C. Tenside,Surfactants, Deterg. 1989, 26, 266.
PE and di-(Cl6:O)-PE/di-(Cl8:O)-PE are completely miscible in both film states. In the system di-(C14:O)-PE/ di-(ClG:O)-PEthe liquid-expanded film state Meexists only within a limited concentration range. In this system we observed only partial miscibility of the components. For the system di-(C12:O)-PE/di-(C16:0)-PE, in which the differences in the number of ACHZunits is ACH2 = 8, we found a miscibility gap. In the region of low film pressures a condensed film coexists with a liquid-expanded film of varying composition. This miscibility gap is extended up to xu = 1, 0 in the case of the system di(C12:O)-PE/di-(C18:O)-PE with ACH2 = 12. This results in a successive demixing trend with increasing chain length differences. It is represented by the appearance of miscibility gaps or by the extension of their region of existence. There is also an influence arising from the film states of the pure components or, in the case of single components forming different film states, from the concentration dependence of the film states created. Since the single components are able to produce different film states, the resulting film polymorphism affects the miscibility behavior of binary systems in different ways. In the system di-(C14:O)-PE/di-(Cl6:O)-PE the components are miscible within the film state Me. Otherwise we find only partial miscibility, if one of the two components produces the condensed film state. This occurs in the systems di-(C14:O)-PE/di-(C16:0)-PE and di-(C12:O)-PE/ di-(C16:0)-PE. Complete or partial miscibility without any miscibility gap within the film state MC and two participating components existing only in the condensed film state are observed solely in the systems di-(C14:0)-PE/di-(C16:0)PE and di-(ClB:O)-PE/di-(Cl8:0)-PE, where ACHZ = 4.
Glossary average area per molecule in the single of binary monolayer molecular area of the single component i in the mixture of the monolayer Boltzmann constant breakpoint of the phase transition: liquidexpanded film state Me condensed film state MC
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monolayer (M) in the liquid-expanded film state (e) monolayer (M) in the condensed film state (c) number of the spread molecules film pressure equilibrium spreading pressure of the single or binary monolayer equilibrium collapse pressure isotherms or compression curves of single or binary monolayers gas constant solid bulk phase temperature mole fraction ( x ) of the component i in the mixed monolayer (a) mole fraction ( x ) of the component i in the mixed bulk phase (b) LA9502781
