Langmuir 1992,8, 3102-3108
3102
Phase Transitions in Phospholipid Foam Bilayers D.Exerowa* and A. Nikolova Institute of Physical Chemistry, Bulgarian Academy of Sciences, Sofia 1040, Bulgaria Received May 26,1992. In Final Form: September 11,1992
Foam bilayers (Newtonianblack films, NBF) obtainedfrom water-alcohol solutionsof two phospholipids (dimyristoylphosphatidylcholine,DMPC,undergoing a phase transition in the bulk phase and egg lecithin, egg PC, which does not undergo a phase transition in the bulk phase) are investigated in the temperature range 15-35 OC. The temperature dependences of parameters characterizing the propertiesof foam bilayers are depicted by the microinterferometricmethod of Scheludko-Exerowa. The critical concentrationsC, of DMPC and of egg PC for formation of foam bilayers are determined at different temperatures. For both phospholipidsa linear dependence of In C, on 1/Tis found. The thicknesses h of the foam bilayers are also measured in the sametemperature range. Sharpchanges in C, and h are detectedat the temperature of the main phase transition (T,= 23 O C ) for DMPC foam bilayers. For egg PC foam bilayers there are no sharp changes either in C, or in h in the temperature range investigated. The binding energies of the DMPC molecule in the foam bilayer Q = 1.93 X J for the gel state and Q = 8.03 X J for the liquid crystalline state are evaluated with the aid of the hole-nucleation theory describing the stability and permeabilityof bilayers. For egg PC foam bilayers, Q = 1.38 X 10-19J. The considerationsshow that the phospholipid molecules in the foam bilayer are able to arrange in different order, corresponding to different short-range interactions in the bilayer and to different phase states in the bulk solution. Introduction One of the most significant achievements in the study of foam films is the detection of the existence of two types of black foam fis-common black films (CBF) and Newtonian black films (NBF)-and the clarification of the difference in their properties. Early investigations of black foam films (Newton,' Plateau? Boys: Johonnot? Rickenbacher: etc.) had a qualitative character, because the films were not obtained under strictly defined conditions, so that the concept of the different types of black films remained unclear. The first profound theoretical and experimental researches were conducted by Perrid and Wells7who studied soap films. The intensity of the light reflected from the film was measured interferometrically by visual comparison of the two halves of the field of vision of the microscope. This made possible the determination of the thickness of a black film. Perrin consideredthe thinnest black fiisas two adsorptionlayers bound together. This was confirmed by his co-worker Wells for foam films from marseillian soap (consisting mainly of sodium oleate),and the measured thickness was h = 4.5-5 nm, which equals the doubled length of a sodium oleate molecule. Of particular interest is the transition region in which a CBF spontaneously turns into a NBF. This region has been the object of special studies using various methods, for example, the determination of the film thicknessg15 and contact angle,16-19longitudinal (1) Newton, I. Optics; Smith and Watford London, 1704. (2) Plateau,J.StatistiqueExpen'mentale et Theoretique des Liquides Soumis aux Seulles Forces Moleculaires;Gauthier-Villars: Paris, 1873. (3) Boys, V. C. Soap Bubbles; Dover: New York, 1959. (4) Johonnot, E.S.Philos. Mag. 1906, 11, 751. (5) Rickenbacher, W. Kolloidchem. Beih. 1916,8, 139. (6) Perrin, J. Ann. Phys. (N.Y.) 1918, 10, 165. (7) Wells, W. P. Ann. Phys. 1921,16,69. (8)Scheludko, A. h o c . K.Ned. Acad. Wet. 1962, B65,86,97. (9) Duyvis, M.E.; Overbeek, J. Th. G. h o c . K.Ned. Acad. Wet.B65, 26. (10) Jones, M. N.; Mysels, J. K.; Scholten, P. C. Trans. Faraday SOC. 1969,65,1146. (11) Scheludko, A. Adu. Colloid Interface Sci. 1967, 1, 391. (12) Krugljakov, P. M.; Exerowa D. Foam and Foam Film; Himija: Moskwa, 1990; Chapter 3. (13) Myeels, J. K.; Jones, M.N. Trans. Faraday SOC.1966,42,42. (14) Kolarov, T.; Cohen, R.; Exerowa, D. Colloids Surf. 1989,42,49. (15) Exerowa, D.; Kolarov, T.;Khristov, Khr. Colloids Surf. 1987,22, 171.
electroconductivity of the film?o*21the film lifetime on a-particle etc. The widely accepted theory explaining the stability of thick liquid films, including foam films-the DLVO theory-in most cases cannot explainthe stability of black foam films. This failure is especiallyclear for foam bilayers (NBF), in which the short-range molecular interactions are decisive. When bilayer films are concerned the definition "liquid" is hardly relevant. It might be expected that they would possess a higher degree of order. Today, Perrin's idea that NBF are bilayer formations without a free water core between the two layers of amphiphile molecules does not raise any doubt. Experimental proofs for the bilayer structure of NBF are, for example, the dependences of their thickness on electrolyteconcentration Cd in the so1ution,8JoJ2J4and on diejoiningpressure 11112-15 which equals the applied external pressure. In both dependences an equal thickness is reached, which does not change with a further increase in C,i or in II, respectively. The fact that NBF are bilayers haa been checked independently by Platikanov et who studied the longitudinalelectrical conductivity of black foam films who investigated the contact and by de Feijter et al.18*23 angles of black foam films. The considerations presented showthat the clarification of the stability of NBF needs a new approach. Such an approach has been proposed in the theory of Kashchiev and E ~ e r o w a ~ where ~ - ~ rupture ~ and permeability of bilayers were considered on the basis of a fluctuation (16) Kolarov,T.;Scheludko,A.; Exerowa, D. Trans,Faraday SOC. 1968, 64,2864.
(17) Huisman, F.;Mysels, J. K. J. Phys. Chem. 1969, 73,489. (18) De Feijter, J. A,; Vrij, A. J. Colloid Interface Sci. 1979, 70,456. (19) Exerowa, D.;Khristov, Khr.; Zacharieva,M. In Surface Forces in ThinF i l m and Disperse System;Dejaguin,B. V.,Ed.;Nauka: Moecow, 1979; p 186. (20) Platikanov,D.; Rangelova,N. C. R. Bulg. Acad. Sci. l%8,21,913. (21) Platikanov, D.;Rangelova, N. In Research in Surface Forces; Derjaguin, B. V., Ed.;Consult Bureau: New York, 1972; Vol. 4, p 246. (22) Exerowa,D.;Khristov, Khr.;Penev, I. In Foam;Akenr,R. J., Ed.; Academic Press: London, 1976; p 109. (23) De Feijter, J. A.; Vrij, A. J. Colloid Interface Sci. 1978,64,269. (24) Kashchiev, D.;Exerowa, D.J. ColloidInterfaceSci. 1980,77,501. (25) Kashchiev, D.; Exerowa, D. Biochim. Biophys. Acta 1988, 732, 133. (26) Exerowa, D.; Kashchiev, D. Contemp. Phys. 1986,27,429. (27) Exerowa,D.; Kaahchiev, D.; Platikanov,D.Adu. Colloid Interface Sci. 1992, 40, 201.
0743-1463/92/2408-3102$03.00/00 1992 American Chemical Society
Langmuir, Vol. 8, No. 12, 1992 3103
Phase Transitions in Phospholipid Foam Bilayers
mechanism of formation of submicroscopic holes in the bilayers. The hole formation was treated as a nucleation process in a two-dimensional system with short-range molecular interactions. The possibility for rupture of NBF by fluctuation formation of holes in them has been demonstrated for the first time by Derjaguin et However, they regarded NBF as a two-dimensional structureless liquid in which a nucleus hole is formed by two-dimensional stretching of the bilayer. The hole-nucleation theory has been experimentally ~ o n f i r m e dwhich ~ ~ t ~permitted ~ the evaluation of several important molecular characteristics of bilayers obtained from different surfactants, for example,the binding energy Q of an amphiphile molecule in the bilayer.30 Obviously the determination of this energy at different arrangements of amphiphilemoleculesin the bilayers is of specialinterest. Such different arrangements of amphiphile molecules in foam bilayers might be expected,when they exist in various phase states in the bulk, for example,gel, liquid crystalline, etc. The article of Balmbraet al.31is one of the pioneer works in which the properties of foam films and the phase state of the surfactant in the bulk solution have been related. A similar idea has been discussed in the review of Clunie et al.32 A correlation between the equilibrium thickness of black foam films and the interlayer spacing of the structurally similar lamellar mesomorphic phase (neat phase) in the decyltrimethylammonium decyl sulfate/ NaBr/water system has been drawn. The investigations were performed using X-ray diffraction and polarization microscopy. Sidorova, Nedyalkov, and Platikan0v3~have found an analogy between the temperature dependence of the specific electroconductivity of highly concentrated surfactant solutions and of black foam films obtained from them. A correspondence between NBF and surfactant-water gel has been found a t low temperatures whereas at high temperatures a correspondence is found between CBF and the liquid crystalline phase. The phase state of vertical black foam films has been studied by FT-IR spectroscopy. For example, Umemura et al.= have shown that for foam films (CBF and NBF) from sodium dodecyl sulfate the methylene chains, located outside the aqueous core of the sandwich structure, were in an oriented liquid crystalline state at 25 OC. Tian35has found that CBF from cetyltrimethylammoniumchloride with addition of methyl orangewere in the liquid crystalline or gel state while NBF were in the liquid crystalline state. For the investigation of the phase state of black films it is very important to chose strictly defined conditions with respect to the properties of foam films. In this sense particularly suitable are NBF in which there is no free aqueous core, as we have already mentioned, and there is a close packing of the adsorption layers as a necessary
AIR
al.28p29
(28)Deryaguin, B. V.;Gutop, Yu. V. Kolloidn. Zh. 1962,24, 431. (29)Derjaguin, B. V.;Prokhorov, A. V. J. Colloid Interface Sci. 1981, 81, 108.
(30)Nikolova, A.; Kashchiev, D.; Exerowa. D. Colloids Surf. 1989.36. 339. (31)Balmbra, R. R.;Clunie, J. S.; Goodman, J. F.; Ingram, B. T. J. Colloid Interface Sei. 1973, 42, 226. (32)Clunie, J.S.;Goodman, J. F.; Ingram, B. T. In Surface and Colloid Science; Matijevic, E., Ed.; Wiley-Interscience Press: New York, 1971; Vol. 3, p 167. (33)Sidorova, M.; Nedyalkov, M.; Platikanov D. Colloid Polym. Sci. 1976. 254. -- --,45. (34)Umemura, J.; Matsumoto, M.; Kawai, T.; Takenaka, T. Can. J . Chem. 1985,63, 1713. (35)Tian, Y. J. Phys. Chem. 1991.95, 9985.
-.
AIR Figure 1. Molecular model of a foam bilayer (schematic presentation).
condition for their f ~ r m a t i o n .The ~ ~ results discussed so far in the literature have shown that NBF obtained from different surfactants do not change in thickness either with an increase in the electrolyte concentration in the solution or with an increase in the applied external p r e ~ s u r e . ' ~ J It ~ -is~ ~interesting to investigate the properties of NBF, for example, their stability and thickness at temperatures in the range where phase transition in the bulk phase takes place, e.g., to search for occurrence of phase transitions in NBF, which is the main aim of the present work. Specifically, foam bilayers obtained from solutionsof two especiallychosen phospholipids(onewhich does and the other which does not undergo a phase transition in the bulk phase in the temperature range studied) have been investigated.
Theoretical Background As already stated, in terms of the theory of Kashchiev and E ~ e r o w a ~the " ~stability ~ and permeability of bilayers are analyzed on the basis of short-range interactions in two-dimensionalsystems. This theory regards the bilayer as consisting of two mutually adsorbed monolayers of amphiphile molecules. In Figure 1 a model of a foam bilayer is schematically presented. The theory assumes that in a bilayer there exist vacancies of amphiphile molecules (the open circles in Figure 1)from which holes with different sizesare formed by fluctuations. The driving force of the hole-nucleation process in the bilayer is the supersaturation which depends on the concentration C of amphiphile molecules in the bulk solution contacting the bilayer. The rupture of bilayers is considered as a result of a two-dimensional phase transition of the "gas of vacancies" into a "condensed phase of vacancies", the latter being equivalent to a ruptured bilayer. A very important parameter in the theory is the equilibrium bulk concentration C, of amphiphile molecules at which the diluted and the condensed phases of vacancies are in thermodynamic equilibrium. This parameter is given by the expre~sion~~~~~*~~
C, = C, exp(-Q/ZkT)
(1)
where Q is the binding energy of a phospholipid molecule in the bilayer, C, is a reference concentration, k is the Boltzmann constant, and T i s the absolute temperature. The adsorption isotherm of vacancies of amphiphile molecules in the bilayer is given by the following equation: 24,37
C/C,= [(l- ev)/evlexp[-Q(l- Bv)/kTl
(2) Here 8, = rV/r,is the degree of filling of the foam bilayer by vacancies, rVis the density of vacancies in each (36)Exerowa, D.; Nikolov,A.; Zacharieva, M. J. Colloid Interface Sci.
----. -. (37)Nikolova, A.;Kashchiev, D.; Exerowa, D. J.Colloid Interface Sei. 1981. 1. 419.
1989,131,598.
Exerowa and Nikolova
3104 Langmuir, Vol. 8, No. 12,1992 monolayer constituting the bilayer, rm = 1/A, is the maximum density of amphiphile molecules in each monolayer constituting the bilayer, and A, is the area of an amphiphile molecule in the bilayer. By definition 8, = 1 - 8, where B is the degree of filling of the foam bilayer by amphiphile molecules. At a low degree of filling by vacancies (0, C,) a jumplike formation of black spotswas detected. They spread allover the f i i , turning it into Critical Concentration for Formation of Egg PC a black one. For each temperature investigated, a series of Foam Bilayers: Effect of Temperature. Figure 4show observations were carried out at different phospholipid concenthe dependence of the critical concentration C, for trations and from them the critical phospholipid concentration formation of foam bilayers from egg PC on the temperature C, was determined, i.e., the minimum concentrationat which a in In C,vs 1/T coordinates. The experimentally obtained foam bilayer is formed. A t each concentration about 25 films were observed, the accuracy of the C, determination being 5%. values of C, are denoted by open circles, and the straight In contrast tothe foam bilayers from syntheticsurfactantastudied line is drawn by the least-squares method, according to eq so far, the phospholipid foam bilayers investigated here were 1, assuming that C, = C, (see eq 4). As seen the infiitely stable at C = C,. In our investigations we have experimental data fit the straight line very well. This conventionally assumed that an infiitely stable foam bilayer is permits calculation of the binding energy of an egg PC the one which does not rupture for 5-6 h. In fact the determiJ molecule in the foam bilayer Q = (1.38f 0.02)X nation of C, is carried out by studying the dependence of the from the slope of the straight line and ale0 the logarithm probability W for observation of a foam bilayer in the foam film of the reference concentration In C, = 20.2 f 0.02from the on the phospholipid concentration C at a certain temperature. segment of the line (C,in pg cmS). Obviously the value The W(0dependence for egg PC foam films obtained at 15 O C of Q is of particular interest. Specifically in this case the is shown in Figure 3. It is seen that at C < C,, W = 0, and at C I
-2
-
~
~
~
~
_
_
_
~~
_
(50) Kolarov, T.; Iliev, L. Ann. Univ. Sofia Fac. Chim. 1974/1976,69,
107. (61)Duyvis, E. M.Ph.D. Thesis, University of Utrecht, 1962. (62)Smart,C.;Senior, W.A. T r a m Faraday SOC.1966,62,3253. (63)Scheludko, A. Colloid Chemistry; Elwvier: Amsterdam, 1966. (54) Radoev, B.P.;Scheludko, A. D.; Manev, E.D. J. Colloid Interface Sci. l988,96,254. (56)Manev, E.;Scheludko, A.; Exerowa, D. Colloid Polym. Sci. 1974, 252,686.
value applies to a phospholipid mixture as far as egg PC is such a mixture. In the temperature range 15-36 OC, the values of Q are respectively 34.7-32.5 kT. These values seem reasonable compared to Q = 16 kT at 30 "C for foam bilayers from sodium dodecyl sulfate (NaDoS),obtained at strictly defined conditions.m It is importantto mention that the values of Q calculated from the best fit of the theoretical dependence (eq 1) with the experimental
Exerowa and Nikoloua
3106 Langmuir, Vol. 8, No. 12, 1992
results, assuming C, = C,, should be regarded as approximate thus far. We have made this assumption because of the "infinite" stability of the foam bilayers investigated, which means that the exponential term in eq 4 equals unity. It might be supposed however that in the case of finite mean lifetime T (even very high) C, # C, and, respectively, the exponential term which is also temperature dependent through parameters A and B should be accounted for. The precise calculation of the exponential term is not possible as the values of these parameters cannot be derived properly (especially the kinetic parameter A), and this applies also to the temperature dependence of A. Nevertheless, an estimation of the influence of the exponential term in eq 4 on the value of Q (calculated by eq 1) is possible. For such an estimation we have used the theoretical d e p e n d e n c e of ~ ~A~and ~ ~B~ and the values of A056157 and of the diffusion coefficient of phospholipid molecules in bilayers.57 A hexagonal packing of molecules in the bilayer has been assumed. The analysis shows that the values of Q obtained at C, = C, are higher than the real ones, the maximum error being 16%. For example, at a temperature of 20 "C the real value of Q might be expected to be in the interval 28.6-33.6 kT. The error in the determination of Q is probably less than the maximum one, because it can be supposed that the temperature dependences of A andB counterbalance each other which leads to a temperature independence of the exponentialterm in eq 4. A reason for such an assumption was brought forward by the experimental values of A and B obtained for NaDoS foam bilayers at different temp e r a t u r e ~ .The ~ ~ adoption of this assumption for our case gives a good argument to conclude that the values of Q determined supposingthat C,= C,should not be corrected. The constant value of Q in the temperature range investigated gives us the grounds to suppose that there is no change in the phase state of the egg PC foam bilayers. This fact is in accordance with the studies of the phase behavior of egg PC in the bulk phase under conditions similar to those in the bilayer (composition,temperature, etc.). Small has obtained the phase diagram of the system egg PC/water by X-ray diffraction.% He has shown that in the range from 15 to 35 "C the egg PC/water system is in the liquid crystalline state in a broad range of egg PC concentrations. Further investigationshave indicatedthat addition of e t h a n 0 1or ~~ alkali-metal ~~ halide# to aqueous solutions of dipalmitoylphosphatidylcholine(DPPC)changes negligiblythe temperature of the main phase transition of the phospholipid. This merits the supposition that in both cases (egg PC foam bilayer and the corresponding bulk phase) no phase transitions take place in the temperature range studied, and the system remains in the liquid crystalline state. Therefore, the determined value of Q refers to the liquid crystalline state of egg PC foam bilayers. Critical Concentration for Formation of DMPC Foam Bilayers: Effect of Temperature. The dependence of the critical concentration C, for formation of DMPC foam bilayers on temperature is plotted in Figure 5 in In C, vs 1/T coordinates. The open circles in the figure present the experimental results, and the straight
\
55
50
'
3.30
3.35
3.LO
I250
3.15
1150
1 o3I T, KFigure 5. Dependence of the critical concentration C, for formation of a DMPC foam bilayer on temperature: open circles, experimental data; straight lines, theoretical dependences, according to eq 1 and assuming C, = C,.
(56) Ivkov, V. G. Biofizika 1986, 31, 784. (57) Yeagle, P. The Membranes of Cells; Academic Press: London, 1987. (58) Small, D. J. Lipid Res. 1967,8, 551. (59) Tenchov, B. G.; Yao, H.; Hatta, I. Biophys. J. 1989, 56, 757.
lines are drawn by the least-squares method, according to eq 1 with the assumption that C, = Ce (similar to the case of foam bilayers from egg PC). A clearly pronounced bend, i.e., a change in the slope of the In C, vs 1/T dependence at 23 "C, is seen in Figure 5. The values of Q calculated from the slope of the straight line are, respectively, (1.93 f 0.19) X J (48.4-47.2 kT) at temperatures lower than 23 "C and (8.03 f 0.19) X J (19.6-19.1 kT) at temperatures higher than 23 "C. The segments of both lines give the values of In C, of, respectively, 29.4 f 0.4 at temperatures lower than 23 "C and 15.6 f 0.2 at temperatures higher than 23 "C. These values of Q should also be regarded as approximate. The analysis of the results obtained when C, = C, and C, # Ce according to eq 4 indicates that the above-mentioned values of Q for DMPC foam bilayers are also probably higher than the real ones. For example, at 28 "C the real value of Q should be in the interval 17.5-19.5 kT while at 19 "C Q should be in the interval 35-45 kT. The clearly pronounced bend in the In C, vs 1/T dependence at 23 "C and the calculated values of the binding energy Q of a DMPC molecule in the foam bilayer provide us with a reason to suggest that the DMPC foam bilayers undergo a phase transition at T, = 23 "C. In favor of this suggestion is the coincidence of the abovementioned temperature of the phase transition in the DMPC foam bilayer and the temperature of the main phase transition in the DMPC/water bulk phase. For example, in the paper of Janiak et al.62the temperature of the main phase transition in the bulk phase was found to be T,= 23 "C. As pointed out earlier the presence of ethanol and soidum chloride in the solution does not influence considerably the temperature of the main phase Therefore, we can conclude that the sharp change in the value of the binding energy Q of a DMPC molecule in the foam bilayer is due to a chain-melting phase transition in the foam bilayer. We can refer the value of Q of about 48 kT to the gel state of the foam bilayer while the value of Q of about 19.5 kT can be referred to the liquid crystalline state. The higher value of Q (nearly twice higher) for the gel state is reasonable, keeping in mind that it applies to a more ordered state of the foam bilayer. A comparison is possible between the value of Q obtained for the gel state of DMPC bilayers with the value of the activation energy for formation of a vacancy of the phospholipid molecule in bilayers from DPPC aqueous dispersions
1988, 163, 1.
(62) Janiak, M. J.; Small,D. M.; Shipley, G. G.Biochemietry 1976,15, 4575.
(60)Tamura-Lis, W.; Lis, L. J.; Qadri, S.;Quinn, P. J. Mol. Cryst. Liq. Cryst. 1990, 178, 79. (61) Cunningham,B. A.; Lis, L. J.; Quinn, P. J. Mol. Cryst. Liq.Cryst.
Phase Tramitions in Phospholipid Foam Bilayers
reported by Leee3-22 kcal mol-' (1.5 X
Langmuir, Vol. 8, No. 12,1992 3107
J)-referring
to temperatures lower than that of the main phase transition. It is important to note that the phase transition in the foam bilayer should be compared with the one taking place in the bulk phase of high DMPC concentration, close to that in the foam bilayer, but not with the one in the bulk phase of the meniscus (of much lower DMPC concentration), which is in equilibrium with the foam bilayer. Thus, the phase transition which we have detected in the foam bilayer is compared with the analogous phase transition in another bulk phase, which is not in equilibrium with the foam bilayer. This comparison does not contradict the existence of the equilibrium between the foam film (in our case, the foam bilayer) and the meniscus, and this equilibrium only means that there should be equality of the corresponding chemical potentials at a certain temperature. Estimation of the Degree of Filling by Vacancies of PhospholipidMolecules in the Foam Bilayer. The values, calculated for the binding energy Q of a phospholipid molecule in the foam bilayer and for the reference concentration C,, together with eq 2 allow one to obtain the dependence of the degree of filling 8, by vacancies of phospholipid molecules on the phospholipid concentration C in the solution, i.e., the adsorption isotherm of vacancies in the foam bilayer. Similar isotherms have been obtained It has been found that with foam bilayers from N~DOS.~O to for the metastable foam bilayers studied 8, is from 4X depending on the concentration of NaDoS and temperature. The calculation of 8, for foam bilayers from egg PC and DMPC is done according to eq 3. For the egg PC foam bilayers, the values of 8, are of the order of and with the increase of temperature 8, increases within the whole temperature range investigated. For the DMPC foam bilayers there is a sharp change in 8, at the temperature of the main phase transition (T,= 23 OC). For the foam bilayers in the gel state 8, = 10-lowhile for the foam bilayers The calculations in the liquid crystalline state 8, E show that in the case when C, # C, the corrected values of 8, are higher than the values pointed out above. The correction of the value of 8, for egg PC foam bilayers is about 1order; i.e., 8, = lo-'. In the case of DMPC foam for the liquid bilayers the corrected values are 8, E for the gel state. crystalline state and 8, = The above estimations give only an idea of the degree of filling by vacancies in the foam bilayers studied. The extremely low values of 8, are determined by the high values of Q and are in accordance with the infinite stability of phospholipid foam bilayers. Thickness of the Phospholipid Foam Bilayer: Effect of Temperature. As already noted, the equivalent equilibrium thickness of the foam bilayers studied has been determined with the aid of the microinterferometric method at different temperatures. The investigation of the equivalent thickness of egg PC foam bilayers has shown that in this case there is no change in the thickness with the increase in temperature (from 15 to 35 "C) within the limits of the experimental error (*0.2 nm). It is worth pointing out that the equivalent thickness measured by us of 6.7 nm is close to the interlamellar spacing of flat lamellae, determined by X-ray diffraction,@' for the bulk phase of the DPPC/water/ethanol system which is in the liquid crystalline state. The dependence of the equivalent thickness of foam bilayers from DMPC on temperature is shown in Figure (63) Lee, A. G . Biochim. Biophys. Acta 1977,472,237.
21 - 0
15
20
23 25
30 t,OC
Figure 6. Dependence of the equivalent thickness h of DMPC foam bilayers on temperature: open circles, experimental data measured at C,.
6. The determination of the equivalent thickness is done at concentrations of DMPC in the solution, C = C,. Two regions are clearly seen: one with equivalent thicknesses about 6.2 nm at temperatures of 24-30 OC and another with equivalent thicknesses about 7.0nm at temperatures of 15-20 OC. Obviously the two temperature regions correspond to the regions above and below the main phase transition of DMPC. The difference between the thicknesses in both regions is 0.8 nm which is larger than the error of the method. It might be supposed that diminishing the foam bilayer thickness by 0.8 nm when increasing the temperature in the range of T,is due mostly to the melting of the hydrocarbon tails of phospholipid molecules, e.g., to the sharp increase in the number of gauche conformations of the C-C bonds. Thus, the sharp change in the foam bilayer thickness within the region of the main phase transition confirms the formation of two types of foam bilayers being, respectively, in liquid crystalline and gel states,and it is in accordance with the change in the values of Q and BV for DMPC foam bilayers obtained from the In C, vs 1/T dependence. It should be mentioned that at high ethanol concentrations and at T < T,there is a very strong difference between the results for the thickness of the DMPC foam bilayers studied and the data for the interlamellar spacing of the bulk phase for the DPPC/water/ethanol system reported by other r e s e a r ~ h e r s . ~ ~This f Q ~ difference is due to the impossibility of formation of an interdigitated phase in the foam bilayer as in the latter the phospholipid molecules in both monolayers are in contact only by polar heads while the hydrophobichydrocarbon tails are directed toward the gas phase. Conclusion The temperature dependencesof the parameters characterizing the properties of the investigated foam bilayers-the critical concentration C, for formation of the foam bilayer and the equivalent thickness h of the foam bilayer-enable the detection of phase transitions in phospholipid foam bilayers. Thus, a phase transition in DMPC foam bilayers at 23 "C is found to take place in contrast to the case of egg PC foam bilayers which do not change their phase statein the temperature range studied. These results are in accordance with the phase behavior of respective bulk phases of concentration close to that in the foam bilayer. Of particular interest is the calculated value of the binding energy Q of a phospholipid molecule in the foam bilayer which for the gel state is about twice as large as for the liquid crystalline state in the case of (64)Simon, S.A.; McIntosh, T.J. Biochim. Biophys. Acta l9R4,773, 169.
3108 Langmuir, Vol. 8, No. 12, 1992
DMPC foam bilayers. The existence of different types of bilayer foam films (NBF) is extremely interesting. This means that the molecules in these foam bilayers are arranged in different manners corresponding to different short-range interactions. For a more detailed study of these interactions it would be very interesting to use other parameters to characterize the properties of foam bilayers and other experimental methods (for example, FT-IR spectroscopy, X-ray diffraction, etc.) which would allow a direct determination of the hydrocarbon chain conformation, of the interaction between polar head groups of phospholipid molecules, etc. The study of the parameters characterizing the phase state of foam bilayers together with the "structure" characteristics of these bilayers would lead to significantprogress in the knowledge of the thinnest molecular formations to which first Jean Perrin paid attention. A parallel study of phase transitions in foam bilayers and of phase diagrams of a bulk solution would be very useful. That is why any data about the phase behavior of systems containing synthetic s u r f a ~ t a n t a , 6phospho~*~ lipids,6'vM etc. are of significant importance.
Exerowa and Nikoloua
It should not be forgotten that foam bilayers are a very good model for the investigation of intermembrane interactions,B cell fusi0n,6~and alveolar surface and stabilit~,'O*~~ so the study of phase transitions in foam bilayera may have wide implications.
Acknowledgment. We thank the Bulgarian Ministry of Education and Science for financial support. WStW
NO. DMPC, 18194-24-6.
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