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12 Thermodynamics of Monolayer Solutions of Lecithin and Cholesterol Mixtures by the Surface Vapor Pressure Method 1

Downloaded by CORNELL UNIV on August 19, 2016 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0144.ch012

KAZUO TAJIMA and N. L. GERSHFELD Laboratory of Physical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Md. 20014

Thermodynamic parameters for the mixing of dimyristoyl lecithin (DML) and dioleoyl lecithin (DOL) with cholesterol (CHOL) in monolayers at the air-water interface were ob tained by using equilibrium surface vapor pressures π , a method first proposed by Adam and Jessop. Typically, π was measured where the condensedfilmis in equilibrium with surface vapor (π < 0.1 ± 0.001 dyne/cm) at 24.5°C; liquid this exceeded the transition temperature of gel crystal for both DOL and DML. Surface solutions of DOL-CHOL and DML-CHOL are completely miscible over the entire range of mole fractions at these low surface pressures, but positive deviations from ideal solution be havior were observed. Activity coefficients of the compo nents in the condensed surface solutions were greater than 1. The results indicate that at some elevated surface pressure, phase separation may occur. In studies of equilibrium spreading pressures with saturated aqueous solutions of DML, DOL, and CHOL only the phospholipid is present in the surface film. Thus at intermediate surface pressures, under equilibrium conditions (40 > π > 0.1 dyne/cm), surface phase separation must occur. ν

ν

C i n c e the initial observation by Leathes ( I ) that cholesterol exerts a ^ condensing effect on spread monomolecular films of natural lecithins, 1

Present address: Tokyo Metropolitan University, Tokyo, Japan.

165

Goddard; Monolayers Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

166

MONOLAYERS


extensive investigations of the condensing phenomenon with synthetic lecithins have been reported (2). These studies have been criticized recently on the basis that the films may be neither homogneous nor at equilibrium (3).

Figure 1. Upper: Schematic of π-Α isotherms in the tran sition region: F-F' represents region where bulk of film material is in the condensed monolayer state; G-G' repre sents the region where virtually all of the film is in the gaseous monolayer state. Area per molecule is the total area occupied by the sum of all lipid molecules in the surface. Lower: π -χ surface phase diagram where data for upper curve containing point a are obtained from F-F' of the π-Α isotherm; lower curve containing point h is obtained from G-G' of the π-Α isotherm. At any value of π , condensed film at point a (x — x(c)) is in equilibrium with vapor film at point a' (x-x(v)). ν

ν

To study the interactions in mixed films of lecithin and cholesterol under conditions of homogeneity and equilibrium, the surface vapor pressure method, first explored by Adam and Jessop ( 4 ), was used. This method uses spread films i n the transition region of the isotherms, where


12.

TA JIMA

AND

Lecithin

GERSHFELD

and Cholesterol

Mixtures

167

the condensed films are in equilibrium with two-dimensional vapor (5, 6). Unlike the high pressure region of the isotherm, where virtually all other studies have been carried out, equilibrium can be established; the phase rule correctly predicts the variance of the system depending on whether two or three surface phases are present (condensed monolayer(s) and surface vapor) (7). The miscibility in the condensed surface phases can be readily specified by this procedure; application of the method to vari ous monolayer systems has been reported (8). Preliminary studies with dipalmitoyl lecithin ( D P L ) at temperatures below the liquid-crystalline transition temperature, T , showed that cholesterol is not miscible with the gel state of this lecithin ( 9 ). W e now use the method to analyze the two-component mixtures of cholesterol with D O L and D M L at tempera tures above T . W e demonstrate that large positive deviations from ideal mixing result from mixing cholesterol with these lecithins, and we predict and verify experimentally that at elevated surface pressures, phase sepa ration in the surface film w i l l occur.


c

c

Experimental Chemical Activities by the Surface Vapor Pressure Method.

Surface

pressure measurements in the transition region between the condensed and gaseous monolayer states of a single lipid component spread as a monolayer on water yield a value of π which is independent of the surface area. This value—the surface vapor pressure, ττ —is analogous to the vapor pressure of a liquid in equilibrium with its vapor. W h e n a second lipid component is in the surface, the limits of miscibility in the con densed phase may be determined on the basis of the surface vapor pres sure dependence on the mole fraction in the condensed phase (8). Figure 1 represents the isotherms for two lipid components which are miscible in the condensed monolayer state. The major feature of the isotherms for the pure components (1:0, 0:1) is the transition region in which the surface pressure is independent of surface area; here the limits of the transition region are at the low area end, A , and at the high area end, A . These areas are characteristic of each lipid and represent the area per molecule of the lipid in the condensed and vapor states (10). For an equimolar mixture of the two components (1:1), the surface pressure in the transition region depends on the surface area; according to the phase rule (11, 12, 13, 14), two surface phases coexist here: a condensed phase of lipids and the surface vapor phase. To obtain the activity coefficient γι of the i component in the condensed phase the following relation may be used: ν

c

v

t h

i, ο

(1)

where the mole fractions (x) in the surface vapor ( ν ) and condensed (c) monolayer states and the surface vapor pressures of the pure π^'° com ponent and of the mixture ?r must be measured. v



168

MONOLAYERS

Equation 1 assumes that Dalton's law of partial pressures applies to the mixture of lipid molecules i n the surface vapor state; studies with various lipid mixtures i n this state support this assumption ( 8 ) . Correction terms for non-ideal behavior of the lipid molecules i n the surface vapor state were calculated from the second virial coefficients of the mixtures using procedures described by Prausnitz (15). T h e con tributions to the activity coefficients i n the vapor were negligible; thus Equation 1 was unchanged. Each term i n Equation 1 is experimentally available. F r o m Figure 1, points along the dotted line F F ' represent the surface vapor pressure i n equilibrium w i t h the mixture i n the condensed film state that is composed of the material deposited on the surface; most of the material is i n the condensed state, and only a small amount is present i n the vapor state. T o obtain the composition of the equilibrium vapor phase one must examine the transition region of the isotherms where the bulk of the lipid is i n the vapor state—i.e., along the line G G ' which joins values of A (Figure 1). Values of ττ along the line G G ' represent 7 r - X i ( v ) data. The data can be represented by a conventional ττ -χ phase diagram (Figure 1). The upper curve represents the ττ data as a function of the composition of the condensed state; the lower curve is for the composition of the vapor state. For the 1:1 mixture the values of 7T on lines F F ' and G G ' are the points a and b ; these are shown i n the T T V - J C phase diagram. The composition of the surface vapor i n equilibrium with the condensed mixture [x = x(c)~\ is at point a' where χ = x( v ) . The millidyne film balance used i n these studies has been described in detail (7, 16). W i t h proper shock mounting, the precision of the balance is ± 0 . 2 m d y n e / c m below 30°C; above this temperature the precision is poorer because of the thermal disturbances caused b y evapo ration from the water surface. W e restricted these studies to below 30°C. The temperature of the trough was controlled to d=0.2°C b y circulating v

ν

v

Ύ

ν

V

O DML • CHOL

I

0

ι

ι

10

ι

ι

20

ι

ι

30

ι

ι

40

ι

1 50

Area/Molecule (A /10 ) 2

Figure

2.

ττ-Α isotherms of DML and CHOL

3

on water, pH 5.8 at


24.5°C


12.

TAjiMA A N D G E R S H F E L D

Figure 3.

π-Α

Lecithin

isotherms for mixtures of DML at 24.5°C

and Cholesterol

and DOL

169

Mixtures

on water, pH

5.8

water from a thermostated bath through coils in the base of the trough. The temperature was monitored by thermistor probes placed at different points in the water surface. The procedure for measuring the very low surface pressures and the evidence for equilibrium i n these systems has been discussed at length (7). Materials. Dimyristoyl lecithin ( D M L ) from Nutritional Biochemical Corp. was purified by column chromatography (Unisil, activated silica gel); solvent elution gradients of chloroform and methanol mixtures were used (17). Pure dioleoyl lecithin ( D O L ) was synthesized and donated by R. E . Pagano. The final purity of the lecithins was confirmed by thin layer chromatography with a mixed solvent system of chloroform:metha nol: water (65:25:4 v / v ) (18). The fatty acid composition and purity of the lecithins were verified by gas chromatography. W e estimate the purity of these samples to be > 9 9 mole % . Cholesterol from Applied Science Laboratories was used without further purification because only the cholesterol peak was observed in the gas chromatogram; the melting point (148.5°C) and melting range ( ± 0 . 1 0 ° C ) were characteristic of purity > 99 mole % . Chloroform and hexane were the spreading solvents and were puri fied by being passed through an activated silica gel column (Florosil). Surface active impurities were detected by spreading 0.1 cc of the solvent on the surface of the millidyne film balance and monitoring the surface pressure after the solvent had evaporated (1-5 m i n ) . No residue was detected with the purified solvents as indicated by the absence of surface pressure after evaporation. If non-purified solvents were used, appre ciable amounts of surface active impurities were detected. A l l solvents were stored under nitrogen to avoid oxidation; the lipid solutions were


170

MONOLAYERS


stored at —20°C but for periods not exceeding three days. Water was purified as described previously (19). A l l studies were at p H 5.8, without the addition of buffer or electrolyte to avoid extraneous contaminations. Isotherms were determined point by point.

Figure 4.

π - Α isotherms for mixtures of DOL at24.5°C

and CHOL

on water, pH

5.8

Results Surface Pressure-Area (A). Isotherms for C H O L , D M L , D O L and their mixtures are shown i n Figures 2-5; their molecular areas were calcu lated by dividing the total surface area by the total number of lipid molecules which had been deposited on the surface. The isotherms for the mixtures extend to approximately 1000 A ; for pure C H O L and D M L the isotherms are shown into the gaseous region. The isotherm for D O L in the gaseous region at the same temperature was indistinguishable from that for D M L and is not shown. 2

For the mixtures of D O L and D M L with cholesterol (Figures 4 and 5) the transition to the liquid-expanded film is represented by line F F ' ; at each point along F F ' the surface vapor pressure is in equilibrium with the condensed phase whose mole fraction is primarily that of the condensed monolayer. The surface vapor pressures in the region of A , where virtually all of the spread film is in the surface vapor state, were obtained by extrapolating the data obtained at about 1000 A to the region of A (about 5000-10,000 A ) assuming a linear decrease i n . This extrapolation is justified on the grounds that it is relatively short (see Figure 2) and that the slopes of the curves are small. v

2

v

2


ν

12.

TAjiMA A N D G E R S H F E L D

Lecithin

and Cholesterol

171

Mixtures

The dependence of the π values on the composition of the vapor and condensed states for D M L - C H O L , D O L - C H O L , and D O L - D M L mixtures is shown i n Figure 6. The upper curve is the surface vapor pressure as a function of the mole fraction of the liquid-expanded film; the lower curve is for the dependence of 7 r on the composition of the gaseous phase. Ideal mixing behavior is given by the linear dotted line which joins the τ τ points for each of the pure compounds. In all cases there was complete miscibility of the components as represented by the continuous function of π with x. In the cholesterol mixtures positive deviations from Raoult's law are observed; for the mixture of lecithins, ideal mixing is observed. These results confirm those obtained with lipid mixtures—i.e., cholesterol mixed with liquid-expanded lipid films forms non-ideal mixtures with positive deviations; for mixtures of lipids which are i n the same monolayer state, as i n the case of the liquid-expanded D O L - D M L mixtures, ideal mixing results ( 8 ) . ν

v

ν

0


ν

Figure 5.

τ τ - Α isotherms for mixtures of DML at 24.5°C

and CHOL

on water, pH

5.8

To calculate activity coefficients, y we apply the data of Figure 6 to Equation 1. Values for the two cholesterol-lecithin mixtures are pre sented in Table I as a function of mole fractions. The activity coefficients are greater than 1 i n all instances of cholesterol mixtures. u

The molecular areas on the transitional line F F ' i n Figures 3—5 are presented as a function of the mole fraction i n the condensed film i n Figure 7; for the cholesterol—lecithin mixtures there is a decrease i n area;



Figure

6.

π -χ surface phase diagrams for lipid mixtures on water, pH at 24.5°C. Dotted line represents ideal mixing.

5.8

ν

for the mixture of D O L and D M L there is no change i n the area of each component caused by mixing. In earlier studies it was concluded that the surface properties of lipid mixtures are determined largely by the hydrocarbon domain of the monolayer and that regular solution theory may be applied to these systems (8). To test the regular solution theory, we compared the partial heats of mixing obtained from the vapor pressure study with values Table I. A c t i v i t y Coefficients of D M L and D O L in Two-Component Mixed Monolayers with C H O L : Comparison with Regular Solution Heats of Mixing

Xi DOL

DML

a

Vi:

X 10

(c)

(1) 0.1 0.25 0.50 0.75 1.0 (1) 0.1 0.25 0.50 0.75 1.0

Xi (v) +

CHOL

CHOL

0.36 0.62 0.82 0.92 1.0

cal/mole RS"

(2)

0.32 0.54 0.75 0.91 1.0 +

AHi,

3

dynes/cm

5.8 6.8 8.0 9.0 9.5

1.91 1.51 1.24 1.13 1.0

384 245 127 73

277 183 74 17

5.5 6.4 7.7 8.8 9.7

2.06 1.65 1.32 1.12

428 297 165 67

223 160 75 20

(2)

R S : regular solution theory ΔΗ D M L = 334, D O L = 442.

ι

=

φ

2

2

Vi

(δι-δ ) , 2

2

δι

=

5.8,


δ

2

=

4.9

12.

T A j i M A A N D GERSHFELD

Lecithin

and Cholesterol

Mixtures

173

calculated from solution theory. For the former Equation 2 may be used, assuming that the entropy of mixing is ideal (20),


where AHi is the partial molar heat of mixing for component i . Values of ΔΗχ are in Table I. For comparison, a calculated value for ΔΗχ, ob-

0

0.2

0.4

0.6

0.8

1.0

X

Figure 7. Average area per molecule in mixed lipid films as a function of composition. Data obtained from Figures 3-5 along F - F ' , i.e., at the low area portion of the transition region in the -π-Α isotherm.


174

MONOLAYERS

tained from regular solution theory, may be obtained from the following relation Δ#!

= φ\ V

1

(δχ-δ ) 2

(3)

2

where Φ is the volume fraction of the component 2 (cholesterol), V i is the molar volume of component 1 (lecithin), and δι, δ are the solubility parameters of each component defined for the monolayer system as 2

2


(4) where Δ Η is the heat of surface vaporization (6, 8). Considering only the hydrocarbon domains of the monolayers we estimated δ = 4.9 for cholesterol and δ = 5.8 for liquid-expanded films (8). F r o m these values and Equation 3, ΔΗ is calculated and compared in Table I for the lecithin-cholesterol mixtures. Mixtures of D O L and D M L form ideal solutions, and ΑΗχ = 0. In each case the theory of regular solutions predicts the sign and magnitude of the partial molar heat of solution. Note that the value of δ for cholesterol is subject to considerable error, and only the magnitude and sign of the calculated values given should be considered significant. θν

1

In addition, it is generally observed for mixtures of aliphatic hydro carbons that if there is a difference in molecular size, a negative excess volume of mixing results (21). F o r the lecithin-cholesterol mixtures molecular area decreases (Figure 7), but it does not decrease with the mixture of the lecithins which forms an ideal mixture. Thus, the mixing of cholesterol and lecithin monolayers is consistent with bulk hydrocarbon mixtures with respect to both positive excess heats and negative excess volumes of mixing. The positive heats of mixing for lecithin-cholesterol mixtures indi cate that interactions between unlike molecules are smaller than the interactions between like molecules, i.e., the hydrocarbon chain interac tions with cholesterol are smaller than in each of the pure phases. If the excess heats of mixing become large enough, phase separation w i l l occur. It may occur when the surface pressure is increased ( i.e., as the films are compressed). The point at which phase separation occurs is difficult to predict, measure, or detect; however, evidence of phase separation can be deduced from the following experiment. If excess amounts of two lipids are placed i n water, the equilibrium surface pressure should reflect whether the surface film is a mixture. According to the phase rule (11,12, 13, 14), if two bulk lipid phases are present, only one surface phase can be present at the air-water surface. Thus the composition of the equi-


12.

T A j i M A A N D GERSHFELD

Lecithin

and Cholesterol

Mixtures

175

librium surface indicates whether there is mixing i n the monolayer at the higher surface pressures. Earlier studies indicated that the surface pres sure is a measure of the composition of the surface (22). Thus for cho lesterol and lecithin dispersed i n water at saturated concentrations, the Table II.

Equilibrium Spreading Pressures," II , of DML, DOL, C H O L , and Mixtures in H 0 at 29.5°C E

&

2

n


Lipid

CHOL DML D M L + C H O L (3/1) DOL D O L + C H O L (3/1)

e

dynes/cm, ±0.1

39.3 49.0 49.0 46.3 46.3

U — 7o — 7 where 70 is the surface tension of water, and 7 is the surface tension of the saturated lipid solution. Π values recorded by the Wilhelmy plate method (23), precision is ± 0 . 1 dynes/cm. At least 0.1 g/1 of each lipid was used to ensure that excess lipid was always present. a

e

β

b

equilibrium surface pressure w i l l be either that for pure lecithin, pure cholesterol, or neither, depending on whether only lecithin, cholesterol, or a mixture is present in the surface. In either of the first two cases, the results w i l l indicate that phase separation has occurred at some inter mediate surface pressure in which the other component or some mixture has been excluded from the surface. In the third case, mixing of the two lipids is obviously present at the elevated surface pressures. The results for D O L , D M L , and C H O L are presented i n Table II. For each of the mixed systems with cholesterol and lecithin, only lecithin is present in the equilibrium monolayer, as evidenced by the surface pressure equal to that of pure lecithin. Thus at some intermediate surface pressure, phase separation of cholesterol must occur. The nature of the separated phase cannot be established from this experiment without a complete analysis of the phase relations in the bulk system. It is likely that it may be a mixture of the two components i n view of the complete miscibility of the components in the surface vapor pressure region of the isotherm. Summary Cholesterol and lecithin form completely miscible solutions in mono layers at very low surface pressures, characterized by excess positive heats and excess negative areas of mixing. A t elevated surface pressures, phase separation occurs. Since these solutions conform to regular solution theory, the hydrocarbon domain of the monolayer makes the major con tribution to the heats of mixing. The polar region of the monolayer may


176

MONOLAYERS

also contribute, but it may not be significant i n the low pressure mono layers. Studies of the condensing effect of cholesterol at elevated surface pressures must recognize that phase separation w i l l occur under equi librium conditions.


Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Leathes, J. B., Lancet (1925) 208, 853. Phillips, M. C., in Progr. Surface Membrane Sci. (1972) 5, 179. Gershfeld, N. L., Pagano, R. E., J. Phys. Chem. (1972) 76, 1244. Adam, Ν. K., Jessop, G., Proc. Roy. Soc. Ser. A (1928) 120, 473. Adam, Ν. K., Jessop, G., Proc. Roy. Soc. Ser. A (1926) 110, 423. Gershfeld, N. L., Pagano, R. E., J. Phys. Chem. (1972) 76, 1231. Pagano, R. E., Gershfeld, N. L., J. Colloid Interface Sci. (1972) 41, 311. Pagano, R. E., Gershfeld, N . L., J. Phys. Chem. (1972) 76, 1238. Pagano, R. E., Gershfeld, N . L., J. Colloid Interface Sci. (1973) 44, 382. Gershfeld, N . L., J. Colloid Interface Sci. (1970) 32, 167. Defay, R., Ph.D. thesis, Brussels (1932). Defay, R., Prigogine, I., Bellemans, Α., Everett, D. H., "Surface Tension and Adsorption," pp. 74-78, Wiley, New York, 1966. Crisp, D. J., in "Surface Chemistry," pp. 17, 23, Supplement to Research, Buttersworth, London, 1949. Gershfeld, N. L., in "Methods in Membrane Biology," Vol. I, E. D. Korn, Ed., p. 77, Plenum Press, 1974. Prausnitz, J. M., "Molecular Thermodynamics of Fluid Phase Equilibria," p. 204, Prentice-Hall, Englewood Cliffs, N. J., 1969. Gershfeld, N. L., Pagano, R. E., Friauff, W. S., Fuhrer, J., Rev. Sci. Instrum. (1970) 41, 1356. Colacicco, G., Biochim. Biophys. Acta (1971) 266, 313. Wagner, H., Horhaurmer, L., Wolff, P., Biochim. Z. (1961) 334, 175. Pak, C. Y. C., Gershfeld, N. L., J. Colloid Sci. (1964) 19, 831. Hildebrand, J. H., Scott, R. L., "The Solubility of Non-Electrolytes," 3rd edition, p. 41, Dover Publications, New York, 1950. Rowlinson, J. S., "Liquids and Liquid Mixtures," pp. 142-146, Butterworths Scientific Publications, London, 1959. Gershfeld, N . L., Pagano, R. E., J. Phys. Chem. (1972) 76, 1244. Gaines, G. L., Jr., "Insoluble Monolayers at Liquid-Gas Interfaces," p. 44, Interscience Publishers, New York, 1969.

RECEIVED

October 24, 1974.


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