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Phase transitions in fatty acid monolayers containing a single double bond in the fatty acid tail
Phase Transitions in Fatty Acid Monolayers Containing a Single Double Bond in the Fatty Acid Tail Y. M. H I F E D A AND G. W. R A Y F I E L D Physics Department, University of Oregon, Eugene, Oregon 97403 Received December 13, 1983; accepted August 24, 1984 The effect on ~r-A isotherms of introducing a single double bond (trans) into the tail of a fatty acid has been studied. When the double bond is introduced at the 11 position the monolayer becomes liquid expanded. Moving the trans double bond to the 6 position (nearer the carboxyl head group) in the fatty acid tail yields monolayers that show a sharp discontinuity in the slope of the 7r-A isotherms. A careful study of the equilibrium state of the monolayer was made. It was found that above the equilibrium spreading pressure (~e) the film is unstable while below ~-~the film is stable. The surface pressure for the discontinuity in the 7r-A curve (~rt) is compared with 7re. When ~t > 7r~ the sharp discontinuity in the slope of the r-A curve disappears. Both solvent spread and crystal spread films were examined. © 1985 Academic Press, Inc. INTRODUCTION Surface pressure versus area isotherms (TrA curves) o f octadecanoic acid (stearic acid) on a water substrate indicate that the m o n o layer is in a liquid condensed state while similar measurements o f trans-octadec-9-enoic acid (elaidic acid) are consistent with a liquid expanded state at r o o m temperature (1). The familiar picture o f these fatty acid monolayers is that their hydrophilic carboxyl head groups are immersed in the water substrate and interact strongly with it while their hydrophobic alkyl chains protrude into the air and interact with each other through van der Waals" forces (2, 3). T h e alkyl chain tends to b e c o m e rigid in the neighborhood of the double b o n d (4). ~r-A isotherms o f an elaidyl alcohol m o n o layer were studied by Glazer and G o d d a r d (5). T h e ~r-A curves are o f the liquid expanded type and show a sharp discontinuity in slope. These authors m a k e the interesting observation that there is no obvious reason why the introduction o f a double b o n d into a long-chain amphipatic molecule results in an expanded film c o m p a r e d to the saturated case. Except for noting that the 7r-A curves
did not depend on the rate o f compression (unspecified) n o studies o f film stability were reported. A quasi-first-order phase transition was assumed and an estimate o f latent heat was made based on the Clausius-Clapeyron equation. T h e y were unable to reach low enough temperatures to produce a fully condensed m o n o l a y e r and such a state had to be assumed for the latent heat calculation. The calculation is also subject to criticism on the grounds that the surface pressure is not constant over a well-defined transition region. We find similar ~--A isotherms when the trans double b o n d is m o v e d from the 9 position in eladic acid to the 6 position. We also find that for this system, a fully condensed film occurs at a temperature o f 4°C. Application o f t h e r m o d y n a m i c s or statistical mechanical theories to the m o n o l a y e r system requires a knowledge o f its state o f equilibrium. T h e stability o f m o n o l a y e r films has been extensively discussed in the literature (3, 6-12, 15, 16). Some o f these studies report that the collapse pressure o f a m o n o molecular film depends on its rate o f compression (3, 6, 9, 10). Therefore, the
2o9 0021-9797/85 $3.00 Journal of Colloid and Interface Science, Vol. 104, No, 1, March 1985
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
HIFEDA AND RAYFIELD
equilibrium state of the monolayer during a slow quasi-static compression is open to question. Several mechanisms have been suggested to explain the onset of film instability. One possibility is that the relaxation of surface pressure is due to the nucleation and growth of bulk solid fragments of surfactant during compression (12). Other investigators (10, 11, 13) propose that above the equilibrium spreading pressure (re) molecules start to eject from the film into a lens. High rates of compression may overcome the rate of lens formation allowing high (nonequilibrium) values of surface pressure to be reached (11). Still other mechanisms have also been suggested to explain pressure loss of monolayers such as evaporation, loss into the subphase, and autoxidation (14). A serious experimental artifact, the leakage of molecules around the surface barrier, can also lead to loss of surface pressure. Regardless of the reason for the instability of the film, a criterion for stability is needed. If the system relaxation time is much larger than the experimental measuring time then the system can be taken to be in equilibrium. Some workers (15) take the film to be stable if the surface pressure decreases by 5% or less during a 30-see interval. Gershfeld (16) has recently examined in detail the equilibrium state of monolayer films formed from both dimyristol phosphatidylcholine (DMPC) and palmitic acid. The equilibrium spreading pressure 7r, is suggested as a criterion for film stability. When ~r < ~r, the film desorbs into the substrate solution and when ~r > ~r~ the film tends to collapse into the bulk (crystalline) phase. Desorption into the subphase involves a high activation energy and is a slow process (on the order of years) for the fatty acid (16, 17) and, therefore, much longer than the measurement times. Collapse of the film when 7r > ~re is, on the other hand, a much more rapid process comparable to the compression time for the film. Based on comparisons between solvent spread films and radioactively labeled palmitic acid films spread from crystals on the surface Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
Gershfeld questions the use of zr-A curves to study phase transitions. However, these studies are somewhat inconclusive in that the comparisons were only made for 7r = 7re and radioactively labeled solvent spread films were not studied. It is further reported that the liquid condensed (LC)/liquid expanded (LE) phase transition reported by other investigators for DMPC depends on solvent spreading of the film and disappears when the film is spread from crystals. Saturated fatty acid isotherm studies have recently been reported by Bois et al. (19). These investigators also report that monolayers with surface pressures in excess of 7r~ are unstable. They question Gershfeld's speculation (16) that the LE/LC transition corresponds to the formation of micelles in an "inner phase." Our results are consistent with the notion that 7re is a critical pressure and represents an upper limit on the surface pressure for stable films. For temperatures below room temperature we find a sharp discontinuity in the 7r-A curves at a surface pressure 7rt such that 7rt < ~re. No difference in ~r-A curves is observed between solvent spread or crystal spread films providing that only stable films (71" < 7re) are studied. MATERIALS AND METHODS
The fatty acids used in this study were grade A, purchased from Sigma Chemical Company, St. Louis, Missouri, Applied Science, Deerfield, Illinois; and Nu Chek Prep, Elysian, Minnesota. Glass-distilled hexanes of high purity were obtained from Burdick and Jackson Laboratories Inc., Muskegon, Michigan. The Langmuir trough, 53 cm long, 15 cm wide, and 1.3 cm deep, was machined out of virgin white Teflon. Temperature regulation was achieved by circulating constant-temperature water through glass tubing fitted into the bottom of the trough. Temperature regulation was better than 0.5°C. A tight-fitting plexiglass cover over the trough provides a seal against dust and foreign matter.
211
PHASE TRANSITION IN M O N O L A Y E R S
The Teflon barrier which confines the monolayer is connected by a lead screw to a stepping motor. This results in a barrier translation of 6.35 X 10 -4 cm per motor step and defines the accuracy of our area measurements. The Wilhelmy plate for our film balance was made from a glass microscope cover slide, 6 cm long, 2 cm wide, and 0.01 cm thick, in order to minimize the wetting problem (3). A linear motion transducer is the main component of the electrobalance. The central magnetic core of the transducer is attached to two springs which determine the balance sensitivity. The stiffness of the springs chosen limited the surface pressure sensitivity to 0.040 dyn/cm. The output signal from the film balance was fed to a 12-bit analog-todigital converter (ADC) connected to a small microcomputer. The pulse generator driving the stepping motor also triggered the ADC so that a correlated record of film area versus surface pressure was stored in the microcomputer. The digitally stored data are easily corrected for experimental artifacts such as plate buoyancy. Unless otherwise noted, freshly prepared triple-quartz-distilled water (pH 7) was used for the monolayer subphase. The substrate surface was cleaned (by aspiration) until repeated sweeping with the barrier produced no change in surface pressure. The trough could then be left overnight and would remain clean providing the plexiglass cover was kept in place. A typical solvent spread monolayer was formed by adding 100 ~1 of 0.5 mg/ml material in organic solvent (hexane) to the free surface of the substrate. Crystal spread monolayers were formed by carefully adding tiny crystals of fatty acid to the freshly cleaned subphase. Typical compression rates for the monolayers were 2 A2/molecule-min. RESULTS
Figure 1 shows stearic acid isotherms taken at room temperature (21°C) and low tem-
>.. ~o
4~
5@ 0J 22 c m
° i~ o o
F
~
J
~ ~o ~ ' 4~ Area per Molecule (sq. ,¢)
FIG. 1. Surface pressure-area isotherms for octadecanoic (stearic) acid on a water substrate are shown. Measurements taken at 21 and at 3°C were identical. The kink in the isotherm occurs near the equilibrium spreading pressure.
perature (3°C). All of the isotherms were consistent with a liquid condensed state and showed no temperature dependence from 3 to 21°C. Near 0°C and at the kink in the isotherm curve (~- = 24 dyn/cm) the Wilhelmy plate moved horizontally, suggesting the formation of a frozen solid sheet of stearic acid film. Introducing a trans ~r bond into the fatty acid chain in place of a single a bond has a marked effect on the shape of the isotherm consistent with similar studies on alcohols (5). Figure 2 shows isotherms of trans-octadec-1 l-enoic acid taken at 21 and at 3°C. The isotherms are indicative of a liquid expanded state with no sign of a liquid condensed state. Changing the position of the double bond in the alkyl chain alters the shape of the isotherm. Figure 3 shows a--A isotherms of trans-octadec-9-enoic acid at both 20 and 4°C. While the 20°C isotherm shows only a liquid expanded state, the slope o f the 4°C isotherm is no longer a monotonically increasing function as the monolayer area is decreased. Further movement of the trans double bond along the alkyl chain (toward the head group) leads to isotherms that suggest a phase transition. Figure 4 shows a family of isotherms for trans-octadec-6-enoic acid taken Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
212
HIFEDA AND RAYFIELD 40
40 (a)
C >-, 3;
a
(c) (b) (c) (d) (e)
°c
(b) 21 ~C 30 %
29~C 27%
25~ 25% 24%
(g) 17%
~
(h) 14%
~i) 4% k 0 L 20
30
40
Ar'eo per- M o l e c u l e
50
fiZ
( ~ q . F) i
FIG. 2. Surface pressure-area isotherms for trans-
octadec-1 l-enoic acid on a water substrate at 3 and 21°C are shown.
at different t e m p e r a t u r e s . D e p e n d i n g o n the t e m p e r a t u r e , a s u d d e n c h a n g e in the slope
0 10
30
40
I
50
60
Areo per Molecule (sq,A°)
FIG. 4. A family of pressure-area isotherms for transoctadec-6-enoic acid on a pure water substrate for nine different temperatures is shown. A discontinuous change in the slope of the isotherm occurs at a unique surface pressure (Tr,) and surface area.
o f the isotherm occurs as the monolayer is compressed.
Further
compression
(Fig.
4)
results only in a gradual increase in the slope. At the l o w e s t t e m p e r a t u r e s the 7r-A curve is fully c o n d e n s e d . It w a s n o t p o s s i b l e to reach
this state in the case o f elaidyl alcohol (5)
A l t h o u g h the results a b o v e are interesting, a m a j o r q u e s t i o n r e m a i n s : D o t h e s e ~r-A curves describe stable films? Stability b e i n g d e f i n e d w i t h i n the c o n t e x t o f 7rt < 7re a n d
a l t h o u g h such a state w a s a s s u m e d to exist
7r(t) = constant (at fixed area) for times, t, o f order hours.
in order to calculate a latent heat o f transformation.
Figure 6 s h o w s a p l o t o f "/re a n d ~r, versus t e m p e r a t u r e . D a t a w e r e corrected to take
Figure 5 s h o w s a p l o t o f the t w o - d i m e n s i o n a l isothermal bulk modulus, B = A*(dTr/
dA)T, in
an expanded region near 7r~. B is very nearly zero for 7r = ~rt.
25,
20 0
0 0 o
o
15 (a)
4 %
(b)
20%
0
3
0 I0
0
~ ze
o
OJ a 0_
I
F
o o
g
o
u)
10
20 Areo
30 per
40 Molecule
50
60
; 0
:55 ' Areo
FIG. 3. Surface pressure-area isotherms for trans-
octadec-9-enoic acid on a water substrate at 4 and 20°C are shown. The slope of the 4°C curve is not a monotonically increasing function of temperature. Journal of Colloid and Interface Science, Vol. 104, No. 1, M a r c h 1985
°20
°
°
4 5'
5 '0
Per Molecule (A°~)
-5
FIG. 5. A plot of the two-dimensional isothermal bulk modulus, B, at 20°C in the region near ~rt is shown.
PHASE TRANSITION IN MONOLAYERS
18' l
~6
t
(o)
"ae;®
~b)
"at ;O
<
/ j ~ /
14i
/
"~ l0
,~ bt i 1 I
/
I J 1
# J
TernpeFoture I(oc)
FIG. 6. The equilibrium spreading pressure ~re and the transition pressure ;rt are plotted versus temperature for trans-octadec-6-erioic acid on water at pH 7.
into a c c o u n t the variation o f water surface tension with temperature. 7re was measured using two methods: (1) A surplus o f fatty acid in solvent was added to the substrate and then the equilibrium spreading pressure was measured after waiting about 1 hr. (2) Crystals o f fatty acid (no solvent) were added to a clean water substrate (t = 30°C) and an equilibrium spreading pressure was allowed to develop. The temperature was then reduced and the equilibrium spreading pressure again measured after waiting 1 hr. Both methods gave the same 71"e ValUeS. 71"t values were obtained f r o m the point o f discontinuity in the ,r-A curves o f Fig. 4. T h e temperature where a-t exceeds ~re agrees well with the temperature where the sharp discontinuity in the slope o f the 7r-A isotherms also disappears. Figure 7 shows the variation in surface pressure with time (a-(t)) at different fixed areas per molecule for a fixed temperature
213
o f 20°C. Each recording was m a d e with a freshly prepared solvent spread monolayer. ~re and ~rt from Fig. 6 are labeled in the plot. The horizontal time scale for curve A is 2.5 hr rather than 60 m i n as for the other curves. The starting pressures were developed by quick compression from a highly expanded film (~r ~ 0). As long as ~r < ~'e the films showed no change in 7r at fixed area even after 8 hr. 7re values taken as the asymptotic limit as ~r decays with time were in g o o d agreement with those o f Fig. 6. The decay time for ~r > 7re was dependent on both the initial starting surface pressure and the temperature o f the substrate. A further check o f the m o n o l a y e r stability was made by first compressing the m o n o l a y e r to just below ~re and then expanding the film by reversing the barrier m o v e m e n t . Little hysteresis was observed. Decreasing the compression rate by a factor o f 10 (to 0.2 ~2/molecule-min) had n o effect on the Ir-A curves (Fig. 4) in the region where a- < 7re. Figure 8 shows a c o m p a r i s o n o f crystal spread and solvent spread films. The solvent spread film was formed in the usual manner. The crystal spread film was formed by first adding a small crystal to the surface at 30°C and then r e m o v i n g it by surface suction. The temperature o f the film was then lowered to
20
s
~t
o~ 0
r g
10
20
Time
i
i
2B
40
p~ 50
60
(rn i rnu6es)
FIG. 7. The decay of surface pressure with time is shown for a monolayer held at constant area but starting with different initial surface pressures. For ri~a > *re the surface pressure eventually relaxes to 7re. If *qma~ < 7re then the surface pressure remains constant for periods in excess of 8 hr. J o u r n a l o f C o l l o i d a n d In te rfa c e Science,
Vol. 104, No. 1, March 1985
214
HIFEDA AND RAYFIELD
£
m
g 10
21~ Are~
3~ per"
48 Molecule
50
6~
( s q . /()
FIG. 8. Typical ~r-A isotherms (20°C) for both crystal spread and solvent spread films are shown.
20°C and compressed. The total n u m b e r of molecules in the crystal spread film is unknown so that the horizontal axis in this case represents a relative area per molecule. Similar results for crystal spread and solvent spread films were found at other temperatures. Substrate p H had little effect on the ar-A curves except when ar >> are. A similar insensitivity to p H of are was also found. The p H of the substrate water was adjusted between p H 2 and p H 7 with HG1. Measurements were also made on 18:1 cis-octadec-6-enoic acid. No temperature dependence of the ar-A curves was observed. ar-A curves are almost identical for the cis and trans forms of the acid at 30°C, where the trans form is fully expanded. Finally, Fig. 9 shows ar-A isotherms taken at room temperature for 6-trans- and 9-lransoctadecenoic acid and a 1:1 mixture of both fatty acids. The isotherms do not show additivity, which indicates an interaction between the two different types of fatty acids.
in the slope of the ar-A curve at a unique surface pressure (art) and area per molecule. Fully condensed ~r-A curves (with no expanded region) develop at moderately low temperature (4°C). For ar < ar~ the ar-A isotherms in Fig. 4 represent an expanded state that is approximately independent of temperature. We speculate that in this region rapid thermal motion of the fatty acid tail dominates the force of interaction (5, 2). The fully condensed ar-A isotherm has a limiting area (29 flk2) which is greater than that of stearic acid (24 A2). This expansion could be due to a reduction in the cohesive force between fatty acid tails when a double bond is introduced. Most theories view the inferred LE/LC phase transition as first order, although there is a lack of experimental support for this view (5, 16, 19). The ar-A isotherms shown in Fig. 4 are very similar to those observed for tetradecanoic acid (myristic acid) (3) and elaidyl alcohol where the trans double bond is at the 9 position. The head group of the alcohol is more cohesive than that o f the acid (5) and the m o v e m e n t of the double bond to the 6 position apparently compensates for this. There are at least two different effects due to the insertion of a double bond into the alkyl chain (5). The first is to limit the flexibility of the chain in this region. The second is to 4Z {a) 1 8 : ~ ttans (b) l:lmixture (c) 1 8 : ~ trans
3~ v
DISCUSSION AND CONCLUSIONS Surface pressures for the fatty acid films studied were very stable at fixed areas providing ar < arc. trans-Octadec-6-enoic acid is found to have a broad temperature spectrum of ar-A curves such that ar < arc. Some of these isotherms show a sharp discontinuity Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
10
20
30
4~
50
6~
APea pep Moleoule (eq.~)
FIG. 9. A surface pressure-area isotherm of a h 1 mixture of 6-trans- and 9-trans-octadecenoic acids is compared with isotherms for the pure fatty acids. Data were taken at a temperature of 20°C.
PHASE TRANSITION IN MONOLAYERS
decrease the adhesion between molecules. Similar effects are seen when double bonds are introduced into the hydrocarbon tails of certain lipid molecules (4). The effect of introducing double bonds into the alkyl chains has not yet been theoretically investigated. Several investigators have postulated that small molecular clusters begin to form when 7r reaches 7rt. Recent calculations by Georgallas and Pink (18) seem to support these conjectures. These investigators find a qualitative shape for 7r-A curves not unlike those of Fig. 4. One interpretation of Fig. 9 is that introducing trans-9 fatty acid into trans-6 inhibits the formation of clusters. The formation of clusters at 7rt is still highly speculative and awaits experimental confirmation. ACKNOWLEDGMENTS We are grateful to J. F. Nagle for very helpful discussions. The work was supported in part by NIH Grant GM 10069. REFERENCES 1. Jalal, 1., and Zografi, G., J. Colloid Interface Sci. 68, 197 (1970). 2. Adam, N., "The Physics and Chemistry of Surfaces." Oxford Univ. Press, London, 1941.
215
3. Gains, G. L., "Insoluble Monolayers at Liquid-Gas Interfaces," pp. 167-172. Interscience, New York, 1966. 4. Seelig, A., and Seelig, J., Biochemistry 16, 45 (1977). 5. Glazer, J., and Goddard, E. D., J. Chem. Soc., part 3, 3406 (1950). 6. Heikkila, R. E., Kwong, C. N., and Cornwell, D. G., J. LipM Res. 11, 190 (1970). 7. Baglioni, P., Gabrielli, G., and Guarini, G. G. T., J. ColloM Interface Sci. 78, 347 (1980). 8. Shuquian Xu, Miyano, H., and Abraham, B. M., J. Colloid Interface Sci. 89, 581 (1982). 9. Rabinovitch, W., Robertson, R. F., and Mason, S. G., Canad. J. Chem. 38, 1881 (1960). 10. Sims, B., and Zografi, G., Chem. Phys. Lipids 6, 109 (1971). 11. Sims, B., and Zografi, G., J. Colloid Interface Sci. 41, 35 (1972). 12. Smith, R. D., and Berg, J. C., J. Colloid Interface Sci. 74, 273 (1980). 13. Motomura, K., J. Colloid Interface ScL 23, 313 (1967). 14. Heikkila, R. E., Deamer, D. W., and Cornwell, D. G., J. Lipid Res. 11, 195 (1970). 15. Horn, L. W., and Gershfeld, N. L., Biophys. J. 18, 301 (1977). 16. Gershfeld, N. L., J. Colloid Interface Sci. 85, 28 (1982). 17. Gershfeld, N. L., Adv. Chem. Ser. 84, 115 (1968). 18. Georgallas, A., and Pink, D. A,, Canad. J. Phys. 60, 1678 (1982). 19. Bois, A. G., Panaiotov, I. I., and Baret, J. F., Chem. Phys. Lipids 34, 265 (1984).
Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
did not depend on the rate o f compression (unspecified) n o studies o f film stability were reported. A quasi-first-order phase transition was assumed and an estimate o f latent heat was made based on the Clausius-Clapeyron equation. T h e y were unable to reach low enough temperatures to produce a fully condensed m o n o l a y e r and such a state had to be assumed for the latent heat calculation. The calculation is also subject to criticism on the grounds that the surface pressure is not constant over a well-defined transition region. We find similar ~--A isotherms when the trans double b o n d is m o v e d from the 9 position in eladic acid to the 6 position. We also find that for this system, a fully condensed film occurs at a temperature o f 4°C. Application o f t h e r m o d y n a m i c s or statistical mechanical theories to the m o n o l a y e r system requires a knowledge o f its state o f equilibrium. T h e stability o f m o n o l a y e r films has been extensively discussed in the literature (3, 6-12, 15, 16). Some o f these studies report that the collapse pressure o f a m o n o molecular film depends on its rate o f compression (3, 6, 9, 10). Therefore, the
2o9 0021-9797/85 $3.00 Journal of Colloid and Interface Science, Vol. 104, No, 1, March 1985
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
HIFEDA AND RAYFIELD
equilibrium state of the monolayer during a slow quasi-static compression is open to question. Several mechanisms have been suggested to explain the onset of film instability. One possibility is that the relaxation of surface pressure is due to the nucleation and growth of bulk solid fragments of surfactant during compression (12). Other investigators (10, 11, 13) propose that above the equilibrium spreading pressure (re) molecules start to eject from the film into a lens. High rates of compression may overcome the rate of lens formation allowing high (nonequilibrium) values of surface pressure to be reached (11). Still other mechanisms have also been suggested to explain pressure loss of monolayers such as evaporation, loss into the subphase, and autoxidation (14). A serious experimental artifact, the leakage of molecules around the surface barrier, can also lead to loss of surface pressure. Regardless of the reason for the instability of the film, a criterion for stability is needed. If the system relaxation time is much larger than the experimental measuring time then the system can be taken to be in equilibrium. Some workers (15) take the film to be stable if the surface pressure decreases by 5% or less during a 30-see interval. Gershfeld (16) has recently examined in detail the equilibrium state of monolayer films formed from both dimyristol phosphatidylcholine (DMPC) and palmitic acid. The equilibrium spreading pressure 7r, is suggested as a criterion for film stability. When ~r < ~r, the film desorbs into the substrate solution and when ~r > ~r~ the film tends to collapse into the bulk (crystalline) phase. Desorption into the subphase involves a high activation energy and is a slow process (on the order of years) for the fatty acid (16, 17) and, therefore, much longer than the measurement times. Collapse of the film when 7r > ~re is, on the other hand, a much more rapid process comparable to the compression time for the film. Based on comparisons between solvent spread films and radioactively labeled palmitic acid films spread from crystals on the surface Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
Gershfeld questions the use of zr-A curves to study phase transitions. However, these studies are somewhat inconclusive in that the comparisons were only made for 7r = 7re and radioactively labeled solvent spread films were not studied. It is further reported that the liquid condensed (LC)/liquid expanded (LE) phase transition reported by other investigators for DMPC depends on solvent spreading of the film and disappears when the film is spread from crystals. Saturated fatty acid isotherm studies have recently been reported by Bois et al. (19). These investigators also report that monolayers with surface pressures in excess of 7r~ are unstable. They question Gershfeld's speculation (16) that the LE/LC transition corresponds to the formation of micelles in an "inner phase." Our results are consistent with the notion that 7re is a critical pressure and represents an upper limit on the surface pressure for stable films. For temperatures below room temperature we find a sharp discontinuity in the 7r-A curves at a surface pressure 7rt such that 7rt < ~re. No difference in ~r-A curves is observed between solvent spread or crystal spread films providing that only stable films (71" < 7re) are studied. MATERIALS AND METHODS
The fatty acids used in this study were grade A, purchased from Sigma Chemical Company, St. Louis, Missouri, Applied Science, Deerfield, Illinois; and Nu Chek Prep, Elysian, Minnesota. Glass-distilled hexanes of high purity were obtained from Burdick and Jackson Laboratories Inc., Muskegon, Michigan. The Langmuir trough, 53 cm long, 15 cm wide, and 1.3 cm deep, was machined out of virgin white Teflon. Temperature regulation was achieved by circulating constant-temperature water through glass tubing fitted into the bottom of the trough. Temperature regulation was better than 0.5°C. A tight-fitting plexiglass cover over the trough provides a seal against dust and foreign matter.
211
PHASE TRANSITION IN M O N O L A Y E R S
The Teflon barrier which confines the monolayer is connected by a lead screw to a stepping motor. This results in a barrier translation of 6.35 X 10 -4 cm per motor step and defines the accuracy of our area measurements. The Wilhelmy plate for our film balance was made from a glass microscope cover slide, 6 cm long, 2 cm wide, and 0.01 cm thick, in order to minimize the wetting problem (3). A linear motion transducer is the main component of the electrobalance. The central magnetic core of the transducer is attached to two springs which determine the balance sensitivity. The stiffness of the springs chosen limited the surface pressure sensitivity to 0.040 dyn/cm. The output signal from the film balance was fed to a 12-bit analog-todigital converter (ADC) connected to a small microcomputer. The pulse generator driving the stepping motor also triggered the ADC so that a correlated record of film area versus surface pressure was stored in the microcomputer. The digitally stored data are easily corrected for experimental artifacts such as plate buoyancy. Unless otherwise noted, freshly prepared triple-quartz-distilled water (pH 7) was used for the monolayer subphase. The substrate surface was cleaned (by aspiration) until repeated sweeping with the barrier produced no change in surface pressure. The trough could then be left overnight and would remain clean providing the plexiglass cover was kept in place. A typical solvent spread monolayer was formed by adding 100 ~1 of 0.5 mg/ml material in organic solvent (hexane) to the free surface of the substrate. Crystal spread monolayers were formed by carefully adding tiny crystals of fatty acid to the freshly cleaned subphase. Typical compression rates for the monolayers were 2 A2/molecule-min. RESULTS
Figure 1 shows stearic acid isotherms taken at room temperature (21°C) and low tem-
>.. ~o
4~
5@ 0J 22 c m
° i~ o o
F
~
J
~ ~o ~ ' 4~ Area per Molecule (sq. ,¢)
FIG. 1. Surface pressure-area isotherms for octadecanoic (stearic) acid on a water substrate are shown. Measurements taken at 21 and at 3°C were identical. The kink in the isotherm occurs near the equilibrium spreading pressure.
perature (3°C). All of the isotherms were consistent with a liquid condensed state and showed no temperature dependence from 3 to 21°C. Near 0°C and at the kink in the isotherm curve (~- = 24 dyn/cm) the Wilhelmy plate moved horizontally, suggesting the formation of a frozen solid sheet of stearic acid film. Introducing a trans ~r bond into the fatty acid chain in place of a single a bond has a marked effect on the shape of the isotherm consistent with similar studies on alcohols (5). Figure 2 shows isotherms of trans-octadec-1 l-enoic acid taken at 21 and at 3°C. The isotherms are indicative of a liquid expanded state with no sign of a liquid condensed state. Changing the position of the double bond in the alkyl chain alters the shape of the isotherm. Figure 3 shows a--A isotherms of trans-octadec-9-enoic acid at both 20 and 4°C. While the 20°C isotherm shows only a liquid expanded state, the slope o f the 4°C isotherm is no longer a monotonically increasing function as the monolayer area is decreased. Further movement of the trans double bond along the alkyl chain (toward the head group) leads to isotherms that suggest a phase transition. Figure 4 shows a family of isotherms for trans-octadec-6-enoic acid taken Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
212
HIFEDA AND RAYFIELD 40
40 (a)
C >-, 3;
a
(c) (b) (c) (d) (e)
°c
(b) 21 ~C 30 %
29~C 27%
25~ 25% 24%
(g) 17%
~
(h) 14%
~i) 4% k 0 L 20
30
40
Ar'eo per- M o l e c u l e
50
fiZ
( ~ q . F) i
FIG. 2. Surface pressure-area isotherms for trans-
octadec-1 l-enoic acid on a water substrate at 3 and 21°C are shown.
at different t e m p e r a t u r e s . D e p e n d i n g o n the t e m p e r a t u r e , a s u d d e n c h a n g e in the slope
0 10
30
40
I
50
60
Areo per Molecule (sq,A°)
FIG. 4. A family of pressure-area isotherms for transoctadec-6-enoic acid on a pure water substrate for nine different temperatures is shown. A discontinuous change in the slope of the isotherm occurs at a unique surface pressure (Tr,) and surface area.
o f the isotherm occurs as the monolayer is compressed.
Further
compression
(Fig.
4)
results only in a gradual increase in the slope. At the l o w e s t t e m p e r a t u r e s the 7r-A curve is fully c o n d e n s e d . It w a s n o t p o s s i b l e to reach
this state in the case o f elaidyl alcohol (5)
A l t h o u g h the results a b o v e are interesting, a m a j o r q u e s t i o n r e m a i n s : D o t h e s e ~r-A curves describe stable films? Stability b e i n g d e f i n e d w i t h i n the c o n t e x t o f 7rt < 7re a n d
a l t h o u g h such a state w a s a s s u m e d to exist
7r(t) = constant (at fixed area) for times, t, o f order hours.
in order to calculate a latent heat o f transformation.
Figure 6 s h o w s a p l o t o f "/re a n d ~r, versus t e m p e r a t u r e . D a t a w e r e corrected to take
Figure 5 s h o w s a p l o t o f the t w o - d i m e n s i o n a l isothermal bulk modulus, B = A*(dTr/
dA)T, in
an expanded region near 7r~. B is very nearly zero for 7r = ~rt.
25,
20 0
0 0 o
o
15 (a)
4 %
(b)
20%
0
3
0 I0
0
~ ze
o
OJ a 0_
I
F
o o
g
o
u)
10
20 Areo
30 per
40 Molecule
50
60
; 0
:55 ' Areo
FIG. 3. Surface pressure-area isotherms for trans-
octadec-9-enoic acid on a water substrate at 4 and 20°C are shown. The slope of the 4°C curve is not a monotonically increasing function of temperature. Journal of Colloid and Interface Science, Vol. 104, No. 1, M a r c h 1985
°20
°
°
4 5'
5 '0
Per Molecule (A°~)
-5
FIG. 5. A plot of the two-dimensional isothermal bulk modulus, B, at 20°C in the region near ~rt is shown.
PHASE TRANSITION IN MONOLAYERS
18' l
~6
t
(o)
"ae;®
~b)
"at ;O
<
/ j ~ /
14i
/
"~ l0
,~ bt i 1 I
/
I J 1
# J
TernpeFoture I(oc)
FIG. 6. The equilibrium spreading pressure ~re and the transition pressure ;rt are plotted versus temperature for trans-octadec-6-erioic acid on water at pH 7.
into a c c o u n t the variation o f water surface tension with temperature. 7re was measured using two methods: (1) A surplus o f fatty acid in solvent was added to the substrate and then the equilibrium spreading pressure was measured after waiting about 1 hr. (2) Crystals o f fatty acid (no solvent) were added to a clean water substrate (t = 30°C) and an equilibrium spreading pressure was allowed to develop. The temperature was then reduced and the equilibrium spreading pressure again measured after waiting 1 hr. Both methods gave the same 71"e ValUeS. 71"t values were obtained f r o m the point o f discontinuity in the ,r-A curves o f Fig. 4. T h e temperature where a-t exceeds ~re agrees well with the temperature where the sharp discontinuity in the slope o f the 7r-A isotherms also disappears. Figure 7 shows the variation in surface pressure with time (a-(t)) at different fixed areas per molecule for a fixed temperature
213
o f 20°C. Each recording was m a d e with a freshly prepared solvent spread monolayer. ~re and ~rt from Fig. 6 are labeled in the plot. The horizontal time scale for curve A is 2.5 hr rather than 60 m i n as for the other curves. The starting pressures were developed by quick compression from a highly expanded film (~r ~ 0). As long as ~r < ~'e the films showed no change in 7r at fixed area even after 8 hr. 7re values taken as the asymptotic limit as ~r decays with time were in g o o d agreement with those o f Fig. 6. The decay time for ~r > 7re was dependent on both the initial starting surface pressure and the temperature o f the substrate. A further check o f the m o n o l a y e r stability was made by first compressing the m o n o l a y e r to just below ~re and then expanding the film by reversing the barrier m o v e m e n t . Little hysteresis was observed. Decreasing the compression rate by a factor o f 10 (to 0.2 ~2/molecule-min) had n o effect on the Ir-A curves (Fig. 4) in the region where a- < 7re. Figure 8 shows a c o m p a r i s o n o f crystal spread and solvent spread films. The solvent spread film was formed in the usual manner. The crystal spread film was formed by first adding a small crystal to the surface at 30°C and then r e m o v i n g it by surface suction. The temperature o f the film was then lowered to
20
s
~t
o~ 0
r g
10
20
Time
i
i
2B
40
p~ 50
60
(rn i rnu6es)
FIG. 7. The decay of surface pressure with time is shown for a monolayer held at constant area but starting with different initial surface pressures. For ri~a > *re the surface pressure eventually relaxes to 7re. If *qma~ < 7re then the surface pressure remains constant for periods in excess of 8 hr. J o u r n a l o f C o l l o i d a n d In te rfa c e Science,
Vol. 104, No. 1, March 1985
214
HIFEDA AND RAYFIELD
£
m
g 10
21~ Are~
3~ per"
48 Molecule
50
6~
( s q . /()
FIG. 8. Typical ~r-A isotherms (20°C) for both crystal spread and solvent spread films are shown.
20°C and compressed. The total n u m b e r of molecules in the crystal spread film is unknown so that the horizontal axis in this case represents a relative area per molecule. Similar results for crystal spread and solvent spread films were found at other temperatures. Substrate p H had little effect on the ar-A curves except when ar >> are. A similar insensitivity to p H of are was also found. The p H of the substrate water was adjusted between p H 2 and p H 7 with HG1. Measurements were also made on 18:1 cis-octadec-6-enoic acid. No temperature dependence of the ar-A curves was observed. ar-A curves are almost identical for the cis and trans forms of the acid at 30°C, where the trans form is fully expanded. Finally, Fig. 9 shows ar-A isotherms taken at room temperature for 6-trans- and 9-lransoctadecenoic acid and a 1:1 mixture of both fatty acids. The isotherms do not show additivity, which indicates an interaction between the two different types of fatty acids.
in the slope of the ar-A curve at a unique surface pressure (art) and area per molecule. Fully condensed ~r-A curves (with no expanded region) develop at moderately low temperature (4°C). For ar < ar~ the ar-A isotherms in Fig. 4 represent an expanded state that is approximately independent of temperature. We speculate that in this region rapid thermal motion of the fatty acid tail dominates the force of interaction (5, 2). The fully condensed ar-A isotherm has a limiting area (29 flk2) which is greater than that of stearic acid (24 A2). This expansion could be due to a reduction in the cohesive force between fatty acid tails when a double bond is introduced. Most theories view the inferred LE/LC phase transition as first order, although there is a lack of experimental support for this view (5, 16, 19). The ar-A isotherms shown in Fig. 4 are very similar to those observed for tetradecanoic acid (myristic acid) (3) and elaidyl alcohol where the trans double bond is at the 9 position. The head group of the alcohol is more cohesive than that o f the acid (5) and the m o v e m e n t of the double bond to the 6 position apparently compensates for this. There are at least two different effects due to the insertion of a double bond into the alkyl chain (5). The first is to limit the flexibility of the chain in this region. The second is to 4Z {a) 1 8 : ~ ttans (b) l:lmixture (c) 1 8 : ~ trans
3~ v
DISCUSSION AND CONCLUSIONS Surface pressures for the fatty acid films studied were very stable at fixed areas providing ar < arc. trans-Octadec-6-enoic acid is found to have a broad temperature spectrum of ar-A curves such that ar < arc. Some of these isotherms show a sharp discontinuity Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985
10
20
30
4~
50
6~
APea pep Moleoule (eq.~)
FIG. 9. A surface pressure-area isotherm of a h 1 mixture of 6-trans- and 9-trans-octadecenoic acids is compared with isotherms for the pure fatty acids. Data were taken at a temperature of 20°C.
PHASE TRANSITION IN MONOLAYERS
decrease the adhesion between molecules. Similar effects are seen when double bonds are introduced into the hydrocarbon tails of certain lipid molecules (4). The effect of introducing double bonds into the alkyl chains has not yet been theoretically investigated. Several investigators have postulated that small molecular clusters begin to form when 7r reaches 7rt. Recent calculations by Georgallas and Pink (18) seem to support these conjectures. These investigators find a qualitative shape for 7r-A curves not unlike those of Fig. 4. One interpretation of Fig. 9 is that introducing trans-9 fatty acid into trans-6 inhibits the formation of clusters. The formation of clusters at 7rt is still highly speculative and awaits experimental confirmation. ACKNOWLEDGMENTS We are grateful to J. F. Nagle for very helpful discussions. The work was supported in part by NIH Grant GM 10069. REFERENCES 1. Jalal, 1., and Zografi, G., J. Colloid Interface Sci. 68, 197 (1970). 2. Adam, N., "The Physics and Chemistry of Surfaces." Oxford Univ. Press, London, 1941.
215
3. Gains, G. L., "Insoluble Monolayers at Liquid-Gas Interfaces," pp. 167-172. Interscience, New York, 1966. 4. Seelig, A., and Seelig, J., Biochemistry 16, 45 (1977). 5. Glazer, J., and Goddard, E. D., J. Chem. Soc., part 3, 3406 (1950). 6. Heikkila, R. E., Kwong, C. N., and Cornwell, D. G., J. LipM Res. 11, 190 (1970). 7. Baglioni, P., Gabrielli, G., and Guarini, G. G. T., J. ColloM Interface Sci. 78, 347 (1980). 8. Shuquian Xu, Miyano, H., and Abraham, B. M., J. Colloid Interface Sci. 89, 581 (1982). 9. Rabinovitch, W., Robertson, R. F., and Mason, S. G., Canad. J. Chem. 38, 1881 (1960). 10. Sims, B., and Zografi, G., Chem. Phys. Lipids 6, 109 (1971). 11. Sims, B., and Zografi, G., J. Colloid Interface Sci. 41, 35 (1972). 12. Smith, R. D., and Berg, J. C., J. Colloid Interface Sci. 74, 273 (1980). 13. Motomura, K., J. Colloid Interface ScL 23, 313 (1967). 14. Heikkila, R. E., Deamer, D. W., and Cornwell, D. G., J. Lipid Res. 11, 195 (1970). 15. Horn, L. W., and Gershfeld, N. L., Biophys. J. 18, 301 (1977). 16. Gershfeld, N. L., J. Colloid Interface Sci. 85, 28 (1982). 17. Gershfeld, N. L., Adv. Chem. Ser. 84, 115 (1968). 18. Georgallas, A., and Pink, D. A,, Canad. J. Phys. 60, 1678 (1982). 19. Bois, A. G., Panaiotov, I. I., and Baret, J. F., Chem. Phys. Lipids 34, 265 (1984).
Journal of Colloid and Interface Science, Vol. 104, No. 1, March 1985