Langmuir 1993,9, 361-362
361
Comments First-Order Pham Transitions and Equilibrium Spreading Pressurea in Lipid and Fatty Acid Monolayers T h e recent paper by Hifeda and RayFeld1gives further confiiation of the finding that the so-called liquid expanded to liquid condensed (le/lc) transition in lipid monolayers at the &/water interfaceis in fact first order.2-5 Since measurementa on phase transitions are important theoretically and as essential standards for experimental studies, several commenta on these welcome new resulta are in order. The conditions for the measurement of spreading pressures, as proposed by the authors, will also be examined. In addition, the experimentalevidence that the fmt-order character of monolayer phase transitions is associated with the presence of macroscopic regions of the coexisting high and low density phases (as expected thermodynamically) will be collated to show that the microscopic fluorescent regions seen after addition of a fluoreecentmarker correspondto a dispersion of the phases and do not describe the one-component firsborder transitions. First,we complimentHifeda and Rayfield for being able to obtain the same resulta with pentadecanoic acid monolayers by very slow continuous compression as by singlashot spreading from crystals or solventa. Their extremely careful cleaning, slow equilibration, and compression techniques are not to be compared with the less exacting routines of compressiondescribed in most papers in this field. Correspondingly, resulta obtained by compression with these more casual methods remain routinely Suspect. Hifeda and Rayfield spread their pentadecanoic monolayers on “water at pH 7”, whereas our studies were on M HC1. The degree of ionization in dense monolayers of fatty acids will be low at pH 7 in the absence of salt, but the isotherms will reflect the ionization at lower densities.6 Furthermore, the surface of water in contact with laboratory air will usually be at a pH lower than 7 due to the carbon dioxide present. In general it would seem preferable to use dilute acid substrates with fatty acid monolayers as a standard, particularly at the liquidvapor transition and lower densities. T h e equilibrium spreadingpressure (esp) of a monolayer substance is a valuable experimental standard. Hifeda and Rayfield corredly state that the esp is a criterion for judging monolayer stability and proposed in an earlier paper’ that the esp be measured as the maximum pressure as observed by adding to the surface successive aliquota of the film substance in a solvent. Somewhat incongruously, it is said that this method applies for compounds that do not spread from crystals below their melting pointa.1 This method of estimating the esp seems to us unacceptable in general and may be grossly in error when the solid substance actually has a very low equilibrium (1) Hifeda, Y. M.; Rayfield, 0. W. Lmgmuir 1992,8, 197. (2) Middleton, S. R.; I w h h i , M.; P h ,N. R.; Pethim, B. A. Proc. R. Soc. London, A 1984,396,143. (3) P h ,N.R.; Pethim, B. A. Langmuir 1986,1,509. (4) Ennuhaw, J. C.; Winch, P. J. J. Phys.: Condem. Matter 1989,1, 7187. (5) Middleton, S. R.; Pethim, B. A. Faraday Symp. 1981,16,109. (6) Betta, J. J.; Pethica, B. A. Trans. Faraday SOC.1966,62, 1581. (7) Hifeda, Y. M.; Rayfield, G. W. J. Colloid Znterjace Sci. 1981,104,
209.
0743-7#3/93/2409-0361$04.00/0
spreading pressure-as appears to be the case for many phospholipids. The actual esp of dihexadecanoylphosphatidylcholine from the solid phase at 25 O C is lower than 0.013 mN m-l, not 42.6 mN m-1 as found by spreading excess solute from organic solventa.8 Low spreading pressures for phospholipids are also recorded by Horn and Ger~hfeld.~ Correspondingly, the relevant phospholipid monolayers reported by Hifeda and Rayfield, ourselves, and others at pressures higher than the esp are presumed to be metastable. However, the kinetics of spreadingof phospholipidsfrom the solid phase have been little studied. Our observations at 25 OC were made out to 24 h.* Above the chain melting temperature, rapid spreading of solid phospholipids to high pressures is expected. The only published esp result in this range that we can find for dihexadecanoylphosphatidylcholineis the 38.9 mN m-l at 45 O C given by Adam and Jessop.l0 We now add a further result of 41.4 mN m-l at the chain melting temperature of 41.5 OC, using the sample and methods described et~lier.~s This spreadingpressurefromthe solid was reached in less than an hour. It was further observed that if the system was cooled over an hour or so to 25 OC, leaving the monolayer in contact with excess solid, the surface pressure remained at 25 mN m-l. Whether this pressure would decrease almost to zero over very long periods remains to be seen. In the absenceof excess solid, as in a spread monolayer, the amount of phospholipid in the monolayer is not sufficient to allow formation of an organized solid phase of significant size. With regard to pentadecanoic acid monolayers, Hifeda and Rayfield appear to have overlooked an earlier comprehensive study of fatty acid spreading pressures11 in which criteria for establishingspreadingequilibriumwere described. In addition, the crystallography of the solid phases was examined, and the subetantial effect of crystal size was demonstrated and correlatedthermodynamically with the sizeeffect on the solubility of the fatty acid crystals in liquid heptane. Since the method of delivery of excess fatty acid solution to the water surface will not produce a crystallographically well-defined large crystal, the resulting “maximum pressure” observed may be expected to be higher than the esp. Hifeda and Rayfield find a “maximum pressure” of 21 mN m-l at 25 “C for pentadecanoic acid spread from hexane, as against the 19.7 mN m-1 for various small crystals and 18.7 mN m-l for large crystals reported earlier.” The value of 18.7 mN m-1 is the equilibriumspreadingpressure for a macroscopic wellformed crystal at 25 OC. Hifeda and Rayfield cite studies on fluorescent domains in monolayers containing small amounts of a fluorescent marker as supporting the first-order character of the le/lc transition. The observations,for example of McConnell et al.,12 frequently show microscopic patches of distinct fluorescence in the monolayer. However, from thermodynamic considerations, a true fmt-order transition should (8)P h , N. R. Ph.D. Thesis, Clarkmn University, NY, 1983. (9) Horn,L. W.; Gershfeld, N. C. Biophys. J. 1977, 18, 301. (10) Adam, N. K.; J w o p , G. Proc. R. SOC.London, A 1926,110,423. (11) Iwahashi, M.; Maehara, N.; Kaneko, Y.; Semiye, T.; Middleton, S. R.; Pallas, N. R.; Pethica, B. A. J. Chem. SOC.,Faraday Trans. 1 1986, 81, 973. (12) McConnell,H.M.; Ta”, L. K.;Weiee, R. M. Proc. Natl. Acad. Sci. U.S.A. 1984,81,3249. (8 1993 American
Chemical Society
Comment8
362 Longmuir, Vol. 9, No. 1, 1993
exhibit macroscopic patches of high density interspersed with macroscopic patches of low density. In this regard, perhaps the clearest demonstration that these onecomponent monolayer transitions are fmt order is that the surface potential in the transition region measured with a macroscopic electrode a few millimeters from the surface show large fluctuations as the electrode is moved parallel to the interface.3~5J3These fluctuations are also observed with a stationary electrode as the macroscopic patches drift slowly over the surface. In thermodynamic accord with these observations are the dramatic density changes observed in fmborder transition regions when the monolayer is polarized by an electric field normal to the interface.5 The microscopic patches seen in the fluorescence experiments cannot produce large fluctuations in surface potential since the air electrode measures a potential integrated over the solid angle subtended at the detector, itself a centimeter in diameter in our experiments. By contrast, a true first-order phase transition will show fluctuations whenever the (macroscopic) regions of low and high density have different dipole strengths. Inaddition to them considerations,the arguments given by McConnelP which suggest that microscopic clusters of point dipoles comprise the structure of the fmborder le/lc transition seem unacceptable for several reasons. In the first place, the isotherms shown for a phospholipid monolayer with and without addition of a marker are distinctly different, and neither is in accord with published rigorous experiments on the phase transition. In fact, it is doubtful whether the isotherm in the presence of the fluorescer shows a transition. Moreover, the theoretical discussion assumes that the molecular dipoles are oriented normal to the interface with no tangential component, contrary to conclusions from surface potentiale,'6 and does not take note of earlier diecussion of the other possible arrangements of phospholipid zwitterions in the interface." The large zwitterions are not realietically treated as point dipoles at high densities, and their orientation is more probably tangential to the interface. It has been pointed that the addition of a fluorescent molecule to the monolayer alters the variance and the phase behavior due to the addition of an extra component and that the marker may change the line tension of the dense phase to give what can be described as a two-dimensional emulsion. McConnell suggeats that some of the shape changes observed in the fluorescence microscope am the result of ~
~~
(13) &, M. W.;Cannell, D. 1976,14, 1299. (14) Pethica, B. A. Faraday Symp. 1981,16, 515. (16) McConnell, H.M. Annu. Rev. Phys. Chem. 1991,42,171. (16) Standbh, M.M.;Pethica, B. A. Tram. Faraday SOC.1968,64, 1113. (17) Pethica, B. A. SOC.Chem. Znd. Monogr. 1%4,19,85. (18) Pallas,N.R.; Pethica, B. A. J. Chem. Soc., Faraday Trona.1 1987,
83,686.
altering the line tension by additives such as cholesterol. The same considerations apply to the marker itself. In conclusion, the contribution of improved techniques in continuous compression by Hifeda and Rayfield and further studies by other critical experimentalists will continue the reform of monolayer studies and eventually upgrade the experimental standarda in this field. Experimentalists in monolayers should at least demonstrate that their techniques canreproduce simplethermodynamic standard pressures at one or other of the available phase transitions before proceeding to publish results having any claim to critical worth. For high prewures, equilibrium spreading pressures of fatty acids seem good practical standarda.11 For intermediate pressures the le/lc transition2ls and for low pressures the liquid vapor transition of pentadecanoicacid appear suitable.lJ8 It is to be hoped that continued efforts will be made to confirm and improve the available surface manometric standards and that editors will correspondingly refuse papers of the poor experimental quality so common in the literature. Note Added in Proof: A recent report describes the development of a Brewster angle microscope, which can visualize surface f h without addition of fluorescer molecules (Henon, S.; Meunier, J. Rev. Sei. Instrum. 1991, 62,936). This important new method will be invaluable in the study of surface microstructures in relation to film composition. Observations with the new device in the gadliquid and le/lc regions of myristic acid show polydisperse patches. The microscopic patches observed in an adsorbed'film of sodium octanoateby the same authors (ThinSolid Film 1992,210/211,121)may be associated with the presence of octadecanoic acid in the sample (Meunier, J., personal communication). Another recent paper from the same laboratory (Bercegol, H.; Meunier, J. Nature 1992,356, 226) shows large microscopic crystallites, with one dimension about 100 Nm, formed from a spread monolayer of a fluorescer at a tranaition apparently at the spreading pressure, which is not quoted. The results raise the question as to the siae range of "patches" above which the surface pressure will be indistinguishable from the ordinary fmborder transition preeeure.
B.P. Research, 44pO
N. R Pallas Warrensville Center Road, Cleveland, Ohio 44118
B. A. Pethica' Langmuir Center for Colloids & Interfaces, Columbia University, 911 S. W.Mudd Building, New York, New York 10027 Received April 13, 1992 In Final Form: August 24,1992
