Advances in Colloid and Interface Science, 35 (1991) 31-138

31

Elsevier Science Publishers B.V., Amsterdam

PHOSPHOLIPIDS AT THE OIL/WATER INTERFACE: ADSORPTION AND INTERFACIAL PHENOMENA IN AN ELECTRIC FIELD YURII A. SHCHIPUNOV

and ALEXANDER

Institute of Chemistry, Far East Department, 690022 Vladivostok (U.S.S.R.)

F. KOLPAKOV

Academy of Sciences of the USSR,

CONTENTS Abstract

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2. General and Experimental ............. 2.1. Electrocapillary phenomena at the oil/water interface ... 2.1.1. Two-component systems .......... 2.1.2. Multicomponent systems .......... 2.2. Specificity of interfacial phenomena under an electric field in phospholipid-containing systems and the scope for investigation 2.2.1. Electrohydrodynamic instability ........ 2.2.2. Emulsification .............. 2.2.3. Structurization ............. 2.2.4. Electrolyte effect ............. 2.3. Materials and methods .............

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3. Phospholipids at Interfaces ............. 3.1. Adsorption ................. 3.1.1. Adsorption kinetics ............ 3.1.2. Adsorption under equilibrium conditions ..... 3.1.3. Electric field effect ............ 3.2. Stratification at the interface and the formation of bimolecular membranes ................. 3.3. Polymorphism at interfaces ........... 3.3.1. Aggregation in nonaqueous media ....... 3.3.2. Hydration of phospholipids ......... 3.3.3. Solubilization of water by nonaqueous PC solutions . 3.3.4. Liquid-crystalline states .......... 3.3.5. Relationship between phospholipid states at the interface 3.3.6. Concluding remarks ............

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4. Effect of Ions on Electrointerfacial 4.1. Inorganic ions ................ 4.2. Organic ions ................

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1. Introduction

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Phenomena

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5. Emulsification and Emulsions under an External Electric Field 5.1. Strong fields ................ 51.1. Dispersion of water in alkane ......... 0001~8686/91/$37.80

0 1991 Elsevier Science Publishers B.V.

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32 5.1.2. Migration of droplets ............... 51.3. Aggregation .................. 5.2. Weaktields .................... 6. Mechanisms for Interfacial Phenomena ............. 6.1. Phospholipid adsorption ................ 6.2. Transport of substances ................ 6.3. Phospholipid-stabilized emulsions ............. 6.4. Microemulsions ................... 7. Proper Perspectives and Possible Biological Relevance Acknowledgements .................... References .......................

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106 106 103 111 112 112 112 113 120 126 127

ABSTRACT Interfacial effects produced in an immiscible liquid system by the action of an external electric field have been considered. The addition of small amounts of neutral phospholipids to the nonaqueous phase has been shown to result in a marked increase in the sensitivity of the interfacial boundary to the voltage applied, which is manifested by: 6) an accelerated decrease of the inter-facial tension after the two immiscible liquid phases have been brought into contact; (ii) reduced inter-facial tension, by 2CG30mN/m, at the oil/water interface at field strengths of l-10 kV/m (the interfacial tension drop in the absence of phospholipids does not exceed 5 mN/m); (iii) development of electrohydrodynamic instability at the planar dividing surface between phases; and (iv) dispersion of water into the nonaqueous phase at smaller field strengths by a factor of about 100 as compared to those normally required in the absence of phospholipids. In order to gain a deeper insight into the mechanisms of inter-facial phenomena, mainly exemplified by the n-heptane/water system containing phosphatidylcholine, three major issues have been considered: (1) Kinetics of the adsorption of phospholipid at the oil/water interface from the nonaqueous phase, and effects produced by exposure to an external electric field; also, the adsorption under equilibrium conditions, and the structure of the adsorption layer formed. (2) Interactions between neutral phospholipid and inorganic or organic ions at the interfacial boundary under the voltage applied. (3) Conditions for the occurrence of electrohydrodynamic instability at the dividing surface between oil and water and the formation of a water-in-oil emulsion; also aggregation and gelation processes induced in the nonaqueous phospholipid solution bulk by the action of a weak external electric field. Throughout the present paper, an attempt has been made to relate the microscopic behaviour of phospholipids under an external electric field to macroscopically observable properties at the movable interfacial boundaries. The adsorption studies have shown that phosphatidylcholine is prone to self-organization into a liquid-crystalline state at an immiscible liquid interface. The disintegration of the interfacial lipid film thus formed by the action of a weak electric field has been explained as due to an enhanced electrohydrodynamic instability of liquid crystals. This results in the formation of either an emulsion, or a microemulsion in the nonaqueous solution bulk. The formation of a microemulsion is manifested by the appearance of an optically anisotropic gel, stable only under an external applied electric field, in the nonaqueous solution bulk. The noticeable drop in inter-facial tension has been attributed to the formation of a microemulsion. The experimental evidence thus obtained has enabled us to suggest a mechanism for electrohydrodynamic instability and for the breakdown of the adsorption layer at the oil/water interface under the applied voltage.

33 F’urther developments for research and potential practical application of interfacial phenomena, induced by an external electric field in ph~pholipid-conning immiscible liquid systems have been indicated, including their use for modeiling processes in the living cell. 1. INTRODUCTION

Phospholipids (PLs) are primarily known for the important role they play in the living cell, where they form a lipid matrix, the structural basis of biological membranes and membranous organelles, and for their wide applications in the food industry, cosmetics, medicine and, in recent years, biotechnolo~ as effective, natural surface-active substances used for the stabilization of emulsions. In biomembranes and emulsions, PLs are either localized or participate in the formation of interfacial boundaries. For this reason, much attention has been given to the study of their properties at movable interfaces, in particular, air/water and, to a lesser extent, oil/ water interfaces. Most commonly, investiga~rs deal with monomolecular lipid films, which are considered as a model for biological membranes and are used for modelling the processes occurring in living cells. The external electric field, as used in physical and colloid chemistry, is an effective tool for studying interfacial phenomena. However, until recently, the effects arising from the action of a voltage applied to movable interfaces filled with PL molecules have not been studied systematically, which regrettably, was indicative of an underestimation of the role the electric field may play in the functioning of the living cell. One can hardly find a plausible explanation why experiments of this kind should be neglected, since biological membranes in vivo are subjected to constant action by a trans-membrane potential difference. In absolute terms this difference amounts to about 100 mV, which certainly is not a large value, but the field strength in a lipid matrix merely 5-7 nm thick can be as high as l,OOO-10,000 kV/m. Undoubtedly, an electric field of such a strength cannot fail to exert a sizeable effect on the processes operating within the living cell. Supposedly, it may also affect the structural org~i~tion of biological membranes. Recent experiments El-51 have provided evidence that the introduction of PLs into a system of immiscible liquids results in a substantially increased sensitivity of the inter-facialboundary to external influences. This is manifested, as the electric field is applied, by a considerable decrease in inter-facial tension, by the occurrence of electrohydrod~amic instability, by dispersion of water in the nonaqueous phase and formation of structures from water-in-oil emulsions in PL solutions at field strengths as low

34

as 10-100 kV/m, which, in fact, is much lower compared to the electric field intensities to which the lipid matrix of the living cell is exposed and at which similar processes take place in both the absence of PLs and in the presence of surface-active substances of other classes. The present review article is intended to provide a possibly exhaustive characterization of inter-facialphenomena, under an applied electric field, in PL-containing liquid/liquid systems with the aim of gaining a deeper insight into the causes leading to the high sensitivity of the interfacial boundary to external influences in the presence of PLs and to suggest, on the basis of the available experimental evidence, a rationale for the mechanisms of interfacial processes. 2. GENERAL AND EXPERIMENTAL

Of the interfacial phenomena produced by an electric field applied to a liquid/liquid system, the electrocapillary phenomena is the best known. The introduction of PLs leads to an alteration in the major properties of a system of two immiscible liquids. One may demonstrate the salient characteristics of PL-containing systems by comparing them with other currently known systems containing surfactants of other classes. To this effect, the present section is concerned with an outlook of the basic concepts on the nature of electrocapillary phenomena at the interface of immiscible liquids, and a brief outline of the major distinctive features of interfacial processes, caused by the introduction of PLs, is given. By comparing PLfree and PL-containing oil/water systems, the salient objectives in the study of interfacial phenomena, induced by the application of a voltage, are formulated and a description of materials used and experimental techniques employed is presented. 2.1. Electrocapillaryphenomena

at the oil/water

interface

The decrease in interfacial tension as produced by an external electric field has been named by the common term “electrocapillary phenomena”. Systematic studies on oil/water system have been initiated over the past decade. The main difficulty that imposes restrictions on a study of electrocapillary phenomena is the measurement of the potential jump at the interface between immiscible liquids 16-91. In a number of instances, this difficulty can be circumvented by using electrolytes which are capable of partly or totally dissociating in organic solvents into ions. Salt of tetrabutylammonium and tetraphenyl boron, first used by Koryta and coworkers [lOI, is the most commonly used organic electrolyte. However,

the salts of organic ions exhibit sufficiently high surface activity and, therefore, make the interfacial tension change markedly, when added to an oil/water system [ill. Moreover, they affect the polarizability of the interface between immiscible liquids due to an increase in its ionic permeability [121. Therefore, with regard to electrocapillary phenomena, whose nature is susceptible to substantial alterations by the action of organic electrolytes, the latter remarks may be regarded as general and used only with provisos. For this reason, salts of organic ions are not universally suitable for the study of inter-facial boundary properties under an applied electric field. Taking into account the above, it seems to be expedient to consider electrocapillary phenomena at the oil/water interface not only in the presence, but also in the absence of organic electrolytes. This will be exemplified by two-component systems composed of water and organic solvents, and by multicomponent systems containing, apart from immiscible liquids, ionic surface-active substances also. 2.1.1. Two-component systems A number of systems, composed of water and organic solvents (both polar and nonpolar) have been studied systematically by Popov 1131.The low conductivity of nonaqueous media makes it impossible to measure the interfacial potential jump. Therefore, Popov chose to study the dependence of the inter-facialtension on the electric field strength in the organic phase, which is responsible for the total voltage drop, because its specific conductance is lower than that of water. The electrocapillary curves for inter-facialboundaries of water with organic solvents such as n-alkane, benzene, nitrobenzene, methylbutylketone and diisopropyl ether are shaped like an inverted parabola (see, for example, curve 1 in Fig. 1). Since their radii of curvature are little different, Popov [131advanced the idea that, in the systems studied, the nature of electrocapillary phenomena at the oil/water interfaces is in essence the same. A similarity in the electrocapillary curves at oil/water and air/water interfaces has also been noted. Therefore, the inter-facialtension drop produced by a potential difference applied to a system of two immiscible liquids has been explained, by analogy, as due to the repulsive interaction between the water dipoles, which are orientated perpendicularly to the boundary surface within the interfacial layer by the action of an electric field. Ion-containing systems, starting from the pioneering work of Gibbs, concerned with electrocapillarity at the mercury/water interface 1141,and a number of recent reports B-8,12,15-191 dealing with the oil/water inter-

18

12

10

8

2 Ofll-w I'

6

I -1

I

I

+l

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E (kV/m)

Fig. 1. Electmcapillary curves for diisopropyl ether/water (curve 1) and n-heptanol/water (curve 2) interfaces. (Adaptedfromthedataof Popov[131.)

face, have become a traditional object of investigation. The problems concerned with the action of an electric field on the inter-facialtension of polar liquids in the absence of free charges have been discussed by Rusanov and Kuz’min 1201. These authors obtained the equation: AyE = A’PEl4z - nE2/2

(1)

where AyE is the difference in inter-facial tension for the states in the presence and the absence of an electric field, E is the electric field strength, AY is the potential jump at the interface, x is the correction factor for dipole-dipole interactions in the interfacial region. The first term in the right-hand side of Eqn (1) is not large in absolute value. With the surface

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potential for water of 0.1-0.2 V (see, for example, Refs [11,121), the value of AYE/4n is about 0.001 mN/m [201. This is markedly inferior to the in&facial tension drop produced by an applied voltage (Fig. 1). For this reason, the first term in Eqn (1) can be omitted. One thus obtains:

Here the term AYEis proportional to the square of the electric field strength, which may, in principle, be accepted as a plausible explanation for the parabolic shape of the electrocapillarycurves measured experimentally. Deviations from the relationship presented by Eqn (2) have been noted by Popov [131 only for two-component systems composed of water and an aliphatic alcohol as the organic solvent. As an example, the interfacial tension versus electric field strength relationship for the n-heptanol/water system is shown in Fig. 1. No effect of the electric field on the AyE is observable at intensities E reaching about 100 kV/m. Popov 1131has interpreted this apparent lack of effect as due to the formation of hydrogen bonds between the molecules of the aliphatic alcohol and water, which may stabilize the structure of liquids in the interfacial region and impede the reorientation of dipolar molecules in response to the applied external field. 21.2. Multicomponent systems The addition of ionic surfactants to a binary system of immiscible liquids produces an alteration in the nature of electrocapillary phenomena. The decrease in interfacial tension occurs as a result of adsorption-desorption of surface-active ions. Besides, the charged organic molecules may transfer across the dividing surface from one phase into the other. The extent of interface permeability for the ions, now present in the system, is a decisive factor for the interfacial boundary polarization state under an applied electric field. This relationship has been used by Grahame [221 as the basis for a classification of the mercury/water interface; however, the adopted classification may be extended to oil/water interfaces also (see, for example, Refs [12,23-251). Three types for phase boundary classification have been proposed in the cited papers. These are: (i) Ideally nonpolarizable phase boundary. The electric current in the system is determined by the ohmic resistance. The interfacial potential jump does not deviate from an equilibrium value as the current passes through the system. For this reason, such an interface between phases may be referred to as a reversible one [121.

(ii) Ideally polarizable phase boundary. No current is observed in the system. The state of the interfacial boundary is defined by the electric charge applied. This charge is generated by the accumulation of ions at the dividing surface (the so-called Koenig surface) which is positionally located at the site of an infinitely high potential barrier, impenetrable to charged molecules. (iii) Real phase boundary. The measured electric current is smaller relative to the value expected from Ohm’s law. The real phase boundary stands midway between the ideally polarizable and the ideally nonpolarizable phase boundaries. Its state is defined by both the current transferred across the dividing surface and the charge applied. The charge, generated at the liquid/liquid interface, allows us to regard the real phase boundary as a partially polarizable one. Now, let us consider in more detail some concrete examples which demonstrate different types of polarizability. 212.1. Ideally nonpolarizablephase boundaT. One can eliminate the interface polarization under an applied electric field by introducing, into the nonaqueous and aqueous phases, electrolytes that compositionally share at least one common ion. This may be exemplified by a system, first considered by Gavach et al. [61;albN+Ph,B- (nitrobenzene)/(water)alk4N+BF,where al&N+ is a symmetric tetraalkylammonium cation, and Ph4B- is a tetraphenylboron anion. The common ion is the tetraalkylammonium cation. The reversible transport of this ion across a dividing surface (which in this particular case acts as an ion-exchange membrane [26-281) generates an exchange current. Under the applied voltage, the system persists in a state of equilibrium as long as the current transferred across the interface does not exceed the exchange current in magnitude. In this regime, the voltage+mrrent characteristic obeys Ohm’s law 1291. The current, as it passes through the oil/water interface subjected to the external electric field, produces a change in the surface-active ion concentration in the phase boundary region, which consequently leads to a change in the interfacial tension. The curvature radii at different points in the parabola-shaped electrocapillary curves are determined by the relative surface ion excess liable to change with voltage variation [6,12,24,281. 2.1.2.2. Ideallypolarizablephase boundary. The interface polarization in a system of immiscible liquids occurs when the inorganic ions and charged organic molecules of surface-active substances, added to one phase, are unable, under an applied electric field, to transfer into the other phase in which they are insoluble. This situation is common to alkane/ water systems whose components differ most of all in polarity in comparison to other investigated pairs of immiscible liquids.

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The electrocapillary curves for the n-heptane/water interface as measured in Refs [ll and [301 in the presence of oil-soluble and watersoluble surface-active substances are shown in Fig. 2. The curves are parabolic in shape and are little affected by surfactants added to the system 113,301.This is presumably indicative of the fact that the decrease in interfacial tension, under an applied electric field, is primarily due, as with pure-solvent two-component systems, to the electric field-induced orientation of water dipoles adjacent to the interfacial boundary [13,16,20,301. If this is the case, Eqn (1) may be applied to the description of electrocapillary phenomena in surfactant-containing alkane/water systems. This presumption appears, to a certain extent, to be borne out by the experimental evidence obtained with cholesterol. As can be seen in Fig. 2 (curve 3), the interfacial tension remains constant when E does not exceed 30 kV/m. This effect has been explained in Ref. [301 by the formation of

50

2 5 F 40

30

I I

I

0

+50 E

(kV/m)

F’ig. 2. Interfacial tension at the n-heptanelwater interface versus electric intensity in the absence of surfactants (curve 1) and in the presence in aqueous solution of 0.005 mM cetyltrimethylammonium bromide (curve 2) and in n-alkane of 1 mM cholesterol (curve 3). (Redrawn from Popov et al. [ll.)

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hydrogen bonds between water molecules and the hydroxyl groups of sterol whose molecule is oriented at the interfacial boundary in a way such that its OH group points to the aqueous phase (see, for example, Ref. 1311,p. 109). It should be mentioned that, since no experimental techniques are available allowing the measurement of the potential differenceat the alkanejwater interface, electrocapillary phenomena in systems containing hydrocarbons have hardly been investigated. A well-studied system may be exemplified by two immiscible electrolytesolutions: BudN+PhdB’-(nitrobenzene)/(water)LiCl. The electrocapillary curves are shown in Fig. 3. Although tetrabutylammonium and tetraphenyl boron ions tend to transfer from the organic phase into the aqueous solution under an applied electric field, the dividing surface in a certain range of potentials seems, nonetheless, to exhibit the characteristic features of ideal polarizability (see, for example, Refs D,!241).This is true for the zero-charge potential region within a narrow voltage interval not extending beyond 0.2-0.3 V when the current in the system does not exceed several tenths of a microampere. The zero-charge potential is easily deter-

28 z jj ?-

I

I

I

I

_

26 24

18

Fig. 3. Electrocapillary curves for the interface between nitrobenzene and water in the presence (in a nonaqueous solution) of 0.1 Mtetrabutylammonium tetraphenyl boron and (in an aqueous solution) 0.01 (curve 11,O.l (curve 2) and 1 (curve 3) Mlithium chloride. Drawn from data of Senda et al. 1241.)

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mined at the maximum of the electrocapillarycurve. Its magnitude is dependent, as is apparent in Fig. 3, on the concentrations of the electrolytes in the respective phases. Gibbs’ classical thermodynamic theory of electrocapillarity can be applied to the description of electrocapillary phenomena at the ideally polarizable phase boundary for two immiscible electrolyte solutions. Its application to oil/water systems have been dealt with in Refs [6,12,X19,24,251. 2.1.2.3. Real phase boundary. The phase boundaries, both ideally polarizable and ideally nonpolarizable, of two immiscible liquids exist within a narrow polarization potential range. Nonideal states are commonly produced by small deviations of the inter-facialjump potential from equilibrium. In the former instance, they are caused by the exchange of current across the dividing surface. In the latter instance, they are due to a small number of available free ions and to the limited conductivity of nonaqueous media. Electrocapillary phenomena at real interfaces are most demonstrably exemplified by cetyltrimethylammonium bromide (CTAB) whose behaviour in oil/water systems has been studied in a large number of cases. Detailed investigations of interfacial phenomena in a CTAB-containing system of immiscible liquids have shown that the inter-facialtension drop, under an applied voltage, is produced by the accumulation of surface-active cetyltrimethylammoniumcations due to adsorption at the inter-facialboundary. It has been established by Senda et al. 1321that the standard energy of adsorption from nitrobenzene for CTAB increases in proportion to the negative electrode potential in an aqueous solution. On the other hand, the desorption of organic cations into water, as reported in Refs 113,23,26,331,is not favored with a high potential barrier at the boundary between immiscible liquids. The combined action of these two factors explains both the accumulation of cetyltrimethylammonium ions at the oil/water interface and the decrease in inter-facialtension when the external electric field is applied. The change of electrode polarity causes the organic cations to desorb into the bulk nonaqueous phase. The maintenance of electroneutrality in the system is provided by the transport of inorganic ions from the water into the nitrobenzene. In this process, the interface between the oil and water acts as an ion-exchange membrane 126-281 and is used for the electrochemical extraction 123,341.The interfacial potential barrier for the transfer of an ion pair composed of an inorganic anion and a cetyltrimethylammonium cation is markedly lower than the potential barrier for desorption of organic ions into the aqueous phase. For this reason, the CTAB concentration at the oil/water interface diminishes with time, which leads to an increase in inter-facialtension.

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It should be emphasized that the pure adsorption-desorption process takes place at low densities of electric current. As the current density reaches as high as 30 A/m2, the initial drop in the interf’acial tension, shown at a negative potential applied to the electrode in water, changes to a sharp increase in y. In addition, fluctuations in the electric parameters measured and a wave-like deformation of the interface are observed. As demonstrated by Popov [26,35,361, these effects are due to the desorption of CTAB into water. The transfer of surfactant from one phase to the other occurs under high electric field strengths only and is accompanied by dispersion at the oil/water interface, manifested by the formation of droplets of organic solvent within the aqueous phase. Turbulent flow near the interface, mechanical oscillations and ripple deformations at the dividing surface between two immiscible solutions become, to all appearances, inevitable at high current densities. These phenomena are responsible for or result in the generation of a tangential interfacial tension gradient, with the resultant occurrence of capillary instability (Marangoni-Gibbs instability [37,381), reported in a number of papers [26,36,39,40-431. In certain instances, a relationship between mechanical and electrical phenomena is easily observable. For example, the appearance of a longitudinal wave at the interface between two immiscible liquids under an applied alternating electric field has been observed by Joos and Bogaert 1391.This effect is reversible: mechanical vibrations of the oil/water interface generate an alternating current 1391. A similar picture has been observed by Guastalla and Bertrand 1441in monolayers of certain surface-active substances subjected to compression and expansion. Dupeyrat et al. [42,431, and Koczorowski and coworkers 1451have suggested the use of mechanoelectric processes at the liquid/liquid interface similar to the piezoelectric effect in crystals for the conversion of mechanical energy into electrical energy. The appearance of electrohydrodynamic instability and dispersion of the organic solvent into the water solution are not observed in the opposite direction of the electric current, i.e., when the polarity for the electrode in the aqueous phase changes from negative to positive. Instead, a solid film of inorganic salt is deposited on the dividing surface. The nature of this effect has been established for a methylbutylketone/water system. As shown by Popov (refs [26,35,361), at high current densities in the saturation region of the volt-ampere curve, CTAB becomes almost completely desorbed from the oil/water interface. For this reason, the bromide anions, incapable of transfer into the nonaqueous phase because of a high potential barrier, concentrate near the dividing surface. This results in an oversaturation of the adjacent layers of the aqueous solution with the inorganic

electrolyte and, ultimately, in the deposition of a salt precipitate at the interf’acial boundary. To briefly summarize the presented material, we would like to emphasize the essential distinctions, from the thermodynamic standpoint, in the properties of real and ideal phase boundaries. While, for the most part in ideally polarizable boundaries electrocapillary phenomena have been studied in sufficient detail, other concomitant inter-facial effects conducive to deviations from ideality under an external electric field (such as, adsorption, formation and behaviour of condensed and liquid-crystalline films at interfaces, convection current of liquids and capillary instability, and emulsification) have not been systematically studied, and the mechanisms involved in these effects are, as yet, largely unclear. Besides, the experiments have been performed on a limited number of surface-active substances. Extending the range of these investigations may help in a deeper understanding of the mechanisms of interfacial processes and may reveal novel features, as has been the case with phospholipids. A more detailed characterization of interfacial phenomena involving PLs will be given in the subsequent section of this review paper. 2.2. Specificity of interfacial phenomena under an electric field in phospholipid-containing systems and the scope for investigation The first brief report on electrocapillary phenomena in a PL-containing system, composed of nitrobenzene and water, was that of Guastalla 1461. The experiments were carried out with egg-yolk lecithin (phosphatidylcholine), but in the main a number of ionic surface-active substances was studied. As has been ascertained, the effects produced by neutral PL are similar to those by cationic surfactants; when the positively charged electrode is placed in nitrobenzene, a decrease in interfacial tension is observed; with the negatively charged electrode, an increase in interfacial tension is observed. However, these changes in y as produced by an external electric field might, presumably, be due to an insufficient purity of the PL samples used and to the occurrence of contaminating acidic lipids in them. In greater detail, electrocapillary phenomena at the methylisobutylketone/water interface in the presence of PLs were studied by Watanabe et al. 123,471. They considered several lipids, including egg-yolk phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and phosphatidylinositol; the first of these was given major attention. As has been evidenced by the reported electrocapillary curves, the neutral PLs behave very much like amphoteric compounds. The major decrease in y occurs just as in the

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case of amino acids, earlier studied by Blank and Feig [481, in either the anode or the cathode branch, depending on the pH of the aqueous solution. At a certain pH, the inter-facial tension, under an applied electric field, remains constant and is independent of the electric field. This pH value has been named by Watanabe and coworkers as the “isoelectric point”. At this point, the authors argued, the lipid molecules are neutral; at any other pH of the aqueous solution, these carry either a positive or a negative net charge. In their study of electrocapillary phenomena, Watanabe et al. 1471 restricted themselves to the measurement of electrocapillary curves only and did not consider the properties of adsorbed lipid layers at the oil/water interface, in both the absence or the presence of an electric field. Besides, the data obtained by these authors are open to criticism from a methodological standpoint. For instance, the measurements of inter-facialtension by the drop volume method were carried out, apparently, under conditions far from equilibrium. To mention but one, the time of squeezing out a drop did not exceed 40 s. Our study of adsorption kinetics has shown (see Section 3.1) that equilibrium in an oil/water system with a PL concentration ofabout 0.01 mM(i.e., that used by Watanabe et al. [471)is attained within several tens of minutes. Besides, the high current density in the methylisobutyl ketone/water system could also lead to deviations. Anyway, Watanabe et al. reported the observation of turbulent flow as the water drop was squeezed out into the nonaqueous solution. Occasionally, a dispersion effect was also observed. A correct study of interfacial processes in an oil/water system under an external electric field has been carried out by Boguslavsky and Metel’sky [491.Their objective was the elucidation of the mechanism of ion transport across a lipid membrane. The n-heptane/water interface, saturated with egg-yolk lecithin molecules, was used as a model system. In designing their experiments, Boguslavsky and Metel’sky rejected the idea of performing typical electrocapillary measurements. They have argued, in support of their decision, that the distribution of an applied voltage within the n-heptane/water system remains largely unknown, while the potential jump at the interface between these immiscible liquids cannot be measured by the currently employed methods. That is why Boguslavsky and Metel’sky have considered the dependence of the interfacial tension on the current density, J, exchange through the dividing surface between phases instead of traditional electrocapillary curves. The Ar versus J relationship obtained was rather uncommon. As the current density was increased and reached 5 mA/m2, the interfacial tension dropped off about 20 mN/m and then remained almost constant. The

45

character of this dependence was unaffected by the change in sign of appliedvoltage. However, the authors 1491ignored the uncommonly high sensitivity of interfacial tension to a weak electric field and preferred not to discuss the unusual course of A? versus J curves. Similar results have been obtained by Popov et al. 111.These authors, in contrast to the procedure of Boguslavsky and Metel’sky 1491,considered the effect of the electric field strength in the nonaqueous phase on the interfacial tension at the n-heptane/water interface. The dependencies they have measured are shown in Fig. 4. A point to be noted is that, on addition of PLs, the shape of the electrocapillary curves becomes transformed from a parabolic form into a concave shape with respect to the Y-axis’. A sharp drop in inter-facial tension, occasionally reaching 20-30 mN/m in magnitude, has been observed at low electric field strengths. As the intensity increases, the tension becomes increasingly less sensitive to the electric field. At high electric intensities, the interfacial tension has even been observed to increase slightly. Girault and Schiffrin [31 and Kakiuchi et al. [501 have studied electrocapillary phenomena with PLs in the systems 1,2-dichloroethane/water and nitrobenzene/water. Unlike Boguslavsky and Metel’sky [491 and Popov et al. 111,they measured the potential difference across the interface of immiscible liquids. For convenience of measurement, salts of organic ions were added to the nonaqueous solution. The electrocapillary curves measured by Girault and Schiffrin, and those obtained by Senda and coworkers are presented in Figs 5 and 6, respectively. The former authors conducted their experiments with egg-yolk PLs and the latter authors, with a synthetic 1,2-dilauroyl-glycero-3-phosphatidylcholine. The electrocapillary curves, obtained for ideally polarizable PL-free phase boundaries, have the typical shape of an inverted parabola. The decrease in inter-facial tension within the studied potential interval of 600 mV is about 5mN/m. The addition of PLs to the nonaqueous phase produces, as in the case of n-heptane/water system (see Fig. 41, a change in the y versus AT relationship. At some points of the electrocapillary cur1 In the thermodynamic sense, the curves in Fig. 4, as well as the inter-facial phenomena with PLs, can be called “electrocapillary” only provisionally. The reason for this statement is that the considerable inter-facial tension decrease under the action of an external voltage is accompanied by a variety of processes, for example, the electrohydrodynamic inter-facial instability and the dispersion of water in the nonaqueous phase. The accompanying processes lead to deviations from thermodynamic equilibrium. Therefore, the interfacial tension versus electrical intensity curves in Fig. 4 have been measured under steady state, rather than under equilibrium conditions.

46

0

-50

+50 E

(kV/m)

0

-50

0

+50 E

(kV/m)

47

E

I

I

0

+50 E

(kV/d

M/m)

Pig. 4. Decrease in the interfacial tension (obtained by subtraction of ywithout the electric field from the yin the electric field) versus electric intensity in the presence of 1 mMpotassium chloride in the aqueous solution. The sign of the electrode potential in the aqueous solution is indicated. Interfacial tension was measured by a drop-volume method. A: nheptane/water system; (1) 0.0; (2) 0.008; (3) 0.033 tiphosphatidylcholine. B: n-heptane/ water system; (11 0.0033; (2) 0.033; (3) 1 mM phosphatidylethanolamine. C: benzene/ water system; (1) 2 mil4 phosphatidylcholine; (2) 0.08 mM sphingomyelin. D: benzene/ water system; (1) 1 m&4phosphatidylethanolamine; (2) 1 mMceramideaminoethylphosphate. (Data for A and B are from Popov et al. [l]; data for C and D are from Drachev et al. unpublished results.)

48

ves of the cathode (or anode) branch the interfacial tension drop may occasionally attain a value of 15 mN/m as the interfacial potential jump is varied within the range 100-200 mV (see Fig. 5). In other regions, the surface tension values are practically unaffected by the applied voltage.

I

I

I

0.4

0.2

I

15

10

5

0.6

0.0 AY (v)

Fig. 5. Electrocapillary curves for the interface between 10 mA4KC1aqueous solution and 1,Zdichloroethane containing 1 n-J4 salt of tetrabutylammonium tetraphenyl boron in the absence (curve 1) and in the presence (curve 2) of 0.025 mM egg yolk phosphatidylcholine. (Adapted from Girault and Schiffrin (31).

The retention of the parabolic shape by the electrocapillary curves within a narrow potential range has been noted by Senda et al. only (see Fig. 6). However, the curve shape tends to become transformed after the first measurements have been carried out, which is indicative of an alteration in the adsorbed lipid layer characteristics by the action of an external electric field.

49

25 3 !! ?-

20

15

syahd4

10

-

5

02

I

0.0

0.2

I

0.4

-

0.6

Fig. 6. Electrocapillary curves for the interface between 60 mM LiCl aqueous solution and nitrobenzene containing 100 mMt.etrapentylammonium tetraphenyl boron in the absence (curve 1) and in the presence (curve 2) of 0.02 mM 1,2-dilauroyl phosphatidylcholine in nonaqueous phase. (Taken from the data of Kakiuchi et al. WI.)

Thus, the introduction of PLs into a system of immiscible liquids leads to an enhanced response of the interfacial tension toward the action of an electric field. Noteworthy, until now no surface-active substances other than those of the PL class that are capable of producing a similar electric field-induced drop in surface tension at the oil/water interface have been described in the literature. Commonly, other surfactants leave the AyE versus E relationship essentially unaffected (see, for example, Fig. 2). On the other hand, the change of one PL for another (see Fig. 4) only has a bearing on the magnitude of the effect; besides, lipid effects are observable in all the systems of immiscible liquids that have been studied to date, including organic solvents such as n-alkanes, benzene, nitrobenzene, and 1,2-dichloroethane. It should also be noted that the sharp interfacial tension drop due to an external electric field is one consequence of the substantial alterations of the immiscible liquid phase boundary properties produced by the absorbed

PLs. Other uncommon effects can also be mentioned, examples of which are: 2.2.1. Electrohydrodynamic instability The occurrence of instability regimes at the liquid/liquid interface under an applied electric field is well known and has been commented on by many authors [26,43-45,51-531. Commonly, in systems containing polar organic solvents, this instability is because of the convective current of liquids near the phase boundary due to considerable interphase mass transfer initiated by the electric current. The field strengths, leading to instability, are rather small and varied within an interval of hundreds to thousands of V/m. The stability of a plane dividing surface between oil and water is seen to drastically increase on changing from polar to nonpolar liquid dielectrics. For example, the electrohydrodynamic instability in alkane/ water systems becomes more marked at field strengths of 50-30 kV/m (see, for example, Ref. 1541).The addition of surface-active substances does not produce any noticeable effect: the instability-inducing electric intensities remain of the same order of magnitude. However, the picture changes with PLs. The addition of lipids into the nonaqueous phase produces a 50-30 fold decrease in the electric field strength that initiates the electrohydrodynamic interf’acial instability. As

Fig. 7. Photographs of an immiscible liquid surface. Electric field intensity: (A) 20; (B) 60 kV/m. Phosphatidylcholine concentration in n-decane is 2 mM, potassium chloride concentration in water, 1 r&T. Magnification X 10. (From Shchipunov and Kolpakov, unpublished observations.)

51

shown in the photographs in Fig. 7, the ripple formation at the dividing surface between alkane and water is observed at a relatively small applied voltage. By its magnitude, E approaches the electric field intensities at which analogous wave-like interface oscillations take place in conducting systems composed, for instance, of mercury and an aqueous solution 1521. Still, the conductivity of PL-containing alkane/water systems remains extremely low. It is smaller by a factor of lO,OOO-100,000as compared to the conductivity of polar oil/water systems with added organic electrolyte. This fact indicates that the mechanisms operative in the induction of instability at the interfacial boundary between immiscible liquids under applied an external electric field are, with reference to the two cases considered, basically different: in the conducting systems, the instability is due to considerable interphase mass transfer, and to field effects in the nonconducting systems containing PLs. The latter has not been investigated. 2.2.2. Emulsification The dispersion of water into a nonaqueous solution is immediately linked to the stability of the liquid/liquid interface (see, for example, Ref. 1551).Since the addition of PLs leads to a substantial increase in electrohydrodynamic instability of the interfacial boundary between liquids, the emulsification processes are also excited by a weak electric field. Figure 8 is illustrative of this effect. As is shown, the water-in-oil emulsion is being formed at the same electric intensities as those that produce the visually observable ripple oscillations at the alkane/water interface (Fig. 7). Under the same conditions, no such effects are observed either in the absence of PLs, or in the presence of surface-active substances of other classes. As has been shown in an earlier paper [21, it is only the addition of PLs that produces the dispersion at the alkane/water interface under low electric fields. The emulsification process, along with the electrohydrodynamic instability and sharp decrease in interfacial tension, are unambiguously indicative of a drastically increased sensitivity of the dividing surface between immiscible liquids to external factors and, consequently, of an essential alteration in the interfacial boundary properties as produced by lipids. 2.2.3. Structurization As the electric field strength falls below a certain critical value, gel-like phases are formed in the PL nonaqueous solution instead of the emulsion. These phases are presented in the photographs in Fig. 9. Preliminary results, reported in an earlier paper 151,have allowed us to draw the conclusion that the gel consists of a microemulsion. Such phases arise only by

52

53

Fig. 9. Gel-like phases generated by a 5 kV/m electric field, confined within the space between platinum electrodes (dark bands at the top and bottom) immersed in a nonaqueous solution. Phosphatidylcholine concentration in n-heptane is 2 n&f. Photographs taken under: (A) indirect lighting; and (B) oblique illumination. Magnification x 20. (B: photograph taken from the paper of Shchipunov and Kolpakov [5].)

the action of an external electric field and are stable as long as the electric field persists. This property makes them basically distinct from microemulsions, produced by conventional techniques, since the latter microemulsions are thermodynamically stable and are not liable to breakdown with time (see, for example, Refs 156-581). Other distinctive features to be noted are that the gel-like phases are formed in diluted PL solutions of concentrations l-10 mM and no cosurfactant is needed for their formation. At present, it is particularly desirable to obtain a better understanding of the mechanisms of lipid gel-formation and stability in an external electric field.

Opposite: Fig. 8. Photographs of the initial stage of the dispersion of water in n-heptane taken in succession within: (A) 10; (B) 20 and (C) 30 s after a 20 kV/m electric field was applied. The minimal spacing between the upper and the lower aqueous solutions is equal to 2 mm. Egg-yolk phosphatidylcholine concentration in the nonaqueous solution was 2 mM. (From Shchipunov and Kolpakov [21.)

.22.4.Electrolyteeffect

Inorganic electrolytes in aqueous solutions at concentrations >l mM may lead to an increase in interfacial tension by a few units of mN/m (see for example, Refs [11,591). They exert no influence on either the electrocapillary phenomena in a system of immiscible liquids [131,or the surface tension, with PL present in the nonaqueous phase, but only in the absence of an electric field [601. With voltage applied to the oil/water system, the picture becomes drastically changed. The sharp decrease in interfacial tension produced by the addition of electrolytes (Fig. 10) is comparable in magnitude to those observed as the PL concentration in nonaqueous solution is varied [601.

Fig. 10. Decreasein interfacial tension at the n-hept.aue/water interface under an applied electric field versus electric intensity in the presence of 0.008 mM phosphatidylcholine in nonqueous solution and in an aqueous solution of: (1) 0.0; (2) 0.001; (3) 0.01; (4) 0.1; (5) 1.0 Mpotaasium chloride. (Redrawn from the data of Shchipunov and Drachev &Xl].)

Thus, a comparison of the effects, produced by external electric field in PL-containing and PL-free liquid/liquid systems shows that the addition

of lipids to the nonaqueous phase leads to a substantial alteration of the oil/water interface properties. The experimental results, for the most part, provide evidence for a markedly increased sensitivity of the interfacial boundary to the voltage applied. This is indicative of both the sharp drop in interfacial tension due to electrocapillary phenomena and the increased electrohydrodynamic instability as well as the effects due to emulsification processes under the specified conditions. The various interfacial phenomena that occur in the presence of PLs are, undoubtedly, interrelated and should be regarded within the framework of a single scheme. The mere fact that they are observed at the same electric field strength is supportive of such an approach. For this reason, it may appear expedient to characterize the sum total of these effects by a single term. In our opinion, the term “electrointerfacial phenomena”, encountered in the literature, is an apt choice. It covers, if one starts from the definition by Friedrichsberg [611,the whole variety of interfacial processes under an external electric field. Thus, the term “electrointerfacial phenomena” may be used for denoting all the interfacial effects under an applied voltage in the PL-containing systems of immiscible liquids. The characteristic features discussed above, of electrointerfacial phenomena with PLs allow a formulation of the major objectives for investigation. These are: (1) The study of adsorption and adsorption layers of PLs at oil/water interfaces. The dividing surface between immiscible liquids acquires abovementioned new specific features in the case of unrestricted saturation with PL molecules due to adsorption from the bulk nonaqueous solution. The behaviour of lipids at the interfacial boundaries has always excited the interest of researchers but their attention has been focused chiefly on monomolecular layers. The properties of adsorbed lipid layers have not been discussed in the literature. At present, they represent a whole unexplored field. (2) The explanation of the role of ions in electrointerfacial phenomena with PLs. (3) The investigation of electrohydrodynamic instability, dispersion of the plane oil/water interface and structurization in the nonaqueous lipid solution under an external electric field. The presentation of material in this review paper follows the above formulation. In doing so, we have intended to proceed along two major lines of reasoning: first, to give the reader both a broad view of the basic physicochemical properties of PLs in oil/water systems and a specific analysis of the nature of the high sensitivity of the dividing surface be-

tween immiscible liquids, covered with adsorbed PL molecules, to the action of an external electric field; second, to suggest mechanisms for the interfacial processes excited by the voltage applied to liquid/liquid systems in the presence of PLs. 23. Materials and methods Since the study of PLs in physical and colloid chemistry has never been carried out on a large scale, it appears opportune to give a brief survey of the properties of lipids and the natural sources of lipid origin. Phospholipids as a class are part of a larger group of biologically essential chemical compounds known under the general name of “lipids”. A distinctive feature of lipids is the occurrence of an aliphatic (or aromatic) hydrocarbon chain in the molecules. For this reason, assigned to the lipid class, excepting PLs (see, for example, Ref. [621), are hydrocarbons, alcohols, aldehydes, ketones, fatty acids, soaps, detergents, glycerides, sphingolipids, steroids, gangliosides and lipopolysaccharides. The major sites for the localization of PLs in the living cell are biomembranes and membranous organelles in which the PLs account for 25 to 40% (by weight) of the total of lipids 163,641.The distribution of PLs and their fatty acid composition are dependent, in a rather complicated manner, on the membrane type, tissue and organ of the living organism, its species, age and systematic position. However, for a given taxonomicgroup of organisms, the PL composition is, as a rule, characterized by a low variability, and the content of individual PLs is maintained at a constant level (see, for example, Refs [63,641). An important consequence of this is the compositional constancy of natural lipid specimens that have been prepared using a similar procedure. Hence, reproducible experimental results can be obtained and allows the comparison of data reported by various authors. In biological membranes and membranous organelles, PLs are, for the most part, represented by neutral lipids which account for 70-90% (by weight) of the total PLs (with the exception of many bacterial and plant membranes [63,641). Included among PLs are: phosphatidylcholine (PC), phosphatidylethanolamine, sphingomyelin and ceramideaminoethylphosphonate. The structural formulas for these lipids, and a Stuart-Breigleb atomic model for PC are shown in Fig. 11. In living organisms, PC exceeds, in amount, all other PLs. The PC content in biomembranes reaches 40~50% (w/w) of the total PL amount [63,641. The next is phosphatidylethanolamine; commonly, its content is lower by a factor of 1.5-2.0 as compared to that of PC [63,641. Sphingo-

57

GLYCEROPHOSPHOLIPIDS Bon-~01~

Polar

region

l-Stearoyl-2-Oleylsn-Glycero-+Phoaphatiidylcholine

e

region

Choline

RI-C-0-CH2

1,2-Diacyl-tm-Glycero-

I R2-f-’ ‘-yH

3-Phosphatidylethanolamine



08 Y@ C%-O+'-CH&-i-H 0

SPHIXWOPHOSPHOLIPIDS

2-Acyl-1-Phosphatidylcholine

Sphingosine

(Sphingomyelin)

08 H8 C&+-I&2-CII;CH-k!H-~-O-~-CH+,_-~-H b-l R-&C 2-Acyl-14minoethylphosphonate Sphingoaine (Cermnideeminoetbylphoephona~e)

Fig. 11. Structural formulas for choline- and ethanolamine-containing a space-filling molecular model of phosphatidylcholine.

phospholipids

and

58

myelin is mostly found in the membranes of vertebrates 163,651, and ceramideaminoethylphosphonate in the membranes of marine invertebrates [64,661. The content of the former in living organisms attains lO20% (w/w), while that of the latter may be as high as 25% (w/w). The structural backbone of a PL molecule is constituted by either a glycerol (three carbon polyalcohol) or a sphingosine (amino alcohol). In glycero-PLs, two hydroxyl groups of glycerol are esterified with fatty acids, and the third hydroxyl group, with phosphoric acid. Besides, the phosphate is linked to various alcohols. When the alcohol is choline, PC is formed; when ethanolamine, then the derived species is phosphatidylethanolamine (see Fig. 11). In sphingo-PLs, phosphorylcholine or aminoethylphosphonate are attached to sphingosine via an OH group at position 1. One hydrocarbon chain in sphingo-PLs is represented by an amino alcohol, sphingosine, which in natural lipids is commonly composed of 18 carbon atoms 1651; the other hydrocarbon chain is a fatty acid residue linked to the amino group at position 2 through an amide bond (Fig. 11). Ceramideaminoethylphosphonate, although considered a member of the PL series, is a phosphonolipid, rather than a phospholipid. The major distinction is the nature of the bond between the phosphorus and carbon atoms: in ceramideaminoethylphosphonate, these are linked directly, while in PLs, they are linked through an oxygen atom, i.e., through an ester bond (Fig. 11). The PL molecules have an extended polar region. For example, in glycero-PLs, this region may be represented by two parts: the glycerol region and the head group region formed from phosphorylcholine or phosphorylethanolamine (Fig. 11). Within a wide pH range of aqueous solution, charges exist in the polar region, but, since the phosphate group and the quarternary nitrogen carry charges of opposite sign, the net charge on the lipid molecules is zero, that is, they are neutral and should be regarded as zwitterions. All of the above neutral PLs have been used in experiments (Fig. 4). They were isolated from natural sources. The chromatographically pure lipid specimens we have used were a gift from Professor E.Ya. Kostetsky and were purified in his laboratory. Glycero-PLs were obtained chromatographically from egg yolk, using standard procedures [671. Sphingomyelin was isolated from bull brain, ceramideaminoethylphosphonate, from the mussel Grenomythilusgruyanas by the method described in Ref. I641. The PL fatty acid composition has been determined by gas-liquid chromatography and mass spectrometry and reported previously [681. Inorganic and organic salts, solvents (twice distilled water, n-heptane and ndecane) were prepared as described in detail earlier [1,2,4,5,11,30,681.

59

Two methods were used in measuring the inter-facial tension: a dropvolume method and the method of a pendant water drop in nonaqueous solution. The former method and the measuring setup were described in detail elsewhere 111,131.A brief comment should be given on the placement of the platinum electrodes used for measuring interfacial tension under an external electric field. One platinum electrode was placed in the water phase and the other in the nonaqueous phase. The latter electrode had a funnel-shaped form. The electrode funnel was designed in such a manner as to provide a radially equidistant location for the water drop within the funnel from the funnel surface. The experimental setup for inter-facialtension measurement by the pendant drop ~chnique included a device for the squeezing out of a liquid drop (similar to the setup used in the volume drop method as described in Ref. [ill), and a pendant drop cell of Teflon. The optical system which was an array of an 01-19 type light source, a condenser, a Teflon cell, and a MFNEIU type photomicrograph attachment, was aligned along a single optical axis. The absence of photographic picture-shape distortions was checked on a number of steel balls of perfectly spherical shape and of precisely known diameter that were placed for control measurements at the same position at which the pendant water drop was formed. The error in the value of interfacial tension caused by instrumental errors was estimated in accordance with the recommendations of Ambwani and Fort 1691and was not greater than 0.12 mN/m, taking into account the uncertainties in the measured quantities. This estimate was conformed with the observed spread in values of the experimental interfacial tensions. The experiments were carried out at a temperature of 20.0 rt0.1%. The funnel electrode could not be used for measuring the interfacial tension by the pendant drop technique since it interfered with the photography. A platinum plate was employed in place of the funnel electrode. It was positioned horizontally beneath the water drop in the nonaqueous solution. A comparison has shown that, at small electric field strengths, the replacement of one electrode for another does not affect the experimental quantities measured. The study of emulsification in a system of immiscible liquids was carried out using either a rectangular spectrophotometer cell equipped with two horizontally positioned platinum plate electrodes or a Teflon cell with two optical glass windows in opposite side walls, schematically shown in Fig. 12(A). Fitted into the cell were: in the bottom, a glass tube 3 of inner diameter 8 mm, and on top, a glass capillary 4 of 2 mm diameter, both containing a sealed-in platinum electrode 5. The glass tube 3 and capillary 4 were filled with 1 mM aqueous potassium chloride solution. The solution

A

B

Fig. 12. Schematic view: (A) the cell assembly for study of emulsification in a system of immiscible liquids; (B) the microscope cell assembly; symbols are defined in the text.

level was maintained by means of two syringes connected to tube 3 and capillary 4 through flexible hoses. The cell was filled with nonaqueous lipid solution. Emulsion formation was observed by transmitted light. An 01-28 type illuminator or high-pressure mercury vapour lamp wwas used as a light source 6. To screen the liquids in the cell from heating, a water heat filter was placed before the light source. For observation and photography, a MFNE-IU photomicrographic attachment 7 equipped with a long focallength lens was used. Microscopic observations were made using a MBI-15 microscope and a special cell shown in Fig. 12(B). The cell of Teflon 3 had a plate 1 from optical glass press-fitted into the bottom. Two platinum wire electrodes 2 of 1 mm in diameter with a spacing of 1 mm between them were fitted into cell wall from opposite sides to supply direct voltage within a maximum value of 300 V from a B5-50 type source. The thinning of nonaqueous films immersed in water was studied using a cell similar in design to that described by Rovin et al. [701.

61 3. PHOSPHOLIPIDS

AT INTERFACES

Lipids at movable interfacial boundaries have been characterized in sufficient detail. Commonly, experiments were carried out on monomolecular films at the air/water interface. Reported studies with PLs at the liquid/liquid interface were less numerous. A common assumption is that stable monomolecular films were also formed on the dividing surface between immiscible liquids (see, for example, Refs 171-731). However, examination of this assumption was not conducted by investigators. One may assume that the monolayers at oil/water and air/water interfaces differ to a significant extent in stability, since lipids are usually soluble in nonaqueous media. The conditions under which an equilibrium between the bulk of nonaqueous solution and the inter-facial boundaries sets in are as yet poorly understood. To our knowledge, the adsorption kinetics for PL molecules at an immiscible liquid interface was studied only in papers by Johnson and Saunders 1741. Unfortunately, the results obtained by these authors are of little value because of computational errors. Moreover, an analysis of their data has allowed us to suggest that in most cases, the equilibrium in the system was not attained even after 10 h, the duration of the kinetic study. Therefore, the equilibrium characteristics of adsorption for PLs at the oil/water interface could not be obtained. This section is concerned mainly with the structural specificity of lipid adsorption layer and the effects produced thereto by external electric field. The material to be discussed will be exemplified by the data obtained for only one PC species; other neutral PLs have been omitted from the discussion. This allows us to ignore the specific inter-facial effects due to polar lipid groups and focus our attention on the major features characteristic of the whole class of PL species. A comparison of the physicochemical properties of PLs with choline and ethanolamine functional groups was carried out by Drachev et al. 1681. 3.1. Adsorption

3.1.1. Adsorption

kinetics

The kinetics of adsorption has been studied by the pendant drop technique. Special measures were taken to prevent leakage in the device for the squeezing out of a water drop. This enabled the measurements to be carried out for an extended (several days) period of observation. A series of photographs of a drop that were taken in succession at different time intervals starting from the initial development of a water drop in a nonaqueous PL solution are shown schematically in the inset in Fig.

62

13.The observed change of the drop shape is indicative of a decrease in y at the oil/water interface. The interf’acial tension versus contact time for the immiscible liquids studied is shown in Fig. 13. The temporal dependence, presented in Fig. 13, has allowed us to conclude that the oil/water system was free of impurities and that the PC samples used were of satisfactory purity. As shown by Mysels 1751,the y versus t relationship exhibits, in the presence of trace amounts of surfaceactive contaminants, an intricate behaviour. The main surfactant produces a drop in interfacial tension at the initial moment of measurement when the immiscible liquids have been brought into contact. Effects due to the presence of contaminants become manifest later. Therefore, following the main initial diminution of y, the secondary exponential drops in interfacial tension produced by surface-active contaminants are delayed in time. No such secondary effects, as evidenced by Fig. 13, have been observed with the PC samples studied. The overall kinetics of adsorption of surface-active substances at an inter-facialboundary may be subdivided into diffusion kinetics and adsorption kinetics [76,771. The former kinetics are determined by the supply of

Fig. 13. Temporal changes of inter-facial tension produced by a water drop formed in: (curve 1) 0.002 and (curve 2) 0.02 mMegg-yolk phosphatidylcholine solutions in n-decane. The interfacial tension was measured by the pendant drop technique. Inset: schematics of photographs taken in succession at: (A) 1; (B) 35; and (C) 90 min from the start of the experiment. (Data taken from Shchipunov and Kolpakov 141.)

63

surfactant from the bulk solution. These kinetics are limited to the diffusion stage in the unstirred layers of liquids adjacent to the dividing surface. The temporal variation of inter-facial pressure is expressed by the equations: l-I = 2RT C (Dt/x)1’2

(3)

where D is the bulk phase diffusion coefficient, t is the time measured from the moment the immiscible liquids are brought into contact. The adsorption kinetics are associated with the penetration of the adsorbed molecules into the adsorption layer and their desorption. In the general case, it is described by the expression [771: lJ=T&Tln

1 + bC 1 + bC exp(-t/T)

(4)

where b is the adsorption equilibrium constant, z is the adsorption relaxation time, I, is the surface excess corresponding to the maximum coverage of the dividing surface with adsorbed molecules. Equation (4) gives no single-value relationship between l-land t. In practice, the expression ll = l-l, exp(-t/d

(5)

is commonly used (see, for example, Refs [76,771), which is a special case of Eqn (4). This expression is also easily obtained in treating the adsorption as a first-order reaction, as was first shown by Zhigach and Rehbinder I781. The decision about which of the two adsorption kinetics regimes is operative is governed by Eqns (3) and (51,and by analyzing the interfacial pressure versus time relationship: under diffusion controlled kinetics, II is proportional to the square root of t, and under adsorption controlled kinetics, log Il is proportional to t. In order to find the limiting stage in the PL adsorption, Il versus the square root oft and log Il versus t have been plotted. These are shown in Figs 14 and 15. As follows from Fig. 14, diffusion kinetics occur at the initial stages of the PC adsorption process. The proportionality between Il and the square root of t remains valid for an extended period of time. For example, the supply of surfactant to the interface by diffusion at PC concentrations in n-heptane of ~0.002 mM is the determining factor for adsorption process within a period of 12-15 h until the equilibrium is attained in the system of immiscible liquids.

64

0

$/2

(o”2)

Fig. 14. Interfacial pressure at the n-decane/wat.er interface versus the square root of time counted from the initial development of a water drop in a nonaqueous solution containing: (1) 0.002; (2) 0.005, (3) 0.02; (4) 0.01 mMphosphatidylcholine. Interfacial tension was measured by a pendant drop technique. (Data from Shchipunov and Kolpakov [41.)

The temporal relationship, presented in Fig. 14, allows the estimation of the diffusion coefficient for PC molecules in alkane. The value of D can be evaluated graphically from the slope of the linear portions in the curves and by making use of Eqn (3). The diffusion coefficient has been found to equal (2.0 f 0.5)*10-10 m2/s (mean value with standard deviation). In dilute solutions (~0.1 ti the diffusion coefficient is independent of the PC concentration in the nonaqueous phase. Johnson and Saunders 1741employed the same method for the evaluation of the PC diffusion coefficient. However, an analysis performed on the

65

. 0

10

30x10-3

20

t

(c)

Fig. 15. The logarithm of interfacial pressure (at the n-decane/wat..er interface) versus time of experiment. Phosphatidylcholine concentrations in alkane are (curve 1) 0.01 and (curve 2) 0.02 mM. Interfacial tension was measured by a pendant drop technique. (Data from Shchipunov and Kolpakov [41.)

measurement results from Ref. 1741has revealed a computational error that led to a nearly l,OOO-foldunderestimation of the diffusion coefficient as compared to our value of D. PC diffusion was also studied by Elworthy and McIntosh [791. The measurements were carried out by making use of a Gouy diffusiometer in a benzene solution containing >1 -PC. At such concentrations, the lipid occurs in a micellar rather than a monomeric form 180,811.For comparison

with our results, we have recalculated the benzene diffusion coefficients from Ref. [791 to the respective n-heptane values by making use of the Stokes-Einstein equation and the tabulated reference data for solvent viscosities. For associations composed of four lipid molecules the value of D is 1.9. 10WIO m 2/s, and for micelles of 70 PC molecules,D is 1.40 10-l’ m2/s. Our adsorption kinetics measurements have led to similar values for the diffusion coefficient. As seen in Fig. 14, at sufficiently long contact time and at PC concentrations larger than 0.002 mM, the experimental points tend to deviate from the straight line characterized by diffusion kinetics. In this case, the temporal dependence of the interfacial pressure is described by means of Eqn (51, see Fig. 15. It follows from this that the limiting stage for the adsorption of PL molecules at the oil/water interface from n-heptane is subject to variation with time: in the initial period after the formation of the water drop in the alkane, the filling of the interfacial boundary is a limiting factor for the migration of lipid molecules from the bulk of the nonaqueous phase. At a later period, after the attainment of a definite PL coverage of the dividing surface between immiscible liquids, the limiting factor is the penetration of lipid molecules into the adsorption layer. This change in the adsorption kinetics regime may be caused by repulsive interactions between molecules within the monomolecular layer that originate at low filling of the oil/water interface 1721. A quantitative characteristic of adsorption kinetics is the adsorption relaxation time. In accordance with Eqn (51, the time constant can be estimated from the tangent to the linear portions of the curves. This is shown in Fig. 15. By way of example, the values of the relation time for PC concentrations in 0.01 and 0.02 mM n-heptane have been found to be 65,000 and 33,000 s, respectively. The adsorption relaxation time includes two components [771: z =

(a +

pc/roarl

(6)

where a and 6 are the rate constants of surfactant desorption and adsorption, respectively. In analyzing the adsorption kinetics, it was assumed by Johnson and Saunders [741 that the filling of the boundary surface with PL molecules took place under conditions close to those of equilibrium. If this is the case, then zz l/a would hold. However, such an assumption was not argumentatively supported. The kinetics took a sufficiently long time to reach equilibrium in the system of immiscible liquids (see Fig. 131,which was rather indicative of the opposite effect. Therefore, a more correct assumption would be a < w/P_ [41.In such a case, z =.l/(cjC/r,). By making

67

use of the experimental values of z, C, and I_, we obtain 141that the adsorption rate p within a wide range of PC concentrations in alkane would be equal to 5*10S7 m/s. 3.1.2. Adsorption

under equilibrium

conditions

Glycero-PLs start forming aggregates in a nonpolar solvent at concentrations of about 0.01 mM 168,811.To provide a study of aggregationfree adsorption, the PC concentrations at which the interfacial tension has been measured under equilibrium conditions were not greater than 0.002 mM. As seen in Fig. 13, the equilibrium sets in only 12-15 h after the water drop has been squeezed out into the nonaqueous lipid solution. Anyway, within the subsequent 4-6 h, the scatter in the values of interfacial tension was within the experimental error of measurement. The y versus t relationships, similar to those in Fig. 13, have been used for estimating the equilibrium interfacial tensions. The inter-facialpressure at the n-heptane/water interface as a function of the logarithm of PC concentration in alkane is given in Fig. 16. The equilibrium values of ll are represented by curve 3. Also shown is curve 4 that has been plotted on the basis of the experimental data of Demel and Joos 1821who studied the adsorption of 1-stearoyl-2-oleoyl-PC (see Fig. 11) at the n-heptane/O.l M aqueous sodium chloride solution interface. A comparison of these two lipids appears to be justifiable, given the similarity in properties of the synthetic PC containing fatty acid residues as specified above and the natural lecithin isolated from egg yolk 1831. A point to be noted is that no adsorption kinetics were studied in Ref. 1821where the interfacial tension was measured by the Wilhelmy plate method. It was merely mentioned that the measurements were carried out 2-3 h after the aqueous and nonaqueous solutions had been brought into contact. However, quite a satisfactory agreement is observed between the interfacial pressure isotherms obtained by us within 12-15 h (curve 3) and those by Demel and Joos (curve 4). The surface excess and the area A occupied by a lipid molecule in the adsorption layer at equilibrium conditions have been obtained graphically in Fig. 16. The calculations have been carried out by making use of the Gibbs’ equation: XI=RTlYalnC where I = l/N&,

(7) NA is the Avogadro number.

68

-7

-6

-5

lg c

-4

(X1

Fig. 16. Interfacial pressure at the n-heptanelwater (curves 1 and 4) and n-decanelwater (curves 2 and 3) interfaces versus the logarithm of egg-yolk phosphatidylcholine (curves l-3) and 1-stearoyl-2-oleoyl-phosphatidylcholine (curve 4) in alkane in the presence of 0.1 Msodium chloride (curve 4). Inter-facial tensions were measured by: (1) dropvolume; (2,3) pendant drop; (4) Wilhelmy plate methods in 3-5 min (curve 1),30 min (curve 2),23 h (curve 4) and under equilibrium conditions within 12-15 h (curve 3) after the immiscible liquids have been brought into contact. (Curve 1 plotted from the data of Shchipunov and Drachev 1811;curves 2 and 3 from Shchipunov and Kolpakov 141;curve 4 adapted from the data of Demel and Joos I821.1

Now, let us compare the characteristics of PL adsorption layer to those of a monomolecular film. The area A, as the PC concentration in alkane approaches 0.002 mM, reaches a value of 0.4 nm2/molecule. This is merely 0.04 nm2/molecule larger than the cross-sectional area for two saturated hydrocarbon chains in a crystal or computed from molecular models [%,76,841. Thermodynamic calculations which make use of Eqn (7) can be verified by data of adsorption kinetics. If we take into account that the adsorption

69

is diffusion-limi~d up to 0.002 m.MPC over the whole time from the beginning of process before an equilibrium is established (see curve 1 in Fig. 14), we can describe an adsorption rate with the following equation (Ref. 1761,p. 165):

Integration of Eqn (8) with n=O when t=Ogives: n = 2C NA(ot/dl”

(99)

Here n is the number of adsorbed molecules per m2 of dividing surface. Putting in Eqn (9), D=2*10W1’ m’/s, C=O.O02m.M,and t=15 h, one obtains for A, the area equated with l/n, a value 0.23 nm2/molecule. Agreement with thermodynamic calculations is satisfactory to within a factor of 1.7. A further feature to be noted is that the area occupied by a lipid molecule at the interface is smaller by this factor as compared to the crosssectional area of two saturated hydrocarbon chains. But it should be taken into account that the natural PC contains unsaturated fatty acid residues (see PL fatty acid composition in Section 2.3.). Its molecule, in a tightly packed monolayer, occupies an area of at least 0.6 nm2/molecule, the in&facial pressure being about 40-50 mN/m (see, for example, Refs 171,85871). As the dividing surface between oil and water becomes filled with lipid molecules by an unrestricted adsorption process, the value of A is about 0.4 nm2/molecule according to Eqn (71, or even 0.23 nm’/molecule according to Eqn (9). Furthermore, the value of ll does not exceed 10 mN/m. In other words, the adsorption layer that is being formed is, by its characteristics to a significant extent different from monomolecular films of the same lipid. This difference was first pointed out by us and the question has been discussed in a previous paper [41. It should be noted, though, that this difference arises not instantaneously when the immiscible liquids have been brought into contact, but takes some time to develop as the s&a&ant is accumulated at the phase boundary. A formal analysis of the inter-facial pressure isotherm that was measured at different time intervals after the water drop formation in the nonaqueous lipid solution and presented in Fig. 16 (curves l-3) reveals that, at the initial stage, the incipient adsorption layer is, by its parameters, close enough to monomolecular films of the same lipids. This picture is commonly observed in 3-5 min after the start of measurements. There is another result that also deserves attention. It was obtained 141 in the course of the determination of an adsorption isotherm which

70

10

2 8. F:

5

0 0

0.01

0.02

Fig. 17. Interfacial pressure (measured under the equilibrium conditions within 12-15 h after the immiscible liquids have been brought into contact) versus the reciprocal area per egg-yolk phosphatidylcholine molecule at the n-decanelwater interface. Values of the area have been computed according to the Gibbs’ equation, Eqn (7), from the dependence of Il on the logarithm of phospholipid concentration presented in Fig. 16. (Drawn from data of Shchipunov and Kolpakov i41.1

describes PC adsorption. Figure 17 illustrates this. Here, the points in the graph are the experimental values for the interfacial pressure at the ndecane/ water interface plotted against the reciprocal area occupied by a lipid molecule in the adsorption layer. The straight line is the theoretical dependence satisfying the equation for an ideal two-dimensional gas: rIA=RT

(10)

As seen in Fig. 17, the experimental points fall on the theoretical line. This means that, between the lipid molecules at the oil/water interface, no sufficiently strong interactions greater than the kinetic energy are operative. If this is the case, then the boundary surface between immiscible liquids must not be completely filled with the adsorbed molecules. However, such a conclusion is at variance with the experimental evidence since the

71

validity of the relationship in Fig. 17 extends to values ofA as small as 0.4 nm2/molecule. Given such an area per lipid molecule at the interfacial boundary the adsorption layer in the form of monomolecular film would have been a closely packed film and, consequently, would not show the properties of an ideal two-dimensional gas. This apparent discrepancy between the adsorption and monolayer data can be removed if the monolayer concept is rejected and multilayer model for filling of the oil/water interface with adsorbed lipid molecules is accepted, as was first suggested in our earlier work [41. A comparison of parameters for PC in the adsorption layer and the monomolecular film permits the estimation of the thickness of the interphase films formed. To achieve this purpose, let us consider the inter-facialboundary of immiscible liquids under specified conditions, for example, at an interfacial pressure of 10 mN/m. Given such II, an egg-yolk lecithin molecule will occupyan area of about 1.3 nm2 within the monolayer (see Ref. 1711).Similar values ofA have been reported in Ref. [721for 1,2-distearoyl- and 1,2-dioleoyl-PC molecules. Simultaneously,A has a value of about 0.4 nm2/molecule (or even 0.23 nm2/molecule) within an adsorption layer formed by unrestricted filling. On assumption that the packing mode for this layer is close to that of a monolayer because they both are at 10 mN/m in the state of an ideal two-dimensional gas, one comes to the conclusion that the PC adsorption layer must be a minimum of three molecules thick 141. The estimates obtained are valid for equilibrium conditions and PC concentrations of 0.001-0.002 mM in alkane. At lower concentrations, an adsorption layer is formed and, by its parameters, is close to a monolayer. On the other hand, an increased concentration of PL in the nonaqueous solution makes the interphase film thickness grow. Thus, the film becomes visually observable after l-2 days at a PC concentration of 0.01 M 141.The formation of an extended adsorption layer with time was also reported in a number of papers (see Refs W3-911). On the basis of the available evidence, it was clearly demonstrated that the adsorption layer has a liquid-crystalline structure. Kakiuchi et al. [921 compared the parameters of an inter-facial film, formed by lipid adsorption at nitrobenzene/water interface from nonaqueous solution, to those of a monolayer that was prepared by spreading a given amount of the same PL (1,2-dilauroyl-PC). The adsorption in this system of immiscible liquids was shown to be extended in time, which basically agrees with our results (Fig. 13). Thus, in a 0.005 mM 1,2-dilauroylPC solution, a state of equilibrium was reached within 1 h. About that long a time was needed for the equilibration of the 1,2-dipalmitoyl-PC system [931.Senda et al. 192,941also observed that the interfacial tension does not

72

noticeably decrease at a concentration of about 0.02 mM when new amounts of PC are added. In the opinion of these authors, the maximum coverage of the oil/water interface with lipid molecules was reached at this concentration. However, there is another possible explanation. In all probability, the effect observed is associated with the incipient aggregation of surfactant in the bulk of the nonaqueous solution. Thus, the formation of PC associates at a concentration of about 0.01 mM in benzene and n-heptane has been shown by Elworthy [801, and Shchipunov and Drachev [68,811.The beginning of aggregation is reflected in the break in the slope of Il against log C (see, as an example, curve 1, in Fig. 16). A comparison of the available parameters for the adsorption layer and monomolecular film, as in Ref. [921,revealed that they agreed fairly well. On the basis of that evidence, Senda et al. drew the conclusion that the 1,2-dilauroyl-PC adsorption led to the formation of a monomolecular film at the nitrobenzene/water interface. However, the measured double-layer capacitance, taken as a criterion for this comparative analysis, was by a factor of 10 greater than the bimolecular lipid membrane capacitance. A similar conclusion may be drawn with reference to the data obtained by Wandlowski et al. 1931.The sizeable difference in capacitance between the putative monomolecular film at the nitrobenzene/water interface and the bimolecular lipid membrane is as yet poorly understood. The more so because no such difference was observed in the capacity values for monolayers at the mercury/water interface (see, for example, Refs [95,961). Kakiuchi et al. [921argued that the observed difference might be due to a change in the dielectric constant of the adsorption layer produced by the insertion of solvent molecules and organic ions. However, such an explanation can hardly be satisfactory, since the addition of organic electrolytes (as can be seen from the data presented in Ref. 1961)did not lead to a sizeable alteration in the monolayer capacitance at the mercury/water interface; as to the insertion of solvent molecules, these must be greater in number than those of PL. Large quantities of solvent can only penetrate into films with a loose packing of the lipid molecules at the interfacial boundary (see, for example, Refs [97-991). But then one should have to reconsider the concept of monomolecular filling of the surface between phases on account of the fact that the PC packing density in the adsorption layer, in accordance with the data by Senda et al. 192,941,is close to 0.75 nm2/molecule. Our studies on PL adsorption from the nonaqueous phase at the alkane/ water interface have shown that the adsorption layer, even for diluted lipid solutions, has a three-dimensional, rather than a two-dimensional, structure. This layer takes a longer time to form. For example, up to a PC con-

73

centration of 0.002 miW,equilibrium in the system is reached in 12-15 h (Fig. 13). In th’IS time interval, the adsorption kinetics proceed by a diffusion mechanism and is described by Eqn (3). This is, however, possible only in an idealized case, when no migration of surfactant away from the adsorbed layer (desorption) takes place (Ref. [761, p. 166 and Ref. [771). Therefore, lipid molecules, in contact with the aqueous solution surface, become bound in one way or another, and desorption into the bulk of the nonaqueous phase is inhibited, which results in a high surface excess. Any strong interactions between lipid molecules within the adsorption layer at low concentrations (~0.002 mM) are absent. The relationship shown in Fig. 17‘supports this statement. The change to which PL molecules are subject to, on approach to the oil/water interface from the nonpolar phase bulk, is due to the addition of water molecules. A comparison between the standard adsorption energy (AGO)and the hydration energy for the polar region of PC lends support to this concept. The values of AGOfor PC have been obtained by making use of the BettsPethica method [ll,lOOl. From a formal standpoint, there are no restrictions imposed on the use of this method, since the PL adsorption layer satisfies the condition for ideality (Fig. 17). The calculations have been carried out by means of the equation: AGO= -RT ln{Kl/Xl~DlO)l

(11)

whereXis the bulk concentration expressed in the molar fraction of the lipid in the nonaqueous solution, the superscript ‘0’ indicates the standard state. The standard energy for adsorption of egg-yolk PC from n-heptane at the oil/ water interface has been found to be equal at l-l= 1 mN/m andX = 1 to -47.6 kJ/mol [4l. It compares well with the standard energy for PC transfer from water to vesicles (-45.7 kJ/mol), measured experimentally by Nichols 11011, but is somewhat less than the hydration energy (-63 to -71 kJ/mol) estimated by Ivkov and Berestovsky from NMR data [1021.The difference, in the latter case, appears to be due to a partial hydration of lipid molecules in the alkane bulk, since the adsorption experiments were carried out with water-saturated n-heptane 141. The hydration shell of an egg-yolk PC molecule is made up of 33 to 39 water molecules [103,1041. According to evidence from Refs 11051 and DO61,in forming the hydration shell, the first 4-5 water molecules attach to the phosphorylcholine region, and the subsequent molecules can reach inside to the glycerol backbone (see Fig. 11). The addition of such a large number of water molecules into the hydration shell results in a substantial volume enlargement of the hydrated lipid molecules. Thus, according

74

to the data by White and King 11071, the volume of the lipid molecule, accepting values of 1.8 to 33.6 water molecules is increased from 1.622 to 2.266 nm3, which is nearly 1.5fold increase in volume. Increasing hydration is also accompanied by changes in other molecular parameters of the lipid phases. For example, the surface area per PC molecule increases by 15-20% [102,108-1101. A point to be emphasized is that the observed increase in both the volume and linear dimensions of the hydrated molecules is effected due to the expansion of their hydrophilic regions. Thus, on PC hydration, the thickness of the polar portion of the bimolecular lipid layer in the liquidcrystalline phase increases from 2.25 to 3.72 nm, while that of the nonpolar portion, in contrast, is decreased from 2.85 to 2.48 nm 11071. Owing to a significant enhancement in the hydrophilicity of the PL molecules, their solubility in the nonaqueous phase drops sharply, which impedes desorption and leads to a gradual accumulation of PL molecules at the interphase boundary. With time, as the number of accumulated PL molecules is in excess of that needed for monomolecular coverage of the dividing surface between phases, the formation of a three-dimensional adsorption layer begins. Within this layer, the hydrated molecules can be induced to form in liquid-crystalline states. The latter are typical of waterlipid mixtures over a very wide range of composition and temperature [62,89,102,109,111-1141. In particular, the formation of PL liquid crystals at the oil/water interface was reported in Refs 188-911. The PL-containing system of immiscible liquids may, in all likelihood, be classified as a self-organized system, and the processes occurring therein may be treated within the framework of the theory of Prigogine et al. (see, for example, Ref. [1151). The following considerations seem to lend support to this statement. Irreversible PC adsorption in the PL-containing oil/water system allows us to regard this system as an open one. It is in a thermodynamic nonequilibrium state starting from the moment the water drop has been formed at the capillary tube in the alkane phase. At first the irreversible lipid adsorption is due to the absence of PL molecules at the freshly prepared dividing surface and then, in the absence of noticeable flow of desorbed PL, there is no migration into the bulk of the nonaqueous phase. This is because of hydration effects and a sharp drop in solubility. With time, the adsorbed lipid forms three-dimensional, rather than two-dimensional, structures. These are localized within a confined space in close vicinity to the interfacial boundary. For this reason, in the system of interest, the immiscible liquid interface acts as a natural boundary for the localization of dissipative lipid structures.

75

3.1.3. Electric field effect Such effects were noted by Senda et al. 150,921and Wandlowski et al. 1931in their studies of PC properties at the nitrobenzene/water interface, but the authors did not consider in such detail the interfacial processes concomitant with the electrocapillary phenomena. In our earlier work 141, we have given more attention to this problem. The application of an electric field has been shown to affect the adsorption kinetics in an alkane/water system; the effect produced is an accelerated decrease in interfacial tension with time. The effect, caused by an electric field on the interfacial tension, is comparable, even at low field strength, to that produced by a substantial increase in PC concentration in the nonaqueous phase. The adsorption kinetics under the voltage applied, are changed markedly. Equations (3) and (5) do not apply any more. The adsorption kinetics becomes a rather mixed type 141,i.e., when the rates at which the surfactant is transferred by diffusion to the boundary surface and the adsorption layer is formed are comparable in their order of magnitude. The effect of an electric field on PC desorption takes some time to become apparent. This may be exemplified by Fig. 18 in which a relationship between the interfacial pressure at the n-heptane/water interface and the square root of the contact time for immiscible liquids is shown. Straight line 1 in Fig. 18 obeys Eqn (3); this relationship has been obtained in the absence of an electric field. With voltage applied, deviations from the diffusion kinetics are observed that become increasingly more pronounced with higher electric fields. However, the deviations take some time to become apparent. Thus, curves 2-4 in Fig. 18 during the initial stage coincide with line 1. It follows, therefore, that the action of an electric field on the interfacial processes involving lipids becomes manifest only as the n-heptane/water interface becomes filled, to a certain extent, with PC molecules. The deviations from the diffusion kinetics do not, however, signify that diffusion kinetics are being replaced by adsorption kinetics, as was the case in the absence of electric field (Figs 14 and 15). Besides, these deviations, as seen by comparing Figs 14 and 18, proceed in the opposite direction: the fast stage is not replaced by a slower one, and the interfacial processes become markedly accelerated under the voltage applied. The greater is the electric intensity, the sooner the accelerating processes set in. In this connection, the adsorption results, obtained in the absence of an electric field, should be recalled. As the oil/water boundary surface become filled with PL molecules, the initial monolayer is gradually transformed into a three-dimensional layer. This process, as evidenced by the altered

76

25 ? 9 c

20

15

10

5

0 0

5

10

20

15

Q/2

25

(c”2)

Fig. 18. Interfacial pressure at the n-heptanelwater interface versus the square root of time counted from the initial development of a water drop in the nonaqueous solution containing 0.02 mM egg-yolk phosphatidylcholine. Electric intensity: (1) 0.0; (2) 0.5; (3) 3.0; (4) 9.0 kV/m. Interfacial tension was measured by a pendant drop technique. (Redrawn from data of Shchipunov and Kolpakov 141.1

adsorption kinetics, is influenced by an electric field (see Fig. 18). The adsorption layer undergoes a structural rearrangement; a simple experiment, reported earlier [1161, supports this fact. Equal portions of a nonaqueous 2mM PC solution and a 1 mM aqueous KC1 solution were transferred into a rectangular spectrophotometer cell. A plane platinum electrode, with an area of about 2/3 that of the boundary surface of the two immiscible liquids, was positioned horizontally in the alkane phase about 10-20 mm above the interface. A platinum wire

77

electrode was placed in the water phase. Light from a halogen lamp was directed parallel to the dividing surface between the oil and water. Alkane and water, when viewed in transmitted light through crossed polarizers placed on opposite sides of the rectangular cell, look opaque, that is, are optically isotropic. A potential difference, applied to the electrodes, with time, makes a transparent layer appear. In other words, an optically anisotropic layer adjacent to the immiscible liquid interface at the nonaqueous solution side. This is illustrated by the photograph in Fig. 19, in which the layer is about 0.1 mm thick. The optically anisotropic layer takes some time to form after the electric field has been applied. Initially it looks like a light reflection on the interfacial boundary, but with time the layer grows in thickness and can be observed visually even under small magnification. It must be emphasized, that such structural forms are observable only when a rather small electric field is applied. No optically anisotropic layer at the oil/water interface as shown in Fig. 19 has been seen to form under zero-field conditions 11161. Now, the application of a voltage to a system of immiscible liquids containing a neutral PC initiates interfacial processes observable only under

Fig. 19. Photograph of the n-decanelwater interface taken in transmitted light with crossed polarizers within 5 min after a 10 kV/m electric field has been applied. The light band is an optically anisotropic layer; it is about 0.1 mm thick. The layer touches, with its lower border, upon the inter-facial boundary. Phosphatidylcholine concentration in the nonaqueous phase is 2 m.M. (Taken from Shchipunov and Kolpakov 11161.)

78

specified experimental conditions. The optically anisotropic layer thus produced is indicative of two essential points: (i) the adsorption layer undergoes a fundamental reorganization under an external electric field; and (ii) the structure of the adsorption layer has a liquid-crystalline nature. 3.2. Stratification membranes

at the interface and the formation of bimolecular

Bimolecular lipid membranes (also bilayers) arise spontaneously in the centre of a biconcave nonaqueous PL solution drop that separates two aqueous solutions. The film thinning proceeds via a number of stages of which the central one is the drainage of liquid by the action of the capillary pressure gradient generated by the surface curvature of the initial film [117-1191. In the formation of a bilayer, the final stage at which the film reaches a critical thickness is also important. The abrupt thinning of the latter causes two results: the rupture of the films or the formation of a thinner film when the adsorbed surfactants covering opposite surfaces of the oil/water interface come into contact. For the monolayers found on the interphase boundaries, the formation of bimolecular membranes is observed and the film thickness changes from a few tens of nm to 5-7 nm. One will easily recognize that the state and the structure of the adsorption layer are the main factors that determine the thickness and the properties of bimolecular lipid membranes. We have made use of this fact for the verification of an earlier hypothesis of a three-dimensional structure of a PC adsorption layer at the oil/water interface. The experimental results have been dealt with in detail in our earlier papers [120,1211. Here, we wish to touch upon some of the salient features concerning the technique for studying the thinning of nonaqueous films down to the bilayer state. We made an estimation of the probability P for the appearance of the so-called black spots (bilayer domains with a diameter of about 0.1 mm which look black in reflected white light) in nonaqueous PL films. The value of P was defined as the ratio of the number of nonaqueous films in which the formation of black spots had been noted to the total number of investigated nonaqueous films (at least 100) produced within a single series of experiments. Now, the buildup of bilayer structures occurs at a sufficiently tight packing of surfactant molecules at the interphase boundary [117,119-1211. The lowest concentration in solution at which black spots are observed to appear for the first time has been named, in current colloid chemistry terminology [119,1221, the “critical concentration for

79

black spot formation”. Commonly, an estimation of this value is made at a probability of several percent. As the surfactant concentration in the nonaqueous solution is raised, the persistence to rupture increases, which leads to a greater probability for the appearance of black spots. The P versus C relationship for egg-yolk PC, as shown in the initial portion of the curve in Fig. 20, has a sigmoidal shape.

0.0 0

1

2

4x704

3

c

00

Fig. 20. Probability of black spot formation versus concentration of phosphatidylcholine in n-heptane. The data, presented by the solid curve, have been obtained 4 h after contact of the immiscible liquids and by the dashed curve, immediately after contact of the aqueous and nonaqueous solutions. (From Shchipunov and Kolpakov D21l.j

As the probability reaches a value of lOO%,the PL concentration in solution is no longer a determining factor for the generation of black spots. In Fig. 20, this is shown by a dotted line. Commonly, the attainment of 100% probability is attributed to a tight filling of the interfacial boundaries and an increased stability of the black spots, which is seen by a large growth

in their number; with time, they expand and merge to cover the whole film surface, This is the moment when the thin nonaqueous film with black spots is transformed into a bimolecular membrane; this effect constitutes the basis for a method suggested by Mueller et al. [117-119,123l. It should be noted that in the method by Mueller et al., bimolecular lipid membranes are formed within a short period of time when the aqueous and nonaqueous solutions are brought into contact. This is easily understood, since the interfacial boundary - as has been shown in the PC adsorption study (see Section 3.2) - is filled with the monomolecular layer. An extended contact time must lead to its transformation into a threedimensional adsorption layer. In an earlier paper 11201, we took a closer look at the transition of thick nonaqueous films to bimolecular membranes. It was established that the black spots cease to form at a certain PL concentration in a nonaqueous solution. The P versus C curve, as seen in Fig. 20, passes through a maximum. In about 4 h after a drop of a 0.4 mMnonaqueous solution has been brought into contact with the aqueous phase, the drop becomes thinned down to a membrane several tens of nanometers thick. Such films are several times larger than the bilayer in thickness. Such thick lipid films are stable and are resistant to rupture for longer periods of time. On the other hand, no obstacles to the transformation of nonaqueous films to bilayer membranes have been observed with surfaceactive substances that were not capable of forming three-dimensional adsorption layers over longer contact times of immiscible liquids El211. Thus, the nonaqueous lipid film thinning experiments have provided unambiguous evidence for the formation, with time, of three-dimensional structures at the immiscible liquid interface, which in an independent way lends support to the conclusion drawn from the PC adsorption study. In addition, there are available experimental data in the literature indicative of the structure of the lipid adsorption layer. Those data were reported by Kruglijakov and Rovin (Ref. [1191, p.150 and Ref. I1241). In their study on nonaqueous drop thinning, they observed the formation of “metastable multilayer films”. These have been shown to form in concentrated PC solutions. A specific feature of metastable multilayer films is their spontaneous thinning down to a bilayer state which is accomplished by a stepwise decrease in thickness. In this process, several layers were “peeled off’ is succession from the boundary surface, the thickness of each layer being about equal to that of the bilayer. Therefore, one may draw the conclusion that nonaqueous lipid films, such as those reported by Kruglijakov and Rovin, possess a multilayer structure. The formation of three-dimensional layers is caused by the adsorption of PC. There are good reasons to recognize that stratification processes take place in the course of the adsorption

81 layer buildup. In such a case, one has to assign both the stratified films and the PC structures at the oil/water interface, formed with time, to liquid crystals. The experiments with a PL-stabilized emulsion (see Refs [88911) are also supportive of this suggestion. To briefly summarize, the ability of PL molecules to form a variety of self-organized interfacial structural forms must be emphasized. Depending on the lipid concentration in a nonaqueous solution and on the contact time of immiscible liquids, a monomolecular layer, a three-dimensional adsorption layer with properties of an ideal two-dimensional gas, or multilayer structures composed of bimolecular layers can form. No systematic investigation of the interfacial processes in all the above-mentioned cases has been made so far. As a matter of major significance, further studies are needed for more detailed information on the stratification effect. The elucidation of reasons for the formation of liquid crystals at the boundary surface allows us to gain a deeper insight into the nature of interfacial processes including those under an external electric field. Therefore, the essential issue appears to be an understanding of the extent to which the liquid-crystalline state is inherent in lipids at movable interfaces. Since such a concept has never been dealt with in the literature, we believe it expedient to discuss this issue in detail in the next section. 3.3. Polymorphism

at interfaces

The disposition of substances in a condensed state to exist in more than one structural form is currently known in the literature as polymorphism. Occasionally, the term “mesomorphism” is also used, but it has a narrower sense and applies to liquid crystals only which are also named, as suggested by Friedel I1251, “mesophases”. Mesomorphism is typical of PLs; commonly, it is classified into two groups, thermotropic and lyotropic. The former type is due to temperatureinduced structural transformations, and the latter is produced in the presence of a solvent. The problems concerned with polymorphic transformation of PL structures have been discussed in a number of circumstantial review papers in greater detail [62,89,108,109,112,1261. Therefore, in this section we shall restrict ourselves only to a brief characterization of lyotropic PL mesomorphism in the presence of water and to the polymorphism of lipid monolayers, and further focus our attention on the aggregation in various media, PL-water interactions, transformation of twodimensional structures to three-dimensional ones, and the eventual formation of liquid-crystalline structures at interfacial boundaries. The

main stress will be on the issues that have been studied in the last few years or have not yet been critically reviewed in the literature. 3.3.1. Aggregation in nonaqueous media PL molecules, owing to the large size of their polar and nonpolar regions (Fig. 111, exhibit amphiphathic properties. By way of example, PLs are soluble in media of different polarity. An exception to the rule among common solvents are acetone and water [62,891. The state of lipid molecules in solution is dependent on the PL concentration. In nonpolar solvents of the alkane or benzene type, PLs persist in a molecular form at concentrations as low as 0.01 m.&f[81,1271. At higher concentrations, lipid molecules associate to form aggregates. For example, PC in benzene at concentrations as low as about 0.1 mM form associates composed of 4-6 lipid molecules M9,1271, and at C 1 1 mM, micelles made up of as many as 70 molecules [79,801. The former have a spherical shape, and the latter, an elliptical shape with an axial ratio of 21 11281. The critical micelle concentration and PL micelle structure are dependent on the dielectric constant Eof the medium. The experiments with PC aqueous ethanolic solutions, carried out by Elworthy and McIntosh [791, have shown that no aggregates are formed in 93% ethanol (E= 29.0). The PC aggregation is observed to occur as the value of dielectric constant either increases (by diluting ethanol with water), or decreases (by adding benzene). The micelles having once started to form, further change of the dielectric constant of the medium in the same direction leads to an increase of the aggregation number. Elworthy and McIntosh I791have suggested that in 93% ethanol, a balance of interactions between polar and nonpolar groups of PC molecules on one side and solvent molecules on the other side, sets in which inhibits micellization. For ethanol diluted with water, the aggregation is determined by the interactions between the functional groups of the lipid molecules and the solvent; in the benzene-ethanol mixture, by interactions involving hydrocarbon radicals. The interaction type is therefore a major factor that determines the formation of two varieties of structurally different micelles: in water-ethanol mixtures, micelles are formed whose outer surface is covered by the functional lipid groups; in benzene-ethanol mixtures, inverted micelles are formed. The transition from PC micelles of one type to those of the other type occurs in a medium with dielectric constant equal to 29.0. The PL micelle size is presumably dependent not only on the dielectric constant of the medium, but also on the chemical nature of the solvent. In organic solvents with a low and little varying dielectric constant (E= 24,

83

egg-yolk lecithin is capable of forming large micelles composed of a few tens of molecules (in benzene, see Ref. [1281 and toluene, see Ref. W91) as well as associates of 3-5 molecules (in chloroform, see Ref. [1301 and nheptane, see Ref. [Sll). However, to what extent the aggregation process is dependent on the nature of the solvent has never been studied in detail. It should be noted though that PLs are not prone to form liquid crystals in organic solvents, the only exceptions being, perhaps, ethylene glycol and glycerol (see, for example, Refs 11311and11321). Under certain conditions, however, liquid-crystalline phases also seem to form in benzene. For example, gel-like structures were shown to form in supercooled, concentrated benzene solutions 11331. A detailed study showed that the gel was made up of a large number of bimolecular lipid layers separated from each other by an interlayer of organic solvent. In such a structure, characteristic of lipid liquid crystals in water, the functional groups of PL molecules point toward the benzene environment. The molecules of organic solvents with a low dielectric constant are usually located in the region of the PL hydrocarbon chains (see, for example, Refs I1341 and 11351). The formation of a benzene interlayer in gel-like structures is, in all likelihood, due to the interactions of benzene molecules with the polar lipid groups via JCelectrons [133,136,1371. 3.3.2. Hydration ofphospholipids The attachment of water molecules to the hydrophilic part of PLs is a multistage process. Each stage corresponds to a definite degree of lipid hydration. The hydration water is conventionally divided into three types: bound, trapped and free. However, the number of molecules constituting each of these types, remains a controversial issue. For example, there is nearly universal acceptance of the fact that the bound water, forming the main hydration shell, is represented by two hydration layers; still, some authors believe that the first hydration layer for PC molecules includes 5 water molecules [133-1401, while other authors contend that this number is 11 1141-1431. The composition of the second hydration layer is also, at present, a matter of dispute: here, the suggested number of water molecules varies from 4 to 10 [109,1441. Besides, out of the bound water, a fourth type of water molecules, named “inner water” (or “tightly bound water”) has been differentiated. According to the evidence from Refs [142,144,1451, this type is formed by the first 1 or 2 water molecules attached at the initial hydration stage. The division of bound water molecules into two hydration shells is based on a difference of the parameters that characterize the interaction of Hz0 molecules with functional PL groups. As an example, in Refs [142,144,1461

84

such a differentiation has been suggested from NMR data that provided plausible evidence for a different degree of binding for water adsorbed on dry lipids from the vapour phase. Therefore, it has been proposed to classify the adsorbed water into tightly and weakly bound types, which actually corresponds to the first and second hydration shells. The Monte Carlo simulation of a PC hydration process, as studied by Klose et al. 11471,has shown that the energy for interaction between the first water molecules and phosphorylcholine is 30-40 kJ/mol, which is greater than the energy for water molecule interaction in the bulk, 25 kJ/mol. As evidenced by the neutron diffraction data reported by Balagurov et al. [1481,the addition of water molecules to PC at the initial stage proceeds at a sufficiently high rate; the time constant for this process is smaller than 1 min. The fast addition of the first water molecules is followed by slower stages. At these stages, the process of water molecule addition is lowered markedly, and this trend becomes increasingly more pronounced as the lipid hydration degree rises. For example, the time constant for addition at the final stage is about 40 min. As has been shown by Klose et al. [1471,the interaction energy for peripheral water molecules with PC is by 1 or even 2 orders of magnitude lower as compared with the binding energy for the first 4 water molecules. The second stage of PC hydration is the addition of trapped water molecules [108,109,142-1441. This type of water molecule is distinct because of certain of its physicochemical properties. Thus, the rotational correlation times for the two former water types are, respectively, 0.3 and 0.8 ns [1431, as compared with 0.003 ns for the bulk water molecules. The trapped water of the hydration shell includes as many as 11 Hz0 molecules [108,109,1431. It serves as an interlayer between the main hydration shell and free water. The free water in liquid-crystalline structures is occasionally referred to as “excess” water. By its properties, it differs little from the bulk water. The complete hydration shell of an egg-yolk PC molecule, as reported by many authors 162,103,104,108,1091, includes 33 to 39 Hz0 molecules. The first 4-5 water molecules are presumably attached to the phosphorylcholine moiety (see Fig. ll), while the subsequent water molecules interdigitate close to the glycerol backbone [105,1061. 3.3.3. Solubilization of water by nonaqueous PC solutions Lipids, even dissolved in nonpolar organic solvents, retain their hygroscopicity. For this reason, nonaqueous PC solutions are capable of absorbing water in sizeable amounts even from humid air 11381. Normally,

85

dissolved PC takes up to 16 HZ0 molecules per lipid molecule [149-1511, but in diethyl ether this proportion is nearly three times as high [1521. Specifically, the amount of solubilized water remains practically proportional to the PC concentration in solution [X01. The mechanism for the hydration of egg-yolk lecithin in benzene and diethyl ether has been studied by IR and NMR techniques [152-1561. Two stages are distinguishable in this process. In the first stage the hydration shell is formed, composed in benzene of l-2 water molecules per PC molecule D551, and of 6-7 molecules in diethyl ether 11521.The attached water molecules are mostly located close to the phosphate lipid group (Fig. 11). This has been named “tightly-bound” water. This definition derives from the fact that at the initial PC hydration stage, the dynamics of both the functional groups and solubilized water molecules becomes retarded. The subsequent increase in their mobilities is commonly regarded as an indication that the formation of the second hydration shell has set in. Water, making up this shell, is by its characteristics very similar to the bulk water, as indicated by the IR spectral evidence 11521.The second PC hydration shell, like the first one, includes different amounts of water in various nonaqueous media: about 9 water molecules per PC molecule in benzene [153,1541, and over 30 molecules, in diethyl ether 11521. The solubilization of water by the nonaqueous solution proceeds through the involvement of reverse PL micelles. These become transformed into swollen reverse micelles. The transition from micelles to swollen micelles is accompanied by altered properties of the system. In particular, the free hydration shell water does not remain permanently bound to definite PL molecules, but rather forms a water pool; such a pool may be of quite significant size. Kumar et al. [1511, in their viscosity, light scattering, electron microscopy and electrical conductivity measurements, have shown that water drops surrounded by a PL layer in benzene are 50 f 15 nm in diameter, and in carbon tetrachloride, 30 + 5 nm. Therefore, the swollen micelles are a lipid-stabilized water dispersion in the nonaqueous phase, rather than simple PC aggregates. Because of the size of the microdroplets, the former dispersions may be assigned to the microemulsion class. A specific water solubilization behaviour has been noted in cyclohexanebased PC solutions. A systematic study of these systems has been carried out by Kumar et al. [151,155,1571 by making use of a variety of physicochemical techniques. Nonaqueous solutions, containing 6-15 moles of water per mole of PC (above which phase separation occurs), have been shown to exhibit anisotropic optical properties. Besides, at the ratio of 6 Hz0 molecules to 1 PC molecule, a discontinuity is observed in the con-

centration dependence for viscosity, conductivity and light scattering. Electron microscopy has allowed, to a certain extent, the distinction of cyclohexane solutions from other nonaqueous PC solutions. As the HzO/ lipid ratio of 6:l is reached, the spherical micelles start transforming to cylindrical or lamellar aggregates, and the formation of tubular structures is observed to occur; these structures with anisotropic optical properties have been assigned by Kumar et al. [151,155,1571 to liquid-crystalline species. Rather uncommon results have been reported by Scartazzini and Luisi 11581.These authors have shown that as water is allowed to dissolve in a nonaqueous solution of purified soya lecithin, thermoreversible viscous gel-like phases, named “organogels”, are formed. A list of organic solvents that have been show to be capable of forming gels contains 41 names. These are fatty acid esters, cyclic and linear hydrocarbons, amines, benzene derivatives, and other compounds. Noteworthy, is that the gelation occurs at low water concentrations. For example, in the case of n-alkanes, the addition of l-3 Hz0 molecules per PC molecule suffices to produce the effect. The structure of organogels has not been established with certainty. Also, little is known about the structure of the lipid shell of swollen inverted micelles. Certain ideas in this respect have been advanced by Malev and coworkers 11591.They used the radioactive tracer method in a study of the kinetics of water transport from aqueous solution to an n-hexanebased PC nonaqueous solution. A sizeable amount of water in the bulk of the nonpolar phase was detected within several hours, while the swelling of micelles took several days to be completed. The calculations that Malev et al. performed, assuming the localization of lipid molecules on the surface of water droplets, showed that the lipid shell was composed of one bimolecular layer and one monomolecular layer, the fact presumably indicative of a liquid-crystalline structure. For confirmation of this conclusion, one may refer to the experiment with the emulsion stabilized with PC. Dispersion formation is to be expected following the solubilization of a maximum amount of water by the nonaqueous PL solution 11501.It is a well-known fact (see, for example, Refs 189-911) that lipids used as emulsifying agents contribute to the high stability of disperse systems. Detailed investigations have provided evidence [88-91,160,1611 that the stabilizing effect is due to the buildup, around the water droplets, of a liquid-crystalline envelope composed of a multitude of bimolecular PC layers and water interlayers. It appears quite probable that such a liquid-crystalline shell already starts forming at the micelle swelling stage.

87

3.3.4. Liquid-crystalline

states

Natural PLs, containing mostly 16 and 18 hydrocarbon chains, in aqueous solution persist in an aggregated state. For example, the critical micelle concentrations for 1,2-dipalmitoyl-PC and 1,2-distearoyl-PC, as reported by Nichols 11621, are, respectively, 32 and 5 nM. However, practically no lipid micelles occur in more concentrated solutions. Instead, liposomes, mostly spherically shaped PL aggregates each composed of a water droplet surrounded by a lamellar liquid-crystalline lipid shell (see Fig. 21), have been found to form. Liposomes are formed in excess water solution. For instance, in the case of egg-yolk PC, over 45% (w/w) of water must be added to the dry lipid to obtain liposomes (see phase diagram in Fig. 21). With added water, PC starts swelling, and initially tubular protrusions called “myelin figures”, precursors to liposomes, are observed to appear [62,114,132,1631. The myelin figures have a positive sign of birefringence, which is explained by a coaxial arrangement of bimolecular layers around the central axis. The appearance of myelin figures is always indicative of the fact that the environmental temperature for PL under the given conditions is higher than the gel-to-liquid crystal phase transition temperature [132,164,1651, and the PL phase has a smectic A structure [164,1661, i.e., the structure of a liquid crystal whose planar bimolecular layers are made up of molecules with their hydrocarbon radicals in a melt-like state perpendicularly oriented to the layer of the plane (in Fig. 21, the smectic A corresponds to mesophase L, devoid of water interlayers). The myelin figures exist only in a concentration gradient of substances in the swollen states 1167,168l. As the partition of substances levels off in the course of swelling, the myelin figures seem to have a tendency to break down into liposomes [132,1671. Mechanical external action produces a similar effect. The swollen PL phases, when submitted to prolonged ultrasonic treatment, yield unilamellar vesicles which, as distinct from liposomes, have the membrane made up of a single bimolecular layer 11691. Egg-yolk PC, on addition of not more than 45% Hz0 (by weight), swells by absorbing the added water. A sufficiently fluid homogeneous transparent phase is thus formed. On examination under the polarizing microscope, a characteristic conic focal texture is distinguished typical of liquid crystals with layered structure [113,114,1671. The mesophase L, (according to the Luzzati classification) produced by absorbing a limited amount of water, belongs to multilamellar structures. As is shown in Fig. 21, it is composed of extended planar bimolecular layers (lamellae) arranged in parallel and separated by water interlayers. The PL molecules are oriented perpendicular to the bilayer surface. The func-

STRUCTURRSAT OIL/WATERIRTRRFACB Spreading

Inverted micelle

Monomer

crystal Solid

STRUCTURES

Lipoeome

SPlectlcA

/

h

,u

60

v

ROB-BIbWER

STRUCT

89

tional groups are exposed to the leaflets of water, while the hydrocarbon radicals point to the lamella interior. Within the lamellae, PL molecules are packed loosely, the degree of packing being dependent on the hydration state and quantity of water associated with the polar region of the PL molecules. So, as the Hz0 weight fraction of the egg-yolk PC mesophase is varied from 0.15 to 0.44, the surface area per lipid molecule within the lamellae arises from 0.593 to 0.717 nm2, in parallel with the bimolecular leaflet thinning from 3.58 to 2.96 nm. Simultaneously, the number of water molecules within the hydration shell increases from 8.3 to 34.5, and the free water interlayer thickness, from 0 to 1.85 nm (data are taken from Table 12-8 in the-monograph by Small [621). In egg-yolk PC, the lamellar L, mesophase is the most typical. As seen in the phase diagram in Fig. 21, this mesophase exists within a wide range of concentration and temperature. The lamellar Lg mesophase is observed to form at low (a few %, w/w) water content. In this mesophase, the PC molecules are tilted at a certain angle with respect to the base plane, as distinct from the mesophase L, perpendicular arrangement. The hydrocarbon chains are all in the trans-conformation, which provides for a more close molecular packing in the Lg, rather than in the L,, lamellar mesophase [62,89,102,108,109,111-1141. The egg-yolk PC exists also in nonbilayer liquid-crystalline states, but these are present at a temperature above 90°C. They are not shown in Fig. 21, since the phase diagram, shown therein, refers to the low-temperature region only; however, structures of hexagonal mesophase Hex, , and cubic mesophase Qa are also sketched schematically. Nonbilayer states are more typical of phosphatidylethanolamine, rather than of PC [62,89,102,111114,136l. 3.3.5. Relationship between phospholipid states at the interface The experiments on PC adsorption at the oil/water interface (see Section 3.1) and on the formation of bimolecular membranes (see Section 3.2) have shown that the monolayer covering of the inter-facialboundary transforms with time and lipid accumulation to a multilayer filling. Therefore, lipid polymorphism at interfaces is not confined only to rearrangements Opposite: Fig. 21. Schematic representation of the structures of the main liquid-crystalline mesophases and inter-facial films. The possible transitions between states and the condensed phase diagram (the low-temperature region) of the egg-yolk lecithin-water system are also shown. Notations: C -crystalline; P - two dimensional oblique or rectangular; R - three dimensional rhombohedric. Further explanations are given in the text. (Phase diagram adapted from Small [62] .)

within the monomolecular films as usually discussed in the literature. (The current concepts on polymorphic transformation of lipid monolayers at interfacial boundaries reported, for instance, in Refs [170-1741). Its mechanism is more intricate and appears to include the transformation of two-dimensional structural forms to three-dimensional ones. Now, we intend to discuss this problem in more detail. To begin with, we would like to emphasize the similarity in structural organization and packing of PL molecules within monomolecular and bimolecular films. This fact has been noted by many authors (see, for example, Refs [102,108,109,117-119,160,173,1751. A feature that adds to this similarity,is that the bilayer in question is composed of two monolayers which are brought together from the apposition of the hydrocarbon radicals. Such a concept, which at first glance appears to be rather speculative, is not in fact built on a shaky foundation. The reasons that argue for this concept have been dealt within detail by Dervichian [1761 and Albon and Baret [1771. These authors compared parameters of PL molecules within the monolayers spread on an aqueous solution surface and within the bilayers of liquid-crystalline phases. As was ascertained, from the standpoint of molecular organization, the monomolecular films in liquidexpanded and liquid-condensed states correspond, respectively, to the L, and L s mesophases. For example, Mitchell and Dluhy 11781 have established that the hydrocarbon chains of 1,2-dipalmitoyl-PC molecules in the liquid-condensed monolayers persist mostly in a trans-trans conformation, that is, as in the gel phase Ll+ On the other hand, Moy and coauthors [1791 are of the opinion that the hydrocarbon chains are tilted in a similar manner with respect to normal to the dividing surface (see Fig. 21). The main gel-to-liquid crystal phase transition is responsible for the transformation of the liquid-condensed state to the liquid-expanded state. The surface area per molecule, for instance, of egg-yolk PC within the monolayer in the liquid-expanded state varies from 0.63 to about 1.00 nm2 EN and in the L, mesophase, from 0.593 to 0.717 nm2, as the water molar fraction grows from 0.15 to 0.44 at 23°C (the latter data taken from Table 12-8 in 1621). Perhaps, this example is not the most illustrative one. Synthetic lipids, as shown by Dervichian [1761 and Albon and Baret [1771, provide a more close similarity between the molecular packing parameters for monolayers and liquid crystals. The establishment of an equilibrium between bimolecular structural forms and monomolecular films may be observed as PL spreading from crystals on the surface of an aqueous solution. This effect was noted both at the air/water [62,160,180-1841 and the oil/water 1731 interfaces. With lipid in excess, a monolayer is formed, within which the interfacial pres-

91

sure l-l, at equilibrium can be as high as 40-50 mN/m. For each individual lipid, the parameter l-l, has a strictly definite value. This value is defined by the maximal inter-facial pressure to which the monolayer can be compressed without breaking down the thermodynamic equilibrium between the monomolecular film and the bulk of the liquid-crystalline lipid phase. In other terms, if l-l = l-l,, the chemical potential of a component is the same at any point of the system [1831. The thermodynamics of PL crystal spreading process has been dealt with in detail by Phillips and Hauser 11831. Two possible routes for the formation of a monomolecular film may be envisioned: (i) from gel phase; and (ii) from liquid-crystalline phase; in other words, at a temperature below (i) and above (ii) the gel-to-liquid crystal phase transition point. In crystal spreading from the gel-like state, heat of adsorption and the monolayer thus formed is less ordered than the bilayer. This process is possible, if the entropy component of the Gibbs free energy is superior to the enthalpy component. This effect may be exemplified by phosphatidylethanolamine. On the other hand, PC crystals at temperatures below the phase transition point do not spread over the surface of an aqueous solution. Under these conditions, the surface pressure does not exceed 0.1 mN/m [183-1871. The spreading mechanism for lipid crystals remains as yet unclear. To date, the available experimental data provide evidence that the process is not confined to the surface only between phases, but extended also to the adjacent layers of the aqueous solution. Thus, for the equilibrium pressure I’l, to be attained within a monolayer, much more of the lipid must be applied than is required to form a condensed monomolecular film 1183,185, 1881. It has been suggested, therefore, in Refs 11831 and 11891 that the spreading of PL crystals at the air/water interface involves dispersion of the substance in the aqueous solution in the form of liposomes. This hypothesis has been borne out by the exchange of lipid molecules between the monolayer and liposomes in aqueous solution [190,1911 and by the formation of monomolecular films in the presence of dispersed PL in aqueous solutions 1192-1951. Gershfeld and coauthors [185,186,196,1971 believe that the transfer of PLs into the substrate is a major factor of monolayer metastability. As is known, because of desorption, a quantity of lipid greater than the theoretical estimate is needed to be placed on the interfacial boundary in order to obtain a monomolecular film. Therefore, monomolecular films should not be considered separately from the subsurface layers of aqueous solution. The true thermodynamic equilibrium, after the monomolecular film has been subjected to compression or allowed to spread freely, sets in only after

92 the film becomes equilibrated with PL molecules in the bulk of the aqueous solution. This equilibrium takes a long enough time to establish, since PL, owing to its poor solubility, is present in the aqueous phase at very low concentrations. The mechanisms of PL adsorption appear, in all likelihood, to be quite similar to those considered [171,198,1991 for the systems with sparingly compressed soluble monolayers. The occurrence of liposomes and vesicles in an aqueous solution facilitates the accelerated establishment of equilibrium in the system. For example, the generation of liposomes and vesicles allows the equilibrium interfacial pressure, as reported by Hernandez et al. [1941, to be attained within 40 min. As has already been mentioned, the lipid liposomes and vesicles in aqueous solution possess a shell built up of bimolecular layers (see Fig. 21). For the reason, the formation of a monomolecular layer entails a rearrangement of the bimolecular films at the interface. According to the data by Pattus et al. 11921, the complete transformation of a bilayer to a monolayer occurs at a surface pressure close to zero. The process proceeds at a slow rate; the increase in Il makes the process slow down or even brings it to a halt. If such is the case, one may expect the occurrence of liquid-crystalline structural forms on the dividing surface between phases. Gershfeld and coauthors [200-2021 made a detailed study of the conditions for formation of a bimolecular layer at the air/water interface for a PC dispersion in aqueous solution. Under equilibrium conditions (occasionally, a period of time of over 10 h was needed to attain the equilibrium surface pressure values), the bilayer, that covers the whole area of the dividing surface, is formed within a very small temperature interval spanning 0.2”C. Actually, this interval may be likened to a point where the condensed monolayer, according to Tajima and Gershfeld t2011, becomes transformed into a bilayer; at a higher temperature, the reverse process takes place. There is experimental evidence that the bimolecular surface film, formed under equilibrium conditions, is a thermodynamically stable structural form. Recently, Exerowa and Lalchev @031, in their study of Newton black foam films stabilized with PLs, have shown the formation of liquid-crystallinestructures from lipids at the air/water interface. The method they applied was similar to that used by us in our studies of nonaqueous films (see Section 3.2). The spontaneously formed foam films were several times thicker than the bimol~ul~ layer. These films were stable but became thin on exposure to external pressure. The film thickness was observed to decrease discretely from 32 to 12 nm by a value of 5.5 nm which was actually equal to the bilayer lipid membrane thickness. The foam film was found to contain at least four such

93

bilayers. One may therefore draw a conclusionthat PLs at the air/water interface were capable of forming not only a monolayer and a single bimolecular lipid layer, but also multilamellar structural forms. This is indicative of a stratification process at the surface of the water solution. As has been mentioned earlier in Section 3.2, the similar self-organized processes leading to the formation of stratified films take place in the oil/water system due to PC adsorption from the nonaqueous phase. The formation of a bilayer on an aqueous solution surface in the above cases occurs spontaneously, within definite concentration and temperature ranges. However, this process is amenable to control by applying compression to the monomolecular film. The transition from monolayer filling of the surface between phases to multilayer structural forms is effected when a state of collapse is reached [31,62,76,84,89,171,1981. As noted by Friberg and Larsson [1601, substances that do not possess a large polar region, such as ethyl stearate, are at first capable of forming a bilayer on the interfacial boundary. Compounds with an extended polar region, like that in PL molecules, are converted after collapse to three-layer films, that is, the thickness growth of the interfacial film occurs at the expense of the bilayer (see Fig. 21). The first evidence for the formation of such folded trilayers as exemplified by fatty acids was obtained by Ries and Kimball [2041 by making use of electron microscopy. Later, this method was improved by combining electron microscopy with the Langmuir-Blodgett technique 12051and applied to studies of the three-layer structure of lipid films, including egg-yolk lecithin. The collapse of a monomolecular film, subjected to compression under thermodynamic equilibrium conditions, is expected to occur as the surface pressure attains a value equal to the equilibrium surface pressure II, at which the PL crystals spread over the aqueous solution surface. This effect, as exemplified by 1,2-dimiristoyl-PC M34,1871, has been observed in the experiments with lipids carried out at a temperature higher than the gel-to-liquid crystal phase transition point. Otherwise, the collapse is reached at a surface pressure greater than the II, value. Gershfeld [185, 186,1971 argues that the failure to provide for the surface pressure correspondence in Il, is major evidence for the absence of thermodynamic equilibrium due to the compression of monolayers. It should be noted that the occurrence of a collapse is indicative only of an incipient formation of multilayer structures. To bring the process to completion, the compression of interface films should be continued. The bilayer that is being formed by the buckling of the monolayer from the surface (see Fig. 21) may be imagined to spread increasingly over the monolayer surface. As the interfacial film is compressed into a trilayer over the whole area, a second

94

collapse occurs followed in succession by other collapses. This process was investigated in detail by Larsson et al. 12061.They have shown that multilayer visually distinguishable films can be obtained on the aqueous solution surface via barrier-assisted compression. The process is completely reversible. The multilayer film-the barrier having been removed-becomes expanded and transformed to a monolayer; this process actually bears a close resemblance to PL spreading from crystals. 3.3.6. Concluding remarks To briefly summarize our understanding of PL behaviour at the inter-facial boundaries, there is some definite reason why lipids are capable of existing in a variety of structural forms, liquid-crystalline included. The liquid-crystalline phases on the aqueous solution surface can coexist with the monomolecular films. A true thermodynamic equilibrium in the system is established if the lipid at the interfacial boundaries attains a state of equilibrium with the lipid in the bulk solution. A change in lipid concentration, temperature, or the compression-expansion of inter-facial lipid films makes the equilibrium shift and elicits a transformation of one structural form to another. This may be illustrated by the following scheme (see also Fig. 21): monolayer

+

\ monomers

bilayer e

+

multilayer

fl liposomes

(interfacial (aqueous

boundary) solution)

mic!lles In turn, each of these structural forms can persist in a variety of phase states and experience phase transformations, which exerts a marked influence on the mechanism of structural transitions and properties of the system in question. The eventual reversible transformation of a monolayer into a bilayer and a liquid-crystalline state diversifies polymorphic transformations of lipids at interfaces as compared, for example, with mesomorphism of liquid crystals or polymorphism of monolayers. PL polymorphism on the aqueous solution surface is at present the most widely studied. Properties of monolayer films have been studied at the oil/water interface only. Our experiments on adsorption from the nonaqueous phase and formation of bimolecular lipid membranes (Sections 3.1 and 3.2, respectively [4,5,116, 1211) and the literature data concerned with emulsion formation [8891,160-1621 and the spreading of PL crystals on the surface between oil and water [73] provide evidence that monolayers are not unique among

95

structural forms; liquid crystals can also exist at immiscible liquid interfaces. However, specific features of transitions between two-dimensional and three-dimensional states have not been studied as yet. One may presume that the structural forms at the oil/water interface are more labile than those at the air/water interface, since PL molecules, in the liquid/liquid system, become desorbed not only into the aqueous phase, but also into the nonaqueous phase (see scheme in Fig. 21) where they are readily soluble (see Sections 3.3.1 and 3.3.3). By way of example, Senda et al. [921 observed a rather rapid disintegration of the PL monolayer in a nitrobenzene/water system apparently caused by the transfer of PL molecules into the bulk of the organic solvent. 4. EFFECT OF IONS ON ELECTROINTERFACIAL

PHENOMENA

Electrolytes exert a marked influence on electrointerfacial phenomena under an external electric field in a system of immiscible liquids contains PLs. Illustrative of this effect is Fig. 10 in which an interfacial tension increment versus electric intensity relationship is shown at varying potassium chloride concentration in an aqueous solution. Effects are observed both with added inorganic and organic salts. The action that the ions produce on electrointerfacial phenomena are, beyond any doubt, dependent on the ion-PL interactions at the boundary surface. The binding of ions by lipids has been considered in detail by many authors [60,68,104, 109,130,207-2161. Therefore, in this review paper we shall focus our attention on the experimental data that are concerned only with the electrointerfacial phenomena observed in a system of immiscible liquids containing PLs and that may be helpful in gaining a deeper understanding of the mechanism of interfacial processes under an external electric field. 4.1. Inorganic

ions

The introduction of inorganic salts of univalent or multicharged ions into an aqueous solution does not produce any significant change in interfacial pressure at the n-heptane/water interface in the presence of PC in the nonaqueous phase 160,681.It should be emphasized, however, that such a state is observed only in the absence of an electric field. As a potential difference is applied to an immiscible liquid system, the electrolytes, as seen in Fig. 10, produce an effect on the interfacial tension. The increment in AyEis proportional to the cube root of inorganic salt and PL concentrations. The graphs shown in Fig. 22 demonstrate these relationships.

96

16

8

6

0

0.0

0.2

0.4

0.6 ,113

0.8

1.0

(H'/3)

Fig.22. CA)Interfacial pressure at the n-~ep~ne/wa~r interface versus the cube root of aqueous potassium chloride concentration. Phosphatidylcholine concentration in alkane is 0.008 mM. Electric field intensity: (1) 0; (2) 5; (3) 10 kV/m.

The dependence of interfacial tension (or pressure) on the cube root of background electrolyte and surfactant concentrations are typical of ionic surface-active substances (Ref. 1761,p.195). If the relationships hold true in the case of neutral PC, this signifies that the lipid molecules become converted, by the action of an external electric field, to a charged form. This result agrees with observations by other authors [3,23,46,471. A plausible explanation of the change in PL properties may be due, as suggested in Ref. 1601,to the reorientation of phospho~lcholi~e (see Fig.

97

0 0

0.02

0.04

0.06 ,1/3

(y'/3)

Fig. 22. (B) Interfacial pressure versus the cube root of nonaqueous phosphatidylcholine concentration. Potassium chloride concentration in water is 1 m&f. Electric field intensity is 10 kV/m. (From Shchipunov and Drachev BOI.)

11) under the applied electric field. Normally, the dipolar functional group of the PC molecule is oriented practically in parallel (within a spread of 30) to the surface of the lamellae [102,108,2161. As shown by Stulen 12171, the phosphorylcholine changes its conformation by the action of an electric field. As the dipolar group becomes reoriented perpendicularly or at an angle to the dividing surface between the oil and water, the inorganic ions are able to penetrate the PL adsorption layer and can thereby affect the interfacial tension. The electric field, treated within the framework of such

an approach, exerts a regulatory action on the lipid-electrolyte interactions at the interphase boundaries. Undoubtedly, this effect, as mentioned in Refs I601and 1681, is essential for the function of the living cell. The mechanism for field-induced PL polarization allows a number of interpretations. One of these will be dealt with in Section 6 concerning electrohydrodynamic instability at the oil/water interface. Noteworthy is that the charging of PL molecules and the introduction of charged contaminants into the neutral PL specimens can produce a significant effect on the structure of mesophases and lipid phase transitions. For example, the addition of an ionic surface-active substance (a few mol %) to egg-yolk PC or synthetic PC leads to an unrestricted and almost infinite swelling of lipids in aqueous solutions [218,2191. The instability of planar multilamellar structures increases as well [2201. They are apt to easily transform into vesicles 12211,spherical drops surrounded by the shell of a single bimolecular lipid layer. A transition to micellar systems is also possible, and was actually observed by Muller et al. 12221, with a mere 1% of cetyltrimethylammonium chloride added to PC. In all cases, a major cause of the transition is, as has been shown by Ohki and Aono 12231,the increase of the Gibbs free energy for the planar bilayer as the surface charge density grows. Within the framework of a similar approach, the disintegration of adsorption layer at the oil/water interface under an applied voltage may be interpreted. This mechanism is discussed in greater detail in Section 6. 4.2. Organic ions Organic ions, as distinct from inorganic ions, are capable of adsorbing at the interface and therefore can afI’ectthe inter-facialtensions of a PLfilled oil/water interface with no electric field applied to the system [11,208-2101. Ions of opposite sign, on interaction with neutral lipids, are both similar and dissimilar in the effects they produce [209,214,216,224,2251. Thus, tetraphenyl boron anions show a specificitytowards the PL functional groups by forming a stronger bond with the choline residue (group -+N(CH&, see Fig. 11) rather than with the ethanolamine residue (group -+NH$ [68,208,2091.Symmetric tetraalkylammoniumand tetraphenylphosphonium cations exhibit no such specificity.Besides, their binding to lipids at the phase boundary is much weaker compared with organic anions 168,208,210,2151. However, in both instances the nature of the ion-PL interaction remains the same: both cations and anions exhibit, in their interactions, hydrophobic effects and a relationship satisfying Coulomb’s law. As shown in Refs 12082101, the hydrophobic effects are major contributorsto the binding of ions by neutral

99

PLs, while the distinctive features between ions of opposite sign, noted above, should be mostly attributed to electrostatic interactions. Precisely these interactions are believed to be responsible for the difference of 0.15 nm in the positioning of adsorption planes for organic ions of opposite sign on the interfacial boundaries filled with lipid molecules [225,2261,as well as for the different conformations the phosphorylcholine region adopts owing to interactions of PL functional groups with tetraphenyl boron anions or tetraphenylphosphonium cations 12161. Distinctions between organic ions of opposite sign also become apparent in the way these ions influence the electrocapillary phenomena involving PLs. The relevant curves are plotted in Figs 23 and 24, obtained, respectively, in experiments with sodium tetraphenylborate and tetraheptylammonium chloride. The addition of a salt of an organic anion to aqueous solution, as is seen in Fig. 23, decreases the response of the inter-facialtension at the n-heptane/water interface to the action of an external voltage. This effect is dependent on the electric field direction [2091.With the anode placed in the alkane phase (cathode branch of electrocapillary curve) or, in other words, when the external field facilitates the passage of anions from the bulk aqueous solution onto the interfacial boundary, the drop in y is minimal. As the polarity of the electrode in the nonaqueous phase is reversed, the presence of sodium tetraphenylborate decreases, to a certain extent, the sensitivity of interfacial tension to the action of an applied voltage, but this effect is less pronounced as compared with that observed in the cathode branch of the electrocapillary curve. Tetraheptylammonium chloride, similar to sodium tetraphenylborate, exerts a marked effect on the electrocapillary phenomena in a system of immiscible liquids containing PLs; however, no considerable asymmetry in the electrocapillary curves has been observed (Fig. 24). A point to be noted is that the AyEagainst E curves become less steep and finally transform into a parabola as the concentration of the salt of the organic cation in aqueous solution is raised. The resultant drop in interfacial tension at an electric intensity of 100 kV/m amounts to several units of mN/m which is of the same order of magnitude as in the absence of PLs in the system. The experimental data presented provide evidence that the action of organic ions of opposite sign on the electrocapillary phenomena with the participation of PLs, while being different in certain aspects, exhibits nonetheless a common property which shows up by inhibiting the interfacial processes in the oil/water system leading to a significant decrease in interfacial tension under an applied electric field. It should be noted in this connection that organic electrolytes increase the lipid membrane conductivity [208,210,212,2271 by penetrating into the interior space formed

100 0

F B !Yl F 4

-10

-20

-30

I -50

0

+50 E

(kV/d

Fig.23.Decrease in the interfacial tension under an external voltage versus electric intensity in the presence of 0.4 n-ubiphosphatidylcholine in n-heptane. Sodium tetraphenylborate concentration in water: (1) 0; (2) 1.0 mkf. (Drawn from data of Shchipunov and Drachev 12091 .I

by the PL hydrocarbon chains (see, for example, Refs [2251and 12281). In a similar manner, the organic electrolytes can affect the lipid adsorption layers at the immiscible liquid interface. For example, a comparison shows that tetraheptylammonium chloride starts influencing the response of the interfacial tension to an external electric field at the same concentrations (Fig. 24) at which the increased conductivity of bimolecular lipid membranes becomes observable 12271.The decrease in resistance of the adsorption layer causes a drop in voltage, which leads to a reduced polarization of the lipid structures at the interfacial boundary. This must affect on the electrointerfacial phenomena in a PL-containing immiscible liquid system.

101

-20 -50

+50

0 E

&V/m)

Fig. 24. Decrease in the interfacial tension under an external voltage versus electric intensity. Phosphatidylcholine concentrations in alkane: (l-3) 0.03; (4) 0 mM. Tetrahep tylammonium chloride concentration in water: (1) 0; (2) 0.0033; (3.4) 0.0075 mM. (Drawn from data of Shchipunov and Drachev [2091.)

The experimental data discussed provide evidence that inorganic and organic ions exert a substantial influence on the properties of PL adsorption layers at the oil/water interface subjected to the action of an external electric field. Therefore, electrolytes cannot be regarded as indifferent additives, as they were presumed to be by a number of authors [3,50,92-941. These authors believed that the salts, added to solutions, produced an increase in solution conductivity, and dismissed the eventual effect of organic and inorganic ions on electrocapillary phenomena with PLs. By contrast, experimental data published in Refs 1601,[681 and [2091 have provided evidence for important involvement of the electrolytes in electrointerfacial phenomena induced by an electric field in a system of immiscible liquids containing PLs. It should be conceded, though, that the interaction between lipids and ions under an external voltage remains poorly understood in many details. Undoubtedly, this field of research deserves an in-depth investigation and offers ample and promising opportunities for systematic studies.

102 5. EMULSIFICATION AND EMULSIONS UNDER AN EXTERNAL ELECTRIC FIELD

The production of emulsions in a system of immiscible liquids by means of an electric field has a long history, and the techniques employed to that effect have found occasional practical applications (see, for example, Refs E31 and [Ed). Commonly, surface-activesubstances are added to a system to stabilize the dispersion. Their use for the production of emulsions in alkane/water systems is not associated with any substantial modifications in the routine experimental technique. However, with lipids, the situation becomes drastically changed. We have established in previous studies D,51that the addition of neutral PLs to the nonaqueous phase leads to a nearly lOO-folddrop in the electric field strength at which an electrohydrodynamic instability emerges and dispersion of the dividing surface between oil and water becomes apparent. This section is concerned with a discussion of the concomitant interfacial processes. It should be noted that the exposure of a PL-containing immiscible liquid system to an electric field produces not only an emulsion (Fig. 81, but also a new gel-like transparent homogeneous phase (Fig. 9). These are formed at different electric field strengths: the former at high, and the latter at low values of E. In particular, at PC concentrations of l-10 mM in alkane, the water-in-oil emulsion starts forming predominantly at electric intensities above 10 kV/m,and the gel-like homogeneous phase below 10 kV/m. These two types of disperse systems will be dealt with separately. For this reason, the electric fields are by convention classified into strong and weak ones, a field strength of 10 kV/m being the dividing borderline. 5.1. Strong fields The experiments were carried out by making use of a cell, schematically sketched in Fig. 12(A). They were reported in more detail in our earlier work 121. Under an applied electric field, emulsion droplets, with a maximum diameter of 0.1 mm at 20 kV/m, are observed to detach themselves from the dividing surface between phases and migrate into the bulk of the nonaqueous solution. A series of photographs taken in succession demonstrate the initial stage of this process (Fig. 81. The emulsion droplets, under an external applied electric field, perform shuttle-like movements within a vertical plane between the lower and upper aqueous solutions [see the cell picture in Fig. 12(A)]. With time, as the emulsification process advances, the droplets start to aggregate and

103

assume an ordered arrangement. At a certain moment, droplet chains are observed to form in the nonaqueous solution; these are made up of water droplets whose number is large enough to build long chains which bridge the lower and upper aqueous solutions. Photographs and a schematic sketch of the ordered array of emulsion droplets in the nonaqueous phase containing PC are shown in Fig. 25.

Fig. 25. Photographs of a water-in-alkane emulsion between upper and lower aqueous solutions taken during electrical treatment and schematic representation of the droplet chains. Egg phosphatidylcholine concentration is 2 mM. Electric field intensity is 20 kV/m. (From Shchipunov and Kolpakov, unpublished observations.)

It is noteworthy that in order to effect an emulsification process, no electric field need be applied directly across the interfacial boundary between immiscible liquids; to this end, it suffices to place both electrodes in the nonaqueous phase. The water-in-oil emulsion, in a manner much similar to that in the previously described experiment, is observed to appear initially in close vicinity to the oil/water interface and to be later pulled into the inter-electrode space. As emulsion droplets grow in number, they start aggregating and ordering into chains oriented perpendicular to the electrode surface. The emulsion photographs, taken at the initial and final stages, are shown in Fig. 26. The experimental evidence shows that the electric field-induced emulsification in a PL-containing immiscible liquid system proceeds via a succession of interrelated stages. The initial stage is dispersion of water in alkane. The aqueous droplets migrate, by the action of an external electric

104

field, into the nonaqueous bulk phase, and aggregate to form chain-like structures. Now, let us consider each of these stages in more detail.

Pig. 26. Photographs: (AI initial stage of dispersion of water in n-heptane; (B) formation of chains from water-in-alkane emulsion into the spacing between two platinum electrodes placed in alkane parallel to the interface. The photographs were taken in succession: 64) 60 and (B) 120 s after a 20 kV/m electric field had been applied. The spacing between the lower electrode and the surface of the water solution is about 2 mm and between the electrodes about 3 mm. Egg-yolk ph~phatidylcho~ne concentration in the nonaqueous phase is 2 m&f. (Similar pho~graphs presented previously by Shc~punov and Kolpakov 121.)

5.1.1. Dispersion of water in alkane Emulsification in an immiscible liquid system is due to the electrohydrodynamic ins~bili~ of the interfacial boundary f23,53,551. The surface of a liquid is commonly agitated by ripples. They are always present on movable boundary surfaces because of thermal fluctuations but, since these interfacial waves are apparent on a microscopic scale, the liquid surface looks perfectly smooth. The application of an electric field perpendicular to the interphase bound~y reduces the velocity of surface-wave propagation. As the electric field strength is increased, a critical value of E may be reached, where there is no wave propagation [229,2301. In such a state, the mechanical stress due to the external electric field is equal to the

105

laplacian pressure. The critical field strength and the interface wavelength (h-1are related by the equation (see, for instance, Ref. [2301):

E2

cr

=

4x(egh+ yA_l)

(12)

where e is the density, g is the gravitational constant, ~J?Lin the righthand side of the Eqn (12) represents a gravitational component, and @-I a capillary component. With the electric field strength even slightly above the critical E value, the electrically-induced stresses become higher than the laplacian pressure, which r,esults in the development of an electrohydrodynamic instability. This instability is manifested by a sharp increase in wave amplitude, which was first demonstrated by Rayleigh 12311in his study of the breakdown or burst of liquid drops by the action of an applied voltage. A very similar process is observed to start at a planar interfacial boundary at a critical electric intensity. In the first stage, the wave amplitude increases exponentially with time [54,230,232,2331; as a result, the dividing surface assumes a chaotic profile 12341.In the second stage, the formation of a dispersion is observed. A study of the development of surface instability in liquid metals under an external electric field has shown 12331 that the crest of a surface wave, as its amplitude becomes sharply increased, is transformed into a narrow wedge-like protrusion. The elongation of the protrusion leads to the formation of a constriction at its base, and finally a drop is formed and becomes detached from the surface. Similar mechanisms are operative in the appearance of instability at a horizontal interface between a conducting liquid such as water and a nonconducting liquid and in spontaneous dispersion of water in oil (see, for example, Ref. 1551).To our knowledge, no papers concerned with a study of the electrohydrodynamic instability in immiscible liquid system containing PLs have been reported. Our experiments with PC have shown that the instability exhibits a temporal development after the electricfield has been applied. The time interval within which the electric field-induced interface wave emerges to reach a maximum amplitude is observed to shorten as the electric field strength is raised. In turn, the wave amplitude and wavelength in a stationary state, as seen in Fig. 7, as well as the size of water droplets dispersed in oil, grows with E.In accordance with Eqn (121,the fact of increasingamplitude and wavelength is suggestive of the gravitational nature of interfacial waves at the alkane/water interface filled with PLs. It should be emphasized once more (see also Section 2.2.) that, in the absence of PC, no visually distinguishable waves at the n-heptane/water interface, similar to those shown in Fig. 7, have been observed under the

106

same experimental conditions. To produce electrohydrodynamic instability in a PL-free system of immiscible liquids or in the presence of surfactants of other classes, a minimum 50-80 fold increase in the electric field intensity is required, according to Taylor and McEwan [541. 51.2. Migration of droplets This phenomenon, called “dielectrophoresis” by Pohl12351, is caused by the polarization of water droplets, induced by an external nonuniform electric field. The force, which acts on a suspended liquid droplet, is expressed by the following equation 12351: Fd = QE

= 2xr3+--- ;-;e)VE2 + 0

(13)

where pe is the induced dipole moment, r is the droplet radius, E, and so are the dielectric constants for water and oil, respectively. The polarization mode is determined by the conductivity ratio of the disperse phase and the dispersion medium. In this particular case, when the water conductivity is much greater than the alkane conductivity, a charge opposite in sign to the electrode potential is developed on the surface of the drop hemisphere facing the electrode 12361.Schematically, this polarization is shown in Fig. 25. The migration of colloid particles by action of an external field is expected to set in, according to Pohl’s estimation 12351,at a field strength E of about 200 kV/m. However, the PC-stabilized water-in-alkane emulsion performs shuttle-like displacements between the upper and lower aqueous solutions in the cell [see Fig. 12(A)], at 10 kV/m electric intensity (Fig. 8). We attribute the observed drop in the threshold value of E to a large difference in the dielectric constants for the dispersed phase and the dispersion medium and to the large size of emulsion droplets whose diameter can reach several tenths of a millimetre. 51.3. Aggregation The emulsion droplets are subject, under an external electric field, to a variety of transformations [53,237,2381: (i) deformation of droplet form from spherical to prolate or oblate; (ii) disintegration to smaller droplets; (iii) aggregation of droplets; and (iv) coalescence of aggregated droplets into larger droplets. Of these effects, the (i) and (ii) are observed at electric field strengths greater than 500 kV/m [235,238-2431, which is much greater than the value of E at which emulsification occurs in a PC-containing alkane/water

107

system. For the emulsion studied, typical aggregation processes are observed in the nonaqueous lipid solution (Figs 25 and 26). The formation of chain-like structures from the dispersed particles in nonaqueous media is well know 1238,2432481. This process is initiated by a drastic increase in the frequency of collisions between the dispersed particles under the voltage applied. For example, the rate constant for emulsion coagulation is proportional to the square of the external electric field strength, as was shown in Refs [2491and I2501.This reaction is in accord with the nature of mutual interactions between the particles (droplets) visualized as dipoles (see schematic diagram in Fig. 25). The force of attractive interactions between the “dipolar” droplets may be expressed by the following equation [2451: F = 4~s0 r2E2 f(r/d)

(14)

where f(r/& is a function of the droplet radius r and the distance d between the centres of interacting droplets. A similar expression has been reported in Ref. [2431. The attractive interactions are maximal when the neighbouring droplets become aligned one after another along the electrical field lines. If the particles are accommodated within a plane perpendicular to the vector of the applied voltage, repulsive forces, produced by identical orientation of the induced dipoles, arise [2381.The final result of this orienting effect due to the external electric field is the formation of chain-like structural forms, as shown in Figs 25 and 26. The attractive interaction energy for neighbouring emulsion droplets, as mentioned by Pearce and Mech 12451,is close in magnitude to the kinetic energy of the original dispersion at an electric field strength of several kV/m. This fact explains the nature of active structuring processes in the above mentioned emulsified system at E 1 10 kV/m (see Figs 25 and 26). Another essential property of an emulsion that facilitates the formation of field-induced chain aggregates in nonaqueous PL solutions is the resistance to droplet coalescence within chains. The fact that the conductivity in gaps between two aqueous solutions (Fig. 25) or platinum electrodes (Fig. 26), short- circuited through emulsion bridges is observed to increase not higher than by a factor of 10 121,which is much less as compared with that in a liquid dielectric breakdown were water tracks are formed [54,242, 243,245,247,251,2521, is indicative of this. Besides, with the voltage removed, the emulsion chains disintegrate into the separate initial dispersion. Occasionally, coalescence is observed to occur, but this process is not the predominant feature 121.

108

Interestingly, an emulsion, exposed to an external electric field and showing the shuttle-like movement between the upper and lower aqueous solutions in the cells shown in Fig. 12(A), does not coalesce on contact with the aqueous support either. However, the electric field having been switched off, the emulsion coalescence proceeds at a greater rate. The resistance of an emulsion to coalescence is determined by the properties of the adsorption layers at the oil/water interface E5,76,119,122,238,242, 2481. Any two neighbouring water droplets, each enclosed within a PL envelope, are separated, at the site of their contact, by a film structurallysimilar to solventcontaining bimolecular lipid membranes. Such a bimolecular lipid film creates a structure-mechanical barrier preventing the droplets from coalescing, which is actually the reason for the high aggregate stability of PLstabilized emulsion systems [88-91,119,159,1601. The stability of bimolecular lipid membranes to electric breakdown is an essential factor that determines the capacity of emulsion droplets to coalesce. Commonly, the electric breakdown of bilayers occurs at electric field intensities of about 10,000 kV/m, or higher [117-119,2531. As chainlike aggregates are formed in a water-in-oil emulsion, the emulsion droplets come into contact with one another, facing the opposite charges of their induced dipoles (see Fig. 25). Such an arrangement of the polarized droplets provides conditions for an electrical breakdown of the nonaqueous solution layer and the interfacial films separated water pools; this electrical treatment technique is widely used in practice for resolving of emulsions [53,55,238,242,247,2481. The high stability of bimolecular lipid membranes under an external electric field prevents the adsorption layers around the water droplets from disintegration. From this standpoint, one may explain the absence of emulsion coalescence and the highly-ordered array of droplets in nonaqueous PL solutions under an applied voltage. 5.2. Weak fields As the voltage is lowered, the emulsification in an alkane/water system is retarded and the water droplets become smaller in size 151.At an electric intensity lower than 10 kV/m, in a nonaqueous solution of l-10 mM PC concentration, the predominant formation of a new phase, rather than an emulsion and chain-like structures, is observed. The photographs are shown in Fig. 9. A different refractive index of this phase allows its identification in the alkane solution. The separated phase is homogeneous and outwardly bears a resemblance to a gel. Its density is higher than that of the alkane, but lower than the water density (note: water constitutes the gel phase 151).The electric field is responsible for the retention of the gel

109

phase within the spacing between two electrodes placed in a nonaqueous solution (see Fig. 9). With the electric field switched off, the gel sediments on the surface between immiscible liquids and dissipates with time. In order to elucidate the nature of the gel phase, the following experiment has been carried out as described in detail in our previous paper [51. The two contacted immiscible liquid phases, each containing a submerged platinum electrode, were exposed to the action of an electric field for 2030 min. Allowing the oil/water system to quieten down for a period of time lo-15 min (which was enough time for the gel to dissipate into the surrounding solution after the electric field was switched off), some 0.1 ml of the nonaqueous solution was sampled with a micropipette in close vicinity to the alkane/water interface. The sample was transferred into a cell for microscopic investigation shown schematically in Fig. 12(B). No gel or emulsion was visible in the sampled nonaqueous solution. However, with a voltage applied to the platinum elect.rodes, the formation of a gel phase in the interelectrode space was observed to occur. At first, the appearance of individual funnel-like structures -tapering down into a thread at the anode and opened wide at the cathode - was observed. They are shown in Fig. 27(A). With time, the threads become thicker and grow in number

Pig. 27. Photographs of the thread-like structures formed in alkane under an external electric field. They were taken: (A) 5 and (B) 10 min after application of a 100 kV/m electric field. When (B) was taken, the field strength was decreased to 30 kV/m. The cathode is on the left side. (A) Magnification x 100; 03) the minimal spacing between electrodes (dark bands on both sides) is 1 mm. ((A) Photograph presented previously by Shchipunov and Kolpakov 151; (B) photograph from Shcbipunov and Kolpakov, unpublished observations.)

111

several days to be completed ~149,150,1591;by contrast, a sizeable amount of gel is observed to form within 10-15 min after the beginning of the experiment (Fig. 9). The solubilized water is localized, in all likelihood, in the gel phase. Therefore, as the gel short-circuits the electrodes, as shown in Figs 9 and 27, an abrupt increase (about lo-fold) in conductivity is observed. As the “gel bridge” between the electrodes becomes broken, the conductivity drops to its initial level. The optical effect, produced by gel formation in the non-aqueous PC solution by the action of an electric field, resembles the Kerr effect. It is known (see, for example, Refs [611and 125411,that this effect is associated with the polarization and orientation of asymmetrical particles in solution and methodologically can be used for determining the particle size. We have made use of this practical application of the Kerr effect in our work [Sl and have shown that the gel is composed of particles ranging in size from several hundred to several thousand &ngstroms. These particles should be classified as microemulsions, since the PC micelles are smaller in size and, besides, no gelation has been observed in their solutions in the absence of solubilized water. The mechanism for the formation of a gel from a microemulsion becomes understood if one makes reference to the experimental evidence obtained from a study of emulsion under an external electric field. As has been shown in Section 5.1., the water droplets, suspended in alkane, tend to aggregate into chain-like structures under an applied voltage (Figs 25 and 26). The aggregation is caused by dielectrophoretic effects 12351 that originate in the field-induced water droplet polarization. It should be noted that at low field strengths, no droplet shape distortion was observed. The microemulsions appear to behave in a similar manner. The chain-like aggregation of microdroplets seems to be a major reason for both the occurrence of thegel as a separate phase and it exhibiting an optical anisotropy. 6. MECHANISMS

FOR INTERFACIAL

PHENOMENA

In Section 2.1., in characterizing the electrointe~acial phenomena with PLs, we have drawn attention to the features of prime importance that require explanation: these are the high sensitivity of the immiscible liquid interface, filled with adsorbed lipid molecules, to the action of an external electric field, and the sharp drop in interfacial tension under an applied voltage. To date, the available experimental data are sufficient enough, in our opinion, to form a general idea of the effects observed and to suggest mechanisms for interfacial processes. We now intend to take a closer look at the causes responsible for the drop in tension at the oil/water interface.

112

6.1. Phospholipid

adsorption

Since surface-active substances produce a diminution of interfacial tension, one may attempt to explain the previously described electrointerfacial effects as due to a regulatory action, exerted by the external electric field on PL adsorption. At first glance, a factor, favourable to this, is the large dipole moment of the lipid molecule; for example, different estimates made in the case of PC are 30 D [1351 and 21 D 11471. However, the water molecules that form the PL hydration shell are arranged in such a manner that their dipoles point in the direction opposite to the direction of the phosphorylcholine dipole. Thus, the effective dipole moment for the lipid molecule in the presence of water is substantially smaller in comparison to its computer value. Commonly, the former dipole moment, as shown by some authors [147,182,2551, is not greater than 4-5 D. The electric field exerts an influence on the adsorption of surfactant molecules with such a dipole moment, and the interfacial tension is lowered by a few mN/m [6,13,21,26,46,256,2571. Nonetheless, this cannot be accepted as an explanation for the interfacial drop of 20-30 mN/m mentioned in case of electrointerfacial phenomena with PLs (Fig. 4). 6.2, Transport

of substances

The low current strength in the alkane/water system suggests the absence of a charge carrier flow across the dividing surface between immiscible liquids. However, there is a substantial water mass transfer due to emulsification processes. The mass transport across the inter-facial boundary affects the interfacial tension, but the major effects are those produced by surface-active substances (Ref. [76], p.319 and Refs 12581and 12591).This is manifested by a decrease in tension on the order of several mN/m. For this reason, the observed inter-facial tension drop of 20-30 mN/m under an external electric field in the presence of PLs cannot be explained as due to the mass transfer across the dividing surface between immiscible liquids. 6.3. Phospholipid-stabilized

emulsions

There has been no evidence reported in the literature suggestive of a strong effect by surfactant-stabilized emulsions on the oil/water interfacial tension. Moreover, the emulsification, as induced by an external electric field, results in an increase, rather than a decrease, of tension which is due to the desorption of substances from the inter-facial boundary. This effect was observed by Popov [13,361 for cetyltrimethylammonium bromide

113

in the systems nitrobenzene/water and methylbutylketone/water as well as by Senda and coworkers 150,921in their experiments with 1,2-dilauroylPC in a nitrobenzene/water system. The drop in tension due to emulsification under an external electric field has also been observed in an alkane/ water system; at field strengths that induce the appearance of visibly distinguishable water droplets in a nonaqueous PC solution, the interfacial tension becomes practically independent of electric intensity (Fig. 4). In certain instances, y has been observed even to slightly increase. Therefore, emulsification cannot be a cause of the sharp drop in interfacial tension observed in PL-containing systems under an applied electric field. 6.4. Microemulsions The gel phase is composed of a microemulsion (Fig. 9) and is formed under an electric field which, even if slightly varied in electric intensity, is capable of producing a substantial alteration of the interfacial tension at the oil/water interface (Fig. 41. Therefore, it seems reasonable to link the field-induced interfacial tension drop to the occurrence of gel in the nonaqueous PL solution. The more so because the extremely low interfacial tension of 0.00001~.001 mN/m at the oil/aqueous solution interface 156-581 is a basically specific feature that distinguishes a microemulsion from an emulsion. The gel forms, as shown in the photograph of an optically anisotropic layer in Fig. 9, at the liquid/liquid interface; therefore, the occurrence of gel structural forms with very low y in the close vicinity of the dividing surface will, undoubtedly, produce a marked decrease in the initial interfacial tension which is equal to 20-40 mN/m under zero external field. In our opinion, the formation of a microemulsion in an immiscible liquid system, containing PLs, is the major reason for the observed oil/water interfacial tension drop under an applied electric field. The mechanism for the occurrence of very low interfacial tension in liquid/liquid systems has been dealt with in a number of review papers (see, for example, Refs 13% 581); therefore we omit their discussion in the present paper. Of special concern, in our opinion, is the elucidation of the mechanism responsible for electrohydrodynamic instability at the liquid/liquid interface and for the formation of microemulsions under weak electric fields. At present, the only reported evidence in the literature was concerned with the preparation of emulsions under strong electric fields. The specific behaviour of an inter-facial boundary, filled with PL molecules, under an applied electric field is associated with definite properties of the adsorption lipid layer. It has been shown above, in discussing

114

adsorption (Section 3.1.) and the thinning of non-aqueous films (Section 3.2.), that PC molecules form a thick interfacial layer, rather than a monolayer, at the oil/water interface. The transition from a monolayer to a multilayer filling of the dividing surface is accompanied by a markedly reduced stability of the liquid/liquid interface under an external electric field. A number of experimental results lend support to this statement. The interfacial tension of monomolecular films of octadecanoic (stearic) and pentadecanoic acids, as well as of sodium dodecyl sulphate, as established by Middleton and Pethica [2601, is not responsible to an applied voltage. At low field strengths, the introduction of cholesterol or cetyltrimethylammonium bromide, capable of forming a monolayer at the oil/water interface [30,120,2611, produced no electrohydrodynamic instability [1,21. It should be noted, that bimolecular lipid membranes are stable under an external electric field; their interfacial tension tends to decrease by several mN/m as the field strength reaches a level of 10,000 kV/m (see, for example, Refs 11191 and 126211. The bilayer breakdown produced by the electric field is observed at roughly the same field strength [117-119,253l. However, the swelling and dispersion of multilayer lipid phases are influenced by small voltages [2631. These processes, as emphasized by Dimitrov 12641, take place under relatively weak electric lields.Their intensities in lipid multilayer structures, based on the experimental conditions and results reported by Dimitrov et a1.12631, may reach lOO--1,000 kV/m. This minimum level is 10-100 times less than the value of the field strength at which structural alterations in the bimolecular lipid membranes are observed. Noteworthy is the fact that the electric field strength at which the lipid dispersion occurs tends to decrease with the growth of lipid multilayer thickness [2631. Therefore, the transition from monomolecular and/or bimolecular films to the multilayer state is concomitant with the exhibition by PLs of novel properties; in particular, an enhanced sensitivity to an applied voltage. We have shown above (see Section 3), with reference to our experimental results and literature data, that PLs at movable interfaces are capable of self-organizing into liquid-crystalline states. Generally speaking, it should be noted that the filling of the boundary surface with the molecules of compounds capable of forming liquid crystals, does not produce an ultralow tension at the air/water or the oil/water interfaces. This is seen in the adsorption data both for PLs 14,81,82,92,941 and for a number of mesogenic compounds of other classes, for instance, those studied by Tamamuschi 12651. However, characteristic of liquid crystals is a dependence of the interfacial tension on the orientation of molecules at interfaces (see, for example, Ref. [2661). As shown by Strula et al. 12671, the value of

115 o changes by 10-15 mN/m with molecular rearrangement. An essential point is that the orientation of molecules in liquid crystals becomes altered on the application of weak mechanical, electric, or magnetic fields [114,266,2681. Therefore, if liquid-crystalline properties are inherent in an adsorption lipid layer, then specific features of those properties may become more manifest under external action. The enhanced sensitivity of the liquid/liquid interface to an electric field, observed in electrointerfacial phenomena with the participation of PLs, should be attributed, in our opinion, to the formation of liquid crystals at the interphase boundary as a result of adsorption. It should be noted that the electric field produces no effect on dry PL samples. The action of an applied voltage shows up on hydrated PL samples, already at the initial swelling stage [2631. In swollen crystals, the external electric field leads to structural instability and to textural alterations [2691. Badalyan and coauthors 12701have shown that the arrangements are accomplished via reorientation of the dipole fragments of PC polar groups, involving large molecular assemblies, rather than single molecules. The rearrangement process has thus got a cooperative character. This cooperativity renders the liquid-crystalline systems more responsive to external factors. A manifest sign of enhanced responsiveness is the electrohydrodynamic instability which is observed at small electric field strengths [114,266,268,2711. As evidenced by our data (see above, Sections 3.1 and 3.2) and those reported in the literature [88-91,119,124,159,1601, lipids on their adsorption at liquid/liquid interfaces, form interfacial films of the smectic type, composed of a number of planar parallel bimolecular layers alternating with layers of water (Fig. 21). The occurrence of electrohydrodynamic instability in smectics has been studied in detail, and an appropriate mechanism has been suggested (see, for example, Ref. I2681). We have made use of this mechanism in formulating our phenomenological model on the origin and development of electrohydrodynamic instability in lipid adsorption layers at the oil/water interface. Figure 29 is illustrative of this. The mechanism for instability in smectics is based on the conductance anisotropy. Inorganic ions are practically incapable of penetrating through a bimolecular or a monomolecular lipid film, but are capable of migrating within the water interlayers in the lateral plane [Fig. 29(A)]. The difference in conductivities, measured perpendicular and parallel to the inter-facial boundary, may reach 5-6 orders of magnitude. It should also be noted that the movement of PL molecules from one bilayer leaflet to another called “flip-flop” is highly restricted (see, for example, Ref. tlO21). The flip-flop becomes discernible only under electric fields with strengths

116

Fig. 29. A schematic representation of: (A) adsorption layer; (B) bending moment as generated by an external field on a wave-like deformation; (C) dispersed adsorption layer and a water-in-oil microemulsion. See text for further explanation. (From Shchipunov, unpublished.)

of 10,000 kV/m [102,108,253,2721. By contrast, lipid molecules exhibit a high lateral mobility in a single leaflet of a bilayer [102,108,2731. Besides, inorganic ions are capable of migrating on the surface of bimolecular and monomolecular films; in particular the surface mobility of the proton is as high as its mobility in the bulk aqueous solution 12741. The instability is associated with microscopic waves on the dividing surface between phases which are due to thermal fluctuations. As shown in Fig. 29(B), an electric field, applied perpendicular to the interfacial boundary, generates local ion currents on the ripples; this is conducive to a spatial separation of oppositely charged ions within the lamellar plane and to a development of local space charges. It should be pointed out that a single bimolecular lipid layer has a high stability towards an external electric field. Thus, the development of defects and disintegration of the bilayer are observed at electric intensities of lo-100 mV/m 1117-119,2531. The electrostriction effects, observed on lipid films, are rather insignificant 12751. However, the bimolecular membranes are susceptible to an easy bending deformation caused by thermal fluctuations, or by the action of an external electric field 12721. The critical potential difference at which the bending of a single bilayer can be observed is high 12761. However, the critical potential tends to decrease significantly on passage to multilayer systems owing to the cooperative nature of processes in liquid crystals [272,2771. In addition, the bilayer bending is facilitated by the difference, however small it may be, in the interfacial tension of the two bilayer surfaces 12781.Thick interfacial layers, produced by PL adsorption at the oil/water interface, are characterized by

117

sizeable concentration gradients for all components, which, undoubtedly, makes these layers more responsible to external factors. The space charges, developed on microscopic surface waves, interact with the external electric field, which results in the occurrence of a bending moment [Fig. 29(B)]. Since bimolecular lipid structures, especially multilayer ones, are easily susceptible to bending even at small field strengths, a deformation of the adsorption layer occurs which finally develops into an electrohydrodynamic instability (Fig. 7). As the wave amplitude reaches a critical value, the adsorption layer, depending on the experimental conditions, breaks down [see Fig. 29(C)] into an emulsion (Figs 8 and 26) and a microemulsion (Fig. 9). The electric field exerts a dual effect on this process: initially, it facilitates the bilayer bending, and then, as evidenced by studies of the mechanism of liposome and vesicle formation [262,2791, their closure into spherical droplets because of a decrease of the boundary energy. The formation of microemulsions is attended by a sharp drop in interfacial tension (Fig. 4). Besides, the aggregation of microdroplets into chains by the action of dielectrophoretic effects is conducive to the formation of an optically anisotropic layer at the oil/water interface (Fig. 19) and of a gel in the bulk of the nonaqueous PL solution (Figs 9,27 and 28). The proposed mechanism for the generation of an electrohydrodynamic instability at a dividing surface between immiscible liquids, filled with PL molecules, and the disintegration of the adsorption layer is not at variance with the available experimental evidence. In addition, the mechanism is an aid to understanding the results obtained in the studies of ion influence on interfacial phenomena under an external electric field (see Section 4). This also argues for the veracity of the suggested mechanism. Indeed, as follows from our experiments with inorganic electrolytes (Section 4.1.), neutral PC, under an applied voltage, exhibits properties similar to those of ionic surface-active substances. An analogous conclusion, although from a different standpoint, has been made by other authors [3,23,46,471. We are inclined to believe that the observed development of the charge on lipid molecules at the interfacial boundary is merely apparent. In fact, this effect is due to a spatial separation of oppositely charged ions within the adsorption layer plane with the ensuing occurrence of space charges in the ripples presented at the oil/water interface [Fig. 29(B)]. Girault and Schiffrin [31 consider this effect from another standpoint. They have suggested that the external electric field facilitates the addition of H-ions to the phosphate group, taking into account an increased concentration of protons near the interphase boundary. The phosphate

118

group having thus become protonated, the PC molecule retains an uncompensated positive charge on the choline residue (see Fig. 11). However, this interpretation of phenomenon can hardly be accepted. An argumentative criticism of this concept has been made by Kakiuchi et al. [SOI.We agree with the reasoning of these authors; in addition, we wish to point out that a PC monolayer, spread on a mercury drop surface, remains neutral under the same experimental conditions 1961.No change in the pK value of the phosphate group that presumably could arise due to the action of electric field has been observed in studies of bimolecular lipid membranes. Anyway, at neutral pH of an aqueous solution, at which, according to Girault and Schiffrin 131, the lipid molecules should have acquired a charge, the membrane remains neutral (see, for example, Refs [68,81,2091). Besides, the drop in interfacial tension most often is not influenced by the sign of the potential difference applied (see Fig. 4), which is difficult to explain from the standpoint of increased proton concentration near the interphase boundary. On the other hand, the direction of electric field is without significance for the interfacial processes when the development of a charge is due to a redistribution of ions within the lamellar plane on the wave-like deformations [Fig. 29(B)]. The suggested mechanism for electrohydrodynamic instability at the immiscible liquid interface has been borne out, in some different ways, by experiments with organic electrolytes. Organic ions, as distinct from inorganic ions (see Section 4.2), are capable of penetrating through the lipid films. Therefore, they can exert an influence on the conductance anisotropy of smectic liquid crystals. This property of organic ions is of decisive importance for the verification of the electrohydrodynamic instability mechanism, since a major idea of this mechanism is the occurrence of space charges arising from the conductance anisotopy of the adsorbed PL layer [see Fig. 29(B)]. The major results are represented in Figs 23 and 24. As seen, the addition and the gradual increase in organic salt concentration in the aqueous solution decreases, and occasionally even completely removes, the effect of high sensitivity of inter-facial tension to an electric field. For example, with tetraheptylammonium chloride, this effect has been achieved at a concentration of 0.0075 mM (Fig. 24). A comparison with the concentrational dependence for bimolecular lipid membrane conductivitypresented in Refs 12271and D301 has shown that the influence of the tetraalkylammonium salt on the electrointerfacialphenomena with PLs correlateswith the, increasinglygrowing, film ionic permeability. The effect of the high sensitivityof interfacial tension to an applied voltage is eliminated as the tetraheptylammonium chloride concentration in aqueous solution approaches a value at which the

119

bimolecular membrane conductivityis observed to increase by about 1Wfold. To the same extent, the conductance anisotropy of lipid multilayers must decrease as well. In liquid crystals, this leads to an increase in the critical potential difference.responsible for development of the electrohydrodynamic instability [268,2711.Presumably, the organic ions exert a similar action on the adsorbed lipid layers at the oil/water interface. This is conducive to a decrease in conductivityanisotropy and, consequently, to a reduced polarization of interfacial films under an applied electricfield. One will easily perceive that this salt effect eliminates the major cause for the generation and development of an electrohydrodynamicinstability in adsorbed PL layers at the immiscible liquid interface. Viewed thermodynamically, the dispersion of interfacial lipid films sets in as the PL chemical potential difference for the dispersion and the planar adsorption layer tends to zero. Commonly, PC in the low-temperature region forms stable bilayer mesophases L, and Lp (see phase diagram in Fig. 21). The transformation to a nonbilayer state, for example, to an inverted hexagonal HexI mesophase, requires a supply of energy. For this reason, this process occurs only at increasing temperature. The observable phase transition from the bilayer state to the hexagonal mesophase has been studied in detail. As proposed by Siegel 12811,it is effected via breakdown of the bilayer into inverted micelles. Apparently, the adsorbed lipid layer at the immiscible liquid interface undergoes dispersion in a similar manner. Distinctive features of this process are that an emulsion and swollen micelles are formed and the driving force is an external electric field, rather than a supply of heat. The free energies for inter-facial lipid films at planar and curved interfaces may be divided into two components as shown below: AG, = AG,, + AG,O

(15)

and AG, = AG,

+ AG,(E)

W-3

Here, in the right-hand side, the first term in both cases refers to the molecular component of free energy, and the second term, to the electrostatic component. The difference between the AGr,,,,of a planar adsorption layer and the AG,, of an emulsion (providing that the composition, packing density and conformation of PL molecules at the interphase boundaries are the same) is equal to 43cnr2,or the free surface energy of dispersion (here r is the droplet radius and n is the number of droplets). The disin-

120

tegration planar interface film in the oil/water system sets in, as the difference between the AGp and the AG, becomes comparable at least with the energy of thermal agitation. For this reason, the contribution of electrostatic components to free energy is an essential factor in establishing the condition for emulsification. The estimation performed by Ohki and Aono [2231provides evidence that, in the case of a bilayer, AG&!?) rises more steeply than AG,O. Their main result is represented graphically in Fig. 30 as the dependence of the electrostatic component of free energy on the net charge of PL molecules in bilayers, cylindrical micelles, and spherical micelles. The point at which the curves intersect thus determines the value of the critical charge for the breakdown of the planar bimolecular film. Within the framework of a similar approach, the disintegration of the adsorption layer at the oil/water interface due to space charge generation under an external electric field (Fig. 29) may be explained. 15

10

5

0

eq

(~4..&10”~

e.6.u.)

Fig. 30. Specific relative free energy versus net charge eq at the surface of: (1) bilayers; (2) cylindrical micelles; (3) spherical micelles. (Redrawn from data of Ohki and Aono [2231.)

7. PROPER PERSPECTIVES AND POSSIBLE BIOLOGICAL RELEVANCE

Our discussion of electrointerfacial phenomena with PLs has, in essence, become focused on the properties and behaviour of egg-yolk PC in an immiscible liquid system. It should be admitted that the behaviour of lipids, exposed to an external electric field, is at present understood only in general. Therefore, the available evidence, both theoretical and ex-

121

perimental, is by no means complete; perhaps, some results and conclusions may happen to be misleading. Nonetheless, the electrointerfacial phenomena, produced in oil/water systems containing PLs by an applied external electric field are quite remarkable and may undoubtedly be classified as a separate group of inter-facialphenomena. The effects of decreased inter-facial tension and electrohydrodynamic instability at the boundary surface and the formation of emulsions and microemulsions by the action of a weak electric field are of importance from the standpoints of both theoretical knowledge and practical application. Of special interest is the self-organizations of PLs into liquid-crystalline structural forms at the oil/water interface and the formations of gels, stabilized by an external electric field, from a microemulsion in the nonaqueous solution bulk. The practical application of electrointerfacial phenomena, as yet little studied, is a matter for the future. However, one may expect that the effect of a markedly reduced inter-facial tension at the oil/water interface under an applied voltage will be of interest for use in practice. An attractive feature of this effect is that it is induced by a weak field at quite small (0.00141 m&f) PL concentrations. Besides, the electric field-induced methods for the production of emulsions and microemulsions may gain acceptance because of their high efficiency. These methods produce disperse systems in large amounts and within a short period of time. An essential feature is that the emulsions and microemulsions are stable only under an external electric field, which provides the possibility, by applying the voItage in an on/off-switch regime, to control the buildup and breakdown of the disperse systems. A handicap to practical applications of the field-induced effects is that they are observed only in the presence of PLs, prohibitively expensive surface-active substances of natural origin. Therefore, a search for synthetic substitutes of natural lipids appears to be an important task at present. Our preliminary experiments have shown that this is a realistic goal since PLs are not the only species capable of exhibiting electrointerfacial phenomena in an immiscible liquid system. A number of compounds, listed in Table 1, exert an influence on the stability of the liqui~iquid interface under an external electric field in a manner similar to that of the reviewed lipids. The electric field, at which wave-like deformation at a dividing surface between oil and water are produced, drops nearly lOO-fold in strength in the presence of the above mentioned surface-active substances [2821. An examination has shown that of the available surfactants only those capable of forming liquid crystals of the smectic type exert an influence on the inter-facialelectrohydrodynamic instability. The said capacity has been estimated by observing the swelling of surface-active substances in water,

77

20-100”

Yes

No

N,N-Dihexadecylethylenediamine (charged form)

N,N-Dihexadecylethylenediamine (uncharged form) -

0.043

0.10

0.01

0.048

0.15

0.12

20-60

45

30

20

T(“c)

No

Yes

Yes

Yes

Effect

1

1

0.1

1

0-60d

60

25

10

25

20

20

20

C(mMD E(kV/m) !KC)

Electrohydrodynamic instability

aCblis the criticxd concentration for black spot formation (see Ref. @831). bCb, is the critical concentration at which the film transforms into a bimolecular state over the whole film area (see Ref. [283]). ‘Temperature range in which properties of amphiphiles were investigated. dElectric strength range in which electrohydrodynamic instability was tested.

20

Yes

Didodecyldimethylammonium bromide

20

Yes

C6m(tib

cbl(mM)’

Formation

WC)

Bimolecular membranes

Myelin figures

Egg-yolk PC

Amphiphile

TABLE 1 Formation of myelin figures and bimolecular membranes; appearance of electrohydrodynamic instability at the n-decane/water interface, (From Shchipunov et al. 12821)

ti

123

the appearance of myelin figures and the formation of planar bimolecular films. The experimental results thus obtained have been reported in Ref. [2831. In part, these are reproduced in Table 1. As seen, the synthetic surfactants that are capable of forming smectic liquid crystals (like egg-yolk PC), enhance the sensitivity of the oil/water interface to the action of an external electric field. Surface waves, similar to those shown in Fig. 7 and produced roughly under the same experimental conditions, were observed on the boundary surface of immiscible liquids. When neither myelin figures, nor bimolecular films were formed (as, for instance, with N,Ndihexadecylethylenediamine at neutral pH in aqueous solution) in a similar fashion, no e~ectrohydrodynamic instability at low field strength was observed, despite the fact that in an acid solution, this compound behaved like egg-yolk PC B821. The observance of PL-assisted electrointerfacial phenomena poses another important question: are weak fields of other than an electric nature capable of affecting the adsorbed lipid layer at the oil/water interface? It is a well-known fact (see, for example, Refs [114,2~,271~) that liquid crystals are exceptionally susceptible to external action; for instance, molecular reorientations and rearrangements in liquid crystals are observable not only by the action of weak electric fields, but also by magnetic fields and mechanical stresses. These features are inherent in lipids, organized into bimolecular layers. For example, effects due to a magnetic field have been reported in planar multilamellar structure 1284-2861, liposomes [285,287-2901, and lipid tubules [2911.A specific behaviour was that spherical structures were distorted to spheroidal ones [287-2891. Other effects, produced by a magnetic field, were an increased surface pressure in PC monolayers [2921, an affected gel-to-liquid crystal phase transition in 1,2-dipalmitoyl-PC vesicles 12931,an increased permeability of liposomes of the same PL samples toward neutral compounds [2941; the magnetic field also produced an effect on the background conductance in inorganic electrolyte solutions and on the electric breakdown of planar bimolecular lipid films [2951, also on the rate of thinning of nonaqueous films to bimolecular membranes depending on the magnetic field direction 12961,and on the contraction of the bilayer area at the expense of the torus and microlenses [2971. An essential point is that the calculated diamagnetic energy for orientation of a single molecule within a lipid bilayer amounts to a mere one millionth of its kinetic energy [284,285,290,2921. For this reason, the noticeably increased sensitivity to a magnetic field was attributed to PL domains or clusters [292-2941 as well as to liquidcrystalline structures at the oil/water interface and to cooperative rearrangements within them [295-2981. One may expect the eventual

124 occurrence of such effects in an adsorbed lipid layer also. Anyway, magnetic and electric fields, as shown by Li and coauthors [2991,produce a practically identical orientation effect on tubules made up of polymerizable diacetylenic PC. A spontaneous orientation of nonaqueous lyotropic liquidcrystalline lipid phases in an external magnetic field [300,3011, and the aggregation of micelles, composed of 1,2-dimyristoyl-PC and sodium glycocholate, into the liquid-crystalline state [3021 have been reported as well. The latter effects appears to bear a resemblance to the gelation in nonaqueous egg-yolk PC solution, produced by an applied voltage (Figs 9, 27 and 28). Therefore, we anticipate the possibility of observing magnetic field-induced interfacial effects in a PL-containing immiscible liquid system similar to those produced by an external electric field. We wish to put special emphasis on the biological relevance of electrointerfacial phenomena with PLs. As is known (see, for example, Ref. [1141), in the living organisms numerous physiological functions are performed by tissues and cells possessing a liquid-crystalline organization. Therefore, the role of liquid crystals in biological systems is an issue of special interest. Model experiments have been widely used in these studies. The oil/water interface in itself is a model of the cell membrane in biophysical and biochemical explorations. In practice, most often monomolecular and bimolecular films are used. The self-organization of lipids into liquid-crystalline states at the interphase boundary opens up new prospects for studying processes of the living cell, since this creates a realistic possibility for designing models of multilayer cellular structures. A number of examples may be cited in support of this statement. Multilayer PL-containing structures in living organisms are, for the most part, represented by nervous tissues and sense organs (see, for example, Refs 13031and 13041). A well-known example is nerve fibre covered with a sheath of myelin; schematically, it is shown in Fig. 31(A). The manifestation of nervous activity in the living organism is associated with the conduction of nervous impulses of an electric nature along the nerve fibre. The velocity of nervous impulse transmission is determined by the medullary sheath surrounding the fibre. In a medulla-covered neuron, the velocity of the nervous impulse propagation is proportional to the fibre diameter, while in a nonmyelinated one, it scales to the power of 0.61 of fibre diameter [3051. Besides, the removal of both the sheath and cytoskeleton from Schwann cells significantly affects the elastic properties of the axons [3061. The ensuing consequences are not favourable, since the movement of a nervous impulse is primarily associated with mechanical deformation of the nerve fibre. Outwardly, these deformations resemble a swollen structure [3071 or a transversal wave [306,3081; in both instances,

125

Fig. 31. Schematic diagram showing the nerve fibre (A) and the Pacinian corpuscle (B). bodied from Prosser 13031and Shepherd [3041.I

they move along the membrane simultaneously with the action potential. A number of interpretations of the observed effect have been suggested. Malev and coauthors 1306,309l have chosen, as the basis of their mathematical model, the membrane electrostriction in the region of a nerve fibre in which an impulse passes. Iwasa and Tasaki [3101believe that the cause of the observed swelling is a conformation change of the membrane proteins as calcium ions are replaced by alkaline metal ions. One might have acquiesced in these authors’ concept, be it supported by model experiments; still, no satisfactorily adequate model for the excitable tissue has been reported in the literature. In our opinion, an appropriate candidate for such a model may be a system of PL-containing immiscible liquids. For example, the application of an external electric field generates an interfacial wave conducted over the oil/water interface (Fig. 71, which in principle is reminiscent of the movement of a nervous impulse. The relationship between mechanical and electrical phenomena in the living organism is manifest in many sense organs. These special structures consist, for the most part, of nerve endings enclosed within a multilayer sheath, similar to that of a myelinated nerve fibre [303,3041. They may be exemplified by a widely spread Pacinian corpuscle, schematically shown in Fig. 31(B). In shape, these resemble a distorted spheroid whose wall is made up of a large number of concentrically arranged, unclosed bimolecular membranes. The multilayer capsule, thus constructed, in all likelihood, plays an important role in the perception of a mechanical

126 stimuli such as, for example, pressure from the external and internal environment. Thus, Loewenstein and Mendelson 13111 showed that the characteristics of the receptor potential changed drastically after the multilayer sheath surrounding nerve endings had been removed. The multilayer capsule appears to play the role of a mechanical filter acting only through transmitted high-frequency stimuli. The sensitivity of the sense organs is exceptionally high. A displacement of the Pacinian corpuscle surface, as small as 1 l_unand even less, suffices to initiate impulses in the nervous system (Ref. [3031, p.352; Ref. [3041, p.330). The appearance of an electric potential in response to mechanical stimulation of a dividing surface between immiscible liquids has been noted in Refs 1391,H-41and 1451.It may be presumed that the filling of the oil/water interface with PL molecules augments the inter-facial sensitivity to simulation not only by an electrical field, but also by a mechanical stress. This finding may constitute a basis for designing highly responsive sensors and for modelling the processes that occur in the sense organs of living organisms. One might have proceeded with further outlining the potential perspectives of practical applications and possible biological relevance of PLmediated electrointerfacial phenomena that have been reviewed in this paper; however, it must be conceded that, even giving the imagination power to wander, one will fail to present an exhaustive list of all the possibilities. For this reason, we have confined ourselves to those that appear to be the most promising ones and with which, in one way or another, our studies are concerned. The practical use of electrointerfacial phenomena may prove to be quite different from their actual prognosis, however, this can emerge only as a result of purposeful and detailed studies of these inter-facial effects under external electric field and their applications. ACKNOWLEDGEMENTS The authors are grateful to Professor E.Ya. Kostetsky for the lipid specimens. We wish to thank Drs A.N. Popov and G.Yu. Drachev for their assistance in the performance of a number of experiments and for stimulating discussion of the material of this review paper. We are grateful to Dr V.V. Malev for his valuable criticism of different problems observed in the manuscript during its evaluation. Thanks as well to our colleagues at the Institute of Electrochemistry (Moscow), Dr L.I. Boguslavsky, Professor Yu.A. Chizmadzev, Professor V.S. Markin and their collaborators, who made it possible to discuss the interfacial phenomena under an external electric field at a number of seminars, that were instructive for us and has allowed us to understand more about the phenomena discussed.

127 REFERENCES 1 2

3

4 5 6

7 8 9

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water interface: adsorption and interfacial phenomena in an electric field.

Interfacial effects produced in an immiscible liquid system by the action of an external electric field have been considered. The addition of small am...
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