The colloidal nature of phospholipid monolayers

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The colloidal nature of phospholipid monolayers

A. Miller, C.A. Helm, H. Möhwald

To cite this version:

A. Miller, C.A. Helm, H. Möhwald. The colloidal nature of phospholipid monolayers. Journal de

Physique, 1987, 48 (4), pp.693-701. �10.1051/jphys:01987004804069300�. �jpa-00210487�

The colloidal nature of phospholipid monolayers

A. Miller, ^{C. A. Helm and H. Möhwald}

TU Munich, Physics Department ^E22 (Biophysics), D-8046 Garching, ^F.R.G.

(Requ ^{le 1er} septembre 1986, révisé le 11 décembre, accepti le 18 d6cembre 1986)

Résumé.

On étudie des monocouches de phospholipides ^{et d’un} acide gras fluoré par des ^mesures de

potentiels ^{et de} pressions ^de surface, ^{et aussi en} observant par microscopie de fluorescence les mouvements de domaines sous l’ effet de champs électriques inhomogènes. Les résultats montrent systématiquement que les forces électrostatiques à longue portée ^sont produites par les dipôles ^{situés sur les esters} glycériques ^{et non} pas par les têtes polaires de la monocouche. Les domaines lipidiques solides observés en coexistence avec une

phase ^{fluide montrent un excès} de densité de dipôles qui ^{varie entre 50 à 200 mD/nm 2 avec des} signes ^différents

pour les phospholipides ^{et les} acides gras. Ceci explique les directions opposées des forces qui s’exercent sur les domaines lipidiques placés ^{dans les} champs électriques. ^Les énergies par domaine pour ^ces forces sont de l’ordre de 104 kT. Nous discutons l’origine moléculaire de ces moments dipolaires de surface.

Abstract.

Monolayers of different phospholipids ^{and of a} fluorinated fatty ^{acid are studied} by ^surface

pressure and potential measurements and by fluorescence microscopic observation of domain movement in

inhomogeneous electric fields. The data consistently ^{show that} long range electrostatic forces result from molecular dipole ^{moments of the} glycerin ^{ester and not} from the head group region ^{of the} monolayer. ^In coexistence with fluid phase ^solid lipid domains exhibit excess dipole density between 50 mD/nm2 and 200 mD/nm2 with different signs ^for phospholipids and fluorinated fatty acids. The latter fact explains ^the opposite direction of forces on lipid domains in electric fields. Energies per domain corresponding ^{to these}

forces were calculated to about 104 kT. The molecular origin of the surface dipole ^moments is discussed.

Classification

Physics ^Abstracts

87.20

68.10

64.70M

1. Introduction.

Lipid monolayers ^{at the air/water} interface ^have

encountered increasing ^{interest as models of} biologi-

cal membranes [1, 2], ^{but also as} models and precursors of well-structured organic ^films promising

many technical applications [3]. Beyond ^that they

also exhibit very interesting physical properties ^as

two-dimensional and interfacial systems. ^{Their struc-}

ture is determined from the amphiphilic ^{character of}

the constituent molecules causing ^a preferential alignment in that the hydrophilic parts point ^towards

the water surface, ^the hydrophobic parts towards the air. As the molecules _possess a dipole ^{moment the} monolayer ^{can be} considered as an array of dipoles

each with a component perpendicular ^{to the water}

surface [4]. ^This gives ^{rise to} long range electrostatic forces and thus to very peculiar condensation

phenomena and formation of periodic superstruc-

tures [5].

Whereas the above features are principally intellig-

ible there rises the central question inhowfar they

dominate other forces such as van-der-Waals attrac- tion and line tension between different phases. ^To

answer this, ^{one has to determine} quantitatively ^the dipole ^moments in different monolayer phases ^and

relate these to molecular and supramolecular ^struc-

tures. This is done within this work by combining

classical surface potential measurements with fluorescence microscopic observations of lipid

domain movement in inhomogeneous electric fields.

The data are interpreted ⁱⁿ conjunction ^with simple

model calculations showing consistently ^{that the}

essential contributions to the surface dipole ^moment

result from a polarization ^{close to the} hydrophobic

membrane region ^for charged ^and uncharged phosp- holipids ^{as well as for} partly fluorinated fatty ^acids.

2. Materials and methods.

The lipids L-a-dilaurylphosphatidylethanolamine (DLPE), L-a-dimyristoylphosphatidylethanolamine (DMPE) ^and L-a-dimyristoylphosphatidic ^acid (DMPA) (Sigma) ^{were used} without further

purification. ^The ethoxylated perfluorinated ^car-

bonic acid (FCS), C6F7(CH2hO(CH2h-COOH, ^was

a gift of Drs. U. Scheunemann and W. Interthal, Hoechst, Frankfurt, ^{BRD. It was} chromatographi-

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphys:01987004804069300

694

cally pure. The dye probe dipalmitoyl-nitrobenzo- oxadiazol-phosphatidylethanolamine (DP-NBD-PE) (Avanti) ^was previously ^{shown to} be insoluble in solid lipid ^{domains, but is soluble in the fluid} phase.

The water used was deionized and filtered using ^a

Milli-Q system. pH ^was established using ^NaOH (Fluka, p.a. grade). Contamination by ^{divalent ions}

was prevented by adding ^{10-5 M EDTA} (ethylenediamine tetra-acetic acid, ^sodium salt, Sigma) ^{to the} subphase. ^All experiments ^were performed ^{at 20 °C. The} phospholipid/dye ^or fatty acid/dye ^{mixtures were} spread ^{from a chlo-}

roform/methanole (9 : 1) mixed solvent.

Surface potential measurements were performed

in the laboratory ^{of Dr. D.} Mobius, Gottingen ^with

the vibrating capacitance technique (6, 7). ^{A con-} ducting ^circular plate (diameter ¹ cm) periodically changes ^its distance d from the surface (frequency

420 Hz, ^{d ~ 0.2} mm, amplitude 0.1 mm). ^{The cur-}

rent towards the counter electrode in the subphase ^is compensated, ^{and the} voltage necessary for this

compared ^{with that for the clean water} surface is the surface potential. The accuracy of potential ^measure-

ments was better than 10 mV. ^{In the} experiments

the molecular area could be varied continuously

while simultaneously measuring potential ^{and sur-}

face _pressure.

The film balance and fluorescence microscope

have been described previously [8]. ^Of special ^rele-

vance for this study ^{is the} fact that the surface texture was observed via an objective ^{lens in the}

bottom of the film balance. Thus manipulations

could be performed by arranging additional tools above the surface. We made use of this by moving ^an

electrode above the surface. The electrode consists of the metal tip ^{of a} syringe, having ^a tip ^{radius of}

about 5 _iLm. It can be moved in three orthogonal

directions by ^a micromanipulator (Bachofer) ^{to an}

accuracy of 5 _Rm. The electrode can be charged ^to yield potentials ^of up ^{to ±} 100 V with respect ^{to the} bulk water that is grounded ^{via the} objective ^{lens in}

the trough bottom. On charging the electrode we

observe a distortion of the surface geometry ^{due to} elevating ^{the water level. However, the} findings reported ^{are not due to} gravitational effects, ^{as the}

latter would be independent ^{of the} sign ^{of the}

electric field. Yet we observe a change ^{in the} sign ^of

force on changing the field direction.

3. Experimental ^results.

Figure ¹ shows surface pressure and surface potential

as a function of molecular ^area for the uncharged phospholipids ^DLPE (Fig. la) ^{and DMPE} (Fig.1b).

Additionally given ^{is also} the molecular dipole

moment u derived- as described in chapter ^{4. Most} pronounced ^{in the} pressure/area ^{isotherms is the} change ^{into a} nearly horizontal slope ^on increasing

the pressure above ^a critical value 7T C. This is known

Fig. ^1.

Surface pressure 7T’, surface potential ^{AV and}

vertical dipole moment A

Eo . OV A as a function of molecular area A for DLPE (Fig. la) ^{and DMPE} (Fig. 1b) monolayers ^{at the} air/water interface. In the phase ^coexist-

ence region ⁱⁿ figure 1b between Af

⁶⁹ A2lmolecule and

At

⁴² A2/molecule the fraction f ^{of fluid} phase may be assumed to depend linearly ^{on A as} also sketched. The dotted non-horizontal line corresponds ^{to an} extrapolation

of thè JL (A) function if the coexistence range would extend towards f

^{0 without} changing ^{the nature of the}

solid phase. (T = 21 °C, ^{10-5 M} EDTA, ^no buffer, ^i.e.

pH 5.5).

to correspond ^{to the onset of a} fluid/gel phase

transition which, ^{due to the} shorter chain length

occurs at a much higher pressure for ^DLPE

(irc

³³ mN/m) ^{than for DMPE} (7T c

⁵ mN/m).

Comparing ^surface pressure and ^surface potential ^as

a function of molecular area there is obviously ^a good correspondence :

For pressures below 1 mN/m and molecular areas

above 90 Å 2/molecule there are large potential ^fluc-

tuations due to extreme surface heterogeneities. ^As

observed in fluorescence microscopic ^studies [9]

there is a coexistence of large domains of high ^and

low lipid density (« gas/fluid» transition). ^These heterogeneities disappear ^on increasing ^the pressure to establish the fluid lipid phase. ^{Then the} potential

increases towards a defined and reproducible ^value.

For pressures between ¹ mN/m and 7T c the potential

increases continuously ^with pressure ^as qualitatively expected ^{for an increase in molecular} density. ^The flattening ^{of the AV versus area isotherm for}

pressures slightly ^above 7T c will be explained ^{as due}

to the transition to a gel phase exhibiting ^{a smaller}

molecular dipole ^moment. Increasing ^{the surface}

pressure beyond ^the plateau region ^{causes the} poten-

tial to rise rather steeply ^with increasing density.

This will be discussed as due to the onset of another

phase transition of the gel phase.

The above features are qualitatively ^{identical for}

the two lipids, the diference being ^{that due to the}

extensive fluid phase region ^{of DLPE this} lipid ^is

more suitable for studying ^this phase ^whereas DMPE, showing ^the pronounced plateau region ^best

serves to study the coexistence region.

Qualitatively ^{similar behaviour of surface} pressure and potential is obtained for monolayers ^{of the} charged lipid DMPA, displayed ⁱⁿ figure ^{2 for}

different subphases. Abrupt ^{and not} well defined

potential changes may appear for very low pressures

( ^{1 mN/m,} Fig. 2a) ^followed by ^a gradual potential

increase with density in the fluid phase. ^{In the}

Fig. ^2.

Surface pressure iT, surface potential ^{AV and}

vertical dipole ^{moment u} calculated as described in

chapter 4.1 ^{for a DMPA} monolayer ^at pH 5.5,

1 mM NaCI (Fig. 2a), ^at pH 11, ^{1 mM NaCI} (Fig. 2b) ^and

at pH 11, ^{10 mM NaCI} (Fig. 2c). (T

^{20 °C,} 10-5 M EDTA).

coexistence region above iTc the potential ^is nearly

flat and at the end of the coexistence phase ^the potential ^{exhibits another} steep ^rise (Figs. 2b, c).

The fluorinated fatty ^acid FCS studied exhibits a

break in the slope ^{of the} pressure/area ^isotherm

analogously ^{to that} corresponding ^{to the} fluid/gel phase transition of phospholipids. ^{In accordance}

with this are also similar features of the OV versus area isotherm. The main difference between FCS and phospholipids resides in the fact that changes ⁱⁿ

the absolute values of the potential ^{are about a}

factor of two larger ^{for FCS} and, ^{what is most} important, ^have opposite signs ^{for the two classes of} lipids (Fig. 3).

Fig. ^3.

Surface pressure ^7r, surface potential ^{AV and}

effective vertical dipole moment u

Eo AV. A for the fluorinated fatty ^{acid FCS.} (T

20 °C,10-5 ^M EDTA, ^no buffer, ^i.e. pH 5.5).

The difference in sign of the surface potential ^is

also reflected in the response ^{to an} electric field as

visualized in figure ^4. Increasing ^{the surface} pressure above iTc gel phase lipid ^{domains are} formed. ^These

domains are observed as dark areas in the fluor-

escence micrographs ^{because the} dye probes ^used

are more soluble in fluid compared ^to gel phase lipid [9, 10]. Placing ^a tip ^{electrode above the water}

surface one expects ^{a field} geometry ^{as sketched at}

the bottom of figure ^4. Upon charging ^{the electrode} gel phase phospholipid ^{domains are} pulled ^{under the}

electrode for negative potential (Fig. 4a) ^and repel-

led for a positive potential (Fig. 4b). ^{This holds for}

the neutral phospholipids ^{DMPE and DLPE as well}

as for the charged lipid ^DMPA irrespective ^{of its} degree ^of dissociation (measured ^{for 0.2 a} 1.5). ^The electrostatic force is directed oppositely

for gel phase ^{domains of FCS} (Fig. 4c).

4. Analysis and discussion.

4.1 EVALUATION OF SURFACE POTENTIAL DATA.

4.1.1 Homogeneous monolayer.

^{In case of an}

uncharged monolayer ^{the surface} potential AV ^can

696

Fig. ^4.

Movement of crystalline (dark) lipid ^domains

under a tip electrode about 20 _um above the water surface. Figure ^{4a :} DMPA, pH 11,

^{= 18} mN/m, A

63 A2lmolecule, ^electrode potential - 100 ^{V ;} Figure ^{4b :} DMPA, pH 11,

^20.5 mN/m, A

⁵⁶ A2lmolecule,

electrode potential + 100 V ; Figure ^{4c :} FCS, pH 5.5,

7T =

29 mN/m, ^A

³³ A2lmolecule, ^electrode potential

-100 V ; Figure ^{4d :} Sketch of geometry assumed for calculation of forces : Circular discs at the water surface

(Z

0) ^move horizontally under the influence of a charge

q smeared ^over the surface of a- sphere with radius rK. (T

20 °C, 10-5 ^M EDTA).

be related to the density p ^of dipoles ^oriented perpendicular ^to the surface according ^to

( Eo

^{8.9 x} 10-12 C2 /m2 N).

As p is proportional ^{to the molecular} density ^{one can}

convert it into a dipole ^moment per molecule u obtaining [6, 11]

ii determined from equation (2) ^is given ⁱⁿ figure ^1.

One realizes u

^const. for surface pressures

betvyeen ^{1 mN/m} and 7Tc ^{where the} monolayer ^{is in a} single homogeneous (fluid) phase. ^{This also means}

that the molecular orientation does not change ^while varying ^{the surface} pressure within this range. One obtains almost identical values of A ^{for the two} lipids showing that u ^{does not} depend ^{on chain} length.

If the monolayer ^is charged ^{one has} additionally ^to

take into account the potential drop qio ⁱⁿ going ^from charged ^{interface to} bulk solution yielding

qf 0 ^{can be} calculated from Gouy-Chapman theory [12, 13] taking ^{into account that the} degree ^of

dissociation of lipids ^{in turn} depends ^on qio. ^The

surface charge density a is related to the density n ^of

monovalent ions in the subphase according ^to

(k

^{Boltzmann constant, T}

^absolute tempera- ture, e

elementary charge).

Neglecting binding of ions other than protons ^the

mass action law for the dissociation of phospholipid

head groups yields

([H+ ]

^bulk proton concentration).

The intrinsic dissociation constants Kl ^and K2 for

dissociation of two protons ^{from the} phosphatidic

acid head groups ^were assumed to be 60 M-1 [13, 14] ^and 108 M-1, respectively. The latter value is not known for phospholipids ^{but deviates} by ^less

than an order of magnitude ^{from the} corresponding

value known for phosphatidic ^acid. Calculations

varying this value did not show a significant ^in-

fluence.

Connecting equations (4) ^and (5) ^{we obtain a non-} analytical equation ^{that can be solved} numerically

for t/10 and then also obtain the surface charge density ^{and the} degree of dissociation [14]. ^{As a}

result of the calculation figure ⁵ gives ^the degree ^of

dissociation and the surface potential ^{as a} function of molecular area A for three different ionic conditions where experiments with DMPA were performed.

Most important for the data evaluation is the poten- tial qio that amounted to between - 175 mV and

-

275 mV. As the measured AV is some hundred mV (Fig. 2) ^but positive, ^one may conclude that the

dipolar contribution to AV exceeds t/10 and is of

comparable magnitude.

One also realizes that the area dependence ^of t/10 ^{can be} linearized over a limited range, e.g.

between 60 and 120 A2/molecule, 4io ^{= a +} bA, ^with parameters a, b given in the first columns of table I.

Experimentally ^these parameters ^can be determined

by assuming ^{also a} linear relation 4/0 = a’ + b’A

and adjusting ^{a’ and b’ in such a} way that u ^obtained

from equation (3) ^is independent ^{of molecular area}

for the monolayer ^{in the fluid} phase. The latter condition is justified ^from the observation for the

uncharged monolayer ^{where u does not} depend ^on

pressure while being ^{in the fluid} phase. Comparison

of parameters ^{derived from the} experiment ^with theoretically calculated values in table I shows a

reasonable agreement. ^{This also} justifies the assump- tions used in the calculations. Among ^{these the most}

critical ones may be the neglect of sodium ion

binding ^and assuming ^{a value of} K2 ^{=108 M-1 1 for}

dissociation of the second proton ^{of DMPA.} Varying

Fig. ^5.

Degree of dissociation and potential qio ^calcu-

lated as described in chapter ^4.1.1 for the DMPA mono-

layers ^of figure ^{2. A :} pH 5.5, ^{1 mM} NaCI ; ^{B :} pH ^11, 1 mM NaCI; ^{C :} pH 11, 10 mM NaCI. Parameters : T

20 °C, K,

⁶⁰ M-I, K2 ^{=108 M-I.}

Table I.

Parameters _a, b derived from the numerical calculation of g/o

^{a + bA and} parameters a’, ^b’

obtained from surface potential ^{data as described in the}

text.

the corresponding parameters ^would yield ^{an even}

better agreement ^between theory ^and experiment,

but this would result in too many adjustable par- ameters without getting ^{a better} physical insight.

The latter statement also holds concerning ^an improvement ^{of the} constancy in 4 ^{while the mono-} layer ^{is in the fluid} phase (see Fig. 2). Nevertheless,

one realizes that the analysis yielded ^{an almost}

constant u and that values determined for the fluid

phase ^of phospholipids ^{are of} comparable mag- nitude. They range ^between 0.64 D and 0.77 D per molecule for DMPA in different ionization states and are 0.52 D and 0.54 D for DLPE and DMPE, respectively. ^This again indicates the consistency ^of

the analysis ^{and also that the} type ^{of head} group does not grossly ^{alter the} dipole ^moment.

The fluorinated fatty ^{acid was studied as an} example ^{of an} amphiphilic compound with surface

potential sign opposite ^{to that of} phospholipids ^and

of fatty ^acids containing exclusively hydrocarbon

chains. However, because there is presently ^much

less information available on this class of compounds

an analysis ^{as extensive as above is not} performed.

Hence we do not discriminate between molecular

charge ^and dipole ^{moment and in} figure ³ give merely ^{an effective} dipole ^moment JLeff defined as

This value describes the behaviour of the monolayer

in electric fields and also allows conclusions on

pressure induced orientations of molecules in the fluid lipid phase. ueff ^{as a} function of molecular area

in figure ^{3 is} nonmonotonous even for the fluid

phase. ^The potential contribution gio ^{due to} partial charging ^{of the} carboxylic ^{acid head} group is negative

and increasing in absolute value with molecular

density. ^{This causes} u I

I geff I - C (A) ^with

C (A ) being positive ^and larger ^for large ^{areas A due}

to the .po (A) relationship ^{which is less} steep ^than

inversely proportional. Thus g ^{increases on increas-} ing the pressure towards ^iT indicating ^a change ^of

molecular orientation with surface pressure. This

can be verified, e.g. by subtracting ^{an estimated}

value of .po

200 mV from the measured value AV and then determining g ^from equation (1). ^{For a}

more refined analysis, however, ^{more refined data}

at different subphase conditions would have to be collected.

4.1.2 Inhomogeneous monolayer.

^As monolayers undergo ^several first order phase transitions ^one

generally expects coexistence of phases ^{with different}

surface potential for certain pressure regimes. ^To

take this into account in the analysis ^one may

envisage ^{two extreme} situations :

(i) The lateral dimensions of coexisting lipid

domains are of comparable ^{size or} larger ^{than the air}

electrode area. The surface potential may then

assume any value between those of the homogeneous coexisting phases depending ^on the fraction of these

phases ^under the electrode. This condition seems to hold .for pressures below 1 mN/m where strong potential fluctuations are encountered (e.g. Fig. 1, Fig. 2a). ^These fluctuations cannot be assigned ^more specifically ^{to definite molecular areas and will}

therefore not be considered further.

(ii) ^{The more} interesting ^{case is that where}

domain dimensions are much smaller than the elec- trode size. This holds for coexistence between fluid and gel phase ^domains for pressures above _{1T, where}

gel phase ^domains with sizes below 100 f.Lm ^are

698

observed [9, 10]. ^{For this case the data} analysis ^is

refined the following way :

Concerning ^the measurement, ^{the two} phases ^can

be represented by ^two parallel capacitors C, and C2.with magnitudes proportional ^{to the} correspond- ing ^areas Fl, F2 ^and by parallel voltage ^sources V1 ^and V2 according ^{to their surface} potentials (see Fig. 6). The external voltage AV necessary ^{to com-} pensate ^{the current I}

Il ⁺ 12 is measured as

surface potential. ^{For I}

^{0 one obtains}

and

Hence

Fig. ^6.

Equivalent electrical circuit to describe the surface potential ^{AV of a} monolayer ^with coexisting regions ^of potentials V1 ^and V2 ^{and areas} proportional ^to C1 ^and C2.

According ^to equation (8) ^{AV is the area} weighted

average of the surface potential ^{of the} coexisting phases. ^The potential ^{can in turn} be converted into

an average dipole moment tt that relates to the

molecular dipole ^moments ILl’ IL2 in the correspond- ing phases according ^to

In equation (9) f

N, + 2 N2 ^{N1 + N2} N 2 ^{is the fractional} number (N2 ) ^{of total molecules} (N1 + N2) ⁱⁿ phase 2, ^{which we} henceforth will consider the fluid

area.

The validity ^of equation (9) ^{is most} clearly ^demon-

strated by inspection ^{of the} coexistence region (above 7Tc) ^{of the DMPE} monolayer (Fig. la).

Assuming ^a coexistence between a

solid » phase ^of

molecular area 42 A2 and a fluid phase ^{of area} Ac corresponding ^to w,, f depends linearly ^on

molecular area as sketched also in figure ^{la. Conse-} quently according ^to equation (9) A depends linearly

on molecular area. This is experimentally ^observed

with DMPE for 0.4 f ¹ and, although ^less pronounced, ^for the other monolayer systems ^for high enough liquid ^{area fraction.} Extrapolation ^of

the linear V versus A plot ^towards f

^{0 then} yields

the dipole moments A ^{in the}

^{solid »} phases given ⁱⁿ

table II.

There are obviously strong deviations from lineari- ty ^on reducing the fraction f of ^{fluid area below 0.4.}

These can be ascribed to a structural change ^{in the}

«

solid » phase, ^{as was deduced from} Synchrotron ^X-

ray diffraction [15] ^and fluorescence microscopic

data [16]. ^{Then it was} also shown that the

solid »

phase coexisting ^{with the fluid one for} pressures only slightly above 7rc is ^one of slightly higher ^molecular

area than expected ^{for the solid} phase. Taking ^this

into account in the analysis ^would yield ^{a correction}

of data on dipole density given ^below by ^{less than}

2 % and is thus negligible.

4.2 DIPOLE DENSITY IN FLUID AND SOLID PHASES AND FORCES IN ELECTRIC FIELDS.

The last column in table II also gives ^an effective difference in dipole density determined with equation (6) ^{and with the}

Table II.

Dipole density p and differences ⁱⁿ dipole ^densities for systems ^{studied in this work.}

model derived to analyse ^the heterogeneous ^mono- layer. This value is given since it is basically ^deter- mining ^solid phase ^{domain movement in in-} homogeneous electric fields discussed below.

Inspection of table II shows that dipole density

differences Ap ^{amount to between 5 % and 20 % of}

the absolute values of _g. This means that errors in the determination of p ^{can lead to errors in} Ap of up ^to 50 %. On the other hand the signs ^of Ap ^{are correct as} they ^are given by ^the sign ^{of the} slope ^{in the} coexistence range of the AV ^{versus area}

diagram.

Values of p ^{do not differ much for} phospholipids

under various ionic conditions indicating again ^that they possess the ^same groups determining ^the dipole

moment.

The situation is entirely ^{different for FCS where}

the sign of p ^and Ap ^{as well as the absolute} magnitude ^{of the} dipole potential ^are distinguished

from that of phospholipids ^{as well as of other} fatty

acids [6] containing exclusively hydrocarbon ^chains.

This proves the ^dominant influence of the fluorocar- bon moiety.

Irrespective of molecular interpretation ^one may

use the data of table II to calculate forces on solid

lipid ^domains. Considering ^the tip ^electrode geomet- ry (Fig. ⁴ bottom) ^we calculated the electric field

assuming ^{the water} surface conducting [17, 18]. ^At

the interface the field has only ^a perpendicular

component E, ^{and the force F on a} dipole ^with

component P. ^is

F points ^{into a direction r} parallel ^{to the surface with}

r

0 being ^the position ^{of maximum field} strength

under the electrode. For a domain of radius ro the effective dipole ^moment Pz is calculated from the difference in dipole density according ^to

If h, ^the electrode distance from the water surface is much larger than ro, the maximum electrostatic energy gain (or loss) Gmax ^{of a domain} pulled ^under (or repelled from) the electrode is given by

The charge q ^{of the electrode} is related to the tip radius rt ^{and the} potential ^U by

To calculate Gmax ^{we use the} following parameters :

-

U = 100 V as applied ^{in the} experiment ^of figure ⁴

- rt

5 _um determined from microscopic inspec-

tion of the tip

-

ro = 5 ^{um as} measured for the domains of

figure ⁴

-

åPeff

⁵⁰ mDlnm2 as given ^{for DMPE in}

table II

-

h

20 _um. This value was estimated from a

vertical movement of the electrode after touching

the surface. It is the uncertainty ⁱⁿ this value that determines the error in Gmax ^which may be ^an overestimate by up ^{to a} factor of 3. With these parameters ^{we obtain} Gmax ^{= 140 eV - 6 000 kT.}

This clearly ^{shows that it is} possible ^{to move solid}

domains against ^{thermal motion,} and in fact it is

generally ^{not thermal but} convective motion which

frequently ^{disturbs the} experiment.

The above value of Gmax ^{is about a factor of}

30 smaller than that estimated previously [17]. ^This

is due to the fact that we now could use measured values of the dipole ^moment density which turned out to be considerably ^{smaller than} anticipated. ^On

the other hand one realizes that APeff ^differs only by

a factor of up ^to 10 for different lipids ^{and enters} linearly ^{in the} energy calculation. This means that the orders of magnitudes ^of the forces will be similar for different systems.

The above calculation showed that due to the colloidal nature of solid lipid domains, essentially being dipolar ^discs, reasonably high forces result in the electric fields applied. ^{This also} implies ^{that we}

do not have to discuss _any other nonquantifiable

effects like trapping ^of charges ^{in head} group ^or

hydrocarbon ^{tails to} explain ^the observed forces. An

especially strong argument ^{in favour of the above} picture also results from a comparison ^of findings

with phospholipids ^and FCS: surface potential

measurements have revealed a different sign ^{of the} dipole ^moment difference between fluid and solid

phase ^{for the two classes of} compounds. ^As expected

within the above model also the forces on solid domains are of opposite orientation.

4.3 MOLECULAR INTERPRETATION OF SURFACE PO- TENTIAL DATA.

4.3.1 Homogeneous fluid phase.

Figure ⁷

sketches a presumed arrangement ^{of the} polar

groups ^{of the} three types of molecules compared ⁱⁿ

this work at the air/water interface. Included by

arrows (from

^to

⁺ ») ^{are also local} dipole

moments due to polarization ^{or due to} charge

accumulation at the interface. Since we want to discuss the experimental findings ⁱⁿ view of the molecular structure we have to exclude the influence of the water structure at the interface that is changed by the presence of surfactants. This is imaginable,

because the surface potential ^{of the free water}

surface amounted to about - 500 mV (for Millipore water) ⁱⁿ our-experiments ^{and we} basically ^measured

the difference from this potential.

700

Fig. ^7.

^Probable arrangement ^{of DMPE and FCS at the} air/water interface. Included by arrows are positions ^of largest dipole ^moment contributions. DMPA is oriented similar to DMPE but lacks the ethanolamine head group and the corresponding dipole.

The strongest argument against the dominance of the water structure results from a comparison ^{of the}

surface potential signs ^{which are} negative ^{for the}

fluorinated fatty ^{acid and} positive ^for fatty ^{acids with} hydrocarbon ^chains [6] ^{as well as} phospholipids.

Hence even for the same hydrophilic group opposite signs ^{of the} potential ^{are measured} excluding ^that

the water structure determines the potential.

Now considering ^the positive sign ^{of the} potential

for charged ^and uncharged phospholipids ^{we deduce}

from figure ^{7 that the} charged phosphatidic ^{acid and}

the uncharged phosphatidylethanolamine ^head

groups would give ^a negative contribution, ^{and the} only positive contributions result from a polarization

of the carbonyl groups. This ^{means that} contribu- tions of the former _groups are of minor relevance.

This conclusion is also valid for phosphatidylcholines

where the same sign ^{of the surface} potential ^was

measured [19] ^{and is obvious} comparing ^{data and}

molecular structure. Yet although ^{there existed}

surface potential ^{data for} phospholipids ^{in the fluid} phase qualitatively ^{identical to ours the} head group

region ^was usually ^{assumed to be} responsible ^{for the} potential ^{and the} origin ^of long range electrostatic forces [4, ^5, 20]. ^{That this is not the case has the} following ^{reasons :}

-

Polar groups protruding ^{into the water} phase

are highly screened due to high conductivity ^and

dielectric constant of water.

-

Due to the large gradient ^{of the dielectric}

constant e across the interface dipoles in the sub-

phase ^{are screened} by image forces above the water level [21]. ^These image forces reduce long range interactions by ^{more than 3 orders of} magnitude ^but

are less effective considering ^short range interac- tions. This fact seems to explain why the influence of the head group charge ^on lipid phase transitions of vesicles [13] ^and -monolayers [14] ^is strong ^and

theoretically well describable.

The strong influence of the carbonyl groups has

already ^been pointed ^out by ^{Paltauf et al.} [19]

comparing phospholipids ^{with and without these}

groups. Using ^the interfacial dipole ^{moment of}

0.36 D for the C

0 bond [22] ^one may estimate that they ^are responsible for the total potential ^if they ^{are tilted} by ^an angle ^{of 20° with} respect ^{to the} surface. On the other hand one realizes that there

are also large in-plane ^{moments which} may give ^rise

to ferroelectric transitions [23].

Looking ^{at the structure of FCS we see that the} potential determining ^{groups are at the interface}

between hydro- ^and fluorocarbons. The electronega-

tive character of fluorine giving ^{rise to this} polariza-

tion has already ^been recognized previously [6, 24].

The fact that the potential determining group ^resides in the hydrophobic ^chain region ^also explains ^the

strong dependence of u ^on surface pressure ^of the fluid monolayer. ^On compression ^{the chains are} aligned ^{to be more} vertically oriented with respect ^to

the surface thus effecting ^{a more} negative potential.

On the other hand groups closer ^to the polar ^head

group region ^{are less sensitive to} any pressure

change ^as long ^{as the} lipid phase ^is preserved. ^{This is} supposedly ^{valid for the} carbonyl groups and ^ex-

plains ^the pressure independence of tt ^{for fluid} phospholipid monolayers.

At the end of this section we should also remark that the conclusion that dipolar contributions of the head group region ^{to the surface} potential ^{are of}

minor relevance is in accordance with the fact that u

measured for phospholipids ^{with different head}

groups ^{does not differ} drastically.

4.3.2 Fluidlsolid phase coexistence region.

^A major contribution of this work results from the fact that experiments ^and analysis ^also considered the fluid/solid phase coexistence region. ^{A molecular} interpretation ^of corresponding dipole ^moment changes will, however, ^remain speculative, ^because

there effects of less than 10 % of the overall values

are discussed. This means that we can no longer

discard the influence of changes ^{in the} head group

region that may result from reorientations ^or from a

change ⁱⁿ water structure affecting ^the screening ^of

forces.

We can still exclude the direct influence of the water polarization that may be different under a

fluid and a solid lipid phase. ^{This may} change ^the potential ^{into one direction} irrespective ^{if the} lipid ^is phospholipid ^{or FCS. However, on} solidifying ^the monolayer u changed ⁱⁿ different directions for these classes of compounds.

On the other hand changes ^{in the absolute values}

of JL ^on increasing ^{the solid area} ratio occurred in a

similar _way suggesting analogous mechanisms : In-

creasing ^the pressure above irc ^one first observes a more or less clearly pronounced linear decrease of JL

with area A and then a steep ^increase in 1L ^{for areas}

below which also the _pressure increases. Yet one has

to be careful in drawing analogies ^too far, ^because

the two classes of compounds ^are vastly ^different.

Considering first the linear decrease in g ^{with A}

for phospholipids ^{the most} appealing ^{idea is that the} carbonyl group orientation changes in the solid

phase ^{where the} hydrocarbon ^{chains are condensed}

and almost vertical to the surface. Assuming ^a

decrease of the angle ^{between bond} orientation and surface from 20° to 3° would explain the observed values. The fluorocarbons in their condensed phase

are distinguished ^from phospholipids ^{in that the} limiting ^area per chain is larger (

³⁰ A2 compared

to 20 A2 for hydrocarbon chains) ^and therefore the

carboxylic ^head group has ^a higher degree ^of

freedom. Assuming that in the solid phase ^the carboxylic head group, due ^to coulombic repulsion

with neighbouring groups would protrude ^further

into the water phase ^{would cause a} potential change

into a direction as measured for _pressures slightly

above _{’TT C.}

At first glance ^one might expect that the conden- sation of fluorocarbons involving ^{a chain} alignment

also effects the chain contribution to the dipole

moment. This, however, ^would correspond ^{to an}

increase of u, in contrast to the findings. ^The

absence of this influence _may be due to the fact that the chains are almost vertically ^oriented already ⁱⁿ

the fluid phase ^near irc.

Another explanation ^{of the} linear decrease of tL

would be that screening of head group ^forces varies

due to a different water structure or water level in different phases. ^{We cannot} exclude this possibility

because it is also consistent with the findings : ^the

head group contributions would be changed ⁱⁿ

absolute values not in sign. Nevertheless, ^this picture

is highly speculative ^and synchrotron X-ray experi-

ments are under way ^to conclude on this.

There is presently ^no consistent picture ^{to con-}

clude on the increase in A ^with increasing ^surface

pressure starting roughly ^at the end of the linear, nearly horizontal, region ^{of the} pressure/area

isotherm. From recent synchrotron X-ray diffraction

experiments [15] ^{we know that this} corresponds ^to

the onset of another phase transition where the

positional ^{coherence length increases} from about 20 to 60 lattice spacings ^at high pressures. One specula-

tion on the transition would be that the monolayer ⁱⁿ

the condensed phase ^{is further} removed from the water surface thus increasing ^the influence of chain

polarization ^{in the} interfacial area. This would give ^a positive contribution to it ^for phospholipids ^{and a} negative ^{one for FCS as observed.}

5. Acknowledgments.

We thank Dr. D. Mobius for allowing ^{us to use his} equipment for surface potential measurements and V. Vogel ^for performing ^the potential measurements with FCS. This work was supported by the Deutsche

Forschungsgemeinschaft.

References

[1] PHILLIPS, ^{M. C. and} CHAPMAN, D., ^Biochim. Bioph-

ys. Acta 163 (1968) ^301.

[2] SACKMAN, E., ^Ber. Bunsenges. Phys. ^{Chem. 82} (1978) ^891.

[3] ROBERTS, ^G. G., ^Adv. Phys. ³⁴ (1985) ^475.

[4] ANDELMAN, D., BROCHARD, F., ^DE GENNES, ^{P. G.}

and JOANNY, ^J. F., Comptes Rendus 301 (1985)

675.

[5] FISCHER, A., LÖSCHE, M., MÖHWALD, ^{H. and} SACKMANN, E., ^J. Physique ^{Lett. 45} (1984) ^L-

785.

[6] GAINES, ^G. L., ^Insoluble Monolayers ^at Liquid-Gas Interfaces, (Interscience, N.Y.) ^1966.

[7] KUHN, H., MÖBIUS, ^{D. and} BÜCHER, ^{H. in} Physical

Methods of Chemistry ^{eds. A.} Weisberger ^and

B. Rossiter, ^Vol.1 (3B) (Wiley, N.Y.) ^1972.

[3] LÖSCHE, ^{M. and} MÖHWALD, H., ^Rev. Sci., ^Instrum..

5 (1984) ^1968.

[9] LÖSCHE, M., SACKMANN, ^{E. and} MÖHWALD, H., Ber. Bunsenges. Phys. ^{Chem. 87} (1983) ^848.

[10] WEISS, ^{R. M. and} McCONNELL, ^H. M., ^{Nature 310} (1984) ^5972.

[11] DAVIES, ^J. T., ^Roc. Roy. ^{Soc. A 208} (1951) ^224.

[12] McLAUGHLIN, ^S. A., in Current Topics in Membrane

Transport, ^{Vol. 9} (1977) p. 71-144.

[13] TRÄUBLE, H., TEUBNER, M., WOOLLEY, ^{P. and} EIBL, H.-J., Biophys. ^{Chem. 4} (1976) ^319.

[14] HELM, ^C. A., LAXHUBER, ^L. A., LÖSCHE, ^{M. and} MÖHWALD, H., ^Coll. Polym. Sci., ²⁶⁴ (1986) ^46.

[15] KJAER, K., ALS-NIELSEN, J., HELM, C. A., .

LAXHUBER, ^{L. A. and} MÖHWALD, H., ^{subm. to} Phys. Rev. Lett.

[16] LÖSCHE, M., THESIS, TU Munich 1986.

[17] MILLER, ^{A. and} MÖHWALD, H., Europhys. ^{Lett. 2} (1986) ^67.

[18] MILLER, A., THESIS, ^TU Munich, ^1986.

[19] PALTAUF, F., HAUSER, ^{H. and} Phillips, ^{M. C.,}

Biochim. Biophys. ^{Acta 249} (1971) ^539.

[20] KELLER, ^D. J., McCONNELL, ^{H. M. and} MOY, ^V. T., J. Phys. ^{Chem. 90} (1986) ^2311.

[21] FLEWELLING, ^{R. F. and} HUBBELL, ^{W. L.} Biophys. ^J.

49 (1986) ^541.

[22] ^{FORT, T. and} ALEXANDER, ^A. E., ^J. Coll. Science 14

(1959) ^190.

[23] HECKL, ^{W. M. and} MÖHWALD, H., ^Ber. Bunsenges., Phys. ^{Chem. 90} (1986) ^1159.

[24] ^{BERNETT, M. K. and ZISMAN, W. A., J. Chem.}

Phys. ⁷ (1963) ^1534.