CHAPTER 4
Phospholipid Monolayers
H. MÖHWALD
Max–Planck-Institut für Kolloid- und Grenzflächenforschung,
Rudower Chaussee 5, D-12489 Berlin, Germany
1995 Elsevier Science B.V.
All rights reserved
Handbook of Biological Physics
Volume 1, edited by R. Lipowsky and E. Sackmann
161
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Experimental methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Thermodynamic measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Surface potential measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Optical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. X-ray studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Neutron scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. Infrared techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7. Surface force measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8. Scanning tunneling and atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9. Electron optical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Theoretical calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Description of the main phase transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Description of ordered phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Description of the hydrophilic membrane region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Description of domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Basic features of phospholipid monolayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Thermodynamic equilibrium? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Phase diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Phase transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. System diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Saturated straight chain lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Unsaturated bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Chain branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Different head group features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. Lipid mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6. Cholesterol/lipid mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7. Protein/lipid mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Future outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Why study phospholipid monolayers? For most of the readers of this book a convincing motivation may be the membrane biophysical aspect. The monolayer, being
half of a membrane, is a very well-defined planar system to study intermolecular
interactions between lipids and also between lipids and proteins. This was also my
perspective when entering the field a decade ago. However, I then realized the many
interesting aspects of physics in two dimensions as well as some technological relevance. Hopefully the reader will also grasp some of these newer aspects which are
related to areas of future research interest.
Most of the basic principles of a phospholipid monolayer are typical for other
insoluble monolayers [1] and hence one may find many ideas now becoming fashionable already in Langmuir’s earlier work [2, 3]. However, whereas these original
ideas were based only on indirect experimental observation and thus were close to
speculations there has been a tremendous development of experimental tools to investigate monolayer structure. These techniques have been to some extent applied
to the best defined monolayers of glycerophosphatidyls with saturated aliphatic tails,
and therefore this chapter will concentrate mostly on these. The discussion of these
results will hopefully help the reader to conclude at least tentatively on other systems
which are not explicitly mentioned here.
This chapter is organized as follows: The main body will contain a description
of experimental and theoretical techniques. It will be shown how they were applied
to phospholipids, what one could learn and where the limitations are. Being myself involved in some very recent developments I feel competent also to comment
on future directions and improvement of our understanding. The next chapter will
then briefly discuss theoretical developments. In a separate chapter the reader will
find an extraction of our present knowledge on phases, phase transitions and on the
structure at length scales between molecular and macroscopic dimensions. I will
also try to correlate results on phospholipid monolayers with those on other surfactant films which will lead to the elaboration of general physical principles and also
suggest extrapolation to other phospholipids and complexer systems not yet studied
as extensively.
2. Experimental methods
2.1. Thermodynamic measurements
The very first and most simple measurement to characterize a surfactant monolayer
is that of the lateral pressure π as a function of molecular area A. π is measured
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H. Möhwald
as the difference in surface pressure comparing the value in the absence (π0 ) and
presence (π1 ) of surfactant at the surface [4]
π = π0 − π1 .
(1)
Since π is the derivative of the surface free energy with respect to the intrinsic
variable A it is a thermodynamic variable.
Measuring π versus A, the so-called pressure/area (π, A)-isotherms, one can determine the isothermal compressibility χ by forming the derivative
1 ∂A
χ=−
.
(2)
A ∂π T
Likewise one can also record area/temperature isobars and from this directly determine the isobaric thermal expansivity
1 ∂A
λ=
.
(3)
A ∂T π
Usually it is more convenient to measure π versus A, but there are situations where
special characteristics of the system are clearer detected in one or the other detection
mode. In any case one measures derivatives of the free energy, and looking for
changes in the slope one may find and characterize phase transitions. Figure 1
shows the π, A-isotherms at different temperatures for the best-known phospholipid
dipalmitoylphosphatidylcholine (DPPC) [4, 5]. At a temperature above 18.6 ◦ C one
observes a distinct change in the slope at a pressure πc and area Ac . On compression
the slope becomes nearly horizontal suggesting a first order phase transition. The
fact that the slope is nearly but not exactly zero, i.e. that χ does not diverge, has
led to much controversy if the transition is really of first order [6].
However, at least more than 90% of the researchers in the field now agree in view
of other experimental data that there is a discontinuous transition. The most probable
explanation for the finite slope are impurities. Thus the number of components is
increased and Gibbs phase rule allows phase coexistence over an extended pressure
range. The influence of impurities has been tested by different ways:
(1) Purifying the material as extensive as possible the isotherm slope could be
reduced to virtually zero [7]. However, the accuracy in these measurements
was only about 0.1 mN/m and thus they are also compatible with a slope of
about 0.2 mN/m over half of the coexistence range.
(2) The above slope could be measured that small even for commercially available dimyristoylphosphatidylethanolamine (DMPE) and the slope increase
on adding impurities could be measured and interpreted within a simple
model [8]. From this one could also estimate that a residual impurity content
of 0.2 mole% would suffice to account for the isotherm slope measured.
(3) The slope increase with impurity content could also be derived in theoretical
calculations [9].
Phospholipid monolayers
165
Fig. 1. Surface pressure π as a function of molecular area A for a monolayer of Dipalmitoylphosphatidylcholine at different temperatures. Indicated are also the pressures (πc , πs ) corresponding to breaks in the
isotherm slope (Data from ref. [4]).
Compressing the monolayer beyond the nearly horizontal section of the isotherm
the slope gradually increases, and a discontinuous change is detected at another
molecular area As and pressure πs [4, 10]. This is indicative of a second order
phase transition. From isobar measurements on this system another second order
transition was postulated for pressures between 1 mN/m and πc [4]. This is not
revealed as a slope change in the isotherm and there are also no other experimental
data confirming it.
At lower temperature (<18.6 ◦ C) the DMPE monolayer does not display the transition at πc , often called main transition (see fig. 1). On the other hand the transition
at πs is retained. One realizes that πs does not depend on temperature [4]. For
charged lipids it was observed that πs depends on the type of divalent counter ion
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H. Möhwald
present in the subphase [10]. However, the transition pressure is independent of ion
concentration if it exceeds a limiting value.
Like in a three-dimensional system one may also derive information on thermodynamic parameters characterizing first-order phase transitions. For a two-dimensional
system the Clausius Clapeyron equation reads [4]:
S1 − S2 =
dπt
dT
· A1 − A2
(4)
where Si , Ai are the molar entropies and molecular areas of the coexisting phases,
respectively. The entropy change is related to the transition enthalpy Qt according to
Qt = S1 − S2 · T .
(5)
Qt and also (S1 − S2 ) often decrease linearly on approaching the critical temperature Tc and hence Tc can be determined from an extrapolation of Qt towards Q = 0
[4, 11].
In order to determine changes in entropy and latent heat corresponding to the
main transition one has to measure the transition pressure πt = πc as a function of
temperature. This value as well as the measurement of the molecular area A1 = Ac
of the LE phase can be derived rather accurately from the abrupt change in the
isotherm slope. A problem, however, presents the accurate determination of A2 , the
molecular area for the ordered phase, since the termination of the phase transition
on compression is not clearly observed in the pressure area isotherm.
Consider as a typical example the main transition of DPPC at 20 ◦ C. According
to the isotherm given by Albrecht et al. [4] Ac = 82 Å2 per molecule. The authors
defined A2 as the molecular area where the isotherm deviates from linearity and thus
obtained A2 = 62 Å2 . Other authors derived A2 from an extrapolation of the very
steep part of the isotherm and in this case would obtain A2 = 50 Å2 . This example
shows that the determination of A1 − A2 and hence of Qt differs by 50% depending
on the way of deriving A2 . The relative error becomes even larger on approaching
the critical temperature Tc . Yet since Q is reduced to zero when Tc is approached,
this temperature can still be determined with reasonable confidence.
Judged from this one can state that it is gratifying that Qt is on the same order as
measured for bilayers [4], but a more detailed comparison is inadequate at least as
long as Qt is determined from isotherm data alone. On the other hand one should
state that, although the definition of A2 in the work of Albrecht et al. appears
arbitrary there is some justification for it in view of recent structural data discussed
later (see II.4): The ordered phase coexisting with the LE phase often has a molecular
area well above that expected for straight aliphatic tails oriented normal to the surface
and thus A2 > 20 Å2 per chain is justified. The lower limit would be derived for A2
from an extrapolation of the nearly vertical isotherm slope for phospholipids with
head groups sufficiently small such that the tails determine the molecular area A2 .
Principally one can also derive A1 − A2 from Raoult’s law:
πc0 − πc = kT
x1 − x2
A1 − A2
,
(6)
Phospholipid monolayers
167
πc0 is the transition pressure measured if there is an impurity present in the monolayer
and if xi are the mole fractions of the impurity in the two coexisting phases. The
linear dependence of πc0 − πc on impurity content has been tested using a dye probe
as impurity [8]. For the latter a negligible concentration in the ordered phase was
demonstrated by fluorescence microscopy (c2 ≈ 0). The data inserted into eq. (6)
yield A1 −A2 = 4.6 Å2 whereas a value near 20 Å2 would have been realistic. Similar
values have also been derived for other impurities like cholesterol [13, 14] or cyanine
dyes [15] indicative of a strong solute/solvent interaction and thus modifying eq. (6).
Measurements of isotherms are also used to measure phase diagrams of lipid
mixtures. The basic physical principle of this is that for a phase separated system
the molecular areas of the coexisting phases are additive and obey a lever rule
A(π) =
x1 − x0
x2 − x1
A1 (π ) +
x0 − x1
x2 − x1
A2 (π).
(7)
In eq. (7) x1 , x2 are the mole fractions of a component in the two phases, x0 its
total content. Plotting A(π) as a function of x0 one thus expects a linear relation and
from extrapolation towards x2 and x1 one derives the composition of the two phases.
As an example fig. 2 (insert) gives the molecular area at 3 constant pressures as a
Fig. 2. Surface pressure π as a function of molecular area A for a monolayer of DMPA and cholesterol
varying in cholesterol content χChol (from right to left): 0,2,5,10,20,30,40,50,60,70,80,90,100 mole%.
T = 29.9 ◦ C, pH 5.5. Insert: Molecular area as a function of χChol for π = 5 (curve 1), 15 (curve 2)
and 35 (curve 3) mN/m. The breaks in the slope mark phase boundaries labelled M, L and C (Data
from ref. [14]).
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H. Möhwald
function of cholesterol mole fraction in a monolayer of dimyristoylphosphatidic acid
(DMPA). One observes especially at the lowest pressure two linear slopes and from
the mole fraction x0 corresponding to the abrupt change in the slope one derives
the composition x0 of the mixture coexisting with one of the nearly pure phases.
This method has been frequently applied on cholesterol/phospholipid mixtures where
solubility limits and corresponding models on complex formation were suggested
and discussed [13, 14, 16]. However, it should be pointed out that it requires very
thorough and careful measurements to locate a phase boundary. Often it is not even
clear if the data follow two straight lines or if the slope changes continuously. The
latter is expected for a homogeneous mixture of noninteracting surfactants. Also one
should stress that A(x) obeys a linear relation over the whole composition range if
the two components are immiscible.
A theoretically as well as experimentally very serious problem arises from the
fact that most phospholipid monolayers are in metastable states [17]. Usually the
‘equilibrium spreading pressure’, the pressure established in equilibrium between a
three-dimensional lipid crystal and the monolayer on the water surface is on the order
of 1 mN/m. This means that for a monolayer at higher pressure there is a tendency
to form a crystal. The more it is surprising that films prepared at different conditions
yield almost the same isotherms. However, there have been differences encountered,
if the monolayer was prepared by spreading from different solvents [18]. These may
be ascribed to metastability or to the inclusion of partly nonevaporating solvent.
The fact that a monolayer can be prepared via injection of bilayer vesicles into the
subphase also indicates that there can be lipid exchange with the subphase. Then
the monolayer cannot be treated thermodynamically as a closed system.
2.2. Surface potential measurements
As a rather simple and traditional tool to probe molecular orientation and changes
at interfaces surface potential measurements have been developed. The surface potential ∆V of an uncharged monolayer is related to the dipole density p normal to
the surface and to the relative dielectric constant ε according to [1]
∆V =
p
ε· ε0
(8)
(ε0 = 8.9 × 10−12 C/Nm2 ).
Hence from the measurement of ∆V one derives the dipole moment µ per molecule
µ = p · A with A being the molecular area, provided ε is known. The latter, however,
is a very serious problem, since in going from water to air ε decreases from 80
to 1, may vary at the interface and therefore µ drastically depends on the local
environment of the polar molecular moieties. Therefore a quantitative interpretation
of surface potential data is very dangerous.
Considering briefly the charged monolayer that can be modeled as a dipolar sheet
existing of one type of charges located at the head groups and the counter charges
in the subphase, there is a second contribution to ∆V . This contribution can be
calculated from Gouy–Chapman–Stern theory [19, 20]. It can also be measured
Phospholipid monolayers
169
spectroscopically using potential sensitive dye probes provided the probe location is
known on an Å level [21].
I will restrict the discussion on uncharged monolayers because it suffices to consider this more simple case to obtain new insight. Looking at the molecular structure
of a phospholipid like DPPC or DMPE one might assume that the groups determining the surface potential are those of phosphocholine or phosphoethanolamine [22].
For these one estimates a dipole moment of 15 Debye, whereas the measurements
yield less than 1 Debye. This discrepancy may be rationalized by the large dielectric
screening of groups embedded in the aqueous phase and also by the assumption that
molecular dipoles are arranged nearly parallel to the surface whereas one measures
the perpendicular components. However, whatever realistic head group arrangement
one may assume, the positive charge on the nitrogen will protrude further into the
subphase than the phosphate group and this would give a negative surface potential
in contrast to the measurements [23–28].
Thus the groups buried deep in the water phase do not dominate the surface
potential, and one has to look for dipolar groups with opposite polarity, i.e. with
the positive charge towards the air. One strong candidate for this are the carbonyl
groups, and indeed it has been emphasized long ago by Hauser et al. [29] that
these groups are very important. The experimental evidence for this results from a
comparison of data obtained with the glycerolester and -ether compound, respectively,
i.e. comparing presence and absence of the carbonyl group. Looking at a probable
molecular arrangement depicted in fig. 3 the oxygen may be deeper in the subphase
than the carbon atom and this yields the correct polarity. Comparing the value of µ
with the one theoretically expected for two carbonyl groups (3.6 Debye) [27] one
estimates that a tilt of the C = 0 axis with respect to the surface plane by about 20
would suffice to comply with the data [25]. The importance of the carbonyl groups
for the electrical surface properties is also supported by the fact that these groups
are closest to the region of low dielectric constant.
Although I have stressed the relevance of the C = 0 moiety I should point to two
other contributions which have at least to be considered when discussing potential
changes in going along an isotherm:
– In elegant work varying the chemical nature of simple aliphatic compounds
Vogel and Möbius [30] have elaborated the contributions of various groups in
the apolar membrane region to the surface potential. From this a contribution
of 0.35 Debye from the methyl groups terminating the aliphatic tails has to
be taken into account.
– A free water surface exhibits a surface potential, indicating a preferential
alignment of water dipole moments with respect to the surface normal. The
value strongly depends on purity but can be as low as −500 mV. ∆V is either
given in absolute values or with respect to the free water surface. Since the
surfactant distorts the water structure this may contribute to ∆V , but must not
be directly related with any dipolar group of the surfactant.
As a consequence of the above discussion I will from now on take the physicist’s
view and talk about surface polarization without digging deep into the molecular
origin. Apparently the latter is not well understood. This is unfortunate since it is
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H. Möhwald
known from experiments with fatty acids and esters that surface potential data carry
very detailed information.
To correlate ∆V with monolayer phases fig. 3 compares pressure/area isotherms
with ∆V /area isotherms continuously measured for the same films of uncharged
phospholipids. For this comparison two lipids with the same head group were selected where the one with the shorter tails (L-α-dilauroylphosphatidylethanolamine,
DLPE) exhibits over a large density range an LE phase whereas the one with longer
tails (DMPE) exhibits a pronounced flat region in the pressure/area isotherm. Measurements of ∆V are meaningless for zero surface pressure: ∆V may be zero or
fluctuate between zero and the value corresponding to the beginning of the surface
pressure rise. These fluctuations can be ascribed to the surface heterogeneity also
seen in optical studies. In the LE phase ∆V changes inversely proportional to A,
i.e. the dipole moment µ per molecule (also given in fig. 3) is constant. This indicates that only the molecular density not the molecular arrangement change on
compression of this monolayer phase. Increasing the surface pressure above πc , ∆V
continues to increase, but with a much smaller slope than below πc and in a linear
way. Supported also by the observations reported later the linear part of the ∆V /A
isotherm can be described by a lever rule assuming two coexisting phases with potentials V1 , V2 , molecular areas A1 , A2 , and the relative area fractions f1 , f2 varying
with A according to f1 = (A − A2 )/(A1 − A2 ) and f2 = (A1 − A)/(A1 − A2 ) [25],
∆V = f1 V1 + f2 V2 .
(9)
One of the phases is the LE phase with V1 (f1 = 1) corresponding to the values
measured at πc . The data for the second phase can then be derived by extrapolation
of the linear ∆V versus A region, although there is a considerable uncertainty. For
example, if A2 corresponding to this phase would be 41 Å2 , V2 would amount to
295 mV, if, however, A2 = 48 Å2 (the value where the linear region is terminated)
V2 = 290 mV would be derived. The difference of 5 mV appears small. Yet it
is important, because V2 − V1 is only 15 mV and this value will be used for the
calculation of electrostatic forces [25]. In any case for the discussion of these forces
it is important to note that V2 − V1 > 0 and 13 mV < V2 − V1 < 18 mV. Again
deriving µ for phase 2 (µ2 ) one realizes that the value is well below µ1 , the value
at πc . We have suggested that this is due to a C = 0 group orientation more parallel
to the surface [25] but cannot rule out different hydration and water structure in this
phase. In any case the expected potential increase in the more condensed phases
due to the higher dipole density is not completely but to a large extent compensated
by the smaller effective molecular dipole moment µ. To make this statement more
quantitative: ∆V = V2 − V1 is about a factor of 10 smaller than the value that was
previously assumed for ∆V from an extrapolation of the value V of the LE phase
to higher densities for a constant µ [25].
Further compressing the monolayer beyond the linear region one realizes the drastic
increase in ∆V accompanied by the rise in lateral pressure. This indicates another
structural change that will be discussed later.
Phospholipid monolayers
171
Fig. 3. Surface pressure π (mN/m), potential ∆V (V) and dipole moment per molecule (D/Mol) as
a function of area per molecule for DLPE (top) and DMPE (bottom) monolayers. Indicated are also
the molecular areas As and Af = Ac and the area fractions if the corresponding phases would coexist.
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2.3. Optical techniques
2.3.1. Fluorescence microscopy
A more direct picture of the phase diagram of phospholipid monolayers has been
gained from fluorescence microscopic studies of the air/water interface. One incorporates a fluorescent dye probe into the monolayer and measures the lateral dye
distribution from analysis of the fluorescence micrographs. Contrast in the images is
obtained either due to different dye solubility, fluorescence quantum yield or molecular density of coexisting phases [31–33].
As an example fig. 4 presents a series of textures observed on compression of a
monolayer above the pressure πc [34]. The dark areas can be ascribed to a more
Fig. 4. Fluorescence micrograph of a DMPA monolayer on increasing the lateral pressure (above πc )
from a to f . The film contains 0.25 mole% of a lipidic dye probe. The images where observed at rather
complicated subphase ionic conditions [34], but are typical for pH = 5 and the absence of divalent ions,
but presence of monovalent ions.
Phospholipid monolayers
173
ordered and denser phase with low dye solubility. As expected the area fraction of
this phase increases on compression. A quantitative analysis of these images shall
illuminate two questions.
(1) What can be learnt about the phase transitions and about the nature of coexisting phases?
(2) Can one understand peculiar domain shapes and superlattices?
Fig. 5. Domain number N (bottom) and condensed phase area fraction φ as a function of molecular area
for a DMPA monolayer. Indicated are also the molecular areas corresponding to the phase transitions
at πc and at πs .
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Ad (1): Figure 5 gives the number of domains, and the dark area fraction φ can
again be described by a lever rule. From an extrapolation to φ = 0 and to φ = 1
one obtains the molecular areas of the coexisting phases. For φ = 0 one obtains,
as expected, a molecular area near Ac , the value of the LE phase. Extrapolation to
φ = 1, however, does not yield a molecular area near As but a value of 48 ± 2 Å2 ,
i.e. distinctly larger than As . This proves that the phase coexisting with the LE phase
is less dense than the solid one.
The fluorescence microscopic technique clearly shows the coexistence of two
phases and thus that the phase transition is of first order. This conclusion has been
challenged since the technique depends on the incorporation of the dye as an impurity. However, the coexistence of phases has been confirmed now also by techniques
not needing surface active dye probes as electron microscopy [35], surface plasmon
microscopy with transferred monolayers [36] and fluorescence microscopy with water soluble dye adsorbing from the subphase [37]. Recently there were developments
of Brewster angle microscopy [38, 39] and imaging ellipsometry [40] proving also
phase coexistence in the absence of dyes. Beyond that the dye concentration used
has been as low as 1%◦ and most monolayers studied hitherto have not been purer.
The dye, however, has an influence on the φ versus A plot for large φ since then the
remaining fluid phase is highly dye enriched and the condensation impeded. This
partly accounts for the deviation of the plot from linearity.
Thus the conclusions on the phase diagram are not invalidated since the dye is an
impurity. This does not contradict the findings below that the numbers and shapes
of domains depend on the type and concentration of surface active impurities.
Ad (2): The number N of domains has been found to be a nonequilibrium property [41]. It depends on the nucleation process and cannot be predicted quantitatively.
However, its qualitative change with variation of a nucleation parameter is as expected. Increasing the compression speed and thus the pressure deviation from πc
during the nucleation period N increases [41]. The free energy to create a critical
nucleus for growth of the ordered phase can be reduced by increasing the temperature
or surface charge density [41, 42] adding cholesterol [43, 44] or proteins [45]. This
reduces line tension, the energy per interface length between the coexisting phases
and thus increases N .
The domain shape can or cannot be an equilibrium property depending on the actual
system. Mostly domains grow far from equilibrium with a rough interface between
the phases. It can be understood as a diffusion limited aggregation process which may
lead to fractal structures [8]. For a special case the process could be analyzed within
the frame work of constitutional supercooling where domain growth is limited by
diffusion of an impurity from the phase boundary [8]. The impurity impedes growth
and is removed fast for a rough boundary which therefore is favoured. Following
this growth period line tension tends to smoothen the boundary. This often leads to
regular shapes like discs, lamellae or spirals which will be discussed below (fig. 6).
In other cases, however, smoothing may not be terminated within observation times
of several hours and then structures like clover leaves or coffee beans may result.
Phospholipid monolayers
175
Fig. 6. Fluorescence micrographs of various lipid monolayers in the LE/LC phase coexistence range.
Upper left: DMPA under conditions of fig. 2. Upper right: DMPA containing 1 mole% cholesterol at
pH 11. Lower left: DMPA at pH 5, no other ions added. Lower right: A diacetylenic lipid in the
unpolymerized state.
2.3.2. Fluorescence spectroscopy
Since phospholipids are themselves nonfluorescent, spectroscopy with monolayers
of them is possible only if suitable dye probes are incorporated. The dependence of
fluorescence emission on the environment has been made use of in many membrane
biophysical studies [46–51] and likewise can be used for monolayer investigations.
By fluorescence recovery after photobleaching (FRAP) local diffusion coefficients
could be measured [52]. In these measurements a circular area or a stripe pattern
are photobleached and recovery of the fluorescence intensity due to diffusion of dye
probes out of nonbleached areas is detected either as change of the total emission
out of the bleached spot [53] or as a change in the contrast comparing previously
irradiated and shaded areas [54]. Thus it was possible to show that the diffusion
coefficient for phospholipid monolayers in the LE phase is larger than 10−8 cm2 /sec
and below 10−10 cm2 /sec in the LC phase, in qualitative agreement with data for
bilayer membranes [32, 55–57]. For studies of diffusion mechanisms the monolayer
has been proven to be extremely useful, since it was possible to vary the molecular
area and compressibility in a well-defined way. In the diffusion model applicable
176
H. Möhwald
for the motion of small molecules in the membrane the diffusion coefficient D is
related to the free area Af per molecule according to [32, 55, 58]
ln D = ln a −
b
,
Af
(10)
Af = A − A0 , A0 being the minimum molecular area is easily determined in monolayer experiments and fig. 7 gives the result of the measurement, varying A while
maintaining the monolayer in the LE phase. The excellent agreement between theory
and experiment proves the adequacy of the diffusion model employed. Measurements
in the coexistence range have also been analyzed within percolation models [55].
There diffusion basically occurs within the LE phase, LC phase domains being obstacles. At high enough density of these obstacles the interconnects between large
LE phase areas become very small and diffusion is thus limited by the passage
through these interconnects. Therefore the effective diffusion coefficient depends on
the area fraction of the LE phase.
In view of this one may expect different values of D if measured over other length
scales. One of these measurements could be the well-known analysis of excimer
fluorescence emission which, using pyrene chromophores attached to amphiphilic
Fig. 7. Translational diffusion coefficient D of a dye probe in DLPC monolayers as a function of free
area Af defined in the text. T = 21 − 22 ◦ C. Also given are the corresponding molecular areas A and
the surface pressure π (from ref. [32]).
Phospholipid monolayers
177
molecules, yields the diffusion coefficient over distances of 100 Å [58]. These studies
have been performed with fatty acid monolayers but not yet with phospholipids [59].
Another way of measuring local concentrations and their changes is via measurement of fluorescence quenching [48–50] or energy transfer [60]. The first technique
is principally simple since it involves merely an intensity measurement at low spectral
resolution. However, it is generally difficult to interpret if there are no supplementary data available: The data analysis assumes the formation of statistical dimers
acting as fluorescence quenchers, but principally also oxygen or impurities in film
or subphase may reduce the emission intensity. Nevertheless, measuring the fluorescence intensity of porphyrin [50] or chlorphyll [46, 47] probes or of phthalocyanine
dyes in phospholipid monolayers as a function of molecular area the onset of the
LE/LC phase transition could be detected from a decrease in fluorescence quantum
yield upon decreasing the molecular area below Ac . This is understood since the dye
probe is less soluble in the LC phase and if its enrichment in the coexisting LE phase
leads to concentration quenching.
These techniques although being indirect provide a means to detect phase separations at length scales below fluorescence microscopic resolution (∼100–1000 Å).
Thus it could be shown via measurements of fluorescence quenching as well as via
energy transfer from a porphyrin to a phthalocyanine that a phase separation existed
in monolayers of DMPE with domains of the ordered phase being too small to be
microscopically detected [60].
2.3.3. Ellipsometry and quasielastic light scattering
Recently it has been become possible to study the monolayer microstructure by
ellipsometry with films on water. With this technique one basically measures an
ellipsometric angle, by which the principal axes of elliptically polarized light are
rotated on reflection and then tries to develop models to extract structural parameters like film thickness, refractive index and its anisotropy [61–68]. Although it
appears highly dangerous to conclude from one measured value on many parameters
the data have been shown to be reasonably realistic since there have been systematic measurements with one class (phosphatidylcholines) of lipids and also going
along an isotherm. Hence further refinement of these models and analysis may elucidate the microstructure, and in special this technique may become very valuable
to observe structural changes accompanying, e.g., a surface reaction. Recently two
papers appeared demonstrating that it is practically impossible to derive more than
one parameter, either thickness or refractive indices from ellipsometric data [65, 66].
In addition even for a known film thickness the anisotropy of refractive index prohibits accurate refractive index measurements for monolayers on water. Although
at this stage ellipsometry has not contributed much it is promising in the following
directions:
– It is sensitive to detect phase boundaries since it enables quasicontinuous
studies going along an isotherm or an isobar [66].
– Refractive index and thickness can be separated in the analysis of thicker
films, e.g., of proteins bound to membranes [67].
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H. Möhwald
– It is sensitive to surface roughness due to capillary waves, although the reason
is not yet understood [68].
As another promising technique becoming more feasible with the development of
more powerful computers quasielastic light scattering has been introduced [69–73].
The technique measures the frequency spectrum of capillary waves and thus the
frequency dependent viscosity and elasticity in the kHz to MHz range. I expect this
technique to become very important in the near future since every researcher in the
field realizes vastly different elastic properties for different lipids and phases.
2.4. X-ray studies
The use of Synchrotrons as brilliant X-ray sources has enabled one to perform X-ray
studies with monolayers at the air/water interface [74, 75]. Since these experiments
are very powerful and new they shall be described in some more detail: A film
balance is placed at the sample stage of a diffractometer [76]. The X-ray beam hits
the surface at an angle below that corresponding to total reflection (∼0.1◦ ). In this
mode the beam penetrates by only 50 Å and, if the surface is laterally periodical at the
nm level, is diffracted. Measuring the diffracted intensity as a function of in-plane
diffraction angle one then derives information on the lattice structure. In studies
performed till now the investigated surface area (∼1 cm2 ) is more than 1000 times
larger than a domain area and these domains are not oriented. Hence the diffraction
pattern corresponds to that of a two-dimensional powder.
Measuring specular reflection as a function of incidence angle one obtains information on the electron density distribution ρ along the surface normal z . The measured
reflectivity R divided by the Fresnel reflectivity RF calculated for an ideal interface
is related to ρ(z ) according to [77]
R
=
RF
Z
dρ
dz
exp iQz dz
2
·
1
ρ2w
(11)
where
Qz =
4π
sin α
λ
is the wave vector transfer in direction of z (λ = wave length, α = incidence angle
with respect to the surface) and ρw is the electron density of water. These measurements can be applied also for non-periodic structures but the analysis depends on
the choice of suitable models. Fortunately it is possible to use very simple models
with only few independent and adjustable parameters and partly the information can
be derived model independent. The experiments can be described with a ‘two-box
model’ where the surface is divided into a slice of length lt and density ρt containing
the tails above another slice (lh , ρh ) containing the head groups, and the density step
between all slices is smeared by a Gaussian function of width σ [77, 78]
Figure 8 gives a series of diffraction peaks on increasing the lateral pressure from
bottom to top. These peaks can be grouped into two regions divided by the pressure πs . For π < πs the lines are weak and broad whereas for π > πs they are much
Phospholipid monolayers
179
Fig. 8. X-ray intensity as a function of in-plane wave vector Qx for a DMPA monolayer at various
surface pressures indicated by arrows in the isotherms (insert). pH 5.5, T ≈ 20 ◦ C.
180
H. Möhwald
narrower and stronger. These results have been confirmed with other phospholipids to
yield the following picture. The lattice constant calculated from the peak maximum
(∼ 4.2 Å) corresponds to the (1,0) spacing of the hexagonal lattice (d10 ) found in
electron diffraction studies with monolayers on solid support [79]. d10 continuously
varies in going through πs whereas the compressibility
χ := −
2 ∂(d10 )
d10 ∂π
(12)
decreases by a factor of 2 on increasing π above πs . Yet χ is close to a factor of
2 smaller than the value of χ derived from the isotherms (eq. (2)).
Although the hexagonal symmetry of the electron diffraction pattern and the observation of merely one X-ray diffraction peak suggest a hexagonal arrangement
of the aliphatic tails this is in many cases not exactly valid. In fact orthorhombic
lattices have frequently been observed for various phases of phospholipid bilayers
[80–82, 86], alkane crystals [83, 84] and fatty acid monolayers [85, 88], and a break
of the hexagonal symmetry is even expected if there is a uniform tilt of the aliphatic
tails with respect to the surface normal. Information on the tilt can be derived from
an analysis of the Bragg reflections along the surface normal z (Bragg rod) [85]. In
case there is uniform tilt of the aliphatic tails, modeled as a cylindrical rod, towards
a lattice plane the maximum of the diffraction intensity is moved out of the surface
is related to the tilt angle t and to
plane. The corresponding wave vector Qmax
z
the azimuth ψ between tail projection on the surface and the normal to the surface
according to
Qmax
= tan t· cos ψ· Qmax
z
x .
(13)
As an example fig. 9 gives in plane diffraction data within different Qz intervals
and for different surface pressures [87]. Thus one can distinguish up to three different
peaks with maximum intensities for different Qz values.
The detailed analysis yields the following:
– At low pressures the aliphatic tails are uniformly tilted and form an oblique
lattice.
– Increasing the lateral pressure the tilt angle is reduced and a centered rectangular lattice with tilt towards a nearest neighbour molecule is established.
– Increasing the pressure above πs the tilt disappears and the lattice becomes
hexagonal.
Figure 10 gives as series of X-ray reflection measurements with DMPE monolayers
increasing the lateral pressure from bottom to top [88]. The corresponding points in
the isotherm are given in the insert. One clearly observes a pronounced minimum
that shifts to lower Q with increasing pressure. The minimum can be understood
as a destructive interference of a wave reflected at the interface hydrocarbon/air
with one reflected from the center of the head groups [88]. This center essentially
Phospholipid monolayers
181
Fig. 9. X-ray scattering intensity as a function of wave vector Qz normal to the surface for an in-plane
diffraction angle corresponding to a diffraction maximum. The corresponding pressures and temperatures
of the DMPE monolayer are given in the isotherm (insert).
corresponds to the position of the phosphatidic acid. Hence one derives for the value
of Qz corresponding to the minimum (Qmin ):
−1
Qmin
1
1
=
lt + lh
2π
2
(14)
and the shift in Qmin is due to an increase in monolayer thickness on compression.
The less pronounced depth of the minimum in curve c is due to the fact that
in this case the reflectivity results from contributions of two phases with different
density profile, and this type of roughness broadens all extrema. In the other cases the
increased depth is qualitatively due to an increase in contrast comparing the maximum
head group density with that of the adjacent moieties. The latter is due to the fact
that the contributions to ρh result from a large value from the phosphatidylglycerol
and a smaller value of hydration water and the latter is squeezed out on compression.
The curves in fig. 10 were obtained from a fit using the two-box model, and a
collection of parameters obtained for a series of measurements including those with
DLPE is given in fig. 11 as a function of molecular area. The right part of fig. 11
gives data for the fluid phase whereas the left one considers in detail the region near
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H. Möhwald
Fig. 10. X-ray reflectivity normalized to the Fresnel reflectivity as a function of incidence angle given
in units of the critical angle Qc for a DMPE monolayer at surface pressures indicated in the isotherm
insert.
πs (As = 41 Å2 /molecule). The following main information can be deduced from
the graphs:
(1) The length of an all-trans chain with n CH2 groups and a terminal CH3 group
Phospholipid monolayers
183
Fig. 11. Electron densities (normalized to that of water) and lengths of head group and aliphatic tail
regions, respectively for DMPE (filled circles) and DLPE (open circles) monolayers at various molecular
areas. Notice the differences in the abscissa scale for the fluid phase (right panel) and for molecular
areas near A ≈ 41 Å2 .
is given by [89, 90].
lt,max = (n· 1, 25 + 1, 265) [Å].
(15)
From this one deduces lt,max = 16.7 Å and 14.2 Å for DMPE and DLPE, respectively.
The fact that at the highest pressure values of lt lower by about 1 Å were measured
cannot be explained by a chain tilt since then the tilt angle would have to be on the
order of 10◦ and have shown up in the rod scans. Instead it is more probable that the
two-box model is too simple to account for all details. For example, it is known that
the carbonyl groups marking the head/tail interface are at different positions with
respect to the surface plane [91, 92]. Hence the assignment of this interface to a
184
H. Möhwald
molecular moiety is somewhat arbitrary. On the other hand comparing DMPE and
DLPE the measured difference in lt of 2.5 Å (at high π) is as expected according to
eq. (15).
(2) Also a good proof of the consistency of the data analysis is the fact that the
head group parameters derived for the two lipids with the same head groups agree.
(3) With the values of ρh , lh measured for the molecular area A one may calculate
the number of electrons Nh in the head group region:
Nh = ρh · lh · A.
(16)
Nh versus molecular area is given in fig. 12. Counting the number of electrons
in the head group moiety yields a value of 140. Hence the additional electrons
Fig. 12. Electron numbers N-head in the head group region as a function of molecular area for DMPE
(closed circles) and DLPE (open circles) monolayers. The lines are best fit to the data for the fluid phase
and for the region near As , respectively.
Phospholipid monolayers
185
must be ascribed to water. The number of water molecules in the head group
therefore amounts to about 15 at πc , to four at 43 Å2 and to 2 above πs , respectively.
Incidentally these are about the hydration numbers determined in vesicle experiments
for the LE (10–20 water), the gel (∼ 6 water) and the solid (1–2 water molecules)
phase [93].
The slope in the graph of fig. 12 for the LE phase agrees within 10% with that
expected if water is squeezed out of the head group region without changing its
thickness. The much larger slope near πs on the other hand is due to a squeezing
out of water plus a decreasing of the head group size. The lowest value obtained for
lh is very close to the value expected from molecular models. This indicates that,
whereas at lower pressures the head groups are disordered along the surface normal
by up to 3 Å, for π > πs they are to better than 1 Å confined within a plane parallel
to the surface.
(4) Changes in lt near πs are only slight and appear smooth. They are therefore
not conclusive on the question if there is a change of tilt angle near πs , but are
compatible with the above finding of a reduced tilt angle on compression.
(5) Comparing ρt at πs and πc one realizes a density change of more than 10%.
Further expanding the fluid phase lt as well as ρt are reduced.
(6) On going to a more ordered phase the smearing parameter is increased reflecting
a larger surface roughness. This will be attributed to a decreased surface tension and
thus increasing amplitude of capillary waves [94].
2.5. Neutron scattering
The vastly different scattering cross section for hydrogen and deuterium has made
neutron scattering a very powerful technique in membrane research [95–97]. Water
can be made invisible to neutrons by index matching for suitable H2 O/D2 O mixtures, and different parts of the membrane can preferentially be studied by selective
deuteration. However, compared to X-ray scattering the high contrast is largely compensated by the much lower fluxes. Therefore neutron scattering still has to prove
its unique features for monolayer research if one is concerned with spatial resolution
better than 1 nm.
Nevertheless it has previously been shown that the technique can yield valuable
structural information on surfactant monolayers [98] and recently also monolayers of
phosphatidylcholines and -glycerols [99] have been studied. Data analysis proceeds
basically using slab models as detailed above for X-ray reflection where the scattering density is proportional to the electron density. Up to now model parameters
determined from neutron scattering have been less accurate than those derived by
X-ray studies, but the technique is brandnew and this was merely a promising start.
Probably it will turn out that the technique is not very valuable used alone, but will
become very important in conjunction with other especially X-ray techniques.
2.6. Infrared techniques
Powerful lasers, detectors and computers have made it possible that Raman and
Fourier-Transform (FT) Infrared spectroscopy have become sensitive enough to study
186
H. Möhwald
monolayers on solids and on water surfaces. Detailed studies with phospholipids
have been performed by FT analysis of the IR reflection from a water surface with a
DPPC monolayer in different phases [100–102]. In analogy to studies with bilayer
membranes shifts of frequency and intensity of the CH2 stretching and bending
vibrations have been detected on going from the LE to the LC phase. This supports
the view that the transition involves an ordering of the aliphatic tails. Changes of
vibrations in the head group region have also been reported, but these are more
difficult to interpret. Yet I believe that the analysis of these vibrations will prove the
unique advantages of the technique. It responds to changes in orientation and local
ordering but does not depend on long-range order. Presumably these changes occur
on going from LC to S and therefore IR spectroscopy will be a very sensitive tool
to characterize them.
2.7. Surface force measurements
With the surface force apparatus initially developed by Tabor and Israelachvili
[103, 104] the forces between two surfaces are measured while changing their distances at the Å-level in a well-defined way. These distances and also the refractive
index in the medium between the surfaces are measured interferometrically. The
surfaces have to be molecularly smooth and thus mostly mica was used. Onto this
a lipid monolayer could be transferred and this enabled studies of interaction between monolayer surfaces in air, varying, e.g., the humidity, or of forces between
the hydrophilic head groups with both surfaces under water [105–107].
The analysis of the measurements on one hand yields structural information as
extension of head group and tail region, on the other hand, and this I consider most
beneficial, it measures local forces over nm distances directly. This in turn has
contributed to conclude on solvent structure near the interface and on the controversies on hydration forces [108–110]. The latter are of entropic origin, dominate
electrostatic and Van der Waals forces at short distance and decay exponentially with
distance with a characteristic length close to 3 Å.
In the case of phospholipid mono- and multilayers deposited on mica the oscillatory
dependence of forces on distance is not observed probably due to the dynamics
in the head group region. On close approach one may also observe monolayer
destabilization followed by interlayer fusion. Hence the technique opens new ways
to microscopically study biologically relevant processes like membrane fusion [111].
2.8. Scanning tunneling and atomic force microscopy
Both these techniques are also applicable to biological samples in water and therefore
have encountered tremendous interest for biophysical studies of microstructure [112].
However, as yet they did not contribute much to our understanding of monolayers
since their basic principles concerning this application are not understood. It has been
possible to obtain STM images of a monolayer of DMPA on oxidized graphite [113]
and most probably these reflect the positions of the aliphatic tails. However, the
electron tunneling through a hydrocarbon chain of more than 20 Å length still remains
a mystery. The interchain distance and the two-dimensional chain lattice can be
Phospholipid monolayers
187
determined more precisely by scattering techniques, but this technique may become
unique as a means to directly visualize and quantify defects. Details of characteristic
defects have not yet been evaluated since the preparation of substrate and monolayer
have not yet been well controlled.
In addition the force exerted by the inhomogeneous field between tip electrode and
substrate distorts the monolayer and leads to surface flow. Therefore results obtained
on chemisorbed monolayers appear more reliable [114] and promising to understand
the underlying mechanisms.
As another technique that does not depend on a conducting substrate or film
atomic force microscopy (AFM) carries many promises [115, 116]. Up to now
the sensitivity of force measurements relies on strong interactions and therefore the
needle scanning the surface distorts it to a large extent. Thus the technique is
applicable only on monolayers that are stabilized either by crystallization, covalent
attachment to the substrate or by polymerization. Presently available data can be
interpreted as indications of a periodic head group arrangement [116]. This was
sometimes postulated and should show up as a superlattice in diffraction studies, but
was never directly observed. It is possible that AFM, not depending on long range
order is more sensitive to observe head group order. Yet at present, where AFM is
still in its infancy one has to be cautious not to overinterprete experimental results.
2.9. Electron optical studies
Since, with rare exceptions [117, 118], electron optical studies have to be performed
in vacuo and not on monolayers in situ they are listed at the end. However, the
development of reliable methods of monolayer transfer has made these studies very
valuable to conclude on structure and phases.
The initial studies were limited by the small contrast provided by the monolayer
and thus had to use staining methods or Pt shadowing and C evaporation to obtain a
replica. These studies helped to conclude on the surface roughness and thus on film
stability and phase coexistence [79, 119]. A big step in the development of electron
microscopic studies of monolayers came through the invention of a charge decoration
technique [119]. It is based on the fact that different monolayer phases are charged
to a different degree by the primary electron beam. Thus a field develops at the
phase boundary and this deflects the electron beam. Due to this the image of the
boundary line appears dark and domains of different lipid phases can be visualized.
The technique can be applied only in the low magnification mode (6 ×5000) but
lipid domains exhibited sizes of several µm and thus could be observed without
using any dye probe.
One prime advantage of this technique is that it can be directly combined with
selected area electron diffraction. Thus one can select specific monolayer areas
of µm dimensions for local structural studies. In many cases the correlation of
the local crystallographic axes (bond orientational order) extends over a domain
with dimensions of more than 10 µm and the pattern then contains distinguished
spots [35, 119]. From a comparison of these profiles with simulations one could
then deduce that the ordered phases (LC,S) of the phospholipids are most probably
hexatic [120].
188
H. Möhwald
For diacetylenic lipids a detailed study of the spot profiles also demonstrated the
existence of chiral structures as a consequence of uniform head group orientation with
respect to a polymer axis [121]. Similarly extended studies have not been performed
for phospholipids where generally merely a hexagonal symmetry (averaged over the
beam area), a lattice spacing of 4.2–4.3 Å and a translational coherence length on
the order of 100 Å were deduced [119].
3. Theoretical calculations
Stimulated by the possibilities of more refined structural analysis and by the interest
in different aspects of low-dimensional systems there has been an increasing activity
to theoretically describe surfactant films. These shall be summarized below classified
not according to the tools used but with respect to the aims.
3.1. Description of the main phase transitions
A prominent feature of the pressure/area isotherms of many phospholipids is the
pronounced break in the slope corresponding to the main transition. Therefore attempts to simulate the isotherms are closely related to an understanding of this
transition. This transition is also of interest in view of the analogy with the main
transition of lipid bilayers. The dominant mechanism driving the transition is the
ordering of aliphatic tails requiring a large entropy change. Therefore tail ordering
is considered as the driving parameter and other forces like head group interactions
are taken into account as secondary effects. These approaches have been reviewed
frequently [6, 122]. They were successful in describing qualitative features of the
isotherms and after introduction of a sufficient number of parameters also a quantitative simulation of isotherms was possible. These parameters were also tested to
yield Raman intensities and order parameters of different tail segments in accordance
with experimental data. As a most interesting new approach a modified cubic lattice
model has recently been introduced to calculate the free energy using merely two
parameters: Contact energy between chain segments and between these segments
and water. Thus it was possible to qualitatively calculate isotherms and predict two
first order phase transitions with critical points, that resemble the gas ⇐⇒ LE and
LE ⇐⇒ LC phase transitions, respectively. Beyond serving the elaboration on entropic and enthalpic contributions to the free energy the calculations also yielded
parameters characterizing the order of tail segments. They could be extended to
enable predictions on the phase diagramme of mixtures of lipids with different chain
lengths [123]. As an interesting result of these calculations there also was the notion
that translational ordering is not necessarily coupled with conformational ordering,
the arrangement of the tails predominantly in all-trans configuration. The latter is
driving the LE/LC transition.
A decoupling of positional and orientational ordering was allowed in the approach
by Mouritsen and Zuckermann [124] who extended Pink’s Potts model [6] by a term
representing the boundary energy between configurationally ordered and disordered
lipid. It was shown that, depending on this energy, the order parameters may be
Phospholipid monolayers
189
coupled or decoupled. The latter was indicated from the line shape analysis of X-ray
diffraction data [74].
The LE ⇒ LC phase transition could be described reasonably well in the first
numerical mean-field calculations where the dominant energy contributions, the internal energy of a molecule with different number and types of kinks and the Van
der Waals interaction between molecules were taken from data experimentally obtained for alcanes. As an extension of these calculations coulombic repulsion has
been taken into account, and the isotherms for charged monolayers were then calculated [125]. Another remarkable result of the calculations is that the free energy is
lowered if the monolayer separates into regions of higher and lower lateral density
with characteristic dimensions on the order of 100 lattice spacings.
One advantage of Pink’s Potts model is that it could also be extended to study the
influence of ‘impurities’ like cholesterol or proteins on the phase diagramme and to
conclude on miscibility and lateral distribution of monolayer components.
3.2. Description of ordered phases
There are well-known theorems that true long-range order cannot exist in one or
two dimensions [126]. However, it was shown by Nelson and Halperin [127] that
there may exist intermediate phases with short range positional order but long range
bond-orientational order. The latter is defined via the correlation of local crystallographic axes and, dependent on the local order, the phases were named hexatic or
tetratic. The existence of hexatic phases has been proven among others for liquid
crystals (LC) [128] and the analogy of lipid layers with smectic LC has often been
pointed out [129]. Hence it is not surprising that one may encounter the richness of
LC phases also in monolayers. Also like molecules used in thermotropic LC phospholipids possess internal and external degrees of freedom and various molecular
parts may order under different conditions. Hence a theoretical description of the
real structure is very demanding and work performed up to now could consider only
a limited fraction of these aspects. These calculations also did not consider phospholipids where two aliphatic chains are linked by the head group but considered
the arrangement of independent aliphatic tails at an interface.
Molecular dynamics calculations of Bareman et al. [130, 131] demonstrated a
nearly vertical arrangement of aliphatic tails with low density of kink defects if
the two-dimensional molecular density is large. Expanding the monolayer the tails
uniformly tilt before at further expansion the system separates into ordered and
disordered regions. These results are in qualitative agreement with diffraction data,
although the absolute values of tilt angle and corresponding molecular area disagree
considerably. In addition it is found that the two different types of tilt azimuth
where the tails tilt towards a nearest neighbor and a next nearest neighbor molecule,
respectively, are not much different in energy. The slightly favoured configuration
of a nearest neighbor tilt is indeed found for fatty acids but not for phospholipids.
Very similar energies of the two qualitatively different tail orientations have also
been found in the Monte Carlo and molecular dynamics calculations of Rice and
coworkers [132, 133]. This group also calculated the free energy as a function of tilt
190
H. Möhwald
angle showing a drastic increase in energy if the tilt angle exceeds 30◦ [134]. This
is remarkable since one experimentally observes a phase separation if the molecular
area is increased such that the tilt angle would exceed 30◦ for a homogeneous film.
One drawback of the calculations where the interactions of every tail segment are
explicitly taken into account is that with realistic effort at most 104 molecules can be
treated. This, however, is insufficient to conclude on long range order correlations.
The latter is possible if the aliphatic tails are considered as rigid rods interacting
via a Lennard–Jones potential. Monte Carlo and molecular dynamics techniques
[135, 136] as well as a variational principle approach [137] were used with this
model to conclude on a question already raised by Langmuir: If the head groups
inhibit a closer approach that would be favoured by the attraction of the tails, does
the system remain uniform or become nonuniform? In the latter case there may
be a tilt which is uniform over small dimensions, small domains (two-dimensional
micelles) may be formed with all tails oriented towards one center. These structures
may be periodic in one or in two dimensions. Results obtained up to now indicate
that the lowest energy state is that with totally uniform tilt, but there may be low
energy excitations or finite size effects which transform the system into such a state
with heterogeneous tilt angle distribution [135–137].
3.3. Description of the hydrophilic membrane region
Although it is apparent that interactions in the head group region are very important
for the structure and for the biological function the theoretical descriptions of these
interactions are scarce.
In order to assess the influence of ionic interactions on the lateral pressure classical
Gouy–Chapman theory has been modified. This theory assumes a diffuse counter
ion distribution and could be extended to describe also the counter ion binding to
the membrane and the dependence of proton dissociation on surface potential [19–
21, 138]. However, it does not yield the specific ion arrangement near the surface
nor its influence on the membrane structure.
As a most interesting recent development simulations have shown the relevance
of entropic contributions to the surface energy [139, 140]. In the case of divalent
ions this may lead to attraction of two surfaces with the same charge.
The complex head group of phospholipids may have to await some time till simulations yield information on the arrangement of water in its environment. The
importance of hydration water for the structure has often been stressed, but up to
now simulations have concentrated on the more simple case of a carboxylic acid
head. For these systems an ordered water arrangement was found [141]. It extends
parallel to the surface, but also about 10 Å in normal direction with the decay length
depending on lipid density.
3.4. Description of domain structure
Motivated by the fluorescence microscopic observations of periodic lamellar or
hexagonal arrangement of lipid domains in the phase coexistence range basically
two different approaches have been applied to explain this unexpected behaviour.
Phospholipid monolayers
191
Their basic feature is that the interface causes an arrangement of molecular dipoles
parallel to the surface normal. This gives rise to long range electrostatic repulsion
competing with short range attraction. Minimization of the free energy in Landau
theory expansion then yields stable phases with periodic density variation [142, 143].
Whereas the above treatment should be valid for a monolayer near a critical point it
should be less accurate for the general situation where sharp phase boundaries exist.
Then the more local description of McConnell et al. appears appropriate [144–148].
Considering the free energy density of a lipid domain the unique feature of the
two-dimensional system is that the electrostatic contribution diverges with domain
size. Hence, whereas in the absence of the long range force a domain with size
above a critical value will grow to infinity it is restricted for a monolayer and an
equilibrium size can develop. Since electrostatic repulsion disfavours compact shapes
one may encounter transitions to elliptical and lamellar shapes and even transitions
into higher two-dimensional modes are predicted [149–151]. It has also been shown
that interdomain repulsion enforces regular arrangement of uniformly sized domains
in hexagonal or lamellar superlattices, but a more quantitative picture in relation to
dedicated experiments is still missing.
4. Basic features of phospholipid monolayers
4.1. Thermodynamic equilibrium?
Attempts to describe a monolayer by equilibrium thermodynamics are frequently
criticized since in most cases the system is not in thermodynamic equilibrium:
(i) The equilibrium spreading pressure generally amounts to at most a few mN/m,
often to the pressure corresponding to the main phase transition [152]. Hence
for higher lateral pressures the monolayer would prefer to form a threedimensional crystal on the water surface. From this follows that generally
the ordered monolayer phases are metastable.
(ii) The lipid density on the surface is on the order of 10−10 moles/cm2 and if all
molecules would dissolve in the subphase of 1 cm depth a lipid concentration
of 10−7 M would result. If the lipid solubility is close to this value, a considerable fraction of the monolayer is dissolved in the subphase in thermodynamic
equilibrium. This is not a problem for the most frequently studied systems
with two saturated long aliphatic tails per head group, but it is critical for
lysolipids and may be critical if there is a large hydrophilic head group or if
the tail hydrophilicity is increased via unsaturated bonds or other hydrophilic
attachments. The latter aspect also raises the question if the monolayer can
be considered a closed system at all.
(iii) Evidence has been provided from measurements of resistivity towards subphase evaporation that the monolayer may be transformed into a bilayer under
specific conditions [17]. This as well as the collapse into a multilayer film is
another mechanism of transforming the monolayer into a more stable threedimensional phase [153].
192
H. Möhwald
In conclusion in many cases the monolayer is not in thermodynamic equilibrium
with the environment, but often nucleation of the more stable phases is very slow
or even unmeasurable during times of days. This means that one may still apply
equilibrium thermodynamics, however, being aware of the restraints imposed to
consider merely local equilibrium. One also has to expect situations where the system
is changed qualitatively not due to a change in parameters strongly influencing the
equilibrium but solely to the conditions for nucleation of a more stable phase. These
parameters may be spurious amounts of impurities, synthesis of impurities by a
surface reaction or local distortions due to protein interactions.
In addition there is also evidence that monolayers are nonergodic and behave like
a glass. Therefore they may not be in equilibrium considering some of their order
parameters, e.g., long range translational order but in equilibrium with respect to
other variables like orientational order. Hence it is principally necessary to specify
for any order parameter separately if it is an equilibrium property.
4.2. Phase diagram
With these cautious remarks we will try to summarize presently available knowledge
about regions of the phase diagram given in fig. 13. At low molecular densities there
is a gaseous phase. This is not completely disordered like in a three-dimensional
system, but the molecules exhibit a preferential orientation relative to the surface
normal. Increasing the molecular density one either enters a liquid expanded (LE)
or a more ordered (LC) phase via a region of phase coexistence or, at higher temperatures, one continuously enters the LC phase.
The LE phase exhibits average molecular areas between 150 Å2 and 50 Å2 depending on type of molecules and surface pressure. Its order is qualitatively identical
to that of the gaseous phase, but its compressibility is considerably larger than that
of a typical liquid or of a bilayer membrane. This concerns the two-dimensional
compressibility but not the three-dimensional compressibility χt of the hydrophobic
membrane moiety. The latter is derived from the pressure dependent measurements
of ρt via X-ray reflectivity
χt =
1 ∆ρt
.
ρt ∆ρ
Typical values determined for DLPE are χt = 5 × 10−5 /atm taking
∆ρ =
∆π
20 Å
as a three-dimensional pressure, and this value is also obtained for a liquid like
water. Viscosity and diffusion coefficient in the LE phase are close to values previously derived for bilayers in their Lα phase. There are indications from indirect
measurements of surface viscosity that there is another fluid phase at the same molecular density [4]. This was suggested to be positionally disordered but to exhibit a
preferential tail arrangement within the surface, equivalent to a smectic c phase.
Phospholipid monolayers
193
Fig. 13. Monolayer phases going along an isotherm. The dashed lines mark coexistence ranges of LE
and gaseous (G) phase and of LE and LC phase. Indicated are also the pressures πc(1) , πc(2) corresponding
to critical points.
The LC phase is reached by compression either directly from the gaseous phase
or through a coexistence range from the LE phase. It exhibits much lower diffusion
coefficients than the LE phase and a low density of kink defects. The aliphatic
tails are aligned parallel and the cross section per tail is close to that of alcanes in
their rotator phases [83, 84]. The tails may be uniformly tilted with tilt angles up
to about 30◦ and with the tilt angle continuously reduced on compression without
changing the volume density ρt . Every tail possesses six nearest neighbors and the
projections of the tails on the surface form a centered rectangular or an oblique
lattice. In the LC phase the orientation of local crystallographic axes is correlated
over long distances. Experimental results are not yet conclusive if the tilt azimuth
is correlated over long ranges. This is suggested from fluorescence polarization
data [154], and electron diffraction results indicate a sixfold degeneration over distances of 10 µm [119]. The positional order extends over merely 10 lattice spacings
and is anisotropic with the larger correlation range normal to the tilt direction [155].
At higher pressures the LC phase is transformed into a phase called S (solid)
without discontinuously changing the density. S is distinguished from LC by a
normal tail alignment and by a larger positional correlation range. Yet this extends
over merely 100 lattice spacings, much less than expected for a crystalline phase.
X-ray reflectivity results suggest that the head groups are rather well aligned and
little hydrated in this phase but there is no clear conclusion on positional and/or
194
H. Möhwald
orientational order of the head groups possible. This could be inferred if it was
possible to detect an X-ray or electron diffraction signal corresponding to a head
group superlattice. In this phase the lattice is hexagonal, and the compressibility is
about a factor of 3 smaller compared to the LC phase.
Recent X-ray diffraction experiments with DMPE, DPPE and DSPE monolayers
reveal the existence of a phase where the centers of the projections of the tails on
the surface form a nonsymmetric triangle. This is only found if the substance is
chirally pure [87]. In case of a racemate the lattice is rectangular under the same
conditions. This indicates positional order of the head groups breaking the symmetry
of an otherwise rectangular lattice of the chirally pure compound. Obviously this
head group order is easily destroyed for the racemate. One also observes phases
where the tail cross section is distinctly below 20 Å2 [156]. This is not surprising in
view of recent results with fatty acid monolayers where also crystalline phases with
cross sections per chain of 18.6 Å2 were detected [157]. These structures correspond
to the so-called herringbone structures of n-alcanes where the chains are hindered
to rotate about their long axes [83, 84]. In contrast to the findings with fatty acids,
however, the diffraction peaks are still as broad as in the S phase. This indicates
that the presence of head groups linking two tails prevents long range positional
ordering. Yet I am convinced that in the near future one will find monolayers
exhibiting the richness of phases now detected and characterized for fatty acids,
alcohols and esters [156–160].
4.3. Phase transitions
Having characterized different monolayer phases we should now concentrate on the
basics of transitions between them. The transition from the gaseous to the LE or
LC phase is clearly of first order for a temperature below a critical one (Tc(1) ). The
corresponding critical pressure πc(1) is below 1 mN/m, and hence the monolayer is
in a liquid phase for pressures above this value. The order parameter describing
the transition obviously is the density. The entropy changes involved are due to a
loss of translational freedom but also of orientational degrees of freedom, since the
molecule in the fluid phase can no longer be parallel to the surface due to steric
reasons.
For temperatures below a tricritical one (Tc(2) ) the LE =⇒ LC transition is of
first order with a latent heat ∆H of the same order of magnitude as for the main
transition of bilayers [4]. Approaching Tc(2) from below ∆H approaches zero often
proportional to Tc(2) − T and from this Tc(2) can also be derived by extrapolation.
The parameter driving the transition surely is the internal ordering of the aliphatic
tails although there are also remarkable changes in the three-dimensional density:
The electron density in the hydrophobic moiety may change by more than 10%
[88] which is to be compared with changes of at most 3% for the main transition
of bilayers [161, 162]. Considering further the coupling of order parameters the
transition to the LC phase also involves a drastic increase in the correlation length of
bond orientational and of tilt orientational order, less so of translational order. Up to
now there is no strong support that the LE =⇒ LC transition also involves head group
Phospholipid monolayers
195
ordering. However, there are indications of systems with direct LE =⇒ S transitions
[163] and these may involve head group ordering.
All available data indicate that the LC =⇒ S transition is of second order. The
lattice spacings change continuously, but the compressibility discontinuously. The
lattice symmetry changes and the positional order correlation range increases on
compression. Yet it is not conclusive if the latter increase is continuous or discontinuous.
Also at this stage a comment should be made on the non-horizontal isotherm slope
going through the LE =⇒ LC phase transition. Very probably it is caused by surface
active impurities in most cases, but even if the system would be infinitely pure the
slope would not be horizontal. This is due to long range electrostatic interactions creating a heterogeneous surface composition and a domain/domain interaction yielding
contributions to the surface free energy that increase with compression [164]. These
repulsive interactions between domains are measurable, but their contribution to the
lateral pressure is immeasurably small [25].
4.4. Domain structure
A unique feature of monolayers is their regular domain structure in the LE/LC phase
coexistence range.
It has been pointed out that this can be understood from an interplay between line
tension and long range electrostatic repulsion. Very crucial is the question to what
extent are these nonequilibrium features and influenced by impurities?
Impurities predominantly reduce line tension and this in turn reduces the free energy to create a critical nucleus and the force that tends to smooth and to shorten
the domain boundary. This can indeed be observed fluorescence microscopically:
Increasing the impurity content the density of domains increases and the boundaries
become rough and do not anneal within hours [8]. Also in increasing the compression speed the domain density increases [41]. Thus the interdomain distance
is a nonequilibrium property that may also be influenced by impurities. The domain shape for some systems depends on the growth history, but there are systems
where one can observe the establishment of local equilibrium after distortion. Thus
the width of lamellar domains is uniform and it can be reversibly varied, e.g., on
temperature cycling. Still the width can be varied via impurity content through the
influence of the latter on line tension [44].
Considering the gas/LC phase coexistence one observes a foamlike structure that
changes dimensions with time [31, 165]. This structure also varies laterally, since it
is very sensitive to gradients of temperature, pressure and concentration at these low
pressures. Also the domain shape in the gas/LC phase coexistence range is difficult
to control. In nearly all practical cases one does not go by continuous compression
from a gaseous to the LC phase but spreads enough material to start compression
in the coexistence range. Hence domains are formed by solvent evaporation and
thus depend on these conditions. On compression the density of these domains
is increased but their structure and shape is fixed. At high domain densities the
interdomain interaction may cause deformation or disruption of domains as classically
expected [34].
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H. Möhwald
5. System diversity
Above we have concentrated on showing basic features of well-defined phospholipid
monolayers and will subsequently try to conclude on more complex systems. For
these there do not exist too many structural data and the extrapolation therefore is
not based on many facts.
5.1. Saturated straight chain lipids
The influence of varying the chain length on latent heat and temperature of the bilayer
main transition is also reviewed within this book. The situation is qualitatively
similar for the monolayer: Extending the aliphatic tail the pressure corresponding to
the LE/LC transition is reduced for a given temperature and head group. This also
holds if head group repulsion is reduced via divalent ion binding or salt addition. In
addition if a lipid possesses a bulky head group impeding tail attraction the transition
pressure is increased (e.g., compare DMPE and DMPC). The qualitative nature of the
LE and LC phase, however, remains unchanged with two exceptions: (1) Repulsive
intermolecular forces may be so large that an LC phase is not formed at all. (2) The
bulky head group may inhibit formation of the S phase or a continuous reduction
of the tilt angle on compression. The latter holds for DPPC (e.g.) forming a ripple
(Pβ 0 ) phase in a bilayer system [78]. In the monolayer the surface is flat and the tilt
angle remains 30◦ up to the collapse pressure.
5.2. Unsaturated bonds
It is known that unsaturated bonds tend to create disorder in the hydrophobic region
and thus hinder LC phase formation. This again is equivalent to a temperature
increase or a reduction of Tc(2) . In many cases this totally removes the ordered phases
unless the tails are considerably elongated (e.g., DOPC).
5.3. Chain branching
Chain branching creates disorder with consequences on the phase diagramme as
listed above, but this depends on the type and position of the branch. A methyl or
ethyl branch near the hydrophobic region/air interface only increases the transition
pressure and the data analysis yields a reduction of transition enthalpies [166–168].
These groups near the head group have only a small influence [11, 12]. In the latter
case they are probably embedded in the head group moiety whereas in the former
case they are in a region where there is chain disorder also in the LC phase. Short
branches connected to the center of the hydrophobic region or branches of medium
length cause a drastic distortion that may even prevent LC phase formation. In case
the branch is nearly as long as the original tail and connected near the head group
the monolayer behaves as if there is an additional tail per head [168]. However,
there are subtle structural differences considering the position of the branched tails.
It affects the shapes of LC phase domains [169] and also the amount of chain tilt in
the LC phase [170].
Phospholipid monolayers
197
5.4. Different head group features
In most cases head group interactions are considered repulsive and these contributions
have been studied extensively for phosphatidic acid, varying ionic conditions [19, 20,
42, 171, 172]. However, beyond electrostatic and steric repulsive forces there may
be attractive interactions, the most prominent one being hydrogen bonding between
ethanolamine, serine or glycerol and the phosphate oxygens. These interactions
may be directional but could not be assessed quantitatively. It is also apparent that
the dipoles connected with the head groups may induce an attractive interaction if
aligned properly [173]. The latter holds for the component parallel to the surface.
However, recent theoretical calculations have shown that counterions and the water
of the subphase screen these interactions resulting in quadrupolar forces [173]. These
forces decay over short distances and thus do not diverge with domain size as known
for dipolar interactions.
There are also many efforts to attach functional groups in the hydrophilic membrane region. These may be dyes, or ligands to specifically bind proteins, or polymerizable groups to link the head groups. The dominant interaction then depends on
the detailed nature of the group, and interesting new features arise from the presence
of a flexible polar group in the head group region. In this case head group interactions can be varied by means known for the swelling of three-dimensional polymers,
but the reduced dimensionality may induce interesting new physical phenomena.
5.5. Lipid mixtures
Natural lipids occur as mixtures of phospholipids with different degrees of unsaturation and with different head groups. There are no direct stuctural studies on these,
and the isotherms appear typical for the LE phase. Thus I presume the structure is
disordered as the LE phase. However, our knowledge with bilayers of defined mixtures suggests that there may be phase separations if one of the components prefers
an ordered phase. This may be induced via divalent ion binding to charged head
groups or occur if one component exhibits long saturated tails. Hence it is possible
but difficult to prove that components and phases are distributed heterogeneously.
Systematic studies on defined mixtures of phospholipids are still scarce. Albrecht
et al. presented a phase diagramme for lipids with the same tail but different head
groups based on careful isotherm measurements [14]. The components are miscible
in their fluid phases but there is a miscibility gap if both are in the LC phase.
Systematic studies with phosphatidylethanolamine mixtures of varying chain length
have been performed by the Halle group [174, 175]. These show miscibility if the
differences in chain lengths are merely 2CH2 units in accordance with measurements
on bilayer vesicles [176]. Concerning miscibility studies with varying chain length
there is an important difference between monolayer and bilayer. In a monolayer the
component with the longer tail separating in a LC phase increases its high energy
interface towards air, whereas in a bilayer the corresponding energy is lower due to
solvation by the opposite monolayer.
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H. Möhwald
5.6. Cholesterol/lipid mixtures
Due to its great importance for understanding the function of biological membranes
mixtures of cholesterol and phospholipids have been studied in great detail. For
these studies phosphatidylcholines were selected, but I will below present arguments
against generalizing the results. A very interesting feature in the phase diagramme
is the existence of fluid/fluid phase separations, at low surface pressures [16, 177].
Sufficiently far from the critical point there coexist two phases, one with a minimum
cholesterol/lipid ratio of about 1: 1 and one with a maximum ratio of 1: 6 (see fig. 14).
The latter value has led to specific molecular pictures of the cholesterol arrangement
in the lipid matrix, resembling complex formation. Such complexes can also explain
the findings with bilayers that cholesterol tends to rigidify a fluid membrane.
The notion that a phase cannot be characterized by a single variable has recently
been introduced into theoretical calculations of the phase diagram [178, 179] (see
fig. 15). There one distinguishes between positional order and conformational order,
the latter concerning the question if the tails are in all-trans configuration or if there
is a substantial fraction of kink defects.
Fig. 14. Phase diagramme of mixed monolayers of DMPC and cholesterol as determined from fluorescence microscopic (×, +) and film balance experiments (from ref. [177]). Phases I, II and IV are
homogeneous and fluid, differing in partial molecular area and hence chain configuration of the phospholipid. Region III corresponds to coexistence of II and IV with an inaccuracy of the phase boundaries
estimated from the right curves. T = 23, 5 ◦ C.
Phospholipid monolayers
199
Fig. 15. Phase diagramme of a bilayer of Dipalmitoylphosphatidylcholine and cholesterol as calculated in ref. [179]. The phases are characterized as follows: so: translationally and configurationally
ordered, ld: translationally and configurationally disordered, lo: translationally disordered and configurationally ordered. The boundaries as well as the phase coexistence ranges are in reasonable agreement
with experimental data.
These types of phase separations have recently gained interest since the fluidity of
coexisting phases allows for domain shapes varying quickly in response to external
forces [180]. Hence domains can be studied at local equilibrium and even thermally
activated shape fluctuations have been analyzed [181, 182].
The incorporation of cholesterol in an ordered lipid phase has been also considered from a solid state physical point of view where the ‘impurity’ tends to enrich
at defect points or lines [183]. This could hold for any impurity, but all effects measured with bilayers are very specific for cholesterol. With monolayers an additional
highly specific influence of cholesterol on the domain shapes was observed. Even
for a cholesterol content below 1 mole% an elongation of lamellar domains was
observed, but this observation could be made only for DPPC and DMPA at high pH
where the chains are tilted uniformly with tilt correlation over µm dimensions. This
can be quantitatively explained assuming that cholesterol arranges at the boundary
200
H. Möhwald
between LE and LC phase and thus reduces line tension, the energy to create the
boundary. The fact that this effect is specific for cholesterol and for lipids with large
tilt suggests that these present suitable steric conditions for their optimum interaction. On the other hand there is only a weak influence of cholesterol on the domain
morphology of other phospholipids. This suggests also a different phase diagram,
but data on these are still missing for monolayers.
5.7. Protein/lipid mixtures
Studies have been performed where the lipid monolayer is used as a matrix to
orient proteins. These have proved to be very useful for membrane proteins that by
themselves are asymmetric and that are involved in directional transport [184, 185].
However, the information gained on these proteins may be of less interest here.
Instead we will concentrate on questions related to the protein/lipid interaction.
The interaction of the protein and other water soluble compounds with the lipid
environment has been mostly studied via the influence on the isotherms [186–189].
Water soluble proteins like cytochrome c or spectrin electrostatically bind to a negatively charged membrane [190–192] and this can lead to a reduction of the pressure
corresponding to the LE/LC phase transition. However, the protein/lipid
interaction
generally is rather local and therefore at low protein/lipid ratios <1: 1000 mole
the
mole
system may be treated like a phase separated one and at higher ratios the features of
the pure monolayer are lost totally [193]. Thus more local probes are needed to detect
these interactions. One more direct probe has become fluorescence microscopy that
was able to show that all proteins preferentially arrange in the LE phase of the monolayer, but the technique did not yield information on local interactions [193, 194].
A crucial problem with many studies of protein/lipid interactions is that proteins
tend to insert into the membrane, unfold irreversibly and form clusters. These clusters can be observed by fluorescence microscopy, but it is very difficult to avoid
them [194]. One way out is to insert a small fraction (∼ 1 mole%) of amphiphilic
ligands into the monolayer and then study specific interactions. Successful examples
on this have been haptens to study antibody interactions [195, 196], glycolipids to
study binding of lectins [194] and biotin lipids for avidin binding [197]. In the latter
case where the binding constant is exceptionally large protein crystallization at the
interface has been observed [198].
For this system new optical, X-ray and neutron reflection techniques to locally
characterize the interface have been established [67, 97]. They show that the protein
is closely attached to the monolayer, forms a dense film, but contains between 0.3
and 1 volume fraction of water.
In very elegant experiments the diffusion of membrane bound antibodies has been
investigated by FRAP [199–201]. The results suggest specific transport mechanisms
due to partial network formation of the protein.
A great deal of effort has been devoted to studies of interactions of phospholipases with phospholipids by means of surface pressure, potential and radioactivity
measurements [202–207]. These experiments led to conclusions on the lytic activity
as regards to monolayer phase and type of lipid. The increased activity observed for
Phospholipid monolayers
201
monolayers in the phase coexistence range has now been verified more directly by
fluorescence microscopy [208]. The preferential lysis by the protein at the boundary
between LE and LC phase has been observed indicating that the protein destroys
LC domains starting from specific defect areas.
The latter example has also demonstrated that the protein prefers a specific lipid
arrangement for its optimum function. This has also been suggested for other proteins
and proved in experiments with bilayers. For the monolayer such an example is still
missing. With respect to this one should be aware of the fact that the monolayer,
being only half of the membrane, is not a good model to study interactions of
membrane proteins. An exception to this surely will be the interaction of the protein
with the head group or with the adjacent water phase, e.g., electron and proton
transport in photosynthesis.
6. Future outlook
This contribution has concentrated on our understanding of some of the most simple phospholipid systems, and I have to apologize with those colleagues working
on complexer systems who are therefore not well represented here. The latter is
unfortunate since the biologically more relevant systems are not the simple ones.
However, lack of space and partly also of knowledge prevented me from simply
listing the wealth of nature. Instead I wanted to spend more effort to elaborate on
physical principles. This could also provide some ground to evaluate future research
directions.
Very important questions will arise around the problems of ordering in the head
group region. New techniques are now emerging to conclude on the head group
arrangement as advanced X-ray and neutron techniques, optical second harmonic
generation, FT-IR spectroscopy, and also computer simulations are able to determine
the head group arrangement and water structure. These questions are important,
since the hydrophilic membrane region mediates interactions with the adjacent water
phase and also carries dipoles that provide long-range interactions.
Having realized that short- and long range order has to be described by many
parameters much work will have to be devoted to the coupling of these parameters.
Clearly answers will be different for different systems and the question how to control
this coupling and how to describe and quantify it will become important. Order
appears to exist on different length scales for some parameters and this theoretically
and experimentally leads to periodic density fluctuations. These may not only be a
theoretical curiosity but also enable long distance information transfer.
Concerning the latter aspect the membrane has not only been considered an electrical but also a mechanical medium. Techniques are now emerging to measure local
and global elasticities and this surely will renew interest in this field.
Controlling the structure is also related to understanding domain formation. This
concerns interesting aspects of physics in two dimensions (with negligible gravity)
but also local and global equilibrium properties. An essential parameter in this field
will be line tension, the energy per length of the boundary between different phases.
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H. Möhwald
Clearly questions following will be how to influence line tension via environmental
parameters and impurities.
Concluding I should stress that I do not think that these areas have to be worked out
before devoting more effort to complexer systems, but answering the above questions
will serve much to an understanding of the complicated biological apparatus.
Acknowledgement
In writing this chapter I have profited from numerous experimental contributions
from my skillful and eager co-workers. However, this review would never have
been completed without the competent and elaborate literature research by Andrea
Dietrich and Ulrike Höhne, by the patient and careful preparation of this manuscript
by Helga Resch.
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