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Advances in Colloid and Interface Science 207 (2014) 93106

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Advances in Colloid and Interface Science


journal homepage: www.elsevier.com/locate/cis

Interactions in lipid stabilised foam lms


Jos Luis Toca-Herrera a, Nadejda Krasteva b, Hans-Joachim Mller c, Rumen Krastev d,e,
a

Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 11, Vienna, Austria
Baldungstr, 70736 Fellbach, Germany
c
Max-Planck Institute of Colloids and Interfaces, 14424 Golm/Potsdam, Germany
d
NMI Natural and Medical Sciences Institute at the University of Tbingen, 72770 Reutlingen, Germany
e
School of Applied Chemistry, Reutlingen University, 72762 Reutlingen, Germany
b

a r t i c l e

i n f o

Available online 22 February 2014


Keywords:
Foam lms
Lipids
Electrostatc interaction
van der Waals interaction
Hydrophilic interaction
DLVO theory

a b s t r a c t
The interaction between lipid bilayers in water has been intensively studied over the last decades. Osmotic stress
was applied to evaluate the forces between two approaching lipid bilayers in aqueous solution. The force
distance relation between lipid mono- or bilayers deposited on mica sheets using a surface force apparatus
(SFA) was also measured. Lipid stabilised foam lms offer another possibility to study the interactions between
lipid monolayers. These lms can be prepared comparatively easy with very good reproducibility. Foam lms
consist usually of two adsorbed surfactant monolayers separated by a layer of the aqueous solution from
which the lm is created. Their thickness can be conveniently measured using microinterferometric techniques.
Studies with foam lms deliver valuable information on the interactions between lipid membranes and especially their stability and permeability. Presenting inverse black lipid membrane (BLM) foam lms supply information
about the properties of the lipid self-organisation in bilayers. The present paper summarises results on microscopic lipid stabilised foam lms by measuring their thickness and contact angle. Most of the presented results
concern foam lms prepared from dispersions of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3phosphorylcholine (DMPC) and some of its mixtures with the anionic lipid 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (DMPG).
The strength of the long range and short range forces between the lipid layers is discussed. The van der Waals
attractive force is calculated. The electrostatic repulsive force is estimated from experiments at different electrolyte concentrations (NaCl, CaCl2) or by modication of the electrostatic double layer surface potential by incorporating charged lipids in the lipid monolayers. The short range interactions are studied and modied by using
small carbohydrates (fructose and sucrose), ethanol (EtOH) or dimethylsulfoxide (DMSO). Some results are compared with the structure of lipid monolayers deposited at the liquid/air interface (monolayers spread in Langmuir
trough), which are one of most studied biomembrane model system. The comparison between the lm thickness
and the free energy of lm formation is used to estimate the contribution of the different components of the
disjoining pressure to the total interaction in the lm and their dependence on the composition of the lm
forming solution.
2014 Elsevier B.V. All rights reserved.

Contents
1.
2.
3.
4.

5.

Introduction . . . . . . . . . . . . . . . . . . .
Forces acting between the foam lm surfaces . . . .
Formation of lipid stabilised foam lms . . . . . .
Long range interactions in lipid stabilised foam lms
4.1.
van der Waals attraction . . . . . . . . . .
4.2.
Electrostatic double layer repulsion . . . . .
Short range interactions in lipid stabilised foam lms
5.1.
Effect of ethanol . . . . . . . . . . . . . .

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94
95
96
97
97
97
100
100

This paper is dedicated to Prof. Helmuth Mhwald. All presented results were obtained at his Department of Interfaces at the Max-Planck Institute of Colloids and Interfaces in Golm/
Potsdam, Germany.
Corresponding author at: NMI, Reutlingen, Germany.
E-mail address: rumen.krastev@nmi.de (R. Krastev).

http://dx.doi.org/10.1016/j.cis.2014.02.016
0001-8686/ 2014 Elsevier B.V. All rights reserved.

94

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

5.2.
Effect of low molecular weight solutes at low ionic strength
5.3.
Effect of electrolyte concentration at high solute content .
5.4.
Effect of solutes at high ionic strength . . . . . . . . . .
6.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . .
7.
Experimental techniques . . . . . . . . . . . . . . . . . . .
7.1.
Contact angle . . . . . . . . . . . . . . . . . . . . .
7.2.
Film thickness . . . . . . . . . . . . . . . . . . . . .
7.3.
Velocity of lm thinning . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
Thin liquid free standing lms or foam lms as they have to be called
according to the IUPAC recommendations [1] have been a fascinating
object of studies for the last 60 years. These lms attract the attention
of children because of the charming play of colours during their thinning (Fig. 1), but they have also played an enormous role to prove
such milestone theories in the mesoscopic and nanometre world like
the theory of DerjaguinLandauVerweyOverbeek (DLVO), which
has been extensively used to predict the stability of lyophobic colloids
[26] and the existence of a disjoining pressure () between two surfaces in close proximity. A specic property of these lms is a very
large difference in size along the lateral and normal directions. Even
though their area is in the macro-world and can be extended even up
to square metres, their thickness can be tuned down to a few
nanometres. This makes them a suitable tool to study the interactions
between surfaces because of the comparatively easy handling, reproducible preparation and their symmetrical geometry.
Foam lms are formed usually from solutions of surfactants [57].
Their thickness is conveniently measured using the microinterferometric
method [59] (Fig. 1). The lms consist of two adsorbed surfactant
monolayers with a thickness h1 separated by a layer of the aqueous
solution from which the lm is created (Fig. 1b) with a thickness
h2. The equivalent solution lm thickness hw is the thickness of the
lm assumed to be a homogeneous leaf of surfactant solution. Indeed
the lm structure is more complicated (sandwich-like structure of
layers with different refractive indices) where the thickness h is
the real lm thickness. The calculation of h and h2 can be easily

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102
103
103
104
104
105
105
105
106
106

obtained from the measured hw (for details see Part 6 Experimental


techniques).
During the lm formation, rst a thick non-equilibrium lm is
formed which becomes thinner upon drainage of the solution due to
the capillary pressure in the meniscus, gravity or action of the surface
forces [57]. Finally, an equilibrium lm is obtained with a uniform
thickness depending on the thermodynamic conditions (pressure, surfactant and salt concentration, temperature, etc.). The very thin lms
appear black in reected light and are called black lms. Two types of
black lms exist (Fig. 1a). The thicker common black lm (CBF) appears
at lower electrolyte concentrations. It shows typical Newton rings in
reected light in the area of the meniscus which surrounds the lm.
The thickness of the CBF and its stability are controlled by the electrostatic double layer repulsion [57] in agreement with the classical
DLVO theory. The electrostatic double layer repulsion is suppressed at
higher electrolyte concentrations, and the equilibrium state in this
case is a very thin Newton black lm (NBF). Once this state is reached,
the lm thickness is independent on the electrolyte concentration and
is only determined by the direct interaction of the surfactant adsorption
layers by short-range surface forces.
The properties of foam lms that have been explored in detail include equilibrium lm thickness, contact angle lm/meniscus, thinning
rate, lm stability, lm elasticity, etc. [5,6,10]. Measurements on the lm
thickness allow establishing a direct relation of the strength of the interactions between the lm surfaces and the distance between them. One
of the most widespread techniques for foam lm formation is the
ScheludkoExerowa glass ring cell technique [6,7] (Fig. 1a, see also
Part 6 Experimental techniques for details). A foam lm is formed

a)

b)
h1
h
h2

Fig. 1. a) Schematic view of a typical experimental set-up with small glass ring where the lms are formed. The photos show images of foam lms at different stages of thinning correlated
with their internal molecular structure CBF (common black lm) or NBF (Newton black lm). M is a microscope used for observing the lms; b) Sandwich-like structure of a foam lm
which consists of two adsorbed lipid monolayers (thickness h1) and a water core (h2), including the region of the hydrated head groups.

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

from a biconcave drop in a glass capillary. The cell allows the lm thickness, contact angle with the surrounding meniscus or lm stability to be
measured.
The interaction between lipid bilayers in water has been intensively
studied over the last decades [1117]. Osmotic stress was applied to lamellar phases and the bilayer distance was measured using small-angle
X-ray diffraction to evaluate the forces between two approaching lipid
bilayers in aqueous solution [11,15,17]. Alternatively, the force and the
distance were measured between lipid mono- or bilayers deposited on
mica sheets using surface force apparatus [12]. Foam lms stabilised
by lipids offer another possibility to study the interactions between
lipid monolayers [1826]. Such studies deliver valuable information
about the interactions between biological lipid membranes and especially their stability and permeability. Presenting inverse black lipid
membrane, the foam lms supply information about the properties of
the self-organised lipid molecules in bilayers.
The present paper summarises the results of our studies on microscopic lipid stabilised foam lms by measuring their thickness and
contact angle. Different methods have been developed for the preparation
of foam lms stabilised by insoluble biosurfactants such as phospholipids
[2729]. The main part of the presented results concerns foam lms prepared from dispersions of the zwitterionic lipid 1,2-dimyristoyl-snglycero-3-phosphorylcholine (DMPC) and some of its mixtures with the
anionic lipid 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
(DMPG). The strength of the long range and short range surface forces
between the lipid molecules as obtained from the thin foam lms experiments is discussed. The van der Waals attractive force is calculated.
The electrostatic repulsive force is estimated from experiments at different electrolyte concentrations (NaCl, CaCl2) or by modication of
the electrostatic double layer surface potential by incorporating
charged lipids in the lipid monolayers. The short range interactions
are studied and modied by using small carbohydrates (fructose and
sucrose), ethanol (EtOH) or dimethylsulfoxide (DMSO). Some results
are compared with the structure of lipid monolayers deposited at the
liquid/air interface (monolayers spread in Langmuir trough), which
are one of most common biomembrane model systems [3034]. The
comparison between the lm thickness and the free energy of lm formation is used to estimate the contribution of the different components
of the disjoining pressure to the total interaction in the lm and their
dependence on the composition of the lm forming solution.

A foam lm is always surrounded by a bulk phase (meniscus). At


equilibrium, the disjoining pressure ( (h)) in the lm equals the
capillary pressure PC in the meniscus.
2
RC

is the van der Waals attraction [46,35] with AH being the Hamaker
constant
 .

2 F

EL dEL 64RT th

4RT

expdEL ;

according to the weak overlap approximation is the double layer electrostatic repulsion [46,36]. R is the universal gas constant, T is the
absolute temperature, is the concentration of ions in the bulk
of the solution (h = ), is the electrostatic double-layer potential,

1 =
z2i e2 =0 kT 2 is the Debye length, F is the Faraday constant,
z is the ionic valence, is the medium permittivity and 0 is the permittivity of the vacuum. The electrostatic interaction is determined
by the double-layer potential of the surfaces [46]. In many cases,
this potential is caused by the adsorption of surface active ions. However, small negative double-layer potentials have been observed
when the lms where formed from solutions of non-ionic
amphiphiles [3739].

HYDR dHYDR H0 expdHYDR =

is the hydration repulsion [4,40] which is a result of the direct interaction between the water molecules in the hydration shell near an interface. H0 takes into account the hydration energy of a surface and is
the decay length of the hydration repulsion. others refers to the other
possible interactions (entropic, ion correlation, etc.).
The values of the disjoining pressure can be directly accessed experimentally by using the porous-plate technique or thin lm pressure balance (TFPB) method rst developed by Mysels and Jones [41] and later
improved by Exerowa [6,7]. Indirectly information about the interactions in the foam lms can be obtained from measurements of the contact angle between the lm and its meniscus (Fig. 2). The contact angle
is connected to the disjoining pressure by the free energy of lm formation per unit of area Ff by the relation [5,7,4244]
Zh
f
F 2 cos1 hdh h:

2. Forces acting between the foam lm surfaces

h PC

95

The Ff is a measure for the difference in the thermodynamic energy


state of the two states of lm formation two fully separated noninteracting solution/air interfaces and the nal equilibrium lm where
two interfaces interact with each other.

where RC is the radius of the curvature of the meniscus which is equal to


the radius of the glass capillary where the lm is formed. is the surface
tension of the solution from which the lm is prepared.
The disjoining pressure has different contributions and it can be
written as
h VW dVW EL dEL HYDR dHYDR others dOTHER

here with dn (n = VW, EL, HYDR and OTHER) we indicate different distances between the interacting interfaces which are situated at different
positions in the lm. This takes into consideration the distinct planes of
interactions active for each type of interfacial forces.
In Eq. (2)
VW dVW

AH
6d3VW

Fig. 2. Denition of the contact angle in a symmetrical lm under mechanical equilibrium between the lm tension f and the surface tension .

96

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

3. Formation of lipid stabilised foam lms


Phospholipids spontaneously form suspensions of vesicles in water
solutions [34]. Depending on their treatment different vesicles can be
formed. Small unilamelar vesicles (SUV) with a diameter of around
100 nm constituted from a single lipid bilayer are one of the most
well-characterised forms of lipid dispersions in water [34]. Stable
foam lms can only be formed when the monolayers of surfactant
which build the lms are saturated and the adsorption density is the
highest possible [6,45]. Because of the very low solubility of the lipids
in water they cannot form densely packed adsorbed lipid monolayer
at the water surface. It is commonly accepted that a condensed monolayer of lipid molecules at the liquid/air interface can be formed by
attachment of SUV to the interface where they rupture and spread to
form a lipid layer [45 - 47]. Different procedures for preparation of
lipid dispersions are described in the literature. We studied the properties of foam lms stabilised by lipid suspensions prepared according to
the following procedures:
Procedure A: Lipid dispersions (solutions) in a water/ethanol mixture
with a constant ratio of 52.5/47.5% vol. containing different amounts of electrolytes. The solutions were prepared
by sonication for 15 min in a sonicator bath and kept before the experiment overnight in refrigerator.
Procedure B: Lipid dispersions in the aqueous solution of the used electrolyte were kept at room temperature overnight. The
foam lms were prepared without further treatment of
the suspension.
Procedure C: Lipid dispersions prepared according to Procedure B but
sonicated for 15 min in a sonicator bath before use.
Such a procedure leads to formation of a suspension of
SUV [48]. The usual lipid concentration used was
0.4 mg/ml.
The time dependence of the surface tension for each type of lipid
dispersions was measured and the dynamics of adsorption at the liquid/
air interface was followed. Fig. 3 shows one such dependence obtained
for suspensions of DMPC in a solution of 0.07 M NaCl and prepared according to each of the three procedures. The surface tension decreased
with time reaching its saturation value after about 10 h for dispersions
prepared according to Procedure C. Similar behaviour was observed for

60
55

, mN/m

50
45

C
40
35

30

200

400

600

800

Time, min
Fig. 3. Time dependence of the surface tension for lipid suspensions (0.4 mg/ml DMPC in
0.07 M NaCl solution) at 26 C for the three preparation Procedures A, B and C as described
in the text.

the dispersions prepared according to the Procedure B (saturation was


achieved in around 10 h), but the surface tension in this case was higher
indicating that the formed lipid monolayers were less densely packed
than those prepared according to Procedure C. The time dependence
of the surface tension for DMPC dispersions in water/ethanol mixture
(Procedure A) shows that equilibrium is reached almost immediately.
These results allowed selecting suitable experimental conditions for
preparation of stable lipid stabilised foam lms.
Films prepared from DMPC solutions in water/ethanol mixtures
(Procedure A) were stable in the temperature range from 20 C to
30 C. Films formed from lipid dispersions in water (Procedures B and
C) were stable only above 22 C. This temperature is very close to the
temperature of the main phase transition (23.5 C) in bulk dispersions
of DMPC [45]. Consequently, all experiments presented in the paper
were carried out at 26 C, a temperature that assured formation of stable lms. Differential scanning calorimetric data show that the temperature of the main transition does not depend on the EtOH concentration
for DMPC dispersions in water/EtOH mixtures. The increase of the EtOH
content shifts only the pre-transition peak to higher temperatures [27].
The transition from gel to liquid crystalline state facilitates the spreading of the lipid vesicles at the air/water interface and is an important factor which governs the lm stability.
The velocity of lm thinning depends on the presence of EtOH in the
lm forming vesicular suspension. A typical lm thinning curve is
shown in Fig. 4a. Initially formed thick lms have a thickness of around
80 nm. They get thinner under the action of the capillary pressure and/
or interfacial forces. A black lm (either CBF or NBF) is formed with a
jump at a certain thickness of the lm where the system lmmeniscus
reaches a local energy minimum. Fig. 4b compares the thinning behaviour of lms prepared according to Procedure A and Procedure C. Different thinning velocities are an indication that different types of
interactions act between the lm surfaces. The lms prepared from
EtOH containing solutions thin faster and smoother. This draining process results in a stable lm of homogeneous and constant thickness.
Fig. 4b also shows a difference in the equilibrium lm thickness
attained after the thinning. Similar to the differences observed in the
surface tension experiments (Fig. 3) the lm thickness is very sensitive
to the preparation procedure of the lipid dispersion, being higher in the
case of EtOH free lipid suspensions (see Table 1). The sonication of the
vesicular suspensions reects in a change in the lm thickness. Films
obtained from Procedure C are slightly thicker than those obtained
from Procedure B. This small thickness difference can be explained by
a larger area per lipid molecule in the monolayer of lms obtained
from Procedure B. The thinnest lms are those prepared from Procedure
A (water/ethanol mixture). The effect of EtOH on the lm thickness was
studied in details and the results are summarised in the Part 5 Short
range interactions in the lm.
An interesting question in the foam lm studies is the density of the
surfactant (lipid) molecules organised in the monolayers which stabilise the lms. Suitable technique to probe the density of the monolayers
is the uorescent microscopy. We studied the distribution of the uorescent dye L--phosphatidylcholine--(NBD-aminohexanoyl)-palmitoyl (NBDDPPC) at the surface of the lm and at the surface of
the meniscus around the lm. The NBDDPPC is located in the expanded
phase of the lipid monolayer and it is not soluble in the condensed
phase [49, 50]. In Fig. 5 the changes of the distribution of the uorescent
dye are shown in the process of lm drainage from thick lm to the thin
equilibrium state. From a) to d) the process of lm formation and thinning is in progress. The contrast is caused by the difference in the concentration of the dye in the different parts of the images. A smaller
intensity of the uorescent light (darker spots) indicates areas with
lower concentration of the dye.
The intensity of uorescence is homogeneously distributed in the
meniscus around the thick lm just after formation (Fig. 5a). The intensity in the centre of the image is lower, due to the formation of the lm.
The lm expands in images b) and c) where only some domains of the

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

a)

97

b)

100
80
80

h, nm

h, nm

60
60
40

40
20

20
0

20

40

60

80

100

120

140

Time, s

1
0

50

100

150

200

Time, s

Fig. 4. a) A typical lm thinning curve for a lm prepared according to Procedure A. The photos show images of the lm at certain thickness. b) Thinning curves for lms prepared according
to Procedure A (1) and Procedure C (2). Films prepared with EtOH thin faster and smoother. Experiments performed in the presence of 0.07 M NaCl at 26 C.

dye can be observed in the area of the lm (Fig. 5c). When the nal state
of the thinning is reached (Fig. 5d) the lm is homogeneously dark and
much contrasted in respect of the surrounding meniscus. This observation indicates an expulsion of the dye from the lm monolayers. We
conclude that this segregation is caused by a higher packing density of
the lipid at the lm surfaces compared to the surface of the bulk. This effect is similar to the case of the formation of domains of different densities of the amphiphile molecules in the two-phase coexistence range of
insoluble monolayers [30]. Such a conclusion is in agreement with the
results [30,51] about lateral diffusion of uorescent dye in the plane of
lipid monolayers which form the lm. The results reveal that the monolayers are more condensed in the case of black lms (CBF or NBF) than
in thick foam lms [52].

4. Long range interactions in lipid stabilised foam lms


4.1. van der Waals attraction
The long range surface forces operating in foam lms are the van der
Waals force and the electrostatic double layer force. The van der Waals
force is always attractive in the case of symmetrical system of thin liquid
layer between two semi-innite gas (air in our case) phases [4]. This force
is the origin of the negative component of the disjoining pressure VW
(Eq. (3)). It tends to decrease the lm thickness and nally leads to its
rupture. The Hamaker constant AH, respectively VW can be easily determined using the dynamic method [57,53] (see Part 6 Experimental
techniques for details) studying the process of lm thinning at high electrolyte concentrations where the electrostatic double layer repulsion
is suppressed. We acquired the Hamaker for lms stabilised with
0.4 mg/ml DMPC. This lipid concentration ensures formation of stable
foam lms. A NaCl concentration of 0.07 M guarantees negligible double
layer repulsion and strength of the Debye length in order of 1 nm. The
experiments were also carried out with addition of sugar or DMSO to
the lm forming solution. The obtained values of AH are presented in
Table 2. They are in good agreement with the published data for similar
systems [4,35]. Actually, the values are effective, as other attractive

Table 1
Inuence of the preparation procedure on the equivalent lm thickness. Procedure A
lipid vesicles in water/EtOH mixture after sonication; Procedure B lipid vesicles in
water without sonication; Procedure C lipid vesicles in water after sonication.
Solutions contained 0.4 mg/ml DMPC and 0.07 M NaCl. Temperature 26 C.
Preparation

Procedure A

Procedure B

Procedure C

h, nm

6.0

7.8

7.9

interactions except the van der Waals, not included in our treatment,
might also act in the lm [4].
AH values slightly decrease with an addition of the considered solutes. According to the Lifshitz theory of van der Waals forces, the
Hamaker constant depends on the dielectric constants and polarizabilities of the interacting media [4]. The addition of sugars or DMSO only
slightly changes these properties. That is why the Hamaker constant
should not vary appreciably with the addition of solutes.
4.2. Electrostatic double layer repulsion
The existence of an electrostatic surface double layer potential creates a repulsive electrostatic component EL of the disjoining pressure
in the lm. Its strength depends on the concentration and the type of
the electrolytes in the lm forming solutions and the surface double
layer potential. The existence of charged interfaces is expected for
lms prepared from ionic surfactants because of the surfactant adsorption. However, the electrostatic component of the disjoining pressure
which is a result of the interaction between charged surfaces was also
found in foam lms prepared from solutions of non-ionic surfactants
[37,53], indicating a weak surface charge. The DLVO theory gives only
the possibility for determining the absolute -value, but it does not explain the origin of this potential. The method does not even allow its
sign to be estimated. The common explanation nowadays of this effect
is the adsorption of hydroxyl ions at the water/air interfaces [38,39],
which results in formation of a negative electrostatic double layer
potential 0. The adsorption of the hydroxyl ions occurs through strong
dipole or hydrogen bonding of the OH ions with the hydrogen atoms
of the interfacial water molecules. The specic adsorption could result
from restrictions in the movement of the water molecules in the
interfacial layer compared to the bulk phase, as shown in [38]. This allows more pronounced hydrogen bonding of the OH ion with the
neighbouring water molecules.
Even though DMPC is a zwitterionic lipid, the charging of the water/
air interface is possible. The fact is rather unexpected, but was experimentally conrmed [24,39]. Experiments were performed to prove
the inuence of two salts (NaCl and CaCl2) on EL and respectively its
effect on the lm thickness and the lm contact angle. The values of
the lm thickness h and contact angle are given in Fig. 6 as a function
of the NaCl concentration for equilibrium lms prepared by Procedure A
and Procedure B. The dependence of the contact angle between the lm
and its meniscus is also shown. Black lms were always formed in the
initially thick foam lm by formation of black spots. The thickness of
the lms was around 7 nm and it varied slightly of about 1 nm on the
addition of NaCl to the lm forming solution. A pronounced difference
in the properties for the lms prepared from the Procedure A or B was

98

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

Fig. 5. Fluorescence microscope images taken at different stages of the lm drainage. From a) to d) the process of lm formation and thinning is in progress. The uorescent dye (NBD
DPPC) which is soluble in the expanded monolayer lipid state is expulsed from the lm when its thickness decreases.

Table 2
Effective Hamaker constants AH for foam lms prepared from DMPC/water and DMPC/
solute/water dispersions always in the presence of 0.07 M NaCl at 26 C.

3.1
1.4
0.7
1.5

a)

h, nm

8.0

7.0

6.0

b)
5.0
4.0
3.0
2.0
1.0
0.001

0.010

0.100

NaCl, M

AH, J
DMPC
DMPC + 0.5 M fructose
DMPC + 0.5 M sucrose
DMPC + 2 M DMSO

The balance of the interaction forces in the lm is changed and a decrease in the contact angle is observed. The further increase of the salt
concentration leads to a decrease in the lm thickness as predicted by
the DLVO theory. It is interesting to note that the lm thickness is larger
with around 1 nm at high CaCl2 concentrations (after the maximum in
the thickness) compared to those at very small concentrations (below
0.003 M CaCl2). The lm thickness of 7.6 nm seems to be the result of
a short-range repulsion between the monolayers bearing layers of
bind Ca2+-ions at this high salt concentration.
In the previous section we demonstrated how the change in the
electrolyte concentration in the lm forming DMPC dispersions changes
the properties of the nal thin lms. Another possible way to understand more about the double layer electrostatic interactions in thin
foam lms is to change the electrostatic double layer potential. Here
we discuss results from the studies with foam lms stabilised by mixtures of DMPC and the charged lipid dimyristoyl phosphatidyl glycerol
(DMPG). The content of the charged lipid in the mixture was varied to
study the role of electrical double layer interaction in the lm. The inuence of the salt (NaCl) concentration on the lm interactions was also

Contact angle, deg

observed. The lms prepared from water/ethanol mixtures were always


thinner than those prepared only from water dispersions. The difference
became more pronounced at high NaCl concentrations. The addition of
ethanol modies nearly all kinds of interactions in the lm. An approach
to explain this behaviour can be given by the interplay of the van der
Waals attraction and short-range repulsion at direct contact of the
monolayers. The effect of the EtOH on the lm thickness will be
discussed in detail later when examining the short range interactions
in the thin lms.
The value of the lm thickness corresponds reasonably well with the
thickness of a bilayer of DMPC molecules together with their hydration
water [48]. Thus, we concluded that only NBF formed by two layers of
lipid molecules in contact with each other was formed. This conclusion
is supported by the lack of inuence of the NaCl concentration on the
lm thickness. The thickness of foam lms with charged surfaces normally decreases with increasing salt concentration due to the screening
of the double-layer repulsion by the ions in the bulk. Probably, the electrostatic double-layer potential at the lm surfaces was weak in this
case and small amount of electrolyte was enough to suppress it, thus reducing the repulsive component of the disjoining pressure and forming
only NBF. The trend of the contact angle and the free energy of lm
formation on the NaCl concentration show similar weak dependency.
The experiments were also performed with CaCl2 used as an electrolyte. The salt concentration was varied in DMPC dispersions prepared
from water/ethanol mixture (Procedure A) (Fig. 7). Films with a thickness of around 6.6 nm where found at small concentrations of CaCl2
(Cel b 0.003 M). These lms were considered to be NBF stabilised by
short-range repulsion between the DMPC monolayers which stabilise
the lms [21]. The contact angles were comparatively large in this region. An increase of the salt concentration above 0.003 M results in an
increase of the lm thickness reaching a maximum of 10.9 nm at
0.015 M CaCl2. A further increase in the salt concentration generates a
continuous decrease in the lm thickness. At the same time, the contact
angle decreases, reaching a very low value close to zero in the range of
0.015 M salt concentration and increases on the further increase of the
salt concentration.
The observed dependence is a result of the specic binding of the
Ca2+ ions to the lipid PC groups. The Ca2+ ions bind to the lipid adsorption monolayers and thus increase the charge at the lm/air interfaces.
This results in a stronger double layer electrostatic repulsion between
the lm interfaces and respectively in an increase of the lm thickness.
The amount of Ca2+ ions adsorbed reaches a maximum which is related
to the higher lm thickness and a minimum in the lm contact angle.

1020
1020
1020
1020

Fig. 6. The lm thickness h and the contact angle as a function of the NaCl concentration
for equilibrium lms stabilised with DMPC prepared according to: () Procedure A
(lipid solution in water/ethanol mixture); () Procedure B (lipid suspension in
water). Temperature was kept constant at 26 C.

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

99

a)
12

h, nm

10

Contact angle, deg

b)
3.0
2.0
1.0
0.0
10-3

10-2

10-1

CCaCl2, M
Fig. 7. Film thickness (a) and contact angle (b) as a function of CaCl2 concentration for
lms prepared according to Procedure A (water/EtOH mixture) at T = 26 C. DMPC concentration was 0.4 mg/ml. The lines are only guide for the eye.

Fig. 8. Film thickness (a) and contact angle (b) vs. amount of DMPG in the DMPG:DMPC
lipid mixture used to stabilise the lms at constant NaCl concentration of 0.07 M and constant total lipid content of 0.4 mg/ml. () dispersions prepared according to Procedure
C; () lipid dispersions in water/EtOH mixtures (Procedure A).

7.5
7.0

h, nm

demonstrated. DMPC foam lms are basically stabilised by structural


forces [22,23,25], but a small effect due to electrical double layer interaction seems also to be involved. The inclusion of DMPG molecules/
ions in the lm forming adsorption layers should result in an increase
of the double layer potential and stronger repulsion [23].
The lm thickness and the contact angle of foam lms prepared from
dispersions of different ratio DMPC/DMPG at constant NaCl concentration (0.07 M) were measured. The total lipid concentration was always
0.4 mg/ml. The lms were prepared either from DMPC:DMPG mixtures
dispersed in water following the Procedure C (sonicated lipid dispersions in water) or from lipid solutions in water/ethanol mixtures. The
results are presented in Fig. 8. The lms prepared from water dispersions of the lipids are thicker than that prepared from water/ethanol
mixture. This difference is similar to that observed for lms prepared
from pure DMPC. The lm thickness increases with increasing content
of DMPG.
Simultaneously, the contact angle shows a decrease in value which
means that the specic interaction lm energy diminishes (see Eq. (6)).
These provide evidence for an increase of the repulsive component of
the disjoining pressure when more DMPG is present in the lipid suspension. Increasing the share of DMPG leads to an increasing number of
DMPG-ions in the lipid monolayers at the lm surfaces. Thus, the electrostatic double layer potential increases and the repulsion between the lm
surfaces is stronger. However, the ratio of the lipids in the monolayers
may deviate from that in the vesicles of the suspension. Different solubilities and surface activities may cause such difference e.g. a stronger surface activity of the charged lipid.
The thickness of foam lms formed from solutions containing 9:1
DMPC/DMPG suspensions in water/EtOH mixture as a function of NaCl
concentration is presented in Fig. 9. The lm thickness decreases
when the electrolyte concentration is increased as expected from the
DLVO theory. This trend is accompanied by an increase of the contact

6.5
6.0
5.5

0.00

0.05

0.10

0.15

CNaCl, M
Fig. 9. Film thickness as a function of NaCl concentration for lms prepared from 1:9
DMPG:DMPC lipid mixture. Films were prepared from lipid dispersions in EtOH/water
mixtures (Procedure A) () or water (Procedure C) (). The lipid content was constant
at 0.4 mg/ml and temperature of 26 C.

100

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

angle (respectively an increase in the absolute value of the free energy


of lm formation).
The disjoining pressure in the lm is equal to the capillary pressure
PC (see Eq. (1)) at equilibrium. This makes possible to calculate the electrostatic component EL of the disjoining pressure (respectively the
electrical double layer potential) if good approximations for other components of the disjoining pressure are made. Such calculations were
usually applied for thick lms (thicker than 30 nm) because in that
case only the classical components of the disjoining pressure are operative and all necessary parameters can be accessed with high precision.
Films stabilised with lipids are usually thin. That is why additional
non-classical terms of the disjoining pressure have to be taken into account when the total disjoining pressure is calculated. We restricted our
calculations to only three terms of the disjoining pressure EL, VW
and HYDR being aware that the values we obtained were only approximations because other components of the disjoining pressure might
also be operative.
The obtained small lm thicknesses at all studied conditions make
necessary to take into account the correct positions of the interacting
planes when calculating (see Fig. 1b). We assume that the double
layer electrostatic interaction occurs between the hydrophilic head
groups of the lipid molecules. We accept that they are positioned at
the border between the aqueous solution in the core of the lm and
the hydrophobic part of the lm formed from the hydrophobic tails of
the lipid molecules. Molecular dynamics simulations of the lipid/water
interface show [54,55] that the charges, for example, of the DMPG
head groups are away from the chain/head group interface by about
0.4 nm. Thus, the distance of electrostatic double layer interaction dEL
we dened as dEL = h2 2 * 0.4. The van der Waals interaction is considered to act between the two lm/air interfaces (see Fig. 1b). We postulate dVW = h assuming a constant dielectric permittivity inside these
planes of the lm. The estimation about the plain of hydration interaction is more difcult. Probably it has to be dened as the distance between the head groups of the lipid molecules. It is difcult to access
such value because of the internal roughness and undulation of the
water/air interface. We accept as a rst approximation that dHYDR =
h2. A value of AH = 5 10 21 J [4] was used to calculate VW. This
value is a good rst approximation and we used it at all experimental
conditions. However, the Hamaker constant can be different for lms
prepared according to the diverse procedures (see Part 3 and Table 1)
because of the presence of EtOH in the lm core and one has to use
more sophisticated methods to estimate it. The hydration part of the
disjoining pressure was calculated using the values for both constants
H0 = 4 107 N m2 and = 0.3 nm [4,16]. The surface tension was
always constant at = 27 mN/m which predicts a value for the capillary
pressure of PC = 27 Pa.
The calculated values of the electrostatic double layer potential as a
function of the amount of charged lipid DMPG present in the lm
forming lipid mixture are shown in Table 3.

Table 3
Film thickness, h, water core lm thickness, h2 and electrostatic double layer potential,
for foam lms prepared from different DMPC:DMPG lipid mixtures. Two procedures were
used to prepare the lipid suspensions. Procedure A: The DMPC:DMPG lipid mixture was
dissolved in 47.5 % vol. EtOH solution in water. Procedure C: The lipid mixture was
dispersed in water by sonication (for details see Part 3 and Table 1). The NaCl
concentration was always 0.07 M and the temperature kept at 26 C.
Procedure A

Procedure C

CDMPG
mol%

h,
nm

h2,
nm

0,
mV

h,
nm

h2,
nm

0,
mV

0
10
20
30
40

5.7
6.4
6.7
8.2
8.5

1.7
2.4
2.7
4.2
4.5

55
63
67
97
105

7.6
10.7
11.0
11.3

3.6
6.7
7.0
7.3

83
198
218
240

Table 3 shows that the increase of the amount of the charged lipid
DMPG leads to the increase of the electrostatic double layer potential.
The increase is continuous when water/ethanol mixtures are used to
prepare the lms and somewhat step wise in the case of lms prepared
from lipid suspension in water. A reason might be the formation of homogeneous mixtures of DMPC and DMPG at the lm interfaces in the
former case because of the better solubility of the lipids in the case of
water/ethanol mixtures. In the latter case DMPG is not even distributed
in the matrix of DMPC and its presence at the interfaces is not regularly
changing.
Our results show that the properties of the foam lms stabilised with
lipids are sensitive to the interplay of dispersion attraction and electrical
double-layer repulsion. These interactions are inuenced by the composition of the lm forming solution. Differences in composition or preparation of the solutions inuence the thickness of the stabilising
monolayers. These lead to variations in the thickness of the lms. How
the lm thickness changes when some low molecular solutes like ethanol, sugars and DMSO are present in the lm forming solution is presented in the next part. The results are explained taking into account
the interplay between short range and long range interactions in the
lipid stabilised foam lms.
5. Short range interactions in lipid stabilised foam lms
Phospholipid bilayers are major structural elements of biological
membranes. Therefore, the study of the interaction of lipids with
water and with water soluble solutes delivers information about the
processes that occur at the membrane/water interface. An addition
of certain solutes, such as small carbohydrates, dimethyl sulfoxide
(DMSO) and ethanol, inuences membrane stability. Soluble sugars stabilise liposomes [56], intact membranes [57] and whole cells [58]
against irreversible desiccation and fusion caused by freezing or
freeze-drying. The incorporation of sugars into the hydrophilic region
of the membrane prevents tight packing of the aliphatic chains [59]. The
inuence of the DMSO on membrane structure and integrity appear to
be controversial. In some cases, DMSO acts as a membrane cryoprotector,
while in others it destabilises the membranes and promotes fusion and
leakage [60,61].
EtOH is a well-known dehydration agent, which decreases the repulsion between lipid membranes and destabilises the contacts between
them [62,63]. The interaction of lipids with water and ethanol is interesting from a medical and biological point of view. Alcohol, anaesthetics
and water compete for the same hydrogen bonds in membrane systems.
Normally, ethanol binds the fatty acid carbonyl region in the lipid molecules. This reduces the water content in the membrane and decreases
the hydrophilic repulsion between membranes. An increase in the
amount of ethanol leads to an interdigitated membrane structure
which is more permeable and forms stable contacts with other membranes [6264].
The aim of the present part is to show the inuence of some low molecular weight solutes (e.g. sugars, DMSO and EtOH) on the interactions
between the adsorbed layers of phospholipids. The dependence of the
lm thickness and the contact angle on the amount of different solutes
is presented. The solutes mainly inuence the short range non-DLVO interactions in the lms, even though an inuence on the van der Waals
attraction and the electrostatic repulsion between phosphatidylcholine
layers is also possible. The results are compared with the properties of
Gibbs adsorbed and Langmuir lipid monolayers. The role of the hydration force on the properties of thin lipid foam lms is shown.
5.1. Effect of ethanol
Foam lms formed from lipid suspensions with a content of ethanol
ranging from zero to 50% vol are stable for at least 30 min. A dependence
of the lm thickness on the ethanol concentration is shown in Fig. 10a.
Complementary dependence of the contact angle between DMPC

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

a)

h, nm

9.0

8.0

7.0

b)
Contact angle, deg

5.0
4.0
3.0
2.0
1.0
0

10

20

30

40

50

EtOH, % vol
Fig. 10. Film thickness (a) and contact angle (b) as a function of EtOH concentration
in water/EtOH mixtures. Lipid solutions were prepared according to Procedure A. All
experiments carried out at a constant lipid concentration of 0.4 mg/ml, salt concentration
0.07 M NaCl and temperature 26 C. The lines are only guide for the eye.

foam lms and the surrounding meniscus is presented in Fig. 10b. The
lm thickness decreases smoothly as the ethanol concentration increases until 40% vol. The thickness drops to a value which does not depend further on the EtOH concentration above this concentration. It is
accompanied by an increase in the contact angle on increasing the
EtOH concentration. There is also an indication for a jump in at ca.
40% vol EtOH. However, after this jump the curve seems to increase further. The contact angles at high EtOH concentrations reach values close
to that obtained for NBF stabilised with other surfactants [6]. This could
mean that the two DMPC adsorbed monolayers are very close to each
other and the lms achieve a bilayer structure. We interpret such behaviour as a transition from a common black lm stabilised by hydration forces to a Newton black lm stabilised by steric forces.
The dependence of the equilibrium lm thickness and the free energy of lm formation on the content of EtOH in the lm forming solution
demonstrate the inuence of EtOH on the interaction between the lm
building DMPC monolayers. An explanation of this effect by modication of the electrical double-layer repulsion can be excluded. It is unlikely that any electrical double layer repulsion according to the classical
DLVO theory plays a signicant role in DMPC stabilised lms. This, for
several reasons: i) the DMPC molecule has no net charge, ii) Na+-ions
do not affect signicantly the range of the repulsion (see Part 4) and
iii) different amounts of EtOH in solution lead to a small change in the
Debye length (from 1.15 nm to 1.03 nm) [65], which cannot explain
the observed results. Therefore, it seems that EtOH affects structural
interactions.
This means that ethanol does not change only the interaction between the DMPC monolayers which form the lm, but also the structure
of these layers. Deeper knowledge of the inuence of EtOH on the structure of these layers could be obtained from studies carried out on lipid
monolayers spread on liquid subphase [25] under the approximation

101

that these monolayers are similar to those which form the lm. Results
show that DMPC monolayers have classical behaviour up to 25% vol
EtOH in the subphase. Above this concentration the monolayers become
soluble which does not allow precise experiments to be performed [22].
Ellipsometric experiments show that the thickness of DMPC monolayer
increases when the EtOH concentration increases (Fig. 11a). Compression with surface pressuremolecular area (/A) isotherms shows that
the DMPC monolayer becomes more compressed as the EtOH concentration in the subphase increases. The mean area per molecule at
which the surface pressure begins to rise (lift-off area) shifts to smaller areas [22]. This correlates with results from grazing incidence X-ray
diffraction (GIXD) experiments which conrm that the tilt angle of the
hydrophobic chains of the lipid molecules decreases [25] (Fig. 11b).
Small amounts of alcohol (up to 4% vol) in the subphase create distortion of the lipid layer lattice. A further increase of the ethanol does not
change the lattice distortion, but reduces continuously the area per
head group. GIXD experiments were performed with 1,2-distearoylphosphatidylcholine (DSPC) because it forms only condensed monolayer at 20 C, whereas DMPC exhibits a liquid-expanded phase without
crystallographic structure up to high surface pressures at this temperature. However, DSPC and DMPC have the same head group with slightly
different chain length. Since the interaction with ethanol is expected to
occur in the head group region, the X-ray experiments performed with
DSPC can be directly compared with the investigations of the inuence
of ethanol on the properties of foam lms stabilised with DMPC. The increased van der Waals interactions between the longer DSPC chains
only shift the liquid-expanded/condensed transition pressure to lower
values but do not change the structure of the condensed phase.
The smaller effective area per lipid molecule at the lift off point can
be explained by a decrease of the diameter of the lipid head groups
with increasing ethanol concentration. The increase of the monolayer
thickness, found in the ellipsometric measurements, should be a result
of a decrease in the tilt of the hydrocarbon chains. This also requires a
decrease of the area per head group in the monolayer. These ndings
are in agreement with results of Klemm [62], Komatsu and Williams
[64] and Komatsu and Okada [64] about the dehydration of phospholipid head groups by ethanol. DMPC head groups in water are highly
hydrated. A DMPC molecule can bind around 1215 water molecules
[62]. Ethanol molecules bind the lipid molecules near the junction of
the alkyl chains and the polar head group at sites normally occupied
by water, displacing hydrogen bonded water molecules [64]. Upon increasing the ethanol content in the subphase, the hydration shell
around the head groups becomes smaller and the average area per molecule decreases.
The parallelism of the decrease of the water core in a lipid-stabilised
foam lm and of the distance of the PC head groups in a monolayer upon
increasing ethanol concentration indicates that the same physical
mechanism is behind these two effects. We conclude that the repulsion
between PC head groups decreases both in the plane of the monolayer
as well as normal to the lm plane with increasing ethanol concentration. These results can be explained by a dehydration of the PC head
groups due to the presence of ethanol in a similar way as ethanol dehydrates proteins, in agreement with the dehydration theory of proteins
by ethanol [62]. Thus, it seems that the effect of dehydration by ethanol
is not restricted to organic hydrophilic groups. A decrease of the range of
the hydration repulsion between two silica surfaces after the addition
of 1020 vol.% ethanol has also been reported [4], with a stronger effect
of methanol. Therefore, we assume that ethanol disturbs the formation
of H-bonds of water molecules with the hydrophilic groups and causes
the effects observed in this work. The comparison of the inuence of
ethanol on the stability of single monolayers at the surface of an extended aqueous phase and of the monolayers in a thin lm shows that the
lipid molecules in the lm are strongly bound to the interface. This is
caused by the interaction in the lm, which decreases the specic
Gibbs energy of the lipid molecules compared to the bulk and to a
monolayer at the surface of an extended bulk phase.

102

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

a)
2.2

b)
40
1.8

Tilt angle, deg

Monolayer thickness, nm

2.0

1.6

38

36

34

1.4
32

1.2

10

20

30

40

50

EtOH concentration, % vol

10

15

20

EtOH concentration, % vol

Fig. 11. a) Thickness of monolayer obtained from ellipsometric experiments and b) tilt angle t from grazing incidence X-ray diffraction (GIXD) experiments of PC lipid monolayers spread
on aqueous subphase containing different amounts of EtOH. The ellipsometric experiments were performed with DMPC while the GIXD experiments were done with DSPC thus assuring
formation of condensed phase at room temperature.

5.2. Effect of low molecular weight solutes at low ionic strength


The thickness of lms prepared from suspensions of DMPC (constant lipid concentration of 0.4 mg/ml) in DMSO or sugar aqueous
solutions with different concentrations were measured in absence
of electrolyte. The concentration of sugars was varied up to 0.5 M
and that of DMSO up to 2 M. The dependence of the lm thickness
on the sugar concentration in the lm forming dispersion is presented in Fig. 12a. The effect of both fructose and sucrose on the lm
thickness is similar. NBF about 7 nm thick is formed at low sugar concentrations. The lm thickness increases steadily with increasing

sugar concentration up to about 0.35 M. After this point the lm


thickness increases with a jump and thick lms (h 50 nm) are observed. In these conditions the lm thickness cannot be measured
with our equipment above concentration of sugar 0.5 M. The dependence of the lm thickness on the DMSO content is shown in Fig. 12b.
NBF about 67 nm thick is formed at concentrations of DMSO lower
than 1 M. The lm thickness varies non-monotonically with the
DMSO concentration in this range. At about 1.5 M DMSO the lm
thickness increases with a jump. On a further increase in DMSO concentration above 2 M the stability of the lm decreases and the lm
ruptures during the thinning.

b)

a)

50.0

70,0
60,0

40.0

h, nm

h, nm

50,0

7,0

7.0

6.5

6,5

0,0

0,1

0,2

0,3

Csugar, M

0,4

0,5

0.0

0.5

1.0

1.5

2.0

CDMSO, M

Fig. 12. Dependence of the lm thickness on the concentration of a) sugar ( fructose; sucrose) and b) DMSO. All experiments performed at constant concentration of DMPC
(0.4 mg/ml) and temperature (26 C). The solutions contained no salt and were prepared according to Procedure C.

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

Two regions in thickness vs. solute concentration dependencies are


distinguished for both solutes: one at lower amount of the additive
where NBF is formed and the lm thickness changes slightly, and another at higher solute concentrations where thick lms are formed. The
small changes in the lm thickness observed at low solute concentrations are probably caused by modied steric-hydration repulsion. The
strong increase in the lm thickness at high solute contents could be
created by an increased electrostatic repulsive contribution to the
disjoining pressure.
5.3. Effect of electrolyte concentration at high solute content
If the formation of stable thick lms is caused by the increased of the
long-range electrostatic double layer repulsion between the lm surfaces, one should expect a dependence of the lm thickness on the electrolyte concentration in the suspension. Such dependency for DMPC
lms in the presence of 0.5 M sucrose or 2 M DMSO on the NaCl concentration is shown in Fig. 13. These are the concentrations of the solutes at
which the thickest lms without electrolyte were obtained (Fig. 12).
The lm thickness gradually decreases with increasing electrolyte
concentration. Formation of CBF is observed in both cases at an electrolyte concentration of around 0.02 M. In the presence of sugar CBF exists
within a wide range of salt concentration up to 0.05 M (Fig. 13a). Their
thickness slightly decreases with increasing NaCl concentration. Abrupt
transition to NBF with a constant thickness of about 7 nm occurs when
the NaCl concentration reaches 0.05 M. In the case of DMSO, formation
of CBF is observed only in a very narrow salt concentration region
(Fig. 13b). Instead, NBF with constant thickness of about 6 nm is already
formed at 0.02 M NaCl.
The dependence of the lm thickness on the electrolyte concentration
depicted above conrms the electrostatic origin of the forces that stabilise
these lms. The forces operating in such lms is successfully described by
the DLVO theory, when only the long-range attractive and repulsive
forces acting in the lm are taken into consideration and the inuence
of short-range interaction terms is omitted. This allows the determination
of the electrostatic double layer potential 0, using Eq. (4), making a reasonable estimation about the van der Waals component of the disjoining
pressure. The thicknesses of the lms measured at the lowest available
electrolyte concentration, Cel = 5 103 M, were taken for the calculations (for details see Part 4). The addition of solutes increases the surface
potential (0) from 21 mV for pure DMPC/water suspensions to about
60 mV for lms obtained in the presence of sugars or DMSO. We should
mention that these 0-values might be overestimated since not all

a)

103

possible repulsive interactions that may exist in a lm are considered


in the calculation [46]. The existence of high surface potential in a
non-charged lipid lm is rather unexpected. The appearance of stronger
repulsion between the lm interfaces in the presence of sugars and
DMSO, which we correlate with increased 0 potentials, remains
unclear.
5.4. Effect of solutes at high ionic strength
Formation of NBF is observed at high electrolyte concentration (the
lowest plateau regions in Fig. 13). The thickness of these NBF depends
on the kind of the solute used. They are about 7 nm thick when formed
in the presence of sugar, while the thickness of NBF in the presence of
DMSO is about 6 nm. The lm thickness varies with the concentration
of the solute as well (Fig. 14). The addition of sugar leads to an increase
in the thickness (Fig. 14a). In contrary, increasing DMSO concentration
decreases the lm thickness (Fig. 14b).
The equilibrium thickness in NBF depends mainly on the short range
steric and hydration repulsion between the lipid monolayers adsorbed
on the both lm surfaces. It can be seen that h increases from about
6.5 nm for the DMPC/water system to more than 12 nm in the presence
of 1 M fructose. The addition of DMSO reduces the h value to about
5.5 nm in the presence of more than 2 M DMSO. Taking into account
the diameter of the water molecule of about 0.2 nm, the observed
changes of h correspond to addition or removal of several water layers
caused by the presence of fructose and DMSO, respectively. Hence, the
observed changes in the DMPC lm thickness in the presence of solutes
in the lm forming suspension can be attributed to altered hydration repulsion between the lm surfaces.
It has been showed [66] that sugars and DMSO inuence the phase
behaviour and the condensed phase structure of insoluble lipid DPPC
monolayers. As in the above discussed comparison between DSPC and
DMPC they possess the same choline head groups and differ only in
the alkyl chain length. Since the polar lipid heads are mainly responsible
for the interactions between the solutes and the phosphocholines, the
results of the DPPC monolayer study can be used to explain the observed variation of the thickness of the black lms. The sugar molecules
penetrate into the polar head group region. The binding of the strongly
hydrated sugars increases the hydration of the lipid. This results in
stronger a repulsion between the PC head groups. The same phenomenon can be responsible for the observed increase in the lm thickness
on the addition of sugar. In contrast, the addition of DMSO into the subphase causes condensation of the DPPC monolayer and reduces

b)
70.0

50.0

60.0
40.0

40.0

h, nm

h, nm

50.0

30.0

30.0

20.0

20.0
10.0

10.0
0.00

0.02

0.04

0.06

CNaCl, M

0.08

0.10

0.00

0.02

0.04

0.06

CNaCl, M

Fig. 13. Foam lm thickness vs. NaCl concentration: a) in the presence of 0.5 M fructose, and b) in the presence of 2 M DMSO in the lm forming lipid dispersion. Films were prepared from
lipid suspensions containing 0.4 mg/ml DMPC at temperature of 26 C according to Procedure C.

104

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

apparently the amount of the water molecules in the vicinity of the


polar lipid head groups. Moreover, the hydrated lipid molecules and
the DMSO molecules compete for the same limited amount of water
at the interface. Thus, certain hydration water is extracted from the region around the lipid head groups. The deciency of hydration water
decreases the lipid head group repulsion and diminishes the effective
volume of the PC moiety. The DMSO induced dehydration can cause
the observed decrease in the lipid lm thickness. The effect of DMSO
is similar to the dehydration effect of ethanol.
6. Conclusions
Phospholipid bilayers are the smallest components of barriers
(i.e. membranes) separating living matter from its external environment.
The lateral organisation of the lipid molecules in bilayers and the interactions between then normal to the lamella plane affect the stability and
the transport processes through membranes. Foam lms stabilised by
lipids offer the possibility to study the interactions between lipid monolayers. Presenting an inverse black lipid membrane the foam lms supply information about the properties of the self-organised in bilayer
lipid molecules at interfaces. The equilibrium thickness of these lms
is determined by the interplay of the dispersion attraction, electrical
double-layer repulsion, and short-range molecular interactions. These
interactions are inuenced by the composition of the lm forming
solution.
The thickness of foam lms stabilised with DMPC is controlled by the
van der Waals attraction and short range hydration or steric repulsive
interactions. The electrostatic double layer cannot be detected, being
very weak. If the lm surfaces are charged either by co-adsorption of
charged lipids (e.g. DMPG) or specic binding of Ca2+ ions the electrostatic component of the disjoining pressure becomes stronger and plays
an important role to determine the lm thickness.
The presence of solutes in the water medium surrounding the membrane inuences both the lateral ordering of the lipid molecules and the
short-range interactions between them in direction normal to the molecular arrays. Results on the inuence of EtOH, low molecular weight
sugars and dimethyl sulfoxide on the short range interactions between
phospholipid molecules in thin free-standing foam lms are reported
and discussed. The dependence of the equilibrium lm thickness on
the content of the solute in the lm forming dispersion demonstrates
the inuence of the solutes on the interaction between the DMPC lm

monolayers. High concentration of solutes leads to a large increase in


the lm thickness. It is caused by modication of the electrostatic
double-layer repulsion between lm interfaces. At low electrolyte and
high solute concentration, the electrostatic disjoining pressure governs
the lm thickness. At high electrolyte concentration, formation of NBF
with thickness of only few nanometres is observed. In these very
thin lms, the equilibrium thickness depends on the short-range interactions between the lipid monolayers adsorbed on both lm surfaces.
Their thickness depends on the type of solute and its concentration in
the lm forming dispersion. The addition of sugar leads to an increase
in the thickness, while the presence of DMSO and EtOH decreases the
lm thickness. The observed changes correspond to the addition or removal of several layers of water molecules which hydrates the PC lipid
groups.
7. Experimental techniques
The ScheludkoExerowa glass ring experimental cell (Fig. 15) is a
suitable tool for studying single foam lms. The lm is formed in a
glass ring (1) with a radius R which is connected via a capillary to the
body of the cell (2). A special piston made of Teon (3) is used to suck
the liquid from the glass capillary and thus forming the foam lm. The
glass ring is placed in a close glass vessel (4) which assures formation
of saturated to water vapour atmosphere. This is an important condition
for the formation of stable foam lms. The whole device is placed
together with a metal thermostatic jacket (5) on experimental table
(6) of an (usually) inverted optical microscope. A horizontal lm is
formed in the glass ring, by suction of the lm forming solution from
the ring through the capillary at low capillary pressure. The lm starts
thinning after its formation due to drainage. It either ruptures during
the thinning process or an equilibrium lm is formed at the end of the
drainage process. Initially a thick coloured lm with a radius r1 is
formed. During the drainage process, due to local uctuation of the
lm thickness black spots appear. These spots grow and nally cover
the whole lm. At that moment the lm expands shortly (in less
than a second) because of the new force balance between the lm
and bulk meniscus. Thereafter the lm size remains constant without any further change. The radius of the thinner equilibrium black
lm r2 is greater than the radius of the thicker lm r 1 just before
the rst black spot appears. A contact angle is formed between
the black lm and the surrounding meniscus.

a)

b)
7.0

12.0

Thickness, nm

Thickness, nm

6.5
10.0

8.0

6.0

5.5
6.0
0.0

0.2

0.4

0.6

CFructose, M

0.8

1.0

0.0

0.5

1.0

1.5

2.0

2.5

CDMSO, M

Fig. 14. Film thickness of Newton black lms of DMPC (0.4 mg/ml concentration) at 0.05 M NaCl and 26 C as a function of the a) fructose and b) DMSO concentration in the lm forming
dispersion. Films were prepared according to Procedure C.

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

105

Fig. 15. Left. A scheme of the glass ring ScheludkoExerova experimental cell used for formation and studies of single foam lms. For full description of the different parts see the text.
Right: Schemes of the cross-section of the glass ring where the foam lm is formed. (a) A thick lm without a contact angle to the surrounding meniscus; and (b) a thin lm with contact
angle . The diameter 2r2 of the thin lm is larger than the diameter 2r1 of the thick one due to the difference between the lm surface tension f and the surface tension on the meniscus
[f b ].

7.1. Contact angle


The contact angle between the black lm and the bulk meniscus
(see Fig. 2) is determined conveniently by the expansion method
[44]. is calculated from the measured values of radii r1 and r2 by
using the expression [43]:
sin

22 21
2

r1 and r2 are the radii of the foam lm immediately after its preparation
(thick lms) and after the lm expansion, respectively, when an equilibrium thin lm is formed. R is the radius of the glass capillary where
the lm is formed and 1 = r1/R, 2 = r2/R. The initial lm radius is
maintained in the range of 0.10.15 mm during the measurement. The
accuracy of the method is 0.2.

are the maximum intensity and the intensity of the reected light in the
thinning process, respectively.
The equivalent lm thickness is the thickness obtained by assuming
an optically homogeneous lm with the same refractive index as that of
the bulk solution from which the lm is formed. Indeed the lm consists
of two surfactant monolayers and an aqueous layer in between, and the
lm thickness h is different from that of the equivalent lm thickness
hw. The lm thickness h is calculated by assuming models with different
complexities. The three-layer model [57] assumes that the lm consists of a solution core of thickness h2 and refractive index n2 anked
by two layers of hydrophobic alkyl chains of the adsorbed surfactant
molecules, with thickness h1 and refractive index n1. The water core includes the hydrophilic head groups of the surfactant molecules. This
three-layer model is used to calculate the lm thickness h from the
experimentally obtained hw values using the relation [69]:

h 2h1 h2 ; h2 hw 2h1
7.2. Film thickness
The lm thickness is measured microinterferometrically [79]. The
method is based on the registration of the intensity of the monochromatic light reected from the lm. A small central part of the lm is illuminated by white light. Part of this light is reected, passes through
a light lter and reaches a photomultiplier. An interferometric pattern
is observed because of the small thickness of the lms comparable
with the wavelength of the illuminating light. The equivalent solution
thickness hw is calculated by the equation [7]:
hw

v
u

I=Im
u
arcsinu
4R
t
2n
1
1I=Im
1R2

where R

n1
n1

2

, n is the refractive index of the bulk solution, Im and I

n21 1
:
n22 1

The lm thickness h is calculated from Eq. (9) using the experimentally obtained values for the equivalent solution thickness hw and
the refractive index of the hydrophobic part assuming values for the
other constants. A value of n1 = 1.4255 is used usually when foam
lms stabilised with DMPC are studied. This value is the same as for
tetradecane at T = 26 C. The thickness of the lm forming adsorption
surfactant layers was assumed to be h1 = 1.5 nm as obtained from independent ellipsometric measurements on liquid/air interfaces [22]. This
value is close to the data reported from X-ray reectometry experiments with foam lms stabilised by different lipids [67].
7.3. Velocity of lm thinning
The lm begins to thin under the action of the meniscus capillary
pressure PC and the lm disjoining pressure (h) until it reaches its

106

J.L. Toca-Herrera et al. / Advances in Colloid and Interface Science 207 (2014) 93106

V/h3, nm-2 sec-1

2.0x10-6

1.5x10-6

1.0x10-6

5.0x10-7

0.01

0.02

1/h,

0.03

nm-1

Fig. 16. Illustration of the application of the dynamic method for lm thinning studies. The
DMPC (0.4 mg/ml) stabilised lm is formed from a suspension that contains 0.07 M NaCl
and 0.5 M sucrose. The points represent the experimental V/h3 vs. 1/h dependence; the
solid line is only a guide for the eye. The dashed line is the best t to the experimental
data for thick lms, where the disjoining pressure is zero.

equilibrium thickness or ruptures. The dynamic method [57] connects


experimentally the measured velocity of lm thinning with the disjoining
pressure operative in the lm. For surfactant stabilised liquid lms, the
relation between the lm thinning velocity V and the thickness h is
given by [5,7,68]:





V
hs
hs
h

PC
h
h
h3

10

where b and hs account for bulk and surface diffusion, respectively, and
is a factor comprising all thickness independent quantities.
The procedure of calculating the disjoining pressure from the experimental thinning curve is to plot the dependence of V/h3 vs. 1/h. Such
dependence for a lm prepared from DMPC suspension in the presence
of 0.5 M sucrose is presented in Fig. 16 as an example. The points represent the experimental values and the solid line is only a guide for the
eye. Eq. (10) predicts a linear dependence of V/h3 on 1/h in thick lms
where both lm surfaces do not interact ( = 0). The linear t to the
experimental data for thick lms is shown as a dashed line on Fig. 16.
The quantities b and hs are determined from the slope and the intercept of this t. At smaller lm thickness, when 0, a deviation from
linearity arises. The value of the disjoining pressure at given lm
thickness h is calculated from the difference between the experimental
data and the linear dependence. Thus, a (h) isotherm is constructed. If
the electrostatic double layer repulsion is suppressed the experimental
(h) isotherms can be tted with the Eq. (3). This procedure allows
estimating experimentally AH.
Acknowledgements
The Max-Planck Society is acknowledged for the nancial support
during the whole period of the project. Parts of the work were nanced
by the Deutsche Forschungsgemeinschaft through projects Sfb 312, Mu
1040/9-1, Mu 1040/4-1 and guest grant 436BUL17/1/99. R.K. was partially supported by the Dr. A. Kalojanoff Stiftung, Mnchen, Germany.
The authors are grateful to Mrs. E. Rtze for technical assistance.
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