Langmuir- Blodgett layers of amphiphilic molecules investigated by Atomic Force Microscopy Langmuir- Blodgett lagen van amfifilische moleculen onderzocht met Atomic Force Microscopy (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. W. H. Gispen, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 23 mei 2007 des middags te 12.45 uur. door Aneliya Nikolova Zdravkova geboren op 10 december 1973, te Silistra, Bulgarije
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Langmuir- Blodgett layers of amphiphilic molecules investigated by Atomic Force Microscopy
Langmuir- Blodgett lagen van amfifilische moleculen onderzocht met Atomic Force Microscopy
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de
Universiteit Utrecht op gezag van de rector
magnificus, prof.dr. W. H. Gispen, ingevolge het
besluit van het college voor promoties in het
openbaar te verdedigen op woensdag 23 mei 2007
des middags te 12.45 uur.
door
Aneliya Nikolova Zdravkova geboren op 10 december 1973, te Silistra, Bulgarije
Promotor: Prof.dr. J.P.J.M. van der Eerden
On the cover: Phase separation in binary mixed system of nuts (almonds and hazelnuts).
CONTENTS
CHAPTER 1 Introduction 1
1.1. Atomic force microscopy 2
1.2. Langmuir – Blodgett technique 4
1.3. Stability of Langmuir monolayer 8
1.4. Crystal structure of triglycerides 9
1.5. Outline of the thesis 12
CHAPTER 2 Phase behaviour in supported mixed monolayers of alkanols,
investigated by Atomic Force Microscopy 15
2.1. Introduction 16
2.2. Materials and methods 16
2.2.1. Chemicals 16
2.2.2. Aπ − isotherms 17
2.2.3. Langmuir - Blodgett film transfer 17
2.2.4. AFM measurement 17
2.3. AFM Observations 17
2.3.1. C16:C22 17
2.3.2. C18:C22 19
2.3.3. C18:C24 19
2.3.4. C16:C24 19
2.4. Thermodynamics 20
2.5. Conclusions 23
CHAPTER 3 Structure and dynamics of Langmuir – Blodgett Tristearin films:
Atomic Force Microscopy and theoretical analysis 25
3.1. Introduction 26
3.2. Materials and methods 27
3.2.1. Chemicals 27
3.2.2 Langmuir method 27
3.2.3. Langmuir - Blodgett film transfer 28
3.2.4. AFM measurements 28
3.3. Langmuir observations 29
3.3.1. Forced compression 29
3.3.2. Isobaric compression 30
3.4. AFM observation 32
3.4.1 Monolayer thickness 32
3.4.2. Initial structure, obtained by forced compression 36
3.4.3. Structural changes during isobaric compression 37
3.4.4. Stability of the transferred LB – film 41
3.4.5. Consistency of Langmuir and AFM data 41
3.5. Theory for nucleation, growth and coalescence of crystals 42
3.5.1. Qualitative interpretation of film evolution observations 42
3.5.2. Parameters and measurable variables 45
3.5.3. Avrami – Kolmogorov theory for coverage 45
3.5.4. Approximate theory for average crystal size and density 47
3.5.5. Interpretation of AFM – images of nucleation and growth 49
3.6. Conclusions 50
CHAPTER 4 Structure and stability of Triglyceride monolayers on water and
mica surfaces 53
4.1. Introduction 54
4.2. Materials and methods 55
4.2.1. Chemicals 55
4.2.2 Langmuir method 56
4.2.3. Langmuir - Blodgett film transfer 56
4.2.4. AFM measurements 57
4.3. Langmuir observations 57
4.3.1. Forced compression 57
4.3.2. Isobaric compression 60
4.4. AFM observations 64
4.4.1 Monolayer thickness 64
4.4.2. Stability of the transferred LB – film 68
4.4.2.1. Initial structure and structural changes of PPP – monolayer 68
4.4.2.2. Initial structure and structural changes of SSS – monolayer 71
4.4.2.1. Initial structure and structural changes of AAA – monolayer 75
4.5. Discussion 76
4.6. Conclusions 79
CHAPTER 5 Phase behaviour in binary mixed Langmuir-Blodgett monolayers of
Triglycerides 83
5.1. Introduction 85
5.2. Materials and methods 86
5.2.1. Chemicals 86
5.2.2 Langmuir method 87
5.2.3. Langmuir - Blodgett film transfer 87
5.2.4. AFM measurements 88
5.3. Langmuir observations 88
5.4. AFM observations 92
5.4.1 PPP – SSS structure 92
5.4.2 SSS – AAA structure 97
5.4.3 PPP – AAA structure 99
5.5. Discussion 102
5.6. Conclusions 106
CHAPTER 6 Summary 109
Samenvatting 113
List of Publications 117
Acknowledgements 119
Curriculum vitae 121
CHAPTER 1
Introduction
“Today…I propose to tell you of a real two-
dimensional world in which phenomena occur that
are analogous to those described in “Flatland”. I plan
to tell you about the behavior of molecules and
atoms that are held at the surface of solids and
liquids.”
I. Langmuir, Science 1936, 84,379
Since Irving Langmuir published his frist work on the study of two-dimensional systems of
molecular films at the gas-liquid interface [1], the interest in this area increased a lot. Many
scientists were fascinated by the idea to assemble individual molecules into highly ordered
architectures. They termed this materials engineering. Even though this is still a dream, the
Langmuir-Blodgett (LB) technique and Self-assembly (SA) process opened a window to the
realization of this goal. Presently LB and SA are widely used in areas like non-linear optics,
nanoelectronics, biosensors and piezoelectric devices [2].
Many molecules can form Langmuir films. We can describe them with one word-
amphiphiles. They have a hydrophilic head group and hydrophobic tail(s).The simplest amphiphilic
molecules are the aliphatic long-chain alcohols (CnH2n+1OH with n = 13-31). They form a
monolayer at the air-water interface, whose stability increases with the chain length. Other materials
like these are fatty acids and their salts, polymers, glycerides, phospholipids, pigments and proteins
[2, 3].
Self- assembled (SA) monolayers are molecular assemblies that are formed spontaneously
by the immersion of an appropriate substrate into a solution of an active surfactant in an organic
solvent [4, 5]. To investigate the surface and bulk properties of thin films, scientists use several
analytical tools. Ellipsometry to measure the thickness and uniformity of freshly prepared films;
1
Fourier transform infrared (FTIR) spectroscopy, in both grazing-angle and attenuated total
reflection (ATR) modes to learn about the direction of transferred dipoles, and to evaluate dichronic
ratios, molecular orientation, packing, and coverage; surface potential measurements to get
information on the coherence of the film at the water-air interface and on metal surfaces. A lot of
surface imaging technologies like X-ray Photoelectron Spectroscopy; Optical, Fluorescence,
Electron and Scanning Microscopy are used to study the surface topography [6].
In this thesis the main analytical tool, which was used for investigation is Atomic Force
Microscopy.
1.1. Atomic force microscopy
Atomic force microscopy (AFM) is one of the scanning probe microcopies. Common to these
techniques is that a probe is moved laterally (in x- and y- direction) across a sample surface, while
the height (z) or other parameters (force) are recorded. The first realization of this kind of
microscopy was the Scanning tunneling microscopy (STM) in 1981 by Binning and Rohrer [7]. An
electric current is measured due to electrons tunneling from a metal tip to a conducting sample. The
disadvantage of STM, that it is useful only for conducting samples, inspired scientists to generalize
this technique. This led to the invention of the atomic force microscopy (AFM) in 1986 [8]. AFM is
capable of scanning non-conductive samples. In an atomic force microscope a small tip on the end
of a cantilever-type spring is used as a probe. As a raster-scan drags the tip over the sample, some
sort of detection apparatus measures the vertical deflection of the cantilever, which indicates the
local sample height. The simplest deflection monitoring system is the laser beam reflection system.
A scheme of atomic force microscope setup is shown in Fig.1.
Fig.1. Schematic presentation of Atomic force microscope setup.
The sample is mounted on top of a piezo crystal, which is used to position the sample very
accurately relative to the tip. A few micrometers above the sample a cantilever with the integrated
pyramidal tip is placed. A horizontally split photodetector detects the reflection of the laser beam
from the back of the cantilever. With the signal from this detector the point of contact of the tip with
the sample can be detected when the tip is lowered. Once the tip is in contact with the sample the
surface can be scanned. The distance the scanner moves vertically at each (x, y) data point is stored
by a computer to form the topographic image of the sample surface.
The AFM mode where the AFM tip is continuously in contact with the sample surface is
called Contact mode. Thus, in contact mode the AFM measures the repulsion force between the tip
and sample. The tip attraction by the capillary force determines the minimal force that can be used
in the AFM measurements, which is a few nanonewtons.
When measuring in air, damage of a sample by the AFM tip can not always be prevented. In
some cases it is useful to remove a small part of the sample material to investigate the thickness of a
complete layer. This can be done by increasing the setpoint, which causes the cantilever to move
3
downwards. From the observed change in the tip deflection the force increase can be calculated by
multiplying the change in distance with the spring constant of the cantilever. The maximum force,
which can be applied, is 200 nN for a cantilever with a spring constant of 0.6 N/m. Because of the
softness of the organic layers, described in this thesis, we did not use scanning forces beyond 30 nN
to make a hole in the layers. To prevent sample damaging, a different way of scanning the sample
with the AFM tip was invented in 1993: Tapping mode AFM [9]. It is a modulated technique where
the tip or the sample is subjected to a periodic vertical oscillation [10]. The advantage of this
technique is that the samples are less damaged by the forces exerted by the tip on the sample. The
disadvantage is that the Tapping mode AFM has slightly slower scan rate than contact mode AFM.
In general AFM has a lot of advantages, like very high resolution (for instance in contact
mode ‘atomic resolution’ images can be obtained). AFM is suitable tool for in-situ measurements,
i.e. materials can be studied in their natural environment [11]. Recently AFM was used for force
measurements in biological systems, for instance the strength of interaction of a membrane protein
in its natural surroundings [12-14].
AFM has also disadvantages. One of them is the heating of the sample by the laser beam
light. Another is the artifacts in the images caused by the interaction of the tip and the sample.
Despite of the disadvantages, AFM is one of the best techniques for observation of surfaces made of
different materials.
1.2. Langmuir-Blodgett technique
It is known that the surface structure of some materials is different from the bulk structure, which
leads to different macroscopic properties as compared to the bulk structure. An example for such
materials is provided by the triglycerides, which in crystals and in bulk solutions adopt a chair or
tuning fork conformation [15], but on the air-water interface they rearrange in a trident
conformation (all hydrocarbon chains pointing toward the same direction) [16, 17]. A detailed
description of the properties of triglycerides at the air-water interface is given in this thesis.
AFM can be used to study surface properties of materials. For this goal thin films are
transformed onto solid substrates via various deposition techniques. The technique we used is
Langmuir-Blodgett technique. This is the commonly used technique for preparation of monolayers
at air-water (or liquid-gas interface in general) interface and their transfer onto solid substrate. It
4
was introduced first by Irving Langmuir [1] and applied extensively by Katharine Blodgett. It
involves the vertical movement of a solid substrate through the monolayer - air interface [18].
In a Langmuir experiment a solution of amphiphilic molecules in an organic solvent is
spread on a liquid-vapor interface. An amphiphile is a molecule that is insoluble in water. One end
is hydrophilic, and, therefore, is preferentially immersed in the water and the other end is
hydrophobic, and preferentially resides in the air. Note that triglycerides, which are the major
substance investigated in this thesis, are lipophilic molecules. However, as an important finding of
our investigations, triglycerides spread as a monolayer on an air-water interface. They adopt a
trident conformation in which glycerol groups are immersed in the water phase and the hydrophobic
tails point into air. Therefore triglycerides behave as amphiphiles in this respect.
In a typical experiment a droplet of triglyceride solution is dripped on a water surface. After
spreading the solvent evaporates and the amphiphiles arrange in monomolecular layer (monolayer).
The molecular layer at the air-water interface is called Langmuir film [6, 19, and 20]. A typical
setup for LB experiments is a Teflon (PTFE) trough with three rigid walls and one movable barrier
(fig.2).
SubstrateWilhelmyplate
Barrier (PTFE)
Trough(PTFE)Amphiphilic molecules
SubstrateWilhelmyplate
Barrier (PTFE)
Trough(PTFE)Amphiphilic molecules
Fig.2. Schematic presentation of Langmuir-Blodgett Trough
By moving the barrier the monolayer can be compressed from an expanded state to a close packing
of the molecules. The amphiphiles have very small interaction, when the distance between them is
large. In this case they have very little effect on the surface tension on the subphase (usual it is
water). When the barrier compresses the layer, the molecules start to interact, which can be
5
regarded as a two dimensional analog of pressure, called surface pressure π . It is defined as
follows:
0π γ γ= − (1)
where 0γ is the surface tension in the absence of a monolayer, and γ the value with the monolayer
present. When the barrier is moved, the area of the film ahead of the barrier changes with , and
the area of the film behind the barrier by
TdA
,0TdA dAT= − . If the compression is isothermal, the Gibbs
free energy, G , of the total surface changes by:
0 ,0 0( )T T TdG dA dA dA dATγ γ γ γ π= + = − ≡ − (2)
The surface tension is measured with a Wilhelmy plate. This is usually a small platinum plate,
which is wetted completely. The downward force on a plate with length l, width w, and thickness t,
with a density pρ , immersed to a depth h in a liquid of density lρ is given by:
2 ( ) cospF glwt t w gtwl hρ γ θ ρ= + + − (3)
Where θ is the contact angle of the liquid on the solid plate, usually taken to be 0, and g is the
gravitational constant. From Eq.(3) changes of the surface tension γ are reflected as changes of the
force . F
π is recorded at constant temperature as a function of the surface area per molecule A , resulting in
a Aπ − isotherm. The measurement of A is straightforward, because it is linearly dependent on the
position of the barrier. A typical Aπ − isotherm is shown in Fig.3.
6
Area per molecule, A
Surfa
ce p
ress
ure,
ΠC
E
G
Phase transition
Phase transition
Fig.3. Schematic presentation of an ideal Aπ − isotherm (G - gaseous phase, E - expanded phase,
C - condensed phase).
A few regions are distinguished, corresponding to several phase transitions. These are,
almost, analogous to the three-dimensional gases, liquids and solids.
In the “gaseous” phase (G in fig.3), the molecules are far enough apart on the water surface
that they exert little force on one another. When the surface area of the monolayer is reduced, the
hydrocarbon chains will begin to interact. The state which is formed is called “expanded “phase
(E).The hydrocarbon chains of the molecules in such a film are in random, rather than regular
orientation, with their polar groups in contact with the subphase. The closest packed state is a state
in which the molecules have a packing resembling the packing in a two dimensional crystal. This is
referred to as the “condensed” phase (C). The area per molecule in such a state will be similar to the
cross-sectional area of the hydrocarbon chain, i.e., ≈ 0.19 nm2 molecule -1. If the monolayer is
compressed even further it collapses, resulting in a sudden decrease in the surface pressure. This is
referred to as collapse.
At and beyond the collapse pressure molecules are forced out of the monolayer and form
other structures, depending of their nature. For example, fatty alcohols and acids form micelles
beyond the collapse pressure. In micelles the molecules are arranged in spheres, with the polar head
groups on the outside and the hydrocarbon chains towards the center. Another arrangement is
characteristic of phospholipids molecules, which is called vesicles. In this arrangement, the double
layers form a shell with water both outside and inside [20]. In some cases multilayers can be
formed, when the monolayer is compressed on interface. E.g. for long-chain esters, up to eight
7
layers on top of each other were obtained [21]. This structure of multilayers on top of the monolayer
is typical also for triglycerides and bile acids [16, 17, and 22]. Recently was found that a single-
chain fatty acid methyl ester forms an unconventional air-stable interdigitated bilayer at the air-
water interface [23].
To investigate these structures the monolayers have to be transferred on a solid substrate,
which is either hydrophilic of hydrophobic. To achieve this, the method developed by Blodgett is
most frequently used and is commonly referred to as the Langmuir-Blodgett technique. With this
technique layers of molecules are deposited on a solid substrate by vertically dipping through the
liquid-vapor interface. During the deposition the surface pressure is kept constant by moving the
barrier to compensate the loss of the material that is transferred on the substrate. The typical dipping
speed is a few mm/s. It must be slow enough to allow the water to drain from the monolayer –
substrate interface and also to let films with a high viscosity adjust in the neighborhood of the
moving substrate. The most commonly used materials as substrates are mica, glass slides, oxidized
silicon wafers and graphite. Before the transfer the substrates can be treated to make them
hydrophilic or hydrophobic. It is possible to create multilayers by repeated dipping of the substrate.
One of the most used techniques for characterization of LB-films is AFM [25-28].
1.3. Stability of Langmuir monolayer
By definition a Langmuir monolayer is thermodynamically stable if under isobaric conditions at air-
water interface it does not change its structure. Conditions for thermodynamic stability can in
principle be established by measuring the equilibrium spreading pressure eqπ , i.e. the pressure at
which the surface area of the film does not change with time [3]. At this point it is important to
clearly discriminate between collapse pressure colπ and equilibrium pressure eqπ . For eqπ we use
the definition of Roberts [3]. The thermodynamic equilibrium (spreading) pressure is the surface
pressure that is spontaneously generated when a sample of solid material in its thermodynamically
stable phase, i.e. in the crystalline phase, is brought in contact with the water surface. Provided that
sufficient time is allowed for equilibration, one can, in principle, be sure that the monolayer which
has been formed by molecules detaching themselves from the crystal surface and spreading over the
subphase is in equilibrium with the crystals themselves. At surface pressures higher than eqπ there
will be a tendency for the monolayer to aggregate into crystals [3].
8
If the monolayer is compressed at a constant rate, at certain pressure it will collapse,
resulting in a sudden decrease in the surface pressure. This pressure is called collapse pressure. The
only way to determine the thermodynamic stability of the monolayer is to investigate it under
isobaric conditions at spreading pressures colπ π< . Note that sometimes one refers to equilibrium
spreading pressure if actually collapse pressure is meant, see e.g. [30].
It was found that some Langmuir monolayers are unstable at air-water interface at surface
pressures below the collapse pressure ( colπ π< ). One of the factors causing the loss of molecules
from the monolayer - “relaxation phenomena” can be desorption in the subphase, e.g. for
monoglycerides [29, 30], evaporation, e.g. for fatty acids. Other mechanisms, such as surface
rheology, surface chemical reaction, polar group hydration, the simultaneous motion of the
monolayer and the liquid substrate as a result of the surface pressure gradient, or structural
relaxation processes in the monolayer itself - such as change in the conformation of the molecules –
are difficult to quantify [24]. By definition these processes occur at pressure eqπ π> .
One of the surprising results of this thesis is that triglycerides, which are the main objects in
this work, also showed a thermodynamic instability at the air-water interface at surface pressures
far below the collapse pressure ( colπ π ). Under isobaric conditions at surface pressures eqπ π> a
molecular rearrangement process takes place which effectively thickens the film. Using Atomic
Force Microscopy for triglycerides we have shown that this process involves the growth of 3D
crystals of triglycerides on top of the monolayer, which is precisely what one should expect for
eqπ π> . For colπ π> similar crystallization processes take place, but in a less controlled and less
reproducible manner.
1.4. Crystal structure of triglycerides
Triglycerides (TAGs) are esterifications of three long-chain fatty acids with glycerol. Many
different types of TAGs exist because the three acids can all differ in chain length and degree of
saturation. The general formula for TAGs is:
CH -O-CO-R2 1
CH -O-CO-R2
CH -O-CO-R2 3
9
TAG molecules are able to pack in different crystalline arrangements or polymorphs, which exhibit
significantly different melting temperatures [15, 31]. It is well known that TAGs may crystallize in
the α (hexagonal, less stable), 'β (orthorhombic), or β (triclinic, most stable) form. However,
some fats display more polymorphs than this [32].
TAG molecules are “three legged” molecules that can pack with the acyl chains(“legs”) in
one of two conformations, neither of which involves all three chains packing alongside each other.
They can pack in a “chair” conformation where the acyl chain in the 2 position is alongside the
chain on either the 1 or 3 positions. Alternatively, a “tuning fork” conformation can be adopted
where the acyl chain in the 2 position is alone and the chains in the 1 and 3 positions pack alongside
each other (Fig.4)
Fig.4. Schematic representation of a tuning fork conformation (a) and a chair conformation (b). Either conformation naturally packs in a chair-like manner. The stacking of these chairs can
be in either a double or triple chain length structure and these stack side by side in crystal planes
(Fig.5).
τ
LL
τ
Double Triple
Fig.5. Schematic arrangement of triglycerides in double and triple layers. Both patterns may lead
to α , 'β or β crystalline phase.
10
The difference between polymorphs is most apparent from a top view of these planes, which
shows the subcell structure (Fig.6). These structures can be identified by X-ray diffraction patterns
[32].
H O T
α β’ β
Fig.6 Schematic presentation of the subcell structure of the three most common polymorphs in
TAGs (viewed from above the crystal plane).
The layer thickness or long spacing (L) gives information on the repeat distance between
crystal planes and obviously depends on the length of the molecules and, furthermore on the tilt
angle (τ ) between the chain axes and the basal plane. In the α phase the chains are oriented
perpendicular to the end-group plane (i.e. ). The o90τ = 'β and β phases have tilted chains (Fig.5).
The short spacing gives information on subcell structure (interchain distances). These
interchain distances depend on how the chains pack together and this is complicated by the “zigzag”
arrangement of successive carbon atoms in aliphatic chains. Closer packing is achieved when the
zigzag of adjacent chains are in step with each other (“parallel”) as opposed to out of step
(“perpendicular”).
In α - phase the chains are arranged in a hexagonal structure (H). They are not tilted and are
far enough apart for the zigzag nature of the chains to not influence packing.
In 'β - phase the chain packing is orthorhombic and perpendicular (O┴). Adjacent chains are
out of step with each other and they do not pack closely. The chains are tilted at 50 - 70o.
In β - phase the chain packing is triclinic (T). Adjacent chains are in step (“parallel”), and
thus pack closely together. This is the densest polymorphic form. The chains are tilted at 50 - 70o
[32].
11
The CnCnCn-type (n = even) TAGs have double chain length structure and the most stable
phase is β . They have asymmetric “tuning-fork” conformation [33]. Because this is the type of
TAGs, which we investigated in this thesis, in the next chapters we will use “tuning-fork”
conformation to describe their crystal structure.
1.5. Outline of the thesis
Langmuir-Blodgett technique and Atomic force microscopy were used to study the phase behaviour
of organic molecules at air-water and air-solid interfaces. Chapter 2 reports the structure of binary
mixed LB monolayers of fatty alcohols. It describes the dependence of phase separation phenomena
on the difference between the chain lengths of the two components and the surface pressure.
Chapter 3 reports the structure and temporal evolution of tristearin (SSS) monolayers at air-water
interface. In order to study the thermodynamic stability of SSS monolayers, they were incubated at
air-water interface, withdrawn and imaged with AFM. During incubation a crystal growth process
took place. A new model was developed to quantitatively describe this process. The crystal growth
theory for tristearin, which we propose was checked by investigating and comparing two more
triglycerides –tripalmitin (PPP) and triarachidin (AAA). In Chapter 4 we show the influence of the
chain length of triglycerides molecules on their stability on water and mica surfaces. Chapter 5
describes the phase behaviour of binary mixed LB- monolayers of triglycerides. We investigated the
relation between phase separation and chain length. In Chapter 6 all results presented in this thesis
are summarized and discussed.
References:
[1] Langmuir, I., The mechanism of the surface phenomenon of floatation, Trans. Faraday Soc.,
15(1920)62-74
[2] Petty, M.C., Langmuir-Blodgett films an introduction, Cambridge University Press, (1996)
[3] Roberts,G., Langmuir-Blodgett film Plenum Press, New York, (1990)
Fig. 3. Excess Gibbs energy for mixed monolayers as a function of spreading pressure π. The
compositions of the mixture are given by the labels at the curves.
From the figure we see that, as in [3], ( ),exG x π is small as compared to RT for all
mixtures, and that the noise is relatively large. Due to noise the sign can not be determined
unambiguously for C mixtures. The fact that with AFM we clearly saw phase separation,
suggests a special interaction between the relatively flexible hydrophobic tails and
alcohols, favouring incorporation of a small amount of in and reverse. The
16 22: C
16C 22C
16C 22C ( )0.5,exG x π=
curve for the C mixture is similar to that for C , suggesting that the difference in chain
length is the main parameter for demixing trends. In line with this G x
16 22: C C
)18 24:
( 0.5,ex π= tends to be
negative for : , which favours homogeneous films and positive for :C , which favours
phase separation.
18C 22C 16C 24
22
2.5. Conclusions
In this study we have obtained AFM images that reveal the structure of mixed alkanol monolayers,
and we applied out thermodynamic measurements and theory to interpret our observations.
As the head groups are the same for all alcohols used in this study, the observed differences
in monolayer structure have to be explained with the methylene-methylene interactions of the tails.
The longer alcohols (C22 and C24) interact more strongly, hence in a condensed layer they adopt a
crystalline, herringbone crystal structure [1, 2, 6] than the shorter ones (C16 and C18), which can be
fluid like. This is in agreement with IR spectra for single alcohol monolayers at 20°C [2].
For surface pressures of π = 10 − 35 mN/m we found phase separation for all systems,
except for C18:C22, with domains of the longer alcohol, embedded in a shorter alcohol film. This
leads to the conclusion that in a condensed monolayer phase separation takes place when the chain
length difference is 6 or more carbon atoms. The greater the length difference is, the more
unfavorable is the mixing free energy, which is also shown from the thermodynamic data.
At high surface pressure, π = 20 − 35 mN/m, the domains get tetragonal shapes. This can be
understood as at higher pressures crystalline packing of molecules is favoured. The π - A isotherms
show an area per molecule 19-20 Å2 for these surface pressures. At lower pressures, π = 10 -
15 mN/m, the excess Gibbs energy is small. Then disordered packing is more favourable and
domains are rounded.
If the chain length difference is only 4 methylene units, both the AFM images and the
thermodynamic data of the C18:C22 mixture indicate no phase separation.
In the case of a chain length difference of 8 units the excess Gibbs energy is so large that the
driving force for phase separation might be beyond the limit where equilibrium structures are
formed. Hence we think that the irregular domain shapes in the C16:C24 mixture are growth shapes,
rather than thermodynamic equilibrium shapes.
The result that we observed phase separation already in the range where our thermodynamic
measurements indicated ∆Gex ≅ 0.1 RT is surprising, since one would expect spontaneous phase
separation only if ∆Gex≥1 RT. This can not be explained yet, but it might be due to a too high
compression rate around the spreading pressure where phase separation starts.
23
References:
[1] Wang, J.L., et al., J. Am. Chem. Soc. 116 (1994) 1192
[2] Popovitz-Biro, R., et al, J. Am. Chem. Soc. 116 (1994) 1179
[3] Kulkarni, V.S., et al., J. Colloid Interface Sci. 89 (1982) 40
[4] Ten Grotenhuis, E., et al., Colloids and Surfaces, A: Physicochemical and Engineering Aspects
105 (1995) 309-318
[5] Gains Jr., G.L., Insoluble Monolayers at Liquid-Gas Interfaces, Interscience, New York, 1966
[6] Gavish, M., et al., Science 250, Issue 4983, (1990) 973
24
CHAPTER 3
Structure and dynamics of Langmuir – Blodgett Tristearin films: Atomic Force Microscopy and theoretical analysis
Abstract
The structure and temporal evolution of tristearin (SSS) monolayers at the air-water interface at
20 ± 1°C are investigated with the Langmuir method. The deposited Langmuir- Blodgett (LB)
layers were investigated with Atomic Force Microscopy (AFM). The LB experiments showed that
adsorption isotherms obtained with commonly used compression rates do not correspond to
thermodynamic equilibrium. Under isobaric conditions at 10 mN/mπ ≥ the film area slowly
decreased ,which corresponded to the formation of crystals on top of the monolayer. The AFM
images reveal that SSS initially form trident monolayers at air-water interface. These layers are
thermodynamically stable at surface pressure 5 mN/mπ ≤ . The thickness of the trident monolayer
was found to be 1.6 to 1.8 nm, corresponding to tilt angles of the molecule chains varying from
at o43τ = 10 mN/mπ = to at o53τ = 40 mN/mπ = . For 10 mN/mπ ≥ growth takes place of
crystals with a tuning fork conformation of the SSS molecules on top of the trident monolayer. The
crystals grow with time, mainly in lateral directions. The growth rate increases with surface
pressure. A new model is developed to quantitatively describe the crystal growth process. A lateral
growth rate of 2.3 nm/min and a vertical growth rate of 0.005 nm/min were calculated for 1
individual crystal at 10 mN/mπ = .The same growth process that was observed on the air-water
interface was also observed in transferred monolayers at room temperature, though the growth was
much slower.
25
3.1. Introduction
Many efforts have been made in investigating the structure of triglycerides. Most of the published
work has been on homogeneous triglycerides (their 3 fatty acid residues are identical). In the solid
state, triglycerides adopt a polymorphic crystalline structure.
Depending on the crystallization procedure, especially the thermal treatment, they may
crystallize in the α (hexagonal, less stable), β’ (orthorhombic), or β (triclinic, most stable) form. In
each of these polymorphic forms the molecules have a tuning fork conformation [1, 2], but the
packing of these tuning forks is different.
However in monolayers at a hydrophilic-hydrophobic interface, triglyceride molecules adopt
a trident conformation (all hydrocarbon chains pointing toward the same direction). This
conformation has been proposed by Bursh and Larsson, based on their Aπ − diagrams for
triglycerides on water at different temperatures [3]. The trident conformation was also found by
Hamilton, using NMR measurements for tripalmitin and triolein at the oil-water interface in
phospholipids vesicles [4, 5] and by Claesson for triolein in contact with mica [6]. In the trident
conformation the hydrophilic glycerol group is in contact with the water or the mica surface, and
the hydrophobic chains point into the air or oil. In some cases multilayers can be formed, when on
an interface a monolayer is compressed laterally [7-9].
Bursh and Larsson investigated what happened when a monolayer of triglyceride at the air-
water interface is compressed beyond the so-called collapse pressure, where the steady increase of
the spreading pressure upon lateral compression is interrupted. They concluded that some molecules
leave the monolayer to form new molecular layers. They proposed a trident conformation for the
first triglyceride monolayer and a tuning fork conformation in the next layers, with a packing
similar to that in the crystalline state [3]. Triple layer formation was reported also for bile acids
[10]. Only a few studies of triglycerides with Atomic force microscopy (AFM) were performed [11,
12]. Michalski investigated Langmuir-Blodgett monolayers on glass of tripalmitin by AFM [12].
The monolayer was compressed and withdrawn at a surface pressure, corresponding to the middle
of the condensed phase in the Aπ − . She suggested that the trident monolayer generally
reorganizes after being transferred to the glass, forming two different structures. The first one
corresponds to bilayers in a regular tuning fork crystalline structure. The second one corresponds to
the triple layer structure, proposed by Bursh and Larsson [12].
26
The aim of this chapter is to better understand the molecular structure and processes in
triglyceride films at the air-water interface (Langmuir film) and on a solid surface like mica
(Langmuir-Blodgett (LB) film). Therefore we measured the Aπ − (spreading pressure π vs area
per molecule A ) diagram of Langmuir films and we investigated LB films with AFM. In this
chapter we focus on tristearin (SSS), in subsequent chapters we extend the investigations to other
triglycerides. Starting with a Langmuir film at very small π , where the film is in a low-density
“gas” phase, we compressed the film, at a constant rate, to the desired pressure π (forced
compression). To investigate whether the Langmuir film was in thermodynamic equilibrium at this
pressure π , we sometimes left the film for some time at pressure t π (isobaric compression). The
Langmuir film was transferred to mica directly after forced compression ( ) or after
or of incubation time at constant pressure
0t =
30 mint = 60 mint = π (isobaric compression).
3.2. Materials and methods
3.2.1. Chemicals
Film material: Tristearin (1, 2, 3, -trioctadecanoylglycerol: SSS) was purchased from Larodan with
a stated purity of >99 mass %. A stock solution of SSS with concentration of 1 mM in distilled
chloroform was prepared.
Subphase: Distilled water was used as a subphase in our Langmuir system for all experiments. The
resistivity of the water was 15 MOhm cm.
Substrates: All monolayers were transferred onto freshly cleaved mica.
3.2.2. Langmuir method
Compression isotherms were measured on a home made instrument, using available components.
The instrument was equipped with a Teflon trough (8.6 ×14.8 cm). The spreading pressure π was
measured with a Wilhelmy type balance consisting of a platinum plate coupled to an electrobalance
(Cahn 1000, Ankersmit), with an accuracy of about 0.1 mN/m. The film material was initially
spread on the water subphase, dropping 20 µL of 1 mM SSS dissolved in chloroform, using a 25 µL
Hamilton syringe. The conditions were chosen such that initially the average area A per molecule is
.We started (asymmetric) film compression 2 min after spreading. In our system two 2110 ÅA ≈
27
modes of operation were available. First forced compression, where the position of the barrier, and
hence the trough length ( )l t ahead of the barrier, is given. Then the resulting spreading pressure
( )tπ is registered. In this mode we chose barrier velocities of the order of 1 cm/min, which
according to the literature should be slow enough that the Langmuir film stays close to
thermodynamic equilibrium.
Second we used the isobaric compression mode, where a constant spreading pressure π is
applied and the resulting trough length ( )l t is monitored. Obviously if the film is in equilibrium at
the applied pressure, then is constant. In practice however we often found the barrier to move
with velocities of the order of 1 m
( )l t
/secµ . This barrier motion reflects rearranging processes in the
Langmuir film. We use AFM images to interpret and quantify this process.
3.2.3. Langmuir-Blodgett film transfer
In order to obtain LB films, first a substrate was immersed perpendicularly in the aqueous subphase.
We started with a very small initial surface pressure ( 0π = mN/m), and compressed the monolayer
slowly (1 cm/min) to the final pressure. To obtain a LB film that is characteristic for forced
compression, the film was then transferred immediately by vertical pulling of the substrate through
the air-water interface at a speed of 2 mm/min. During the transfer the surface pressure was kept
constant by appropriately moving the barrier. The transfer process takes a few minutes.
In order to study the structural changes of the Langmuir film during isobaric compression
the film was left at constant pressure for 30 or 60 min before it was transferred to the substrate.
After deposition the LB-films were dried in air and kept in close containers until use. All
experiments were done at . o20 1 C±
3.2.4. AFM measurements
The samples were examined with AFM within about 5 hours after preparation. We checked that the
length of this delay time is not critical. Imaging was done with a Nanoscope(R) IIIa (Digital
Instruments, Santa Barbara, CA) in contact mode with oxide-sharpened silicon nitride tip (k = 0.06
N/m). The AFM was equipped with a J scanner (176 x176 µm; z limit = 5.349 µm). All images
were processed using procedures for flattening in Nanoscope III software version 5.12r5 without
28
any filtering. To check if the monolayer is successfully transferred to the mica surface we measured
at least five different spots (each 150 µm2 ) of every sample. In order to detect structural changes in
the adsorbed film in contact with air we studied LB films several days after preparation as well.
3.3. Langmuir observations
3.3.1 Forced compression
0
10
20
30
40
50
60
0 20 40 60 80 100 120
2Area / molecule A (Å )
Surf
ace
pres
sure
π (m
N/m
) Fig.1.Example of surface pressure vs
area isotherm of tristearin (SSS) at air-
water interface, at 20o C, obtained by
forced compression at a rate of
1cm/min (x - observed data; - fit using
Eq. (1) with ,
260.4ÅcondA =
258ÅcolA = and 40.5mN/mcolπ =
Fig. 1 shows a typical Aπ − isotherm of tristearin (SSS), recorded at a barrier velocity of
1 cm/min. Three different regimes can be recognized. Starting at a large area per molecule A the
pressure is low and increases only slowly with decreasing A. Upon decreasing A further the
condensation area is reached and the pressure starts to increase more rapidly. Compressing
further it is seen that for A below the collapse area the increase of the pressure is slow again.
The explanation of this characteristic dependence is that for
condA
colA
cond colA A A= = the SSS molecules are
close enough together to form a condensed monolayer, whereas for this monolayer
collapses to form multilayer structures. The measured
colA A<
Aπ − data showed that the transition from
one regime to another were not very sharp. It order to get reliable and unbiased estimations for , and the collapse pressure , we fitted the isotherms with: condA colA colπ
( ) ( ) ( , ) ( , )col colcol col cond
col cond col cond
A s h A A a h AA A A A
π ππ ≈ − − + −
− −A b (1)
29
where , , , , a and b are fitting parameters, representing the slope of the
isotherm during collapse, i.e. for A < a and b characterize the smoothness of the transitions
from one regime to the other. The function
condA colA colπ cols cols
colA and
( ) ( )2 21,2
h x a x x a≡ − + (2)
is a hyperbola interpolating between ( ),h x a x≈ for large negative and x ( ),h x a ≈ 0 for large
positive . This function has no direct physical interpretation and was introduced for practical
purposes only. As shown in Fig.1 satisfactory fits were obtained. Fitting a number of isotherms that
were obtained at compression velocities varying from 0.5 cm/min to 2 cm/min we found
, and
x
262 2ÅcondA = ± 57.8 0.3ÅcolA = ± 41 1mN/mcolπ = ± . These values did not vary significantly
within the range of the barrier velocities that we applied .The is consistent with
the trident conformation of the SSS molecules in a monolayer film at the air-water interface. The
cross-sectional area per hydrocarbon chain for tristearin at 20
262 2ÅcondA = ±
oC in the α phase (α phase has the
most mobile acyl chains) is [13].Our isotherms are consistent with earlier reports [3, 12]. 219.7Å
3.3.2 Isobaric compression
Even though we found that the forced compression isotherms did not change appreciably for barrier
velocities between 0.5 and 2 cm/min, under isobaric conditions we did observe further compression
though at velocities that were one or two orders of magnitude smaller. We stopped the forced
compression when a certain surface pressure π was reached. Next we kept the surface pressure
constant at that value, allowing the barrier to move. This is shown in Fig.2.
30
Barrier position vs time
13.50
14.00
14.50
15.00
15.50
16.00
16.50
0 1000 2000 3000 4000 5000
time t (sec)
l(t)
(cm
)
π = 10mN/mπ = 35mN/m
Fig.2. Two examples of the measured barrier position as a function of time during forced and
isobaric compression. The almost vertical parts of the curves correspond to forced compression at
a rate of 1 cm/min. The slowly decreasing parts correspond to small residual isobaric compression
rates at the spreading pressure given in the figure.
After several minutes a constant velocity was reached usually. The evolution of the trough
length was fitted to
l t (3) ( ) ( ) ( ) ( )0 0 ,f 0l v t t v v h t t a≈ − − − − −
Here the five fitting parameters are l , the trough length at the start of the isobaric compression, t ,
the starting time of the isobaric period,
0 0
fv and v , the forced and isobaric barrier velocity
respectively, and a , characterizing the transition from the forced to the isobaric regime. The
accuracy of the fits typically was 0.2%. In all cases the fitted forced velocity fv was very close to
the applied barrier velocity.
Isobaric compression
0
5
10
15
20
25
0 10 20 30 40 50
Spreading pressure π (mN/m)
Vel
ocity
v (
um/s
ec)
Fig.3. Isobaric velocity ν (µm/sec) as a
function of spreading pressure π as obtained
by fitting the measured barrier position to
Eq.(3). Note the sharp increase of ν for
spreading pressure close to the collapse
pressure colπ .
31
In Fig. 3 we show the dependence of the isobaric velocity v on the surface pressure π . It
can be noted that for and depends linearly on for
. For pressures
0v ≈ 5 mN/mπ ≤ π
5 mN/m 35 mN/mπ≤ ≤ 42 mN/mπ = (the collapse pressure) a much faster
compression is found. These results show that the isotherm shown in Fig. 1, can be considered as an
equilibrium isotherm only for . For larger pressures the equilibrium value of A is
smaller than the value displayed in Fig.1. At this point it is worth wale to clearly discriminate
between collapse pressure
5 mN/mπ ≤
colπ and equilibrium pressure eqπ .We use the definition of Roberts in his
book [14],whereas sometimes in the literature one manes equilibrium spreading pressure what we
call collapse pressure, see e.g.[15]. Equilibrium (spreading) pressure is the surface pressure that is
spontaneously generated when a crystalline sample of the solid material is placed in contact with
the water surface. Provided that sufficient time is allowed for equilibration to occur one can, in
principle, be sure that the monolayer which has been formed by molecules detaching themselves
from the crystal surface and spreading over the subphase is in equilibrium with the crystals
themselves. At any surface pressure higher than this there should be a tendency for the monolayer
to aggregate into crystals [14]. According to our results (Fig.3) for tristearin at air-water interface is
5mN/meqπ = .
In the isobaric conditions some rearrangement must take place which effectively thickens
the film. We assume that this process involves the growth of 3D crystals of SSS, and we investigate
this hypothesis using AFM-imaging. To this end we compare LB-films obtained by transfer at
with films transferred 30 or 60 min after . 0t t= 0t
3.4. AFM observations
3.4.1. Monolayer thickness
From the AFM images of LB-films, withdrawn at 5 mN/mπ = (data not shown) it is seen that the
mica is covered with a homogeneous monolayer. The monolayer can be successfully transferred to
a mica surface and it is quite stable in the course of time. When the Langmuir film was prepared at
higher pressures a monolayer was observed as well, but now with embedded higher domains. After
1 day storage at room temperature of the withdrawn LB- film the monolayer is still present, though
with slightly higher thickness (fig.4, C, F).
32
A B C
D1.68 nm 1.73 nm
µm0 2.50 5.00
-2.0
00
2.00 E
1.53 nm 1.44 nm
µm2.50
-2.0
02.
00
5.000
0F
0
1.86 nm 1.78 nm
µm
-2.0
02.
00
0.50 1.501.00
0
AAA BB CCC
D1.68 nm 1.73 nm
µm0 2.50 5.00
-2.0
00
2.00D
1.68 nm 1.73 nm
µm0 2.50 5.00
-2.0
00
2.00 E
1.53 nm 1.44 nm
µm2.50
-2.0
02.
00
5.000
0E
1.53 nm 1.44 nm
µm2.50
-2.0
02.
00
5.000
0
1.53 nm 1.44 nm
µm2.50
-2.0
02.
00
5.000
0F
0
1.86 nm 1.78 nm
µm
-2.0
02.
00
0.50 1.501.00
0
F
0
1.86 nm 1.78 nm
µm
-2.0
02.
00
0.50 1.501.00
0
Fig.4. AFM height image of an SSS monolayer transferred immediately after forced compression to
surface pressure π = 30mN/m. The black squares are holes in the monolayer produced by scanning
at a high force (~30 nN). (A) image scanned at AFM force F = 1nN with corresponding cross
section (D). (B) same area as in (A) scanned with AFM force F = 7.6nN and the corresponding
cross section (E). (C, F) same sample exposed to air at room temperature for 1 day at F = 1nN.
The scale bar is 2 µm (A, B) and 500nm (C) and the vertical scale is 5 nm for all images.
We estimated the monolayer thickness using the following procedure. We first
scratched a rectangular hole in the monolayer with the AFM tip by scanning with a relatively large
force
( )0 0d d π=
30 nNF ≈ . Then a larger image, including the hole was scanned with small forces
1 8 nNF = − (fig.4). The height difference between the hole and the surrounding gives an
apparent thickness d . The fact that d turned out to depend on the scanning force F, shows that
the real monolayer thickness
′ ′
0 ( )d π depends on d . ′
In Fig.5 we show data, together with an overall fit of the form
( ),d F a b cF d Fπ π′ ≈ + + + π (4)
From this fit we can estimate the real thickness ( ) ( )0 ,d d Fπ π′≈ = 0 , corresponding to scanning
force , which is presented in Fig. 6. 0F =
33
Apparent monolayer thickness
1.0
1.2
1.4
1.6
1.8
2.0
0 5 10AFM force F ( nN )
AFM
thic
knes
s d'
(n
m)
10 mN/m10 mN/m20 mN/m20 mN/m30 mN/m30 mN/m
Fig.5. Measured layer thickness d as a
function of applied AFM force F and surface
pressure π. The surface pressures (π) at
which the monolayer was compressed are
given by the labels at the curves. The symbols
correspond to the measured data and the lines
are the fit according to Eq. (4).
′
0 '( 0)d d F
Real monolayer thickness
1.5
1.6
1.7
1.8
1.9
0 10 20 30 40
Spreading pressure (mN/m)
Laye
r thi
ckne
ss
d o (
nm)
Fig.6. Variation of the real thickness
= =
'( , )F
of the monolayer with varying
spreading pressures. Line: from the combined
fit with Eq. (4), squares: from independent
linear fits of d π at fixed π .
Note that the monolayer thickness varies from about 1.6 to 1.8 nm over the pressure range
that we study here. We interpret this change in thickness as reflecting a change in the tilt angle τ
between the alkyl chains and the substrate surface. Such a change in the tilt angle of amphiphilic
molecules on air-water interface due to compression was reported before [16, 17].
To translate the thickness into a tilt angle we need to estimate the effective chain length. A
first estimation we get from crystal data on the hexagonal α-phase [18, 19]. In this phase the SSS
molecules, in tuning fork conformation, are parallel to the c-axis. Then the interplanar distance d
(001), which is often referred to as long spacing, is equal to the length of the SSS molecule in
tuning fork conformation. This length is built up from two times the chain length plus the length of
the glycerol group, plus a small contribution from the contact region between SSS layers. Since
in the hexagonal α-phase, the alkyl chain length must be about 2.5 nm. A more
precise analysis and interpretation of crystallographic data of SSS in the stable β′-phase [20], where
and
( )001 5.06 nmd =
( )001 4.48 nmd = 60.8τ = ° , allows us to estimate an effective length of 5.13 nm of an SSS
molecule in tuning fork conformation. Correcting this for the length of the glycerol and the
34
contribution from the contact region in that phase, the alkyl chain length can be estimated as 2.31
nm.
We have no detailed information on the molecular conformation of the triglyceride
molecules in the monolayer. In order to estimate the tilt angle in the monolayer, we assume that the
glycerol part of the molecule makes close contact with the (hydrophilic) substrate. The alkyl chains
are stretched similar as in the α , β and 'β phases, though in different orientation with respect to
the glycerol group. This leads to a structure where alkane chains of 2.31 nm extend from the
substrate to the monolayer surface at a height above the substrate. Thus in the monolayer SSS
molecules adopt a trident conformation we get a simple relation:
0d
( ) 0sin /(2.31 nm)dτ = (5)
Interpreting our monolayer thickness data with Eq. (5), we see that the tilt angle varies from
43τ = ° at 10 mN/mπ = to 53τ = ° at 40 mN/mπ = . It is known that in the crystalline β′ and β-
phases of triglycerides the chains adopt specific tilt angles, which are characteristic for the chain-
packing in the given triglyceride. In these phases tilt angles always are above about 50o. Smaller tilt
angles are energetically unfavourable [1, 19]. Since presumably in the trident monolayer the alkyl
chains are less densely packed than the crystalline phases, a smaller tilt angle seems acceptable.
35
3.4.2. Initial structure, obtained by forced compression
A B
C D
AA BB
CC DD
Fig.7. AFM height image showing monolayers of SSS transferred immediately after forced
compression to surface pressure (A) 10 mN/mπ = , (B) 20 mN/mπ = , (C) 30 mN/mπ = and (D)
42 mN/mπ = . The density of higher domains, embedded in the monolayer, increases with the
surface pressure. The scale bar is 2 and the vertical scale is 20 nm for all images. µm
Figure 7 shows AFM images of SSS-layers that we transferred from the water-air surface to
mica, immediately after the spreading pressure π was reached by forced compression. Domains are
found that extend 3.5 nm or more above the monolayer level. Their density increases with
increasing π as shown in fig.8. We suggest that they are small initial crystals, formed in the period
where the spreading pressure increases from the small values at which the film is in a two-
dimensional gas state, to the final pressure π at which the condensed phase has formed. In this
period SSS molecules undergo major orientation and packing changes. Since the molecular surface
density of the adsorbed film is already high, in the last part of this period such motions are hindered
36
considerably. As a result the formation process of the domains will not be strictly deterministic and
a metastable film structure may form. We suppose that the domains serve as crystal nuclei from
which bigger crystals can grow when the Langmuir film is further compressed isobarically at
constant pressure π .
Initial coverage and density
0.00
0.04
0.08
0.12
0.16
0 10 20 30 40 50
Spreading pressure (mN/m)
Cov
erag
e
0.0
0.2
0.4
0.6
0.8
1.0
Den
sity
( um
-2)
Fig.8. Fraction θ of the film area, covered with crystals (▲), formed during the forced
compression to spreading pressure (π ) and crystal density ρ (♦). The curves are results obtained
fitting all forced and isobaric compression image data to the model described in Section 3.5.
3.4.3. Structural changes during isobaric compression
To investigate the structural changes of the Langmuir film in time, we transferred the Langmuir
film to the mica surface 0, 30 and 60 min after isobaric compression started. At surface pressure
5 mN/mπ = we observed no significant differences between the monolayers withdrawn 0 or 30
min after the start of isobaric compression.
37
B CA
D
1.59 nm
0 5.0 10.0
0-5
.05.
0
µm
E
10.05.00
-5.0
5.0
0
µm
3.49 nm 3.52 nm F0.192 nm
µm5.00 1
-5.0
5.0
0tmm m m
0.0
B CA
D
1.59 nm
0 5.0 10.0
0-5
.05.
0
µm
E
10.05.00
-5.0
5.0
0
µm
3.49 nm 3.52 nm F0.192 nm
µm5.00 1
-5.0
5.0
0
0.0
BB CCAAA
D
1.59 nm
0 5.0 10.0
0-5
.05.
0
µm
D
1.59 nm
0 5.0 10.0
0-5
.05.
0
µm
1.59 nm
0 5.0 10.0
0-5
.05.
0
1.59 nm
0 5.0 10.0
0-5
.05.
0
µm
E
10.05.00
-5.0
5.0
0
µm
3.49 nm 3.52 nmE
10.05.00
-5.0
5.0
0
µm
3.49 nm 3.52 nm
10.05.00
-5.0
5.0
0
10.05.00
-5.0
5.0
0
µm
3.49 nm 3.52 nm F0.192 nm
µm5.00 1
-5.0
5.0
0
F
0.0
0.192 nm
µm5.00 1
-5.0
5.0
0
0.0
0.192 nm
µm5.00 1
-5.0
5.0
0tmm m m
0.0
Fig.9. AFM height image of SSS monolayers transferred at π = 10 mN/m. (A) immediately after
forced compression, (B) after 30 min isobaric compression at air-water interface. (C) the same area
as in (B) after several scans with AFM force ~2nN. The scale bar is 2 µm and the vertical scale is
10 nm for all images. The corresponding cross sections are given in (D, E and F). Length
differences are given by the numbers at the markers. The symbols below the lines give our proposed
structure of the crystals (m - trident conformation; t – top layer tuning fork conformation)
At a surface pressure π = 10 mN/m, the AFM images show a homogeneous monolayer with
small defects when the LB-film was transferred to mica immediately after forced compression, as
shown in Fig.9A and D. After 30 min isobaric compression we observed a few higher domains,
embedded in the monolayer (fig. 9B, E). These domains were soft and could be scratched away
with the AFM tip, even at the normal scanning forces F that are normally used for imaging. After
several scans with F = 1-2 nN the second layer disappeared, leaving a flat film with the same
thickness as the trident monolayer, Fig.9C and F. The thickness of the domains, measured from the
monolayer, was 3.5 – 3.6 nm.
38
A D
µm
5.1 nm 4.9 nm
3.750 7
-9.5
09.
5
mαm
.50
B
µm
4.9 nm8.2 nm
3.75 7.500
-30.
030
.00
m αm
tαm
E
C F
µm3.75 7.500
-30.
030
.00
8.2 nm
5.0 nm15 nm
m tαm
tααm
tαααm
mica
AA D
µm
5.1 nm 4.9 nm
3.750 7
-9.5
09.
5
mαm
D
µm.50
5.1 nm 4.9 nm
3.750 7
-9.5
09.
5
mαm
.50
BB
µm
4.9 nm8.2 nm
3.75 7.500
-30.
030
.00
m αm
tαm
E
µm
4.9 nm8.2 nm
3.75 7.500
-30.
030
.00
m αm
tαm
µm
4.9 nm8.2 nm
3.75 7.500
-30.
030
.00
m αm
tαm
E
CC F
µm3.75 7.500
-30.
030
.00
8.2 nm
5.0 nm15 nm
m tαm
tααm
tαααm
mica
F
µm3.75 7.500
-30.
030
.00
8.2 nm
5.0 nm15 nm
m tαm
tααm
tαααm
mica
8.2 nm
5.0 nm15 nm
m tαm
tααm
tαααm
mica
Fig.10. AFM height image of SSS monolayers transferred at 20 mN/mπ = . (A) immediately after
forced compression, (B) after 30 min isobaric compression at air-water interface and (C) after 60
min isobaric compression. The corresponding cross sections are given in (D, E and F). The scale
bar is 2 µm for all images and the vertical scale is 20 nm for (A) and 70 nm for (B, C). Length
differences are given by the numbers at the markers. The symbols below the lines give our proposed
structure of the crystals (m – trident conformation; α - crystal tuning fork conformation; t – top
layer tuning fork conformation).
39
At surface pressure 20 mN/mπ = we found that the directly transferred LB-film consisted
of an almost defect free trident monolayer, in which many small domains were embedded. The
thickness of the domains was found to be 4.8 - 5.1 nm, as shown in Fig.10A and D. On LB-films
transferred after 30 min isobaric compression, the domains within the trident monolayer were
higher and bigger. The maximum measured thickness from the monolayer was 8.2 ± 0.2 nm
(fig.10B and E). After 60 min incubation even higher domains were found with thickness up to 20 ±
0.2 nm measured from the mica (Fig.10 C, F). On the highest domains we found terraces separated
by steps of height 4.9 ± 0.1 nm. In all cases, the domains were surrounded by the trident monolayer.
The same growth process was observed for LB-layers obtained at surface pressure
30 mN/mπ = . A closer AFM observation showed us that on the bigger crystals formed at
20 mN/mπ ≥ two different terraces can be found, with height thicknesses 3.5 nm and 5.1 nm from
the monolayer (fig.11).
A
B5.2 nm
3.5 nm5.1 nm
µm2.50 5.000
-20.
020
.00
mαm
αm
tm
A
B5.2 nm
3.5 nm5.1 nm
µm2.50 5.000
-20.
020
.00
AA
B5.2 nm
3.5 nm5.1 nm
µm2.50 5.000
-20.
020
.00
B5.2 nm
3.5 nm5.1 nm
µm2.50 5.000
-20.
020
.00
5.2 nm3.5 nm
5.1 nm5.2 nm3.5 nm
5.1 nm
µm2.50 5.000
-20.
020
.00
mαm
αm
tm
Fig.11. AFM height image of SSS monolayer
transferred at 30 mN/mπ = after 30 min isobaric
compression at air-water interface (A). The
corresponding cross section is given in (B). The scale
bar is 2 µm and the vertical scale is 50 nm.
40
3.4.4. Stability of the transferred LB-film
To check the stability of SSS-layer in air, we transferred it immediately after forced compression to
30 mN/mπ = and left it for 1 day at room temperature (fig.12). The monolayer became grainy and
slightly higher. The crystals grew slightly and the newly grown parts of the crystals were 3.5 ± 0.1
nm above the monolayer level. In some crystals higher domains (5.0 ± 0.1 nm) were observed.
During the incubation in air of the transferred LB- film not only the present already crystals were
growing, but also new very small nuclei appeared. We suggest that the grainy character of the
monolayer is due to molecules, which leave the monolayer to form the new nuclei and the new parts
of the present crystals.
Fig.11. AFM height image of a monolayer of SSS that
was transferred to mica immediately after forced
compression to surface pressure 30 mN/mπ =
and that
was left for 1 day in air at room temperature. The cross
section is shown in (B). The scale bar is 2 µm and the
vertical scale is 20 nm.
A
B
1.86 nm3.3 nm
5.1 nm
3.75 7.500
-10.
010
.00
µm
mtm
αm
AA
B
1.86 nm3.3 nm
5.1 nm
3.75 7.500
-10.
010
.00
µm
mtm
αm
B
1.86 nm3.3 nm
5.1 nm
3.75 7.500
-10.
010
.00
µm
1.86 nm3.3 nm
5.1 nm
1.86 nm3.3 nm
5.1 nm
3.75 7.500
-10.
010
.00
µm
mtm
αm m
tm
αm
3.4.5. Consistency of Langmuir and AFM data
If the densities of the monolayer and the higher domains were exactly the same, then the total film
volume ( )V t should remain constant during the isobaric compression process (SSS is not volatile).
In the Langmuir system we measure the temporal change of the film area ( )A t . From the AFM
41
images we can estimate the average film thickness ( )d t . Ideally ( ) ( )A t d t V= is constant,
whence ( ) ( )0 /A A t , which can be obtained from the Langmuir experiment should be equal to
as measured by AFM. ( ) ( )/ 0d t d
Fig.13 shows that, within the experimental accuracy this is true. The relatively large
uncertainty of 5 - 10% of as estimated from AFM is due to the inherent inaccuracy of the
standard Nanoscope “bearing analysis” software for estimating crystal volumes. The uncertainty in
the Langmuir estimation of 2 - 4% is caused by the differences in the observed isobaric velocities
as obtained from the fits in section 3.3.2. As the individual fits are accurate, these velocity
differences reflect accidental differences in the structure of the film that was being compressed.
( )d t
v
Scaled film thickness
1
1.2
1.4
1.6
0 10 20 30 40 50Spreading pressure (mN/m)
d(t)
/ d(0
)
Langmuir 30 min
Langmuir 60 min
AFM f it 30 min
AFM f it 60 min
AFM data 30 min
AFM data 60 min
Fig.13. Scaled film thickness estimated by Langmuir machine and AFM
3.5. Theory for nucleation, growth and coalescence of crystals
3.5.1 Qualitative interpretation of film evolution observations
In the sequel we shall interpret the observed film structure on the basis of a model that is
schematically presented in figure 14.
42
~1.75 nm
4.8 - 5.1 nm~ 3.5 nm
~ 3.5 nm
4.8 - 5.1 nm
45 2± o
40 -45 o
40 -45 o
~ 90 o
~ 90 o
3.5 0.1nm±43 -45 o
43o
A B C D E
Fig.14. Schematic illustration of the structures proposed for thin layers of SSS molecules.
(A) Monolayer structure. At 5 mN/mπ ≤ this is the only structure found, at 10 mN/mπ ≥ it is the
structure around the higher domains, (B) Structure of stable thin crystals. At 10 mN/mπ = all
observed crystals have this structure. (C) Structure of metastable crystals. Such crystals are found
on films that are withdrawn immediately after forced compression to 20 mN/mπ = . (D, E)
structure of higher crystals. Such crystals are observed after 30 or 60 min isobaric compression at 20 mN/mπ ≥ .
Our observations suggest that an SSS trident monolayer is thermodynamically unstable for
spreading pressure 5 mN/mπ . Therefore during isobaric compression at 5 mN/mπ , some
SSS molecules move to the top of the monolayer. These molecules rearrange in higher domains
where they presumably adopt the more stable tuning fork conformation and pack similar as in the
crystalline α and β crystal forms. This film structure was first proposed by Bursh and Larsson to
interpret the triple chain LB-film thickness that they observed for LB-films that were compressed
beyond the collapse pressure [3, 21]. Based on a careful analysis of the observed domain height we
propose a new model for the structure and packing characteristics of the domains.
Using, as above the estimated effective length of 5.13 nm for an SSS molecule in the tuning
fork conformation, the observed domain thickness of 3.5 nm at 10 mN/mπ = , corresponds to a tilt
angle 43 44.5τ = − ° , i.e. somewhere between the estimated tilt angle in the trident monolayer and
43
the tilt angle in the stable β′ phase, Fig.9B, E. We may suppose that the structure of these layers can
be described as a slightly deformed β or β′ phase. As we observed this layer thickness always at the
upper film layer, we refer to this structure as the top layer structure (‘t’ in the figures). The fact that
we have observed this structure to be common for top layers shows that the increased tilt is a form
of surface relaxation, caused by the different interaction with other crystals layers.
At surface pressures 20 mN/mπ ≥ some domains extended as much as 5.0 ± 0.1 nm above
the surrounding monolayer. This suggests that in these domains the molecules are fully stretched
(5.13 nm) and oriented perpendicular to the monolayer, i.e. the structure of these domains is similar
to the crystalline α phase (fig.14, C). During forced compression of the Langmuir film at
20 mN/mπ ≥ , the crystal growth process is so fast that this metastable, α -like , polymorph with
layer thicknesses d(001) ~ 5 nm is formed. Domains that are grown in the α - phase will not
spontaneously transform to the β or 'β phase because this involves a very slow solid-solid
transformation process.
For the slow growth process at 10 mN/mπ = the stable β - phase is grown immediately,
though with a top layer slightly thinner than the interplanar distance d(001) = 4.48 nm of the real
β - phase (fig.9).
The higher domains (8.2 ± 0.2 nm) found after isobaric compression can be explained with
the formation of a second layer on top of the first one (fig.14, D). This measured thickness does not
correspond to two fully stretched layers (~ 10 nm). We assume that the first layer is in α - phase
(5.1 nm) and the second layer, which in this case is the top layer, has the ‘t’- structure, having a tilt
of 40-45o and a thickness of 3.4 ± 0.2 nm .The reproducibility of the step height in the next layers,
which is 4.8-5.1 nm, supports this interpretation.
Combining our observations we concluded that for 5 mN/mπ crystals of SSS in tuning
fork conformation are growing on top of a trident monolayer at the air-water interface. If the growth
is slow enough (e.g. at 10 mN/mπ = or in the last stages of growth at 15 mN/mπ > ) the crystals
grow in the β or 'β phase. The top layer is tilted at 43-45o, i.e. the molecules are somewhat more
flat than in the β or 'β phase. At larger growth rates (e.g. the initial stages of growth at
15 mN/mπ > ) crystals are formed with a metastable α - like structure. The transformation of α -
like crystals to a β or 'β -like crystal structure is too slow to be observed. Only the top layer may
relax to an inclined molecular orientation. The same crystal growth processes occur at the mica-air
44
interface of the transferred LB-film as at the air-water interface of the isobarricaly compressed
Langmuir film, though much slower.
The type of crystal growth process proposed here, where the structure of the first layer is
very different from that of the subsequent layers, is known as the Stranski - Krastanov growth
mode.
3.5.2 Parameters and measurable variables
At this point we know that the size of the crystals increases with time, the growth being mainly in
lateral directions, and that the growth rate increases with surface pressure. The number of crystals
too, increases initially with time and with increasing surface pressure. In later stages, when a
significant fraction of the monolayer film is covered by crystals, crystals start to coalesce, and their
number decreases again. To interpret these observations more quantitatively we develop a simple
model.
Our model provides us with the dependence on time and on surface pressure of the crystal
density the average crystal area and the fraction (dimensionless) of the film
that is covered by crystals (area of the crystals divided to the total area of the image). At
random nucleation of crystals starts at a rate I . We assume that the crystals have a roughly
cylindrical form, with initial radius and height , and that they grow with rates and in the
lateral and vertical direction respectively. We consider , , , and as time independent
physical parameters that may depend on the spreading pressure of the film. Inspection of the data
suggests that some nucleation of crystals takes place before the sample is removed from the
Langmuir through. This means that a non-zero initial substrate coverage
2( mρ µ − ) )2(a mµ θ
0t =
0R 0h lv vv
I 0R 0h lv vv
( )0tθ = and crystal
density have to be considered. ( 0tρ = )
3.5.3 Avrami - Kolmogorov theory for coverage
In the beginning of the nucleation and growth process the crystals are far enough away from each
other to grow independently. We shall refer to the crystal density and the covered fraction in the
initial stages as “free” values fρ and fθ
45
(7) ( ) ( ) ( ) ( )0
0t
f f ft I t dtρ ρ ρ′ ′= + = +∫ 0 It
( ) ( ) ( ) ( )( ) ( )2 2 2
0 0 0 0 00
13
t
f f l f l lt t I t R v t t dt t I R t v R t vθ θ π θ π ⎛ ⎞⎟′ ′ ′ ⎜≡ + ⋅ + − = + + + ⎟⎜ ⎟⎜⎝ ⎠∫ 2 3t (8)
The first terms in Eq(7) and Eq.(8) describe the effect of crystals that were already present at
the beginning, , of the isobaric compression. Their density does not change in time, but their
coverage grows according to
0t =
( ) ( )( )( )
2
0
00
0f
f ff
tθ
θ ρ ππρ
⎛ ⎞⎟⎜ ⎟⎜= ⎟⎜ ⎟⎜ ⎟⎜⎝ ⎠lv t+
)
(9)
This expression can be found by using that the average area of the crystals at
equals
( 20R tπ = 0t =
( ) ( )0 / 0f fϑ ρ .
In the later stages of film growth, the actual values of and will be smaller than
the free values,
( )tρ ( )tθ
( ) ( )ft tρ ρ< and . The fact that crystals can only grow and nucleate in
the uncovered film area between the already existing crystals, is captured by the Avrami -
Kolmogorov theory, leading to
( ) ( )ftθ θ< t
)tθ (10) ( ) ( )(1 exp ftθ = − −
This expression gives us the actual coverage of the film by crystals. It depends however, on
too many physically important parameters to hope that all these parameters can be derived from
observed curves alone. Therefore we want to use observed crystal sizes and crystal densities
as well. What we need is an expression for the number
( )tθ
( )c cN N t= of free crystals that have
merged to form one actual crystal. Then the experimental crystal density and crystal size are found
from
/f cNρ ρ= (11)
(12) / /c fa Nθ ρ θ ρ= =
Unfortunately, no general theory to obtain is available. In the next section we develop an
approach to the problem.
cN
46
3.5.4 Approximate theory for average crystal size and density
In the spirit of the Avrami-Kolmogorov theory the first step is to consider the growing film as if
circular crystals nucleate and grow independently. For the free crystal coverage and density we use
Eqs.(7) and (8). The average area fa of freely growing crystals is not simply the area ( )20 lR v tπ +
of crystals that nucleate at time . Crystals that nucleate later have a smaller area at time .
Taking this into account we obtain
0t = t
( ) ( ) ( ) 20 0
1/3f f f l la t t t R R v t v tθ ρ π⎛ ⎟⎜= = + + ⎟⎜ ⎟⎜⎝
2 2⎞⎠ (13)
( ) 20 0
13
ff
aR t R R v t v t
π≡ = + + 2 2
l l (14)
for the average area fa and radius fR of freely growing crystals.
In the next step we take the merging and overlapping of these free crystals into account.
Two crystals merge to form one new crystal if they are located close enough together. Two circular
crystals with radius fR touch each other, and will probably coalesce, if their (centre-to-centre)
distance is 2 fR or less. Generally, we assume that two crystals merge if one is located within the
merging region A of the other. The area of is written as + A+ 2fa η+ = a , with . 2η ≈
The key idea is to define as the density of original crystals that are, directly or
indirectly, connected to a original crystal in the origin. This density satisfies
( )cρ r
( ) ( )( )01c f Pρ ρ= −r r (15)
Here ( is the probability that an original crystal at r is not connected to the crystal in
the origin. Let be the merging region of a crystal at . All original crystals in the merging
region
)0P r
( )A+ r r
( )0A+ of the central original crystal are connected to this crystal, hence ( )c fρ ρ=r for
inside . Further away the probability of a given crystal at r to be consider to the central crystal
is equal to the probability to find at least one connected crystal in its merging region .
into the same direction). In the trident conformation the hydrophilic glycerol group is in
contact with the water or the mica surface, and the hydrophobic chains point into the air or the
oil [18-22].
In previous work [Chapter 4] we investigated monolayers of tristearin (SSS, chain
length 18 C atoms), tripalmitin (PPP chain length 16 C atoms) and triarachidin (AAA chain
length 20 C atoms), at air-water interface (Langmuir film) and on solid surface like mica
(Langmuir- Blodgett film). We established the relation between their molecular structure and
their monolayer stability. We found that the trident monolayer is the less mobile and the
crystal phase is the more stable the longer the acyl chains are. Using AFM carefully the
thickness of the trident monolayers was measured. It is 1.49 nm for PPP, 1.75 nm for SSS and
2.2 nm for AAA, corresponding to tilt angles of the molecules of 46o, 49o and 59o
respectively.
The aim of the work, presented in this chapter is to understand the phase behavior of
binary mixed TAGs: PPP-SSS, PPP-AAA and SSS-AAA at air-water interface (Langmuir
film) and on solid surface like mica (Langmuir-Blodgett film). We measured the Aπ −
(spreading pressure π vs area per molecule A ) diagram of Langmuir films. Starting with a
Langmuir film at very small π , where the film is in a low-density “gas” phase, we
compressed the film, at a constant rate, to the desired pressure π (forced compression). The
Langmuir film was transferred to mica directly after forced compression ( ) and
investigated with AFM immediately.
0t =
5.2. Materials and methods
5.2.1. Chemicals
Film material: In our experiments we used saturated monoacid triglycerides (their three acyl
chains are the same). Tripalmitin (1, 2, 3-Propanetriyl trihexadecanoate: PPP, chain length 16
86
C atoms), Tristearin (1, 2, 3, -trioctadecanoylglycerol: SSS, chain length 18 C atoms) and
Triarachidin (trieicosonoin: AAA, chain length 20 C atoms) were purchased from Larodan
(Sweden) with a stated purity of >99 mass %. Stock solutions of PPP, SSS and AAA with
concentration of 1 mM in distilled chloroform were prepared. The stock solutions were mixed
in ratios 1:1, 1:3 and 3:1.
Subphase: Distilled water was used as a subphase in our Langmuir system for all
experiments. The resistivity of the water was 15 MOhm cm.
Substrates: All monolayers were transferred onto freshly cleaved mica.
5.2.2. Langmuir method
Compression isotherms were measured on a commercial, fully automated Langmuir Blodgett
Trough (model: 311D, Nima Technology Ltd., England). The instrument was equipped with a
Teflon trough (283.0 cm2) and one Delrin barrier. The spreading pressure π was measured
with an accuracy of about 0.1 mN/m. The film material was initially spread on the water
subphase, dropping 30 µL of 1 mM stock solution dissolved in chloroform, using a 100 µL
Hamilton syringe. The conditions were chosen such that initially the average area A per
molecule is . We started (asymmetric) film compression 2 min after spreading. In
our system we used the forced compression operation mode, where the position of the barrier,
and hence the trough length ahead of the barrier, is given. Then the resulting spreading
pressure
2110 ÅA ∼
( )l t
( )tπ is registered. In this mode we chose barrier velocities of the order of 1 cm/min,
which according to the literature should be slow enough that the Langmuir film stays close to
thermodynamic equilibrium.
5.2.3. Langmuir-Blodgett film transfer
In order to obtain LB films, first a substrate was immersed perpendicularly in the aqueous
subphase. We started with a very small initial surface pressure ( 0π = mN/m), and
compressed the monolayer slowly (1 cm/min) to the final pressure. To obtain a LB film that is
characteristic for forced compression, the film was transferred immediately by vertical pulling
of the substrate through the air-water interface at a speed of 2 mm/min. During the transfer the
surface pressure was kept constant by appropriately moving the barrier. The transfer process
87
takes a few minutes. After deposition the LB-films were dried in air and kept in closed
containers until use. All experiments were done at 20 ± 1°C.
5.2.4. AFM measurements
The samples were examined with AFM immediately after preparation. Imaging was done with
a Nanoscope (R) IIIa (Digital Instruments, Santa Barbara, CA) in contact mode with oxide-
sharpened silicon nitride tip (k = 0.06 N/m). The AFM was equipped with a J scanner
(176 x176 µm; z limit = 5.349 µm). All images were processed using procedures for
flattening in Nanoscope III software version 5.12r5 without any filtering. To check if the
monolayer is successfully transferred to the mica surface we measured at least five different
spots (each 150 µm 2) of every sample.
5.3. Langmuir observations
A
0
5
10
15
20
25
30
35
40
50 60 70 80 90
Area/molecule A (A2)
Sur
face
pre
ssur
e (m
N/m
)
PPPSSSPPP-SSS (1:1)
88
B
0
5
10
15
20
25
30
35
40
50 60 70 80 90Area/molecule A (A2)
Sur
face
pre
ssur
e (m
N/m
)
PPPAAAPPP-AAA (1:1)
C
0
5
10
15
20
25
30
35
40
50 60 70 80 90
Area/molecule A ( A2)
Sur
face
pre
ssur
e (m
N/m
)
SSSAAASSS-AAA (1:1)
Fig.1. Surface pressure vs area isotherms of tripalmitin (PPP), tristearin (SSS) and
triarachidin (AAA) and their mixtures at air-water interface, at 20oC, obtained by forced
compression at a rate of 1cm/min.
In the previous Chapter 4 we already discussed the shape of the typical Aπ −
isotherms of PPP, SSS and AAA, where two different regimes can be recognized for the three
triglycerides. The condensation area and condensation pressure condA condπ have been
described as values at which the transfer from “gaseous” to “condensed” phase occur. The
collapse pressure colπ is the surface pressure at which the monolayer collapses to form
multilayer structures. For the studied triglycerides it was in the range of 40 48 mN/mπ = −
and it increased in order: (AAA) (SSS) (PPP)col col colπ π π< < . With our LB instrument the
collapse pressure was difficult to reproduce because of details in its construction. For the
mixtures we measured similar
colπ
Aπ − isotherms as for the single components (Fig.1). The
89
measured Aπ − data for the mixtures showed that the pressure range, where the transition
from one regime to another takes place, was rather wide. For the mixtures the adsorption
isotherm was always between the isotherms of the single components. An exception was the
mixture SSS-AAA, for which the Aπ − isotherm sometimes almost coincided with the
isotherm of AAA (Fig.1C). In order to get reliable and unbiased estimations for and condA
condπ , we fitted the isotherms with:
( ) ( , )condA ch A A aπ ≈ − (1)
where , a , and h are fitting parameters. The function condA c
( )2 21( , )2
h x a x x a≡ − + (2)
is a hyperbola interpolating between for large negative x and for
large positive x . This function has no direct physical interpretation and was introduced for
practical purposes only, i.e. to arrive at an unambiguous definition and evaluation of
( ),h x a x≈ ( ), 0h x a ≈
/ 2cond caπ = and . Fitting a number of isotherms (15) that were obtained at compression
velocity 1cm/min we found and
condA
264 1 ÅcondA = ± 9 3 mN/mcondπ = ± for SSS-AAA;
and 263 1 ÅcondA = ± 10 3 mN/mcondπ = ± for PPP-SSS and and 263 3 ÅcondA = ±
11 2 mN/mcondπ = ± for PPP-AAA. Together with the corresponding data for the pure PPP,
SSS and AAA systems, these results are presented in Figure 2.
A Condensation area A cond
56
58
60
62
64
66
68
70
14 16 18 20 22
Average chain lenght (C atoms)
Aco
nd (Ǻ
2 )
90
B Condensation pressure πcond
2
4
6
8
10
12
14
14 16 18 20 22
Average chain lenght (C atoms)
Con
dens
atio
n pr
essu
re (m
N/m
)
C Fitting parameter a
0
2
4
6
8
14 16 18 20 22
Average chain lenght (C atoms)
a
Fig.2. Condensation area (A), condensation pressure condA condπ (B) and fitting parameter
(C) for triglycerides (▲) and their mixtures (■). X axis presents the number of the carbon
atoms in the triglyceride chains: PPP (16), SSS (18) and AAA (20). For the mixtures it was
calculated as follow: 17 = PPP-SSS (1:1), 18 = PPP-AAA (1:1) and 19 = SSS-AAA (1:1).
a
The fact that is around for all studied triglycerides and their mixtures is
consistent with a trident conformation of triglyceride molecules in a monolayer film at the air-
water interface. Indeed, the cross-sectional area per hydrocarbon chain for tristearin at 20
condA 263 Å
oC in
the α phase (the α phase has the most mobile acyl chains) is [23]. 219.7Å
The fact that condπ is almost the same for the investigated pure triglycerides and their
mixtures as well (8 1 ), is consistent with the idea that the packing properties of the
hydrocarbon chains is mainly determined by short range repulsive interactions. The effective
repulsion is quite independent of the chain length and compositions, which shows that mixing
of triglycerides does not change their packing properties drastically. The tendency of
0 mN/m−
condA
91
and condπ to increase slightly with increasing chain length reflects a slightly enhanced
repulsion of longer chains.
The value of the fitting parameter (Eq.1) describes the sharpness of the gas -
condensed transition and is found to depend strongly on the chain length ( the smaller , the
sharper is the transition). This is also seen in Fig.1 where the
a
a
Aπ − isotherm for PPP is
sharper than those for SSS and AAA. This observation can be understood if one realizes that
the shorter PPP molecules are stiffer than the longer SSS and AAA. The longer chains will
spread somewhat more in lateral direction. The isotherms in Fig.1 suggest that in a
moderately dense packed monolayer at the air-water interface the longer triglycerides interact
already at significantly larger intermolecular distances than the shorter ones. The fitting
parameter is rapidly increasing with increasing chain length. Apparently the presence of
PPP in a mixture reduces the hindering of the motion of the longer molecules and thus
sharpens the transition from the gas to the condensed phase.
a
In general the Aπ − isotherms of the mixtures interpolate linearly between the
isotherms of the pure components. E.g. the isotherm of the PPP-AAA mixture (average chain
length 18) is very similar to the isotherm of pure SSS (chain length 18). Thus from the Aπ −
isotherms alone one would be tempted to conclude that the triglycerides mix (almost) ideally.
In the remains of this chapter, we show that this conclusion is incorrect. We investigated
Langmuir – Blodgett monolayers of the mixtures with AFM. Our experimental results clearly
show non-ideal behaviour, and even phase separation.
5.4. AFM observations
To investigate the structure of the three mixtures we withdrew Langmuir monolayers
immediately after forced compression to 20 mN/mπ = .We chose this surface pressure
because is in the middle of the condensed region of the Aπ − isotherms. We know from the
Aπ − isotherms that it is well above the condensation pressure condπ , but still below the
collapse pressure colπ .
5.4.1. PPP-SSS structure
92
0 2.5
0
5.0
5.0
-5.0
µm
1.7 nm 1.6nm
DA
C
0 1.0-1.5
01.
5
0.21 nm 0.25 nm
µm
F
2.0
B
0 2.5
2.5
-2.5
0
µm
1.69 nm3.52 nm
E
5.0
0 2.5
0
5.0
5.0
-5.0
µm
1.7 nm 1.6nm
D
0 2.5
0
5.0
5.0
-5.0
µm
1.7 nm 1.6nm
DAA
CC
0 1.0-1.5
01.
5
0.21 nm 0.25 nm
µm
F
2.00 1.0-1.5
01.
5
0.21 nm 0.25 nm
µm
F
2.0
BBB
0 2.5
2.5
-2.5
0
µm
1.69 nm3.52 nm
E
5.00 2.5
2.5
-2.5
0
µm
1.69 nm3.52 nm
E
5.0
Fig.3. (A) AFM height image of PPP-SSS monolayer transferred immediately after forced
compression to π = 20 mN/m. The black square is a hole in the monolayer produced by
scanning at a high AFM force (F~30 nN). The monolayer is scanned at AFM force F~1 nN.
(B) another area of the same sample, where the onset of phase separation was observed. (C)
zoomed image of (B). The corresponding cross sections are given in (D, E and F). The scale
bar is 2 µm for (A and B) and 1 µm for (C). Height differences are given by the numbers at
the markers.
93
The AFM images of PPP-SSS (1:1) showed a homogeneous monolayer with thickness
1.6 ± 0.1 nm, i.e. somewhere between the measured thicknesses of PPP and SSS (Fig.3A, D).
The monolayer contains more holes than monolayers of the pure systems, which are almost
defect free. This is the first indication that due to the difference in the chain length of the two
components in the mixture, random packing of longer and shorter molecules is
thermodynamically not optimal. In some regions of the samples the onset of phase separation
was observed (Fig.3B, C). It was difficult to measure directly the height difference by the
AFM. We estimated a height difference of 0.2 ± 0.1 nm (Fig.3C, F). The carbon chain length
of PPP and SSS differs by 2 carbon atoms, which is ~ 0.5 nm ( sc = 0.254) [24]. The thickness
of the monolayers, which we obtained by extrapolation to zero scanning force, is = 1.49
nm
0d for PPP, = 1.75 nm for SSS and = 2.19 nm for AAA. These monolayer thicknesses
correspond to tilt angles of the molecules of 46
0d 0do, 49o and 59o respectively [Chapter 4]. The
measured height difference of 0.2 ± 0.1 nm in the mixture PPP-SSS (1:1) is slightly below the
expected height difference of ~ 0.3 nm between tilted PPP and SSS monolayers. A reasonable
explanation of the small height difference is that PPP and SSS are not completely separated.
The fact that most of the AFM images of PPP-SSS (1:1) showed a homogeneous monolayer
supports the idea that PPP and SSS have only a weak tendency to phase separate.
In Chapter 4 we demonstrated that the apparent thickness depends strongly on the
AFM scanning force. Even relatively small scanning forces may compress triglyceride
monolayer. We showed that the compressibility varies little between the investigated pure
triglycerides. To measure the real monolayer thickness of the mixture PPP-SSS (1:1) we
used the same procedure as in Chapter 4. By scanning with a relatively large force
0d
30 nNF ≈ we scratched a rectangular hole in the monolayer with the AFM tip. Then a
larger area, including the hole, was scanned with small forces 1 8 nNF = − (fig.4). The
height difference between the hole and the surrounding film gives an apparent thickness d for each strength of the scanning force . We investigated three different holes in one sample
(Fig.4).
′
F
94
Apparent monolayer thickness of PPP- SSS
0.8
1
1.2
1.4
1.6
1.8
2
0 2 4 6 8 10AFM force F (nN)
AFM
thic
knes
s d'
(nm
)
hole 1hole 2hole 3SSSPPP
Fig.4. Measured layer thickness d for PPP, SSS and PPP-SSS (1:1) as a function of applied
AFM force F at surface pressure
′
20 mN/mπ = .
Real monolayer thickness for PPP-SSS
1.4
1.5
1.6
1.7
1.8
0 50SSS in the mixture (%)
Laye
r thi
ckne
ss d
0 (n
m)
100
Fig.5. Real monolayer thickness for PPP, SSS and PPP-SSS (1:1) for three different holes
at surface pressure
0d
20 mN/mπ = . The values are found by extrapolation of the apparent
monolayer thickness in Fig.4 to AFM force F = 0 nN.
0d
'd
In Fig.4 we see that the dependence of d on the scanning force F for the mixtures of
triglycerides and for the pure phases is very similar. By definition the isothermal
compressibility of 3 dimensional materials is:
′
95
1T
T
VKV P
∂⎛ ⎞= − ⎜ ⎟∂⎝ ⎠ (3)
Analogously the isothermal film compressibility can be defined as
0
0
131
film TT
dKd P
∂⎛ ⎞= − ≈⎜ ⎟∂⎝ ⎠K (4)
where the last approximation is valid if the material properties of the film are the same as of
the bulk material. In our system we measure the AFM force . The pressure in this case
would be
F
/P F contact area= (5)
but unfortunately we cannot accurately estimate the contact area. For practical reasons we
define the quantity as K
0
0
1 dKd F
∂≡ −
∂ (6)
and, somewhat loosely, we shall refer to as film compressibility from now on. K
The film compressibility of the monolayer, given by the slope of curves, is
slightly higher for PPP-SSS (1:1) ( ) than for pure PPP and SSS
( ). The real thickness , corresponding to scanning force is
presented in Fig. 5. As shown in Fig.5 for PPP-SSS (1:1) we found two distinct results,
and . These values are close to
and
'( )d F
-10.08 0.01 nNK = ±
-10.07 0.01 nNK = ± 0d 0F =
0 1.50 0.02 nmd = ± 0 1.69 0.01 nmd = ±
0 (PPP) 1.49 0.02 nmd = ± 0 (SSS) 1.75 0.02 nmd = ± respectively, and we suppose that
they are the thickness of PPP-rich and SSS-rich areas in the monolayer respectively.
We conclude that in the mixture PPP-SSS (1:1) phase separation takes place, which is
not complete. The PPP-rich regions contain dissolved SSS molecules and SSS-rich regions
contain dissolved PPP molecules. As the dissolved molecules will influence the average
thickness it is now clear why the height difference of the domains in Fig.3, as well as the
difference in monolayer thickness does not correspond to the length of 2 carbon atoms but 0d
96
is slightly smaller. Note that we can not completely exclude the possibility that PPP and SSS
are well separated in very small domains, which cannot be detected by the AFM.
Like in our previous investigations for the single components [Chapter 3 and 4] we
found higher domains on top of the PPP-SSS monolayer. Most of them had a thickness of 3.5
± 0.1 nm (Fig.3 B and E). This corresponds to molecules of PPP or SSS in tuning fork
conformation. Similar domains were formed when a Langmuir monolayer of the single
component was transferred immediately at 20 mN/mπ = (3.3 ± 0.1 nm for PPP and 3.5 ± 0.1
nm for SSS) [Chapter 4]. The composition of the crystals on top of the PPP-SSS monolayer is
not clear. They could contain either PPP or SSS or both types of molecules.
5.4.2. SSS-AAA structure
A
0 2.5
5.0
-5.0
0
µm
1.93 nm 1.86 nm
B
5.0
AA
0 2.5
5.0
-5.0
0
µm
1.93 nm 1.86 nm
B
5.00 2.5
5.0
-5.0
0
µm
1.93 nm 1.86 nm
5.00 2.5
5.0
-5.0
0
µm
1.93 nm 1.86 nm
B
5.0
Fig.6. (A) AFM height image of SSS-AAA (1:1) monolayer transferred immediately after
forced compression to 20 mN/mπ = and the corresponding cross section in (B). The black
square is a hole in the monolayer produced by scanning at a high AFM force (F~30 nN). The
monolayer is scanned at AFM force F~1 nN. The scale bar is 2 µm and the vertical scale is
10 nm. Height differences are given by the numbers at the markers.
The AFM images of SSS-AAA (1:1) showed a homogeneous monolayer (Fig.6). To
measure the thickness of the monolayer we used the same procedure as described in Section
5.4.1. In order to get reliable and unbiased estimations for this procedure was repeated for
3 independent holes in one sample (Fig.7). For SSS-AAA (1:1) we found
by extrapolation of the apparent monolayer thickness d in Fig.7 to AFM force .
0d
0 1.95 0.02 nmd = ±
′ 0 nNF =
97
The film compressibility of the mixture monolayer was very close to
that of the pure monolayers ( ). We did not observe any crystals on top of
the monolayer. Contrary to PPP-AAA we saw no indications for phase separation. No
domains were observed. This suggests the absence of phase separation in PPP-AAA. To
investigate this further we also studied (1:3) and (3:1) mixtures. The same absence of domains
was observed for 1:3 and 3:1 mixtures (data not shown). The monolayer thickness
depended linear on composition (Fig.8). The film compressibility was the same for all SSS-
AAA mixtures. All these observations support that PPP-AAA forms (almost) ideal mixtures.
-10.08 0.01 nNK = ±
-10.07 0.01 nNK = ±
0d
Apparent monolayer thickness for SSS - AAA (1:1)
1
1.2
1.4
1.6
1.8
2
2.2
2.4
0 2 4 6 8 10AFM force F (nN)
AFM
thic
knes
s d'
(nm
)
hole1
hole 2
hole 3
SSS
AAA
Fig.7. Measured layer thickness ' for SSS, AAA and SSS-AAA (1:1) as a function of applied
AFM force at surface pressure
d
F 20 mN/mπ = .
Real monolayer thickness of SSS-AAA
1
1.2
1.4
1.6
1.8
2
2.2
2.4
0 25 50 75 100
AAA in the mixture (%)
Laye
r thi
ckne
ss d
0 (nm
)
98
Fig.8. Real monolayer thickness for SSS-AAA at surface pressure 0d 20 mN/mπ = as a
function of the mole fraction of AAA in the mixture. The values are found by extrapolation of
the apparent monolayer thickness to AFM force F = 0 nN. 'd
5.4.3. PPP-AAA structure
In some cases AFM images of LB-films of PPP-AAA (1:1) show areas where phase
separation is hardly visible (Fig.9A, B and C) as in the SSS-PPP (1:1) mixture. There are
other areas however, with very well separated domains (Fig.9D, E and F). To measure
monolayer thicknesses for different domains, we used the same procedure as described in
section 5.4.1. We measure the monolayer thickness in different areas independently. As
before we corrected for the compression of the AFM at low scanning force (Fig.10).
0 2.5
5.0
-5.0
0
µm
1.72 nm1.65 nm
C
5.0
BA
D E
0 2.5-5.0
5.0
0
µm
1.98 nm1.49 nm
F
5.0
0 2.5
5.0
-5.0
0
µm
1.72 nm1.65 nm
C
5.00 2.5
5.0
-5.0
0
µm
1.72 nm1.65 nm
C
5.0
BBAA
DD EE
0 2.5-5.0
5.0
0
µm
1.98 nm1.49 nm
F
5.00 2.5-5.0
5.0
0
µm
1.98 nm1.49 nm
5.00 2.5-5.0
5.0
0
µm
1.98 nm1.49 nm
F
5.0
Fig.9. (A and B) AFM height images of PPP-AAA (1:1) monolayer transferred immediately
after forced compression to 20 mN/mπ = with little indication of phase separation. The
corresponding cross section is given in (C). (D and E) present areas of the same sample,
99
where phase separation is clearly visible. The corresponding cross section is given in (F).
Black squares are holes in the monolayer produced by scanning at a high AFM force (F~30
nN). The monolayers are imaged at AFM force F~1nN. The scale bar is 2 µm and the vertical
scale is 10 nm for all images. Height differences are given by the numbers at the markers.
The thickness of the PPP-AAA (1:1) monolayer, at positions where the phase
separation is not obvious, is , i.e. between the monolayer thicknesses of
the single components. The film compressibility at such positions is
larger than for PPP and AAA separately ( ).
0 1.86 0.05 nmd = ±
-10.11 0.02 nNK = ±
-10.07 0.01 nNK = ±
Apparent monolayer thickness for PPP-AAA
0.5
1
1.5
2
2.5
0 2 4 6 8AFM Force F (nN)
AFM
thic
knes
s d'
(nm
)
AAA-rich domain
AAA-pure
PPP-rich domain
PPP pure
PPP-AAA (1:1)
Fig.10. Measured layer thickness for PPP, AAA from the monolayers of the pure
components and in the mixture PPP-AAA (1:1) as a function of applied AFM force F at
surface pressure
'd
20 mN/mπ = .
At positions with clear phase separation the two different domains had thicknesses
0 1.42 0.01 nmd = ± and (Fig.9E, F). These heights are close to the height