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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;
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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.
2
DetectorElectronics
AB
SplitPhotodiodeDetector
X ,Y
Z
Sample
Cantilever & Tip
Scanner
Laser
ControllerElectronics
Feedback Loop MaintainsConstant Cantilever Deflection
Measures
A + Bof deflection signal
A - B
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
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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
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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
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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].
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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)
[4] Bigerow, W.C., Pickett, D.L., Zisman, W.A., J. Colloid Interface Sci. 1(1946) 513
[5] Zisman, W.A., Adv. Chem. Ser. 1 (1964) 43
[6] Ulman, A., An introduction to ultrathin organic films, Academic Press, London, (1991)
[7] Binning, G. and Rohrer, H., Helv.Phys. Acta 55 (1982) 726
[8] Binning, G., Quate, C.F. and Gerber, C., Phys. Rev. Lett. 56 (1986) 930
12
[9] TappingModeTm is patented by Digital Instruments, Santa Barbara CA, USA
[10] Radmacher, M., Tillman, R.W., Fritz, M . and Gaub, H.E., , Sciences 257 (1992) 409
[11] Rinia, H.A., Atomic force microscopy on domains in biological model membranes, Ph.D.
Thesis (2001), Utrecht University, The Netherlands
[12] Merkel, R. Phys. Rep. 346 (2001) 343-385
[13] Maeda, N., Senden, T.J., and Di Meglio, J.M., Micromanipulation of phospholipids bilayers by
AFM, Biochem. Biophys. Acta 1564 (2002) 165-172
[14] Ganchev, Dragomir N., Rijkers, D.T.S., Snel, M.M.E., Killian, J.A. and de Kruijff,
B.,Biochemistry 43(2004) 14987-14993
[15] Garti, N., Sato, K., In Crystallization and polymorphism of fats and Fatty Acids; Dekker, M.
New York (USA) 1988
[16] Bursh, T., Larsson, K., Chem. Phys. Lipids 2 (1968) 102-113
[17] Hamilton, J. A., Small, D.M., In Proc. Nat. Acad. Sci. USA 78 (1981) 6878
[18] Blodgett, K. B., Monomolecular films of fatty acids on glass, J. Am. Chem. Soc.,56 (1934) 495
[19] Gaines G.L., Insoluble monolayers at liquid gas interfaces, Wiley, New York, (1966)
[20] Petty, M.C., Langmuir-Blodgett films: an introduction, Cambridge University Press, 1996
[21] Lundquist, M., In Surface chemistry, Copenhagen. Munksgaard (1966) p.294
[22] Ekwall, P., Ekholm, R. and Norman, A., Acta Chem. Scand. 11 (1957) 703
[23] Chen X., et al., J.Phys. Chem. B 109 (2005) 19866-19875
[24] Chi, L.F. et al, Langmuir 8 (1992) 2255-61
[25] Flörsheimer, M., et al, Thin Solid Films 244 (1994) 1078-82
[26] Zasadzinski, J.A. et al., Science 263 (1994) 1726
[27] Sparr. E., Langmuir 17 (2001) 164-172
[28] Porter, M.D., Bright, T. B., Allara, D.L., Chidsey, C.F.D., J. Am. Chem. Soc. 109 (1987) 3559
[29] Fuente, J.F. and Rodriguez Patino, J.M. Langmuir, 10 (1994) 2317-2324
[30] Sanchez, C.C., Rodriguez Nino, M., Rodriguez Patino, J.M., Colloids and Surfaces B:
Biointerfaces 12 (1999) 175-192
[31] Chapman, D., Chem Rev 62 (1962) 433
[32] Hamawan, C., Starov, V.M., Stapey, A., Advances in Colloid and Interface Science 122 (2006)
3-33
[33] De Jong, S., PhD thesis (1980) University of Utrecht, The Netherlands.
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14
CHAPTER 2
Phase behaviour in supported mixed monolayers of alkanols, investigated by Atomic Force Microscopy
Abstract
The structure of several mixed Langmuir-Blodgett monolayers of fatty alcohols, CnH2n+1OH
with even n = 16-24, was investigated by AFM at 20-22°C. Phase separation was found for
compressed films, if the chain length of the two components differed at least with six carbon atoms.
A strong dependence of the domain shape on the surface pressure was observed. The excess Gibbs
energy vs. surface pressure and mole fraction was calculated from π-A isotherms. In line with
thermodynamic expectation, the tendency of phase separation increased with increasing . A
surprising and as yet unexplained result was that we sometimes observed clear phase separation
already in the range
exG∆exG∆
0.1exG R∆ ≅ T
15
2.1. Introduction
Aliphatic long-chain alcohols Cn (CnH2n+1OH with n = 16–31) can be adsorbed on water surface.
Interestingly, adsorbed Cn turns out to enhance ice nucleation. Grazing-incidence X-ray diffraction
(GID) studies of Cn monolayers on water at 5°C revealed two-dimensional structure formation.
Wang et al. [1] concluded that the molecules in the Cn monolayers adopt a herringbone pattern.
According to the GID data, monolayers with n = 16 and 20 contain less crystalline material than
monolayers with n = 23, 30, 31 [2]. IR spectra of the same alcohol monolayers at an area per
molecule of 20 Å2 have been measured at the air/water interface at 20°C. These measurements also
showed that the hydrocarbon chains become more ordered with increasing length. It was found that
only alcohols with molecular areas of 18.5-20 Å2 significantly enhance nucleation of ice [2].
Combining these two types of experiments we expect that 2D layers of long alcohols (n > 20)
crystallize, when the molecular area is about 20 Å2. Kulkarni et al. [3] investigated mixed
monolayers of C16 and C22 at 25°C, studying surface viscosity and the area per molecule. Isotherms
of the system at five different mole fractions showed that all mixtures were thermodynamically non-
ideal.
In order to better understand mixed monolayers and to study the effect of chain length on
mixing, we investigated six mixed monolayer films thermodynamically and with AFM: C16:C22
with stoichiometry 1:1, 1:3 and 3:1, C18:C22 (1:1), C18:C24 (1:1) and C16:C24 (1:1). We used
Langmuir-Blodgett technique to transfer at several surface pressures binary mixed monolayers from
the water/air interface onto a mica substrate. Equilibrium layers were obtained by using a very small
initial surface pressure (π = 0 mN/m) of the Langmuir layer, and compressing slowly to the final
pressure.
2.2. Materials and methods
2.2.1. Chemicals:
Film material: Fatty alcohols (CnH2n+1OH, with n = 16, 18, 22, 24) were obtained from Merck and
used without further purification. Separate stock solutions of each alcohol with concentration of
5 mM in distilled chloroform were prepared. Solutions containing 1 mM mixtures of the alcohols in
mole ratios 1:1, 1:3 and 3:1 were prepared by proper mixing and diluting of stock solutions.
16
Subphase: MiliQ water was used as a subphase in our Langmuir system for all experiments. The
resistivity of the water is 18 MOhm*cm.
Substrates: All monolayers were transferred onto freshly cleaved mica.
2.2.2. π - A isotherms
Compression isotherms were measured on a Teflon trough (17.2×35.7 cm). The spreading pressure
π was measured with a Wilhelmy type balance consisting of a platinum plate coupled to an
electrobalance (Cahn Ankersmit 2000), with an accuracy of about 0.1 mN/m. The film material was
spread on the water subphase, using a 100 µL Hamilton syringe. The area per molecule A was
controlled by a moving barrier, at an accuracy of 1-2 Å2 per molecule. Spreading took place at
Å100A ≈ 2. Film compression started almost immediately after spreading, at a rate of 1 cm/min.
2.2.3. Langmuir-Blodgett film transfer
In order to obtain LB films, first a substrate was immersed perpendicularly in the aqueous subphase.
Equilibrium layers were obtained by using a very small initial surface pressur 0e (π = mN/m) of
the monolayer, and compressing slowly (1 cm/min) to the final pressure. Film transfer was then
accomplished by vertically lifting the substrate through the air-water interface at a speed of
2 mm/min. After deposition the monolayers were dried in air and kept in close containers until use.
All experiments were done at 20-22°C.
2.2.4. AFM measurements
The samples were examined with AFM within about 5 hours after preparation. Imaging was done
with a Nanoscope III (Digital Instruments) in contact mode with oxide-sharpened silicon nitride tip
(k = 0.06 N/m). The AFM was equipped with E scanner.
2.3. AFM Observations
2.3.1. C16:C22
17
A B
C D
E F
µm
1.038 nm 0.983 nm
0 1.25 2.5
02.
0-2
.0
AA BB
CC DD
EE F
µm
1.038 nm 0.983 nm
0 1.25 2.5
02.
0-2
.0
F
µm
1.038 nm 0.983 nm
0 1.25 2.5
02.
0-2
.0
Fig.1. AFM height image showing C16:C22 (1:1) mixed monolayers transferred at surface pressure
(A) π = 10 mN/m, (B) π = 15 mN/m, (C) π = 20 mN/m and (D) π = 35 mN/m. In panel E, an
enlarged height image is given, showing the tetragonal shape of C22 domains with corresponding
cross section in (F). The height difference between both alcohols is given by the vertical distance
between the markers. The scale bar is 1µm and the vertical scale is 4 nm for all images.
18
The AFM images in Fig.1 clearly show phase separation at surface pressures π ≥ 10 mN/m.
The thicker domains presumably mainly consist of C22, the thinner mainly of C16. The thickness of
the C22 domains and of the surrounding C16 film was found to be less than the thickness calculated
from X-ray data of crystals with vertically extended alcohols. The measured values are 1.0 nm (1.87
nm in crystals) for C16 and 2.0 nm (2.51 nm in crystals) for C22. This effect was observed before and
explained as monolayer depression, caused by the AFM tip [4]. Fig.1 F shows the height difference
between C16 and C22, to be 0.9 - 1.0 nm.
Phase separation in domains with the same height difference was found for C16:C22 mixtures with
(1:3) and (3:1) stoichiometry (data not shown).
2.3.2. C18:C22
The AFM images of this system showed a homogeneous monolayer at all surface pressures at
which the monolayer was compressed (π = 10, 20 and 35 mN/m). The measured thickness of the
monolayer was ~ 2.1 nm at π = 35 mN/m, as for C22.
2.3.3. C18:C24
This mixture with 6 carbon atoms length difference behaved similar to C16:C22. The AFM images
showed phase separation with C24 domains embedded in C18. At π = 35 mN/m the domains have
tetragonal shapes and they are more ordered than in the C16:C22 mixture. The measured thickness
for C18 is ~1.6 nm (2.09 nm in crystals) and for C24 it is 2.2~2.3 nm (2.7 nm in crystals) (Fig.2 A,C).
2.3.4. C16:C24
In this mixture we observed different C24 domain shapes as in other mixtures, they were very
irregular, at all final surface pressures. The height difference between the C24 domains and the C16
film is 1.1~1.2 nm (Fig.2 B, D).
19
B
D
0 1.25 2.5
0
-2.0
2.0
µm
1.198 nm 1.144 nm
A
C0.600 nm
0 1.25 2.5µm
0
2.0
-2.0
0.600 nm
BB
D
0 1.25 2.5
0
-2.0
2.0
µm
1.198 nm 1.144 nm
D
0 1.25 2.5
0
-2.0
2.0
µm
1.198 nm 1.144 nm
0 1.25 2.5
0
-2.0
2.0
µm
1.198 nm 1.144 nm1.198 nm 1.144 nm
A
C0.600 nm
0 1.25 2.5µm
0
2.0
-2.0
0.600 nm
C0.600 nm
0 1.25 2.5µm
0
2.0
-2.0
0.600 nm0.600 nm
0 1.25 2.5µm
0
2.0
-2.0
0.600 nm
Fig.2. AFM height image showing mixed monolayers transferred at surface pressure π = 35 mN/m.
(A ) C18:C24 and (B) C16:C24 (A ) with the corresponding cross sections in (C and D). The scale bar
is 1µm and the vertical scale is 4 nm for all images. The black lines show the area in the image,
where the cross section was taken.
2.4. Thermodynamics
In order to interpret the observed structures of mixed alcohol films, we introduce
thermodynamic information. The films were formed at the same temperature T 294 K, hence we
drop the temperature from the formulation. At given spreading pressure
≈
π and mole fraction the
structure with the lowest possible Gibbs energy (in J/mol) will be formed. Let be the Gibbs
energy for a homogenous, uniform film. If
x
G homG
( ),homG x π is a concave function of then a
homogeneous film is thermodynamically stable and . If
x
homG G= ( ),homG x π has a convex part then
a homogeneous film is unstable for a composition interval ( )0 1,x x x∈ that includes the convex part.
A homogeneous film with ( )0 1,x x x∈ can decrease its Gibbs energy by phase separating in
20
fractions ( ) ( )0 1 0/x x x x x= − − and ( ) ( )1 11 / 0x x x x x− = − − with composition 1x and
0x respectively:
( ) ( ) ( ) ( ) ( ) ( )0 1 0 1, , 1 , , hom ,x x x G x x G x xG x G xπ π π∈ → = − + < π (1)
The points ( )0 0x x π= and ( )1 1x x π= are the common tangent points to . A completely
immiscible film separates in pure phases, i.e.
homG
0 0x = , 1 1x = and hence x x= .
The Gibbs energy per mol can be determined, using ( )/x
G Aπ∂ ∂ = from Aπ − diagrams
(2) ( ) ( ) ( ) ( ) ( )( )A
G G A d A RT A dπ
π π
π π π π π π π∞
∞
∞ ′ ′− = ≈ − −∫ A∫
where π ∞ is the reference spreading pressure, which is chosen small enough that the film is
thermodynamically ideal at π ∞ . The Gibbs energy is split into an ideal and an excess part:
( ) ( ) ( ), ,id exG x G x G xπ π= + ,π (3)
( ) ( ) ( ) ( ) ( ) ( ) ( )0 1,1 ln 1
idG x G Gln 1x x x x x x
RT RT RTπ π π
= − + − − − − (4)
The Gibbs energy of mixing is defined as
( ) ( ) ( ) ( ) ( )0 1, , 1mixG x G x x G xGπ π π= − − − π (5)
Since , we get ( ) 0exG π ∞ = ( ),exG x π of a mixture using the right hand side of Eq. (2) for the mixed
and pure films and substituting in Eq. (5). By definition a mixture is non-ideal if ( ), 0exG x π ≠ .
Phase separation only occurs in non-ideal mixtures. Using Eqs. (3)-(5) it is seen that for ideal
mixtures the so-called additivity rule
( ) ( ) ( ) ( )0 1, 1A x x A xAπ π= − + π (6)
holds [5]. The reverse is not true. Indeed, Eq. (6) holds for completely immiscible films as well. It is
often believed that ( ),exG x π > 0 is necessary for phase separation to occur. In the most common
case where phase separation is driven by energetically unfavourable mixing, i.e. for all
,
, 0hom exG ≥
x ( ),exG x π > 0 indeed. But phase separation may occur also if energetical or entropical reasons
favour incorporation of a small fraction of the other component in a pure phase. Then may
have negative minima near
,hom exG
0x and 1x , and a maximum in between. This can cause phase separation
with ( ), 0exG x π < .
21
From the discussion so far it is clear that we need to measure ( ),exG x π as accurately as
possible. At the low reference pressure 0π the mixed film is ideal. We assume that upon decreasing
the film area, it stays ideal down to the molar area ( )A A x∗= where π starts to increase. The
results are given in Figure 3.
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0 5 10 15 20 25 30 35 40 45
surface pressure π (mN/m)
Gex
/RT
C16/C22(1:1)C16/C24C18/C22C18/C24C16/22(1:3)C16/22(3:1)
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 .
Therefore
r
(0)A+
( )A+ r
( )cρ r satisfies the implicit equation
(16) ( )
( )
( )( )
( )2
0
1 exp 0
f
cf c
A r
for r A
rr d r for r A
ρ
ρρ ρ
+
+
+
⎧ ∈⎪⎪⎪⎪⎪ ⎡ ⎤⎛ ⎞= ⎨ ⎟⎜⎢ ⎥⎪ ⎟′ ′⎜− − ∉⎟⎪ ⎢ ⎥⎜ ⎟⎪ ⎜ ⎟⎢ ⎥⎝ ⎠⎪ ⎣ ⎦⎪⎩∫
47
The latter expression derives from to the case of the probability 0N =
1 ( ) exp( )!
NNP a
Naρ ρ= − (17)
to find N objects within an area a ,when the average density of these objects is ρ .
Eq.(16) is an integral equation for , which can not be solved exactly. We make the
following observations. If the free crystal density
( )cρ r
fρ is so high that the summed merging area is
larger than the total film area, , then far enough outside 2 1f fa ρ η θ+ = > ( )0A+ a constant solution
(18) ( ) ((1 expc c f aρ ρ ρ ρ+= = − −r ))cexists. In this case merging of the original crystals leads to an infinite crystal.
For smaller free crystal densities fρ , the connected crystal density decays to 0 with
increasing distance from the central crystal. Assuming that varies slowly over the circular
region of radius
( )cρ r
( )cρ r
( )A+ r fRη and area 2 2fa πη+ = R , we have for the integrand in Eq.(16)
( )( )
( ) ( ) ( )22 21 .......
8c c cA
d r a aρ ρ ρ+
+ +′ ′ ≈ + ∇ +∫r
r r r (19)
where is the two-dimensional Laplace operator. Here the first two terms of a Taylor expansion
of have been used.
2∇
( )cρ r
Substituting Eq.(19) in Eq.(16) for , we get for a linear differential
equation
( )0A+∉r 1ca ρ+
( ) ( ) ( ) ( )2 21
8c f c ca aρ ρ ρ ρ+ +⎛ ⎟⎜= + ∇ ⎟⎜ ⎟⎜⎝r r ⎞⎠r (20)
The general solution that vanishes at infinity is
( ) ( )( )( )( )
0
0
K /
Kf
c c f
r Rr c
β ηρ ρ ρ
β= =r (21)
where is a Bessel function. The scaling factor can be expressed in terms of the free coverage 0K β
fθ as given by Eqs.(8)-(13)
( )2
2
8 1 f
f
η θβ
πη θ−
≡ (22)
The constant c is obtained from a matching criterion at the edge of the merging region
of the central crystal, i.e. at (0)A+fr Rη= .Of the circular region with the same area ( )A+ r a+ at
48
the edge of , a fraction (0)A+ ( )2/3 3 / 2γ π= − overlaps with .In that part all crystals
are connected to the central crystal, whence
(0)A+
( )c fρ ρ=r . On the remaining fraction 1 γ− of
we approximate the density of connected crystals by the density at the edge:
( )A+ r
( ) ( )c c fRρ ρ η≈r . Thus
we approximate the surface integral in Eq.(16) by
(23) ( )( )
( ) (2 1c fA
d r a Rρ γρ γ ρ+
+ ⎡′ ′ ≈ + −⎢⎣∫r
r )c fη ⎤⎥⎦
Substituting Eq. (20) with fr Rη= into Eq. (15) leads to an implicit equation for c :
(24) ( )[( 21 exp 1fc η θ γ γ= − − + − ])cfrom which c can be solved by numerical iteration.
Upon integrating ( over the whole surface we obtain the average number of original
crystals that are, directly or indirectly connected to a given cluster, i.e. for the average number
of original crystals that merge to form a new crystal:
)cρ r
cN
( ) ( )( )( )
22 2 1
200 0
K1 1 2 2 1 1
K
f fR R
c c f c fcN d r rdr r rdr
η ηβηρ π ρ π ρ η θ
β β⎛ ⎞⎟⎜ ⎟≡ + ≈ + + = + +⎜ ⎟⎜ ⎟⎜⎝ ⎠∫ ∫ ∫r (25)
Note that this average crystal size diverges if the free coverage fθ approaches the limiting
value where we found that Eq.(16) had an infinite cluster solution Eq.(18). 2 1fη θ →
3.5.5 Interpretation of AFM-images of nucleation and growth
Combining Eq.(25) with Eqs.(7)-(12), we can express the observed film coverage, crystal density
and crystal size in terms of the nucleation rate and the lateral growth rate. To interpret data on the
individual crystal volumes as well, we assume that the nuclei have a monolayer height and that
vertical growth takes place on top of these with a constant velocity . We have simultaneously
fitted all our AFM data to Eqs.(7)-(12), (25). The results of the fit are given in the table below.
vv
49
Symbol in text Value Unit
Equilibrium pressure eqπ 5.0 0.5± mN/m
Initial density ( )0f tρ = ( )( )0.020 0.002 eqπ π± − µm-2
Initial radius 0R ( )( )4.5 0.5 eqπ π± − nm
Nucleation rate I ( )0.0002 eqπ π< − µm-2 min-1
Lateral growth rate lv ( )( )0.45 0.05 eqπ π± − nm/min
Vertical growth rate vv ( )( )0.0009 0.0001 eqπ π± − nm/min
The first fit parameter is the equilibrium value eqπ of the spreading pressure. Nucleation and
growth of crystals is expected to take place in the film only for pressures eqπ π> . For the other
model parameters we compared the quality of the fit for the two cases that the parameter was taken
independent of π or proportional to eqπ π− . In all cases the second choice performed better. We
also found that the fitting model did not produce a significantly positive value for the nucleation
rate (after withdrawal of the film from the through). As a matter of fact, direct inspection of the
AFM images confirmed that only rarely the number of crystal increased with increasing with time
after withdrawal. The decrease in the number of crystals due to coalescence and merging was a
more dominant process. Therefore we took
t
0I = to estimate the other model parameters (thus
leaving us with a 5-parameter fit to 4 independent observables. We used 10 different combinations
of time and spreading pressure (altogether 78 AFM images were taken into account). For the
nucleation rate we give an upper limit.
In view of the uncertainty and experimental spread of the AFM images the fits are quiet
satisfactory. Using more elaborate models does not seem justified or useful.
3.6. Conclusions
In this study we have obtained AFM images that reveal the structure of thin tristearin (SSS) films,
formed at air-water interface. Based on Langmuir and AFM experiments we estimated crystal
growth rates in the lateral and vertical direction. Our investigations lead to the following
conclusions.
50
Compressing an extended SSS film at the air-water interface slowly, starting at very low
surface pressure, monolayers of SSS molecules in trident conformation are formed. These
monolayers are thermodynamically stable at surface pressure .This is the equilibrium
spreading pressure for this system. At higher surface pressures a monolayer with a trident
molecular conformation is still formed, but it is metastable and transforms slowly to form
crystalline domains. The monolayer can be successfully transferred onto a mica surface and the
resulting Langmuir-Blodgett film is quite stable in the course of time. Using AFM-imaging the
monolayer thickness of the trident monolayer can be measured. We find that the thickness depends
on the surface pressure, which we interpret as a change in the tilt angle between the average chain
direction and the crystal surface. From thicknesses between 1.6 and 1.8 nm, we conclude that the tilt
angle gradually increases from
5 mN/mπ ≤
43τ = ° at 10 mN/mπ = to 53τ = ° at 40 mN/mπ = .
At pressures the trident monolayer is thermodynamically metastable.
Macroscopically it is immediately determined by the behaviour of Langmuir films during isobaric
compression. The film area decreases slowly but steadily when the film is subject to a constant
surface pressure. We investigated this process on the molecular scale by comparison of AFM
images of LB-films that were produced after different periods of isobaric compression. We
concluded that crystal growth takes place under these conditions. Within the crystals the molecules
adopt a tuning fork conformation, except for the bottom layer where the molecules stay in the
trident conformation. The crystal sizes increase with time, the growth being mainly in lateral
directions. The growth rate increases with surface pressure.
10 mN/mπ ≥
We developed a new model to describe the growth and coalescence of crystals in the film.
Fitting the AFM-data to this model we estimate a lateral growth rate of 2.3 nm/min and a vertical
growth rate of 0.005 nm/min at 10 mN/mπ = . The data were insufficient to get a reliable estimate
of the nucleation rate of new crystals, we merely estimate an upper limit of 0.001 new nuclei per
µm2 and per minute.
At and above the collapse pressure the crystal growth process is the same, though faster than
in the intermediate regime. The isobaric compression velocity increases rapidly from 3 µm/sec at
to 18 µm/sec at . mN/m35π = mN/m42π =
The transformation processes that took place in the Langmuir film (floating on water), were
observed in the transferred LB-film (on mica) as well. In the latter case however, the processes were
orders of magnitude slower.
51
References:
[1] Garti, N. and Sato, K., In Crystallization and polymorphism of fats and Fatty Acids; Dekker, M.
New York (USA) 1988
[2] Ollivon, M., Triglycerides. In Manuel des Corps Gras. Ed.A.Karieskind, Lavoisier, Paris
(France) 1992; p. 469
[3] Bursh, T., Larsson, K. and Lundquist, M., Chem. Phys. Lipids 2 (1968) 102-113
[4] Hamilton, J.A., Small, D.M., In Proc. Nat. Acad. Sci. USA 78 (1981) 6878
[5] Hamilton, J.A., Biochem. 28 (1989) 2514
[6] Claesson, P.M., Dedinaite, A., Bergenstahl, B., Campbell B. and Christenson, H., Langmuir, 13
(1997) 1682
[7] Lundquist, M., In Surface chemistry, Copenhagen. Munksgaard, 1966 p.294
[8] Kuzmenko, I., Buller, R., Bouwman, W.G., Kjaer, K., AlsNielsen, J., Lahav, M. and
Leiserowitz, L., Science 274 (1996) 20046-20049
[9] Xue, J.Z., Jung, C.S., Kim, M.W., Phys. Rev. Lett. 69 (1992) 474-477
[10] Ekwall, P., Ekholm, R., and Norman, A., Acta Chem. Scand. 11 (1957) 703
[11] Birker, P.J. and Blonk, J.C., J. Am. Oil Chem. Soc. 70 (1993) 319-321
[12] Michalski, M., Brogueira, P., Goncalves da Silva, A. and Saramago, B., Eur. J. Lipid Sci.
Technol. 103 (2001) 677-682
[13] Akita, C., Kawaguchi, T., Kaneko, F., Yamamuro, O., Akita, H., Ono, M. and Suzuki, M.,
Journal of Crystal Growth 275 (2005) 2187-2193
[14] Roberts, G., Langmuir-Blodgett Films Plenum Press, New York (1990) p.21
[15] Gaines, G.L., Insoluble Monolayers at Liquid-Gas Interface, Wiley, New York, 1966
[16] Karaborni S. and Toxvaerd, S., J.Chem.Phys. 97 (8) (1992) 5876-5883
[17] Lin, B., Shih, M.C., Bohanon, T.M., Ice, G.E. and Dutta, P., Physical Review Letters Vol.65,
No.2 (1990) 191-194
[18] Yase, K., Ogihara, S., Sano, M., Okada, M., Journal of Crystal Growth 116 (1992) 333-339
[19] Takeuchi, M., Ueno, S. and Sato, K., Crystal Growth & Design Vol.3, NO.3 (2003) 369-374
[20] De Jong, S., Triacylglycerol crystal structures and fatty acid conformations, a theoretical
approach- PhD thesis (1980).University of Utrecht, The Netherlands
[21] Larsson, K., Physical Properties - Structural and Physical Characteristics. In: The Lipid
Handbook. Eds. Gunstone, F.D.; Harwood, J.L.; Padley, F.B. Chapman & Hall, London (UK) 1986;
p.321
52
CHAPTER 4
Structure and stability of Triglyceride monolayers on water and mica surfaces
Abstract
The structure and the stability of tripalmitin (PPP), tristearin (SSS) and triarachidin (AAA)
monolayers at the air-water are investigated with the Langmuir method. The Langmuir-
Blodgett (LB) layers obtained by deposition on mica were investigated with Atomic force
microscopy (AFM). Our experiments show that the three triglycerides can form monolayers
with molecules in trident conformation at the air-water interface. We determined the
equilibrium spreading pressure eqπ below which such monolayers are thermodynamically
stable. Under isobaric conditions at 0π π> the film area decreased slowly for PPP and SSS,
corresponding to crystal formation with molecules in tuning fork conformation on top of the
monolayer. Here 0π may be significantly larger than eqπ . In the intermediate range
0eqπ π π< < film area decrease was not measured. The isobaric compression rate was highest
for PPP and almost zero for AAA. Using carefully AFM 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 46.4o, 49.2o and 59.0o respectively. The LB-
monolayers of PPP and SSS, which were transferred at 0π π≥ are thermodynamically
unstable in air. Small crystals form on top of the monolayer, presumably in β -phase for SSS.
Contrary to SSS, domains with α -like and β -like structure coexist in the LB film of PPP.
The nucleation rate increases with increasing surface pressure π and with decreasing chain
length of the triglyceride. For AAA no well - defined crystals were found on top of the LB-
monolayer during the investigated period of days. The trident monolayer is the less mobile
and the crystal phase is the more stable the longer the alkyl chains are.
53
4.1. Introduction
The interfacial behaviour of surfactants and their mixtures is of importance in a wide range of
applications. The most commonly used emulsifiers in the food industry are the
monoglycerides [1]. Spread monolayers at air-water interface can show relaxation phenomena
mainly because of instability due to desorption or collapse [2].
For monoglyceride monolayers, it was observed that the main causes of instability are
desorption in subphase competing with collapse followed by nuclei formation. It was found
that the stability of the monolayers depends on the film structure, subphase composition, the
temperature, the surface pressure [3, 4] and aqueous phase pH [5]. Some of the investigated
monoglycerides were unstable at surface pressures π below the so-called collapse pressure
colπ . The rate of monolayer molecular loss due to desorption increased with surface pressure.
Molecular loss at the interface depended also on the hydrocarbon chain length. The longer
monoglycerides were more stable than the shorter [3, 5].
The triglycerides are another class of molecules of great importance in the food
industry. They are isolated from plant seeds or animal tissues and processed into edible-fat
products, of which they are the main constituents. The crystallization of triglycerides is a key
step both during manufacturing fat products and fractionating fats and oils. In all cases, the
crystallization behaviour is very complex due to the intricate composition of fat blends and
the tendency of triglycerides to crystallize in a variety of morphological forms. 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. Each of these
polymorphic forms consist of layers in which the molecules have a tuning fork conformation
but the orientation of the tuning forks within the layers, as well as the packing of the layers
is different [6, 7].
On the other hand, in monolayers at a hydrophilic-hydrophobic interface, triglyceride
molecules adopt a trident conformation (all hydrocarbon chains pointing toward 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 oil [8-12].
In previous work [Chapter 3] we investigated monolayers of tristearin (SSS, chain
length 18 C atoms) floating on water in a Langmuir system and deposited on mica with AFM.
The Langmuir experiments showed that adsorption isotherms obtained with commonly used
compression rates do not correspond to thermodynamic equilibrium. Under isobaric
54
conditions the film area decreased slowly, corresponding to the formation of crystals on top of
the monolayer. The AFM images revealed that SSS initially forms trident monolayers at air-
water interface. These layers are thermodynamically stable only at surface pressure
5mN/mπ ≤ . For 10mN/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 was
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 one individual
crystal at 10mN/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.
The aim of this work is to understand the behavior of different triglycerides
(tripalmitin-PPP chain length 16 C atoms, tristearin-SSS and triarachidin- AAA chain length
20 C atoms) at air-water interface (Langmuir film) and on solid surface like mica (Langmuir-
Blodgett film) and to establish the relation between their molecular structure and their
monolayer stability. Two kinds of experiments have been done. First, 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). To
investigate whether the Langmuir film was in thermodynamic equilibrium at this pressure π ,
we sometimes left the film for some time t at pressure π (isobaric compression). In the
second type of experiment the Langmuir film was transferred to mica directly after forced
compression ( ). We investigated LB films with AFM immediately and a few days after
incubation in air at room temperature.
0t =
4.2. Materials and methods
4.2.1. 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
C atoms), Tristearin (1, 2, 3, -trioctadecanoylglycerol: SSS, chain length 18 C atoms) and
55
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.
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.
4.2.2. 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 two modes of operation were available. First forced compression, where the
position of the barrier, and hence the trough length
2110 ÅA ∼
( )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 ( )l t is constant. In practice however we often found
the barrier to move with velocities of the order of 1 m/secµ .
4.2.3. 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
56
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
takes a few minutes. After deposition the LB-films were dried in air and kept in close
containers until use. All experiments were done at 20 ± 1°C.
4.2.4. 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 x176µm; 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. In order to detect structural changes in the adsorbed film in
contact with air we studied LB films several days after incubation at room temperature (20 ±
1°C) as well.
4.3. Langmuir observations
4.3.1. Forced compression
Fig.1. Surface pressure vs area
isotherms of tripalmitin (PPP),
tristearin (SSS) and triarachidin
(AAA) at air-water interface, at
20oC, obtained by forced
compression at a rate of 1cm/min
(the thermodynamic equilibrium
isotherms are obtained, in
principle, at very slow
compression rates).
02468
101214161820
40 50 60 70 80 90 100 110Area/molecule A ( A2)
Surfa
ce p
ress
ure
(mN
/m)
PPP
SSS
AAA
57
Fig. 1 shows typical Aπ − isotherms of tripalmitin (PPP), tristearin (SSS) and
triarachidin (AAA), recorded at a barrier velocity of 1 cm/min. Two different regimes can be
recognized for the three triglycerides. 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. The pressure at which
was reached we will call condensation pressure
condA
condA condπ .We interpret the low pressure
phase as “gaseous”, and the high pressure phase as “condensed”. On the basis of Langmuir
experiment alone we can not conclude whether the condensed phase is liquid-like or solid-
like. For condπ π≥ the molecules are close enough together to form a condensed monolayer.
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 48mN/mπ = −
and it increases in order: (AAA) (SSS) (PPP)col col colπ π π< < . With our LB instrument the
collapse pressure was difficult to reproduce because of technical problems. colπ
The measured Aπ − data showed that the transition from one regime to another was
not very sharp and it was different for the investigated triglycerides. In order to get reliable
and unbiased estimations for and condA condπ , we fitted the isotherms with:
( ) ( , )condA ch A A aπ ≈ − (1)
here , , c and are fitting parameters. The function condA a h
( )2 21( , )2
h x a x x a≡ − + (2)
is a hyperbola interpolating between for large negative 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≈ x ( ), 0h x a ≈
/ 2cond caπ = and . Fitting a number of isotherms that were obtained at compression
velocity 1cm/min we found and
condA
262 1ÅcondA = ± 6 2mN/mcondπ = ± for PPP;
and
262 1ÅcondA = ±
9 2 for SSS and and 265 1ÅcondA = ± 8 1mN/mcondπ = ± for AAA (fig.2). mN/mcondπ = ±
58
B Condensation pressure πcond
0
5
10
15
20
25
14 16 18 20 22
Number carbon atoms
πco
nd (m
N/m
)
Fig.2. Condensation area (A) and condensation pressure condA condπ (B) as a function of
number of carbon atoms in the alkyl chains of the triglycerides: 16 (PPP), 18 (SSS) and
A Condensation area A cond
60
61
62
63
64
65
66
67
14 16 18 20 22
Number carbon atoms
A con
d (Ag
stro
m s
quar
ed)
20 (AAA).
The fact that is around for all studied triglycerides 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 [13]. Our isotherms are consistent
with earlier reports [8, 12] and the results in Chapter 3.
219.7Å
The fact that condπ is almost the same for the investigated triglycerides as well (Fig.2
B), 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.
The value of the fitting parameter (Eq.1) describes the sharpness of the gas -
condense transition and depends strongly on the chain length. This is also seen in Fig.1 where
the
a
Aπ − isotherm for PPP is sharper than those for SSS and AAA. This effect is of a kinetic
nature, i.e. PPP realizes the transition from gas to condensed phase faster than the longer SSS
and AAA. The longer chains need more time to transform and rearrange in perpendicular
position. If this was solely due to the flexibility and mobility of individual triglycerides, the
sharpness of the adsorption isotherms would be strongly dependent on the compression rate.
As we did not observe such dependence we conclude that the sharpness of the PPP isotherm
also is influenced by interactions between the molecules. The isotherms in Fig.1 suggest that
in a moderately dense packed monolayer at the air-water interface the longer triglycerides
59
(SSS and AAA) repel each other significantly at larger intermolecular distances than the
shorter PPP. From this perspective we shall discuss the experimental results in the next
section.
4.3.2. Isobaric compression
To investigate the stability of the triglyceride films at an air-water interface we made the
following experiment. We stopped the forced compression at a constant compression rate fν
when a certain surface pressure was reached. Next we kept the surface pressure constant at
that value, allowing the barrier to move. After several minutes a constant isobaric velocity
π
ν
was reached usually. Due to molecular loss from the monolayer the barrier moved forward to
keep the surface pressure constant and the through length l(t) decreased. This is shown in
Fig.3.
Barrier position vs time for speading pressure π = 20mN/m
9.50
10.00
10.50
11.00
11.50
0 1000 2000 3000 4000 5000time t (sec)
l(t)
(cm
)
AAASSSPPP
Fig .3. Examples of the measured barrier position as a function of time during forced isobaric
compression for ( )PPP, (□) SSS and (○) AAA. The almost vertical parts of the curves
correspond to forced compression rate of
∆
fν ≈ 1 cm/min. The slowly decreasing parts
correspond to small residual isobaric compression rates at velocity ν at the spreading
pressure 20mN/mπ = . Note that the molecular loss, presented by l(t) is higher for PPP.
60
The evolution of the trough length was fitted to
(3) ( ) ( ) ( ) ( )0 0 ,fl t l v t t v v h t t a≈ − − − − − 0
Here the five fitting parameters are l , the trough length at the start of the isobaric
compression, , the starting time of the isobaric period,
0
0t 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.
Fig.4. Isobaric velocity
ν (µm/sec) as a function
of the spreading
pressure π, as obtained
by fitting the measured
barrier position to Eq.
(3) for PPP, SSS and
AAA. Note the sharp
increase of ν for PPP for
spreading pressure close
to the collapse pressure
48mN/mcolπ ≈
In previous work [Chapter 3] we found that for SSS the adsorption isotherms that were
Isobaric compression
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20 25 30 35 40 45
Spreading pressure ( mN/m)
Vel
ocity
v (u
m/s
ec)
PPP
SSS
AAA
obtained by forced compression did not change appreciably when increasing the barrier
velocities from 0.5 to 2 cm/min. Nevertheless under isobaric conditions we observed further
compression. We noted that the isobaric velocities were one or two orders of magnitude
smaller than commonly used forced velocities of ~ 1cm/min. The same behavior we observe
here for PPP and AAA. In Fig. 4 we show the dependence of the isobaric velocity v on the
surface pressure π for the three triglycerides. It can be noted that for PPP 0v for
10mN/m
≈
π ≤ and ν depends linearly on π for 10mN/m 25mN/mπ≤ ≤ . For pressures
61
30mN/mπ ≥ , i.e. close to the collapse pressure, a m is found. These
that the PPP isotherm in Fig.1 cannot be considered as an equilibrium isotherm
for 10mN/m
uch faster compression
results show
π > . For such pressures the equilibrium value of A is smaller than the value
give
At this p
n in Fig.1.
oint it is important to clearly discriminate between collapse pressure colπ and
equilibrium pressure eqπ . In isobaric conditions a molecular rearrangement process takes
place which effectively thickens the film. Using Atomic Force Microscopy for SSS we have
shown that this process involves the growth of 3D crystals of SSS on top of the monolayer,
which is precisely what one should expect for eqπ π> . For colπ π> the same crystallization
process takes place, but in a less controlled and less reproduc nner. This interpretation
is in line with the definition of Roberts [14]. Note however that sometimes one names
equilibrium spreading pressure what we call collapse pressure, see e.g. [5]. According to
thermodynamics equilibrium (spreading) pressure is the surface pressure that is
spontaneously generated when a sample of triglyceride in its thermodynamically stable phase,
i.e. in the crystalline
ible ma
β 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
[14]. Another way to express this, is that the chemical potential of the triglycerides in the β
phase, βµ , is equal to the chemical potential Lµ of triglycerides in the Langmuir layer on th
water surface:
e
( )L eq βµ π π µ= ≡
Therefore we can, in principle estimate eqπ from the dependence of the isobaric
velocity ν on π . In Fig.4 we suggest that there exist a surface pressure 0π , such that ν is
proportional to 0π π− for 0π π> . If we assume that 0 eqπ π≈ we get an estimation 0π of
eqπ . On the other hand, we know from our AFM observation that the process taking place for
0π π is crystal growth and therefore a non-linear dependence of > ν on eqπ π− is expected.
stal growth theory for faceted crystals 0 eqIn cry π π− would be called “nucleation gap”,
62
meaning that for π in the regime 0eqπ π π≤ ≤ no observable growth takes place. Thus we
should realize that the real equilibri equm pressure π may be well below the linear estimate
0π . According to our results (Fig.4) for tripalmitin (PPP) at air-water interface 0 10mN/mπ = .
e instability of the monolayer of PPP at 0Th π π> increases with the surface
same dependence was reported before for m almitin at air-water interface [3, 5]. The
isobaric velocity for SSS given here is slightly smaller than what we measured before
[Chapter 3] and 0 15mN/m
pressure. The
onop
π = is higher (fig.4). The difference in the results probably is
caused by the bett trough that we used in this work. It is known that the use of
Delrin barrier in our present system drastically improves the quality and reproducibility of the
experiments [15]. It is also been observed that simple Langmuir systems (like the one we used
before) tend to underestimate the spreading pressure in condensed films. From our
experiments it was difficult to estimate 0
er Langmuir
π for AAA. The measured isobaric velocity was too
close to zero for all investigated surface pressures (fig.4). From our experiments it can be
concluded that both the mobility, characterized by ν and the effective stability, characterized
by 0π of triglycerides monolayers at an air-water interface depend on the film composition,
notably on the length of the triglycerides. For PPP and SSS we can determine an upper bound
0π for the equilibrium pressure eqπ and we see that 0π is lower for PPP than for SSS. This
e mobility and flexibility will decrease with increasing of
the chain length.
The increa
dependence can be understood as th
sing interactio th will also influence the thermodynamic stability of n streng
the crystal phase, as is signified both by an increasing melting point and by decreasing
equilibrium vapor pressure. Therefore one would expect ( ) ( ) ( )eq eq eqPPP SSS AAAπ π π≥ ≥ .
This seems contradictory to the inequality 0 0( ) (PPP )SSSπ π< that we found above. It i
lar i on gen
s
well-known however, that a stronger molecu erally leads to stronger non-
linearity of the dependence of the growth rate and the nucleation rate on eq
nteracti
π π− . As a
consequence the nucleation gap 0 eqπ π− will be larger for SSS than for PPP. Only if we could
make full non-linear ( )ν π fits to data like those in Fig.4, we might find the real dependence
of eqπ on the chain length.
One should also expect that the stronger interaction reduces the mobility of
triglyceride molecules in a condensed phase. This effect is seen by the decrease of the isobaric
63
velocity when changing from PPP to SSS and to AAA. The isobaric velocity for PPP and
SSS, and presumably for AAA as well, depends roughly linearly on the surface pressure when
π is far enough above eqπ , i.e. for 0π π> . The slope /d dν π is positive and increases with
decreasing chain length (Fig.4).
4.4. AFM observations
ess
In es form a trident monolayer at an air-water
interface, which can be transferred to mica. We found that the monolayer thickness varied
4.4.1. Monolayer thickn
Chapter 3 we showed that SSS molecul
from 1.6 to 1.8 nm over the pressure range we studied. The molecules were tilted and the tilt
angle varied from o43τ = at 10mN/mπ = to o53τ = at 40mN/mπ = .
From the AFM images of LB-films of PPP, SSS and AAA, withdrawn at
20 mN/mπ = (fig. is s e m5) it een that th ica substrate is covered by a homogeneous
water surface
monolayer. Apparently the Langmuir monolayer can be successfully transferred from the
in the Langmuir trough to a mica surface to form a Langmuir-Blodgett film
there. When the LB-films were prepared at lower pressures (data not shown) a monolayer was
observed as well, but with a lot of holes in it.
64
A B C
0
0
2.5-5.0
5.0
5.0
1.35 nm 1.39 nm
µm
D
µm2.50-5
.0
5.0
5.0
0
1.57 nm 1.61 nm
E
0µm
0
2.5 5.0
5.0
-5.0
2.12 nm 2.12 nm
F
AA BB CC
0
0
2.5-5.0
5.0
5.0
1.35 nm 1.39 nm
µm
D
0
0
2.5-5.0
5.0
5.0
1.35 nm 1.39 nm
0
0
2.5-5.0
5.0
5.0
1.35 nm 1.39 nm
µm
D
µm2.50-5
.0
5.0
5.0
0
1.57 nm 1.61 nm
E
µm2.50-5
.0
5.0
5.0
0
1.57 nm 1.61 nm
2.50-5.0
5.0
5.0
0
1.57 nm 1.61 nm
E
0µm
0
2.5 5.0
5.0
-5.0
2.12 nm 2.12 nm
F
0µm
0
2.5 5.0
5.0
-5.0
2.12 nm 2.12 nm
0
2.5 5.0
5.0
-5.0
2.12 nm 2.12 nm
F
Fig.5. AFM height image of monolayers transferred immediately after forced compression to
surface pressure π = 20mN/m. The black squares are holes in the monolayer produced by
scanning at a high AFM force (F~30 nN). The monolayers are scanned at AFM force F =
1nN. (A) PPP monolayer with corresponding cross section (D). (B) SSS monolayer and the
corresponding cross section (E). (C, F) AAA monolayer. The scale bar is 2 µm and the
vertical scale is 10 nm for all images.
We estimated the monolayer thicknesses using the following procedure. 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.5). The height difference between the hole and the surrounding film
gives an apparent thickness d . Analyzing our data carefully we found that d turned out to
depend on the scanning force F for all investigated triglycerides. Therefore the real monolayer
thickness differs from d . In Fig.6 we present the observed dependence of d on . It is
seen that d is larger for larger chain length, as expected. It is also seen that the vertical
compressibility of the monolayer, given by the slope of curves, is the same for PPP,
SSS and AAA. The real thickness
′ ′
0d ′ ′ F
′
'( )d F
0 '( )d d F= , corresponding to scanning force is
presented in Fig. 7. It is linear dependent on the length of the chains in the triglyceride
molecules.
0F =
65
Apparent monolayer thickness at spreading pressure π = 20 mN/m
0
0.5
1
1.5
2
2.5
3
0 2 4 6 8 10
AFM force F (nN)
AFM
thic
knes
s d'
(nm
)
PPP
SSS
AAA
Fig.6. Measured layer thickness d for PPP, SSS and AAA as a function of applied AFM
force F at surface pressure
′
20mN/mπ = .
Real monolayer thickness at spreading pressure π =20 mN/m
1
1.2
1.4
1.6
1.8
2
2.2
2.4
12 14 16 18 20 22 24
number of carbon atoms in the chain
Laye
r thi
ckne
ss d
0 (n
m)
Fig.7. Real monolayer thickness for PPP (16), SSS (18) and AAA (20) at surface pressure 0d
20mN/mπ = . The values are found by extrapolation of the apparent monolayer thickness
in Fig.6 to AFM force F = 0 nN.
'd
To translate the thickness into a tilt angle τ of the acyl chains with the mica surface
we need to estimate the effective chain length. A precise analysis and interpretation of
crystallographic data for the long spacing of PPP, SSS and AAA in the stable β-phase
[16], allows us to estimate an effective length of PPP, SSS and AAA-molecules in tuning
fork conformation. Correcting this for the length of the glycerol group and the contribution
(001)d
effd
66
from the contact region of (001) layers in the β phase, the alkyl chain length can be
estimated. When carrying out an analogous procedure for the α and β’ phase the same lengths
are found.
chaind
chaind
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 α , β and 'β phases, though in different
orientation with respect to the glycerol group. This leads to a structure where alkyl chains
extend from the substrate to the monolayer surface at a height above the substrate.
Thus in the monolayer the triglyceride molecules adopt a trident conformation we get a
simple relation:
chaind 0d
0sin( ) / chaind dτ = (4)
The estimated chain lengths and the tilt angles are given in the table below.
triglyceride (001)d
β - phase (nm) effd (nm) chaind (nm) 0d (nm) of
monolayer
Tilt angle τ
PPP 4.03 4.62 1.80 1.49 46.4o
SSS 4.48 5.13 2.31 1.75 49.2o
AAA 4.92 5.62 2.82 2.20 59.0o
In the stable β-phase the triglyceride molecules are tilted at tilt angle [16].
Since presumably in the trident monolayer the alkyl chains are less densely packed than the
crystalline phases, a smaller tilt angle seems acceptable. Interpreting our monolayer thickness
data with Eq.4, we see that the tilt angle increases with increasing the length of the alkyl
chains, i.e. the longer chains in the monolayer are more perpendicular to the substrate than the
shorter. In the aliphatic alcohol monolayers on water the same dependence of the tilt angle on
chain length was found. IR spectra of these alcohol monolayers showed that the hydrocarbon
chains become more ordered with increasing length [17].
o60.8τ =
67
4.4.2. Stability of the transferred LB-film
In our previous investigation we found that during incubation in air transferred SSS - films
become slightly thicker and grainy. Crystals that were present directly after the transfer were
growing and new very small nuclei appeared [Chapter 3]. We suggested that the grainy
character of the monolayer is due to small clusters of molecules, which leave the monolayer
to form new crystals or contribute to the growth of existing crystals. To investigate this
mechanism further we transferred PPP, SSS and AAA Langmuir layers to mica, immediately
after forced compression to spreading pressure π, and left them in closed containers for a few
days at room temperature ( C). o20 1±
4.4.2.1. Initial structure and structural changes of PPP-monolayer
After 30 min incubation on water surface at surface pressure 10mN/mπ = (this is 0π for
PPP), which we expect to be close to eqπ for PPP, the AFM images show a homogeneous
monolayer with small holes when the LB-film was transferred to mica (data not shown). At
surface pressure 20mN/mπ = ( 0π π> ) we found that the directly transferred PPP-film
consists of a closed monolayer, onto which many small domains were positioned. The
thickness of the domains ranged from 3.3 nm to 4.6 nm above the monolayer level (Fig.8A,
D). These domains were soft and could be easily scratched away with the AFM tip, even at
the low AFM forces that are normally used for imaging. Indeed, after a few scans with
these domains usually had disappeared, leaving a flat film with the thickness of
the trident monolayer. The maximum measured height of the domains 4.6 ± 0.1nm
corresponds to fully extended PPP molecules in tuning fork conformation perpendicular to the
substrate, i.e. to one crystalline layer in the
1 2 nNF = −
α phase. This result is consistent with the growth
of crystals in tuning fork conformation on top of the trident monolayer, reported for SSS
[Chapter 3]. We therefore may assume that the observed domains are small crystals, formed
in the period where the spreading pressure increased from the small values at which the film
is in a two-dimensional gas state, to the final pressure 0π π− at which the condensed phase
has formed. In this period PPP - molecules undergo major orientation and packing changes.
As a result the formation process of the domains will not be strictly deterministic and a
68
metastable film structure may form. The domains serve as crystal seeds which can grow into
bigger crystals when the LB - film is incubated longer time in air (Fig.8).
B
C
3.3 nm4.5 nm
tm
αm
m
10.0
-10.
00
10.05.00µm
D
0 5.0 10.0
10.0
-10.
0
0
3.3 nm 4.6 nm
m tm
αm
µm
E
10.0-10.
0
0
10.0
5.00
tm
αm
αm
m
4.5 nm4.6 nm3.3 nm
µm
F
A
BB
CC
3.3 nm4.5 nm
tm
αm
m
10.0
-10.
00
10.05.00µm
D3.3 nm4.5 nm
tm
αm
m
10.0
-10.
00
10.05.00µm
D
0 5.0 10.0
10.0
-10.
0
0
3.3 nm 4.6 nm
m tm
αm
µm
E
0 5.0 10.0
10.0
-10.
0
0
3.3 nm 4.6 nm
m tm
αm
µm0 5.0 10.0
10.0
-10.
0
0
3.3 nm 4.6 nm
m tm
αm
µm
E
10.0-10.
0
0
10.0
5.00
tm
αm
αm
m
4.5 nm4.6 nm3.3 nm
µm
F
10.0-10.
0
0
10.0
5.00
tm
αm
αm
m
4.5 nm4.6 nm3.3 nm
µm10.0-1
0.0
0
10.0
5.00
tm
αm
αm
m
4.5 nm4.6 nm3.3 nm
µm
F
AAA
Fig.8. AFM height image of PPP monolayers transferred at 20 mN/mπ = . (A) immediately
after forced compression, (B) the same sample after 1 day incubation in air at room
temperature, (C) the same sample after 2 days incubation in air. The corresponding cross
sections are given in (D, E and F). The scale bar is 2 µm and the vertical scale is 20 nm for
all images. Height differences are given by the numbers at the markers. The symbols below
the lines give our proposed structure of the crystals (m – monolayer in trident conformation;
t – top layer in tuning fork conformation; α - crystal in tuning fork conformation).
69
The AFM images of LB-films of PPP incubated for 1 day in air showed that the
monolayer is still present and the density of the crystals is higher. Most of the new crystals
were small and with thickness 4.6 ± 0.1nm. Some bigger domains were found with thickness
3.3 ± 0.1 nm (Fig.8B, E). After 2 days incubation the domains were bigger, mostly with
thickness 4.6 ± 0.1 nm and still surrounded by the trident monolayer (Fig.8C, F). Apparently
immediately after the transfer a few crystals are present, some in α phase, other in β phase. In
air these crystals grow and new crystals, mostly in α phase, nucleate. The same processes
were observed for PPP monolayer transferred at 10 mN/mπ = after a few days incubation in
air, but the speed of nucleation is lower (Fig.9).
A Crystal density for PPP
0
0.5
1
1.5
2
2.5
3
3.5
0 1 2 3days
Cry
stal
s pe
r µm
2
π = 10mN/mπ = 20mN/m
B Volume per total area for PPP
0
0.00002
0.00004
0.00006
0.00008
0.0001
0.00012
0 1 2 3days
Volu
me
per t
otal
are
a ( µ
m)
π = 10mN/mπ = 20mN/m
Fig.9. Crystals per area (A) and average crystal layer thickness (B), formed during
incubation of PPP LB-monolayer in air at room temperature.
70
From these observations we conclude that the LB monolayer is unstable both with
respect to the α -phase and to the β -phase:
( ) ( ) (LB PPP PPP PPPα β )µ µ µ> > (5)
where ( )LB PPPµ is the chemical potential of PPP molecules in a monolayer on the mica
surface. Note that from the LB layer mainly the less stable α -phase grows. This is a
manifestation of Ostwalds rule, which states that if two (or more) phases can grow in
principle, then the least stable of these phases usually will dominate because it is less ordered
and hence its growth kinetics are faster.
4.4.2.2. Initial structure and structural changes of SSS-monolayer
When the SSS-film was withdrawn after 30 min incubation at air-water interface at
15mN/mπ = (this is 0π for SSS) we observed only a homogeneous (trident) monolayer with
a lot of holes. After 2 days incubation in air the LB - monolayer was covered with small
domains with thickness 3.6 ± 0.1nm (data not shown). At surface pressure
20mN/mπ = ( 0π π> ) we found that the directly transferred SSS film consisted of an almost
defect free monolayer, in which a few domains were imbedded. The thickness of the domains
was 3.6 ± 0.1nm (fig.10A, D). After 1 day incubation in air the LB-monolayer was covered
with many new nuclei with thickness 3.6 ± 0.1nm (fig10B, E). After 2 days incubation in air
the observed nuclei were bigger, all with the same thickness (fig.10C, F).
71
B
C
5.0µm
tm
mtm
tm
3.6 nm10.0
10.0-10.
00
0
3.7 nm 3.7 nmE
5.00
10.0
-10.
0
10.0
0
µm
3.6 nm
mtm
D
3.6 nm
1.7 nmtmm
mica
µm5.0 10.00-1
0.0
010
.0F
1.7 nm3.6 nm
micam
tm
A
BB
CC
5.0µm
tm
mtm
tm
3.6 nm10.0
10.0-10.
00
0
3.7 nm 3.7 nmE
5.0µm
tm
mtm
tm
3.6 nm10.0
10.0-10.
00
0
3.7 nm 3.7 nm
5.0µm
tm
mtm
tm
3.6 nm10.0
10.0-10.
00
0
3.7 nm 3.7 nmE
5.00
10.0
-10.
0
10.0
0
µm
3.6 nm
mtm
D
5.00
10.0
-10.
0
10.0
0
µm
3.6 nm
mtm
5.00
10.0
-10.
0
10.0
0
µm
3.6 nm
mtm
D
3.6 nm
1.7 nmtmm
mica
µm5.0 10.00-1
0.0
010
.0F
1.7 nm3.6 nm
micam
tm
3.6 nm
1.7 nmtmm
mica
µm5.0 10.00-1
0.0
010
.0F3.6 nm
1.7 nmtmm
mica
µm5.0 10.00-1
0.0
010
.0
3.6 nm
1.7 nmtmm
mica
µm5.0 10.00-1
0.0
010
.0F
1.7 nm3.6 nm
micam
tm
AA
Fig.10. AFM height image of SSS monolayers transferred at 20mN/mπ = . (A) immediately
after forced compression, (B) the same sample after 1 day incubation in air at room
temperature, (C) the same sample after 2 days incubation in air. The corresponding cross
sections are given in (D, E and F). The scale bar is 2 µm and the vertical scale is 20 nm for
all images. Length differences are given by the numbers at the markers. The symbols below
the lines give our proposed structure of the crystals (m – monolayer in trident conformation;
α - crystal in tuning fork conformation; t – top layer in tuning fork conformation).
72
The structure of SSS-films immediately transferred after forced compression to
30mN/mπ = is reported in Chapter 3.
A
10.0
µm10.0
D4.8 nm
mαm
αm
5.00
0-1
0.0
4.7 nm
B
3.5 nm 4.7 nm
0 5.0µm
-10.
010
.00
αm
tm
m
E
10.0
3.8 nm 3.6 nm 3.6 nm
tm
tm
tmm
10.05.0µm
0-10.
00
10.0F
C
AA
10.0
µm10.0
D4.8 nm
mαm
αm
5.00
0-1
0.0
4.7 nm10.0
µm10.0
D4.8 nm
mαm
αm
5.00
0-1
0.0
4.7 nm
BB
3.5 nm 4.7 nm
0 5.0µm
-10.
010
.00
αm
tm
m
E
10.0
3.5 nm 4.7 nm
0 5.0µm
-10.
010
.00
αm
tm
m
10.0
3.5 nm 4.7 nm
0 5.0µm
-10.
010
.00
αm
tm
m
E
10.0
3.8 nm 3.6 nm 3.6 nm
tm
tm
tmm
10.05.0µm
0-10.
00
10.0F
3.8 nm 3.6 nm 3.6 nm
tm
tm
tmm
10.05.0µm
0-10.
00
10.0
3.8 nm 3.6 nm 3.6 nm
tm
tm
tmm
10.05.0µm
0-10.
00
10.0F
CC
Fig.11. AFM height image of SSS monolayers transferred at π = 30 mN/m. (A) immediately
after forced compression, (B) the same sample after 1 day incubation in air at room
temperature, (C) the same sample after 2 days incubation in air. The corresponding cross
sections are given in (D, E and F). The scale bar is 2 µm and the vertical scale is 20 nm for
all images. Length differences are given by the numbers at the markers. The symbols below
the lines give our proposed structure of the crystals (m – monolayer in trident conformation;
α - crystal in tuning fork conformation; t – top layer in tuning fork conformation).
73
Most of the observed crystals had a thickness 4.9 ± 0.1 nm above the monolayer level. This
thickness corresponds to fully extended alkyl chains of SSS (fig.11A, D). When the LB-
monolayer was incubated in air for 1 day the existing crystals were growing and new very
small nuclei appeared. The thickness of the new nuclei and the newly grown parts of the
crystals was 3.6 ± 0.1(fig.11B, E). After 2 days incubation in air the new nuclei became
bigger (fig.11C, F).
The density of the crystals of SSS formed during incubation in air is initially growing
with time (fig.12A).
A Crystal density for SSS
00.20.40.60.8
11.21.41.6
0 1 2 3days
Cry
stal
s pe
r µm
2
π = 15mN/m
π = 20mN/m
π = 30mN/m
B Volume per total area for SSS
0
0.00001
0.00002
0.00003
0.00004
0.00005
0.00006
0.00007
0 1 2 3days
Vol
ume
per a
rea
( µm
)
π = 15mN/m
π = 20mN/m
π = 30mN/m
Fig.12. Crystals per area (A) and average crystal layer thickness (B), formed during
incubation of SSS LB-monolayer in air at room temperature.
Note however that the density decreased at 30mN/mπ = after 2 days of incubation.
This is due to coalescence of the large number of relatively large crystals at this pressure. The
74
average crystal layer thickness during the incubation increases with the surface pressure and
the time (Fig.12B).
Summarizing, we found that for SSS immediately after transfer, several crystals are
observed which are mainly in the β - phase for 20mN/mπ ≤ and mainly in α - phase for
30mN/mπ ≥ . This suggests that 0 (SSS, )π β is 15mN/m or smaller, and
0 (SSS, ) 25mN/mπ α ∼ . The fact that 0 0( ) ( )π α π β> could have been explained
since ( ) ( )eq eqπ α π β> , because the β -phase is more stable than the α -phase, and the
nucleation gap 0 eqπ π− probably is not much different for α and β . After transfer to mica
surface the existing crystals grow further and new crystals appear, all mainly in the β -
phase, but not with respect to the α - phase. In terms of chemical potentials this means that
( ) ( ) (LBSSS SSS SSSα )βµ µ µ> > (6)
Note that the location of the LB chemical potential LBµ with respect to αµ and βµ is
different for SSS and for PPP.
4.4.2.3. Initial structure and structural changes of AAA-monolayer
Fig.13 presents a LB-film of AAA transferred immediately after forced compression at
20mN/mπ = and incubated 2 days in air. We did not observe any crystals, but the monolayer
was somewhat coarse. We suppose that this coarsening was due to very small nuclei, which
were difficult to detect directly with the AFM. If we would have incubated the monolayer for
a longer time, then probably the existing nuclei would grow into crystals. The absence of well
developed crystals after two days incubation is due to very slow kinetics of AAA. In term of
chemical potentials we hypothesize the same relative positions as for SSS:
( ) ( ) (LB )AAA AAA AAAα βµ µ µ> > (7)
The slower kinetics of the AAA monolayer (as compared to SSS and PPP) is expected in
view of the stronger interaction between the longer alkyl chains of AAA.
75
A
10.05.00
10.0
-10.
00
µm
2.1 nm
m
BAA
10.05.00
10.0
-10.
00
µm
2.1 nm
m
B
10.05.00
10.0
-10.
00
µm
2.1 nm
m
10.05.00
10.0
-10.
00
µm10.05.00
10.0
-10.
00
µm
2.1 nm
m
B
Fig.13. AFM height image of AAA monolayer transferred at 20mN/mπ = immediately after
forced compression and incubated 2 days in air (A) with the corresponding cross section (B).
The scale bar is 2 µm and the vertical scale is 20 nm (m – monolayer in trident
conformation).
4.5. Discussion
By definition trident monolayers formed of PPP, SSS and AAA are thermodynamically stable
for eqπ π≤ at the air-water interface. We discussed several methods to estimate eqπ . First
from the collapse pressure colπ . This leads to a large overestimation, col eqπ π . A practical
estimate is condπ , obtained from fitting experimental isotherms. We found 8mN/mcondπ ∼ for
all three triglycerides. The reliability of the assumption cond eqπ π≈ however, is unclear, both
theoretically and experimentally as condπ may depend on the forced compression rate, eqπ
not. From isobaric compression we obtained the pressure 0π below which compression was
absent or too slow to be measured. It is clear that 0 eqπ π≥ , but unfortunately the amount of
overestimation, i.e. the “nucleation gap” 0 eqπ π− , can not be deduced from our data. We
found that 0 10mN/mπ = for PPP, 0 15mN/mπ = for SSS and 0 20mN/mπ = for AAA. We did
not observe any changes in the structure of the trident monolayers on the air-water interface in
the regime 0π π≤ . Combining all our observations with a physically reasonable picture we
conclude that 10mN/meqπ ≤ for all three triglycerides, thus supporting the idea eq condπ π≈ .
For all eqπ π> the Langmuir monolayers are thermodynamically unstable though this
becomes evident in the compression data only for 0π π> .
76
Under isobaric conditions at 0π π> slow, but observable compression rates were
found for PPP and SSS. The isobaric velocity was highest for PPP and almost zero for AAA.
We demonstrated that the stability and the kinetics of Langmuir monolayer depend strongly
on the length of the triglycerides alkyl chains. The trident monolayer is the less mobile, and
the crystal phase is the more stable, the longer the alkyl chains are.
The AFM images of LB-films transferred immediately after forced compression at
0π π> for PPP and SSS showed some domains on top of the monolayer. The molecules in
these domains presumably adopt the tuning fork conformation and pack similar as in the
crystalline α and β crystal forms. This film structure for SSS was explained with the model
we proposed in Chapter 3. The observed domain thickness of 3.6 ± 0.1 nm at 20mN/mπ =
for SSS, corresponds to a tilt angle , i.e. somewhere between the estimated tilt
angle in the trident monolayer and the tilt angle in the stable
o43 44.5τ = −
β phase (fig.10A, D). We
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 crystal layer, also for
multilayer crystals, we refer to this structure as the top layer structure (‘t’ in the figures).
At surface pressure 30mN/mπ = some domains extended as much as 5.0 ± 0.1 nm
above the surrounding SSS monolayer (fig.11A, D). 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. The fact that we did not
observe such α -domains for 20mN/mπ ≤ shows that 0 (SSS, ) 20mN/mπ α > , which is
consistent with the higher stability of the β -phase, since 0 (SSS, ) 15mN/mπ β ≈ .
Even though the SSS trident monolayer was stable at eqπ π≤ ( 15mN/mπ = ) at the
air-water interface, a LB-monolayer that was transferred changed its structure during
incubation in air. Small nuclei with thickness 3.6 ± 0.1nm ( β - phase structure) appeared on
top of the monolayer (data not shown). The same crystal growth process takes place during
incubation in air of LB-monolayer of SSS that was transferred at 20mN/mπ = (Fig.10 B, E).
Again the thickness of the newly formed nuclei was 3.6 ± 0.1nm. After 2 days incubation the
density of the nuclei was higher, but their thickness remained the same (fig.10 C, F). On a
LB-film of SSS transferred at surface pressure 30mN/mπ = initially some α - like structures
were found, but they did not change in time. The newly formed parts around them, and all
new nuclei as well, have a thickness of 3.6 ± 0.1nm, which corresponds to the β or 'β phase
77
(fig.11). Domains that were grown in the metastable, α - like, polymorph phase on the water
surface, do not spontaneously transform to the β or 'β phase because this would involve a
very slow solid - solid transformation process. We conclude that, on mica, β is more stable
than a trident monolayer, and α probably not. Stated in terms of chemical potentials this is
expressed in Eq.6 in section 4.4.2.2.
PPP behaves similar to SSS. The Langmuir monolayer of PPP does not change at the
air-water interface at 0 10mN/mπ ≤ and it is thermodynamically unstable at 0 10mN/mπ ≥ .
Domains with different thickness were found in LB monolayer, that was transferred
immediately after forced compression at 20mN/mπ = (fig.8A, D). Judging from their
thickness most of them were in α - phase, some in the β - phase. Using the estimated
effective length of 4.62 nm for a PPP molecule in tuning fork conformation, the observed
domain thickness of 3.3 ± 0.1 nm corresponds to a tilt angle , which is
between the estimated tilt angle in the trident monolayer and the tilt angle in the stable
o43.8 47.3τ = −
β
phase (‘t’ in the figures). The height of 4.5 ± 0.1 nm corresponds to fully extended PPP
molecules (4.62 nm) almost perpendicular to the substrate, similar to the crystalline α - phase.
Contrary to SSS, domains with α - like and β - like structure coexist in the LB film of PPP.
All transferred LB monolayers at 0 10mN/mπ ≥ change during incubation in air. The AFM
images showed that after 1 day in air small nuclei in α - phase and the β - phase appeared on
top of the PPP monolayer and the density of the crystals extremely increased. After 2 days the
crystals become bigger and their density decreased due to the crystals coalescence (fig.9 A).
Comparing figures 9 (in 4.4.2.1.) and 12 (in 4.4.2.2.) we see that the growth and
nucleation rates depend on the surface pressure and the nature of the triglyceride. The LB-
monolayers, which were transferred at eqπ π≥ are thermodynamically unstable in air. The
different mobility of the molecules in the trident monolayer of SSS and PPP is the main
reason for the different rate of nucleation. The increase of the nucleation rate with increasing
π reflects that the initial monolayer on mica is denser, and hence more unstable, when the
Langmuir layer is transferred at higher π . Indeed we may expect that the chemical potential
of monolayer on mica is close to the chemical potential of the monolayer on water, i.e.
( 0) (LB Lt )µ µ π= ≈ . The monolayer molecules thus can reduce their free energy from about
( )Lµ π to αµ or βµ by moving to the top of the monolayer to form new crystals in tuning
fork conformation.
78
Based on our results for isobaric velocity at the air-water interface (section 4.3.2.) we
concluded that PPP has a smaller 0π (corresponding to faster kinetics) than SSS even though
eqπ is larger. The same will be true for layers on mica. For PPP both the α and β phase are
more stable than the LB monolayer. The driving force for β - formation is larger than for α -
formation, but the kinetics are faster for α , therefore we observe both domains with α - like
and domains with β - like structure. For SSS only the β - phase seems more stable. Also for
the longest triglyceride AAA, the monolayer is thermodynamically unstable at the air-mica
interface, but the crystallization kinetics are so slow that on the time scale of days, or even
weeks, they behaves as if they were stable.
4.6. Conclusions
In this study, we investigated the behavior of three triglycerides: PPP, SSS and AAA at the
air-water interface and on a solid substrate. Based on Langmuir and AFM experiments, we
established the relation between the molecular structure and the stability of the monolayers.
Our investigations lead to the following conclusions.
At the air-water interface all investigated triglycerides form monolayers of molecules
in trident conformation. These monolayers are kinetically stable at air-water interface at
surface pressure 0π π≤ . 0π is the surface pressure below which we did not observed any
changes in the Langmuir monolayer under isobaric cnditions. We know that 0 eqπ π≥ , where
eqπ is the thermodynamic equilibrium pressure, but the amount of overestimation, i.e. the
“nucleation gap” 0 eqπ π− , can not be deduced from our data. We found that 0 10mN/mπ =
for PPP, 0 15mN/mπ = for SSS and 0 20mN/mπ = for AAA. From dynamic adsorption
isotherms, obtained at a compression rate of 1 cm/min we find a condensation pressure
8mN/mcondπ ∼ for all three triglycerides. Since eqπ must be smaller for AAA and larger for
PPP we conclude that 10mN/meqπ ≤ for all three triglycerides. Thus we arrive at the
conclusion that eq condπ π≈ .
For eqπ π> the Langmuir monolayers are thermodynamically unstable at air-water
interface. Under isobaric conditions at 0π π> slow compression was found for PPP and SSS.
The isobaric compression rate is highest for PPP and almost zero for AAA.
79
LB-monolayers can be successfully transferred onto a mica surface. Using the AFM
imaging, the thickness of the trident monolayers can be measured. We demonstrated that the
apparent thickness depends strongly on the AFM scanning force and we showed that the
compressibility of the investigated triglycerides is the same. The thickness of the monolayers,
obtained by extrapolation to zero scanning force is 1.49 nm for PPP, 1.75 nm for SSS and 2.2
nm for AAA. These monolayer thicknesses correspond to tilt angles of the molecules of 46.4o,
49.2o and 59.0o respectively. We conclude that the tilt angle increases with increasing the
length of the alkyl chains, i.e. the longer chains in the monolayer are more perpendicular to
the substrate.
The LB-monolayers transferred immediately at surface pressure 0π π> for PPP and
SSS contain domains on top of the monolayer. The molecules in these domains adopt the
tuning fork conformation and pack similar as in the crystalline α and β crystal phase.
The LB-monolayers of PPP and SSS, which were transferred at 0π π≥ are
thermodynamically unstable in air. Small nuclei in tuning fork conformation form on top of
the monolayer. For SSS they are all in β -phase, for PPP, domains with α - like and β - like
structures coexist in the LB film. The density of the crystals increases in time. We conclude
that the different mobility of the molecules in the trident monolayer of SSS and PPP is the
main reason for the different rate of nucleation and growth. For AAA, the monolayer is
thermodynamically unstable at the air-mica interface, but the crystallization kinetics are so
slow that on the time scale of days they behaves as if they were stable.
We conclude that the stability and the kinetics of Langmuir-Blodgett monolayer depend
strongly on the length of the triglycerides alkyl chains and also on the surface pressure at
which the deposition took place. The trident monolayer is the less mobile and the crystal
phase is more stable the longer the alkyl chains are. The nucleation rate increases with
increasing π , due to the fact that the LB-monolayer is denser , and hence more unstable,
when the Langmuir layer is transferred at higher π .
80
References:
[1] Charalambous, G., Doxatakis, G., In Food Emulsifiers: Chemistry, Technology,
Functional Properties and Applications; Elsvier, Amsterdam, 1989
[2] Smith, R. and Berg, J. J. Colloid Interface Sci. 74 (1980)273-286
[3] Fuente, J.F. and Rodriguez Patino, J.M. Langmuir 10 (1994) 2317-2324
[4] Fuente, J.F. and Rodriguez Patino, J.M. Langmuir 11 (1995) 2090-2097
[5] Sanchez, C.C., Rodriguez Nino, M., Rodriguez Patino, J.M., Colloids and Surfaces B:
Biointerfaces 12 (1999)175-192
[6] Garti, N. and Sato, K., In Crystallization and polymorphism of fats and Fatty Acids;
Dekker, M. New York (USA) (1988)
[7] Ollivon, M., Triglycerides. In Manuel des Corps Gras. Ed.A.Karieskind, Lavoisier, Paris
(France) (1992) p. 469
[8] Bursh, T., Larsson, K. and Lundquist, M., Chem. Phys. Lipids 2 (1968) 102-113
[9] Hamilton, J.A., Small, D.M., In Proc. Nat. Acad. Sci. USA 78 (1981) 6878
[10] Hamilton, J.A., Biochem. 28 (1989) 2514-2520
[11] Claesson, P.M., Dedinaite, A., Bergenstahl, B., Campbell B. and Christenson, H.,
Langmuir 13 (1997)1682-1688
[12] Michalski, M., Brogueira, P., Goncalves da Silva, A. and Saramago, B., Eur. J. Lipid Sci.
Technol. 103 (2001) 677-682
[13] Akita, C., Kawaguchi, T., Kaneko, F., Yamamuro, O., Akita, H., Ono, M. and Suzuki,
M., Journal of Crystal Growth 275 (2005) 2187-2193
[14] Roberts, G., Langmuir-Blodgett Films Plenum Press, New York (1990) p.21
[15] Hardy, N.J., Richardson, T.H. and Grunfeld, F., Colloids and Surfaces A:
Physicochemical and Engineering Aspects 284-285 (2006) 202-206
[16] De Jong, S., Triacylglycerol crystal structures and fatty acid conformations, a theoretical
approach- PhD thesis (1980) University of Utrecht, The Netherlands
[17] Popovitz-Biro, R., Wang, J.L., Majewski, J., Shavit, E., Leiserowitz, L. and Lahav, M.,
J. Am. Chem. Soc. 116 (1994) 1179
81
82
CHAPTER 5
Phase behaviour in binary mixed Langmuir-Blodgett monolayers of Triglycerides
Abstract
The structure of binary mixed monolayers of triglycerides: tripalmitin (PPP), tristearin (SSS)
and triarachidin (AAA) at air-water interface are investigated with the Langmuir method. The
Langmuir-Blodgett (LB) layers obtained by deposition on mica were investigated by Atomic
Force Microscopy. Based on Langmuir and AFM results the relation between the phase
behavior of binary mixed TAGs and the difference in the chain length is established. Our
experiments show that TAGs mixtures form monolayers with molecules in trident
conformation at the air-water interface, like pure TAGs. The condensation area
and the condensation pressure
263 ÅcondA =
8 10 mN/mcondπ = − are found to be the same for all mixtures
and pure systems. The sharpness of the transition from “gas” to “condensed” phase in the
Aπ − isotherms decreases linearly with the average chain length for all systems. Using AFM
carefully the monolayers thicknesses for the mixtures were measured and compared to
those of the pure systems. We found that is linear dependent on the average chain length
of the TAGs molecules. We determined the relative film compressibility and found that it
is higher for mixed monolayers ( ) than for pure systems
( ). The AFM results show phase separation in the systems PPP-SSS and
PPP-AAA, which is not complete. The solubility of the shorter PPP molecules in the “long”
(SSS-rich and AAA-rich) phase is significant. For the mixture SSS-AAA, phase separation
was not observed. In that mixture the monolayer thickness varies linearly with
composition, supporting the conclusion that SSS and AAA mix almost ideally. In general the
0d
0d
K-10.08 0.01 nNK = ±
-10.07 0.01 nNK = ±
0d
83
main driving force for phase separation is the difference in the alkyl chain length. Indeed
PPP-AAA (length difference 4 C atoms) shows the most clear phase separation. The relatively
weak phase separation in PPP-SSS and the absence of phase separation in SSS-AAA shows
that the influence of chain length difference decreases with increasing average chain length.
In air the PPP-SSS and PPP-AAA mixtures monolayer are unstable and crystals with
α - like and β - like structure are formed on top of the monolayer as in pure PPP and SSS
systems.
84
5.1. Introduction
Because triglycerides (TAGs) are the main components in the natural fats, they have been
studied for many years. Most of the research focuses on investigating the melting and
crystallization properties of TAGs. An overview of the thermodynamic and kinetic aspects of
fat crystallization was published recently [1]. It is well known that TAGs may crystallize in
the α (hexagonal, less stable), 'β (orthorhombic), or β (triclinic, most stable) form; the
nomenclature scheme following Larsson [2] as reviewed in Hagemann [3], Hernqvist [4],
Wesdorp [5], Sato [6], and Ghotra [7]. Each of these polymorphic forms consists of layers in
which the molecules have a tuning fork conformation but the orientation of the tuning forks
within the layers, as well as the packing of the layers is different. This polymorphic behavior
of TAGs strongly determines the physical properties of the fats. The monoacid saturated
TAGs (the three acyl chains are identical) are the simplest in the TAGs family. Because of
the simplicity of their structure this group has been examined in more detail than other
groups. The three basic α , 'β and β polymorphic forms have been formed [3]. Generally,
the polymorphic behaviour of TAGs with an even carbon number n are well represented by
the behaviour of PPP (n = 16) and SSS (n = 18) [3, 8-13]. The crystallization and the phase
transformation properties of these two triglycerides were found to be very similar. They only
differ with respect to the rate at which these processes occur. Both TAGs exhibit a
preferential tendency for β - crystallization. The 'β - form can only be crystallized from the
isotropic melt within a narrow temperature range and shows the typical 'β - wide-angle
diffraction pattern [13].
Binary mixtures of TAGs show far more complex polymorphic behaviour as
compared to pure TAGs. For binary TAG mixtures, the primary factors determining phase
behaviour are differences between the TAGs in chain length, the degree of saturation and
position of the fatty acid moieties, and which polymorphs are involved. Different phase
behaviour is frequently observed for different polymorphs. For example in the mixture PPP-
SSS the triglycerides are completely miscible in the less stable phases (α and 'β ) but they
form a eutectic system in the stable β - form [14-16]. The same behaviour was observed by
Takeuchi et al. [17] for the mixture LLL-MMM, where the carbon numbers in the fatty acid
chains differ by 2. When the difference in the carbon chain length differs by 4 or 6 like in the
mixtures LLL-PPP and LLL-SSS respectively, the metastable phases (α and 'β ) turn out to
85
be immiscible. Eutectic and monotectic behaviour is observed in the β - form for the LLL-
PPP and LLL-SSS systems, respectively, with the α form of SSS co-existing with the β
form of LLL under certain conditions [17].
In monolayers at a hydrophilic-hydrophobic interface, e.g. water/oil, water/air or
mica/air triglyceride molecules adopt a trident conformation (all hydrocarbon chains pointing
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
of PPP ( ) and AAA (
0 1.96 0.03 nmd = ±
0 1.49 0.02 nmd = ± 0 2.19 0.04 nmd = ± ) monolayers, respectively
[Chapter 4]. The thickness of the higher domains is somewhat less than the thickness of pure
AAA. We assume that in regions like those in Fig. 9 D and E the higher domains are AAA-
rich. The film compressibility of AAA-rich areas is higher than the one
of the pure AAA ( ). This is consistent with the hypothesis that the AAA-
rich domains contain some PPP molecules, which lower the packing density of AAA
-10.10 0.01 nNK = ±
-10.07 0.01 nNK = ±
100
molecules and thus make the monolayer more compressible. The lower domains are similar in
thickness and have the same film compressibility as pure PPP monolayers
( ).We assume that they are PPP-rich. The fact that in the phase separated
regions the AAA-rich domains occupy a significantly larger fraction of the surface area than
the PPP-rich domains (Fig.9) indicates that PPP is more soluble in AAA than AAA in PPP.
Moreover, the fact that the lower, PPP-rich, domains are similar in thickness to pure PPP
monolayers suggests that AAA is hardly soluble in PPP. Such a tendency is reasonable, since
it will be energetically more unfavourable to dissolve long molecules in a thin layer than
reverse. In some of the AFM images of PPP-AAA (1:1) monolayer we observed three
different levels (Fig.11). The highest and lowest levels corresponded to the thickness of
AAA-rich and PPP-rich layers respectively. The area at the middle level was very irregular,
showing the onset of phase separation as for PPP-SSS in Fig.3. Its thickness of about 1.65 nm
corresponds to the thickness of PPP-AAA monolayers before demixing, see Fig.9A-C.
-10.07 0.01 nNK = ±
A B
0 2.5
0
2.5
-2.5
µm
1.65 nm 1.48 nm1.92 nm
mica level
5.0
AAA B
0 2.5
0
2.5
-2.5
µm
1.65 nm 1.48 nm1.92 nm
mica level
B
5.00 2.5
0
2.5
-2.5
µm
1.65 nm 1.48 nm1.92 nm
mica level
5.00 2.5
0
2.5
-2.5
µm
1.65 nm 1.48 nm1.92 nm
mica level
5.0
Fig.11. (A) AFM height image of PPP-AAA (1:1) monolayer transferred immediately after
forced compression to 20 mN/mπ = . The corresponding cross section in (B) presents three
different domains in the mixture. 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 ~1nN.
The scale bar is 2 µm. Height differences are given by the numbers at the markers.
On top of the PPP-AAA (1:1) monolayer we observed crystals with thickness vary
from 3.5 to 4.7 nm (Fig.12). These values coincide with the measured values for PPP crystals
on top of monolayer in α and β - like phases. We also know that PPP crystallizes faster from
101
a monolayer than the longer AAA [Chapter 4]. In view of these facts we suppose that the
crystals, formed on top of PPP-AAA monolayer, are (almost) pure PPP. This conclusion
however, can not be drawn with certainty from the present AFM observation alone.
0 2.50
-5.0
5.0
µm
4.75 nm 3.5 nm
PPP level
B
5.0
A
0 2.50
-5.0
5.0
µm
4.75 nm 3.5 nm
PPP level
B
5.00 2.50
-5.0
5.0
µm
4.75 nm 3.5 nm
PPP level
5.00 2.50
-5.0
5.0
µm
4.75 nm 3.5 nm
PPP level
B
5.0
AAA
Fig.12. AFM height image of PPP-AAA (1:1) monolayer transferred immediately after forced
compression to 20 mN/mπ = (A) and the corresponding cross section in (B). The image
presents crystals on top of the monolayer. Their height is measured from the PPP level. The
scale bar is 2 µm. Height differences are given by the numbers at the markers.
5.5. Discussion
It is known from the literature that the phase behaviour in binary mixed TAGs in 3D crystal
structure strongly depends on the difference between the TAGs chain length [14-17]. When
the carbon numbers in the alkyl chains differ by 2 the mixtures are complete miscible in the
less stable forms (α and 'β ) and they form a eutectic system for the β - form. When the
difference in the carbon chain length is 4 or 6 also the metastable phases (α and 'β ) are
immiscible. Eutectic and monotectic behaviour is observed in the β - form for the LLL-PPP
and LLL-SSS systems, respectively. The α - form of SSS may co-exist with the β - form of
LLL under certain conditions [17].
In our 2D systems of binary mixed TAGs we have found a similar dependence on the
difference between the TAGs chain length. In order to compare quantitatively the
condensation area , the condensation pressure condA condπ and the isotherm sharpness , a
102
Langmuir isotherms were fitted for all mixtures and single components. At an air-water
interface we expect the binary mixed TAGs to form a trident monolayer like the single
components. This is confirmed with the Langmuir adsorption isotherms, which show that
for all investigated mixtures. The condensed pressure 63 2 ÅcondA 2= ± condπ also turned out
to be very similar for all systems (see Section 5.3.).
The value of the fitting parameter , which characterizes the sharpness of the
transition from “gas” to “condensed” phase in the
a
Aπ − isotherm, varied considerably for
different systems. In the mixtures was larger for SSS-AAA, than for PPP-SSS and PPP-
AAA. In the single components was smaller for PPP, than for SSS and AAA. We found a
linear increase of with average chain length for all systems (Fig.2C).
a
a
a
Based on our results we conclude that the smaller molecules increase the mobility in
the film and make the transition from one phase to another sharper. This was clear for the
single component films and for all mixtures. Judging on Langmuir data alone, one could be
tempted to conclude that PPP, SSS and AAA are well miscible in the monolayer regime. Our
AFM analysis however, shows that this conclusion would be incorrect. AFM thus is shown to
be essential for a sound interpretation of monolayer data.
From the AFM images of LB-films of PPP-SSS, SSS-AAA and PPP-AAA, withdrawn
at 20 mN/mπ = it is seen that the mica substrate is covered by a monolayer. Apparently the
Langmuir monolayer can be successfully transferred from the water surface in the Langmuir
trough to a mica surface to form a Langmuir-Blodgett film there.
The AFM images of LB-monolayers transferred immediately after forced compression
at 20 mN/mπ = for PPP-SSS (1:1) showed a monolayer, which in some areas was quite
homogeneous. In other areas of the same sample the onset of phase separation was observed.
In the areas with the onset of phase separation two different layer thicknesses were found. The
estimated height difference (0.2 ± 0.1nm) was somewhat smaller than the length of a two
carbon atom chain at a tilt angle of about 50o (0.3 nm) and considerably smaller than the
length of a perpendicular two carbon atom chain (~ 0.5 nm). The thickness of the monolayer
, measured from the homogeneous areas of the sample varied from 1.5 to 1.7 nm (Fig.5). 0d
We concluded that in the mixture PPP-SSS (1:1) phase separation takes place, but it is
not complete, i.e. the difference in composition of the PPP-rich and SSS-rich phases is small.
The alternative hypothesis, that the phase separation is complete, but PPP and SSS form very
103
small domains, which cannot be detected by the AFM, seems unlikely but cannot be excluded
with certainty.
The other mixture SSS-AAA, where the difference in the carbon chain length is also
two atoms behaved different from PPP-SSS (1:1). All AFM images of SSS-AAA (1:1, 1:3
and 3:1), transferred immediately after forced compression to surface pressure 20 mN/mπ = ,
showed homogeneous monolayers. We never observed the onset of phase separation. The
monolayer thickness varied linearly with composition between the monolayer thicknesses
of the single components (Fig.8). Based on these observations we concluded that SSS and
AAA mix (almost) ideally. We suppose that, due to stronger interactions between longer alkyl
chains, the sensitivity for differences in the chain length decreases with increasing alkyl chain
length.
0d
In the mixture PPP-AAA (1:1) (4 carbon atoms difference) phase separation was
observed. From the AFM images it is seen that there were areas, where only the onset of
phase separation was clearly visible and areas, where it was well defined (Fig.9). The
measured height difference between the higher and the lower domains was 0.6 ± 0.1 nm. This
difference is close to the difference in monolayer thickness of the single components PPP and
AAA with and respectively. One could assume that the highest
domains in the mixture correspond to AAA and the lowest to PPP. Precise measurement of
the thickness of the different domains in the mixture as a function of the AFM force showed
that the thickness of the higher domains is somewhat smaller than the thickness of pure AAA
monolayers. The film compressibility of the higher domains is slightly larger than for pure
AAA monolayers. The thickness of the lower domains was close to the thickness of a PPP
monolayer (Fig.10). The area occupied by higher domains in PPP-AAA (1:1) monolayer was
more than 50%. Based on these results we concluded that the higher domains in the mixture
are not “pure” AAA but are rich in AAA, with a significant fraction of dissolved PPP
molecules. Probably the lower domains are PPP with little dissolved AAA. Some AFM
images of PPP-AAA (1:1) (Fig.11) showed even other domains with thicknesses between the
highest and lowest measured thickness. These regions may correspond to PPP-AAA systems
that are in initial state of phase separation.
0 1.49 nmd = 0 2.19 nmd =
Our results for the film compressibility (Fig.13) showed that the monolayers of the
mixtures have higher compressibility than those of the pure components. We suppose that in
104
the pure systems due to the identity in the chain length the molecules pack better and thus
make the monolayer less compressible.
Film compressibility K
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
15 16 17 18 19 20 21
Average chain length (C atoms)
Film
com
pres
sibi
lity
K (n
N-1
)
Fig.13. Film compressibility as a function of the chain length for the pure components (■)
and their mixtures (○).
K
From Chapter 4 we know that at surface pressure 20 mN/mπ = PPP and SSS form
crystals in fork conformation on top of the monolayer. For SSS they are all in β - phase, for
PPP, crystals with α -like and β -like structures coexist in the LB-film. We also know that
the rate of crystallization on top of the monolayer is highest for PPP, lower for SSS and
almost zero for AAA.
The AFM images of LB-films, withdrawn at surface pressure 20 mN/mπ = of PPP-
SSS and PPP-AAA mixtures often showed domains on top of the monolayer as well. On SSS-
AAA, like on AAA, such domains were not observed. The thickness of the domains in PPP-
SSS and PPP-AAA vary from 3.5 to 4.7 nm (Fig.3 B, E). For PPP-AAA the thickness of the
crystals was measured from the PPP level (Fig.12). These thicknesses correspond to those of
PPP and SSS crystals in fork conformation in α -like and β -like structures, found in the LB-
films of the pure components. From these data we cannot deduce the composition of the
crystals that are formed in the second layer.
105
5.6. Conclusions
In this study we have obtained AFM images that reveal the structure of binary mixed TAGs
films (PPP-SSS, PPP-AAA and SSS-AAA), formed at air-water interfaces. Based on
Langmuir and AFM experiments we established the relation between their phase behavior and
the chain length of the two components. We compared the results with those for the pure
components. Our investigations lead to the following conclusions.
Compressing an extended binary mixed TAGs film at the air-water interface slowly,
starting at very low surface pressure, monolayers of TAGs molecules in trident conformation
are formed. We obtained reproducible Aπ − isotherms for all mixtures. After comparing with
the Aπ − isotherms of the single components we find that the condensation area
and the condensation pressure263 ÅcondA = 8 10 mN/mcondπ = − are the same for all
investigated systems. We find that the value of the fitting parameter , which characterizes
the sharpness of the transition from “gas” to “condensed” phase in the
a
Aπ − isotherms
increases linearly with the average chain length for all systems. For the mixtures it is larger
for SSS-AAA than for PPP-SSS and PPP-AAA. In the single components a is smaller for
PPP, than for SSS and AAA. We conclude that the smaller molecules increase the mobility in
the film and make the transition from one phase to another sharper. From the Langmuir
results we see that the thermodynamics (i.e. Aπ − isotherms) is insufficient to determine the
monolayer phase behaviour. For better understanding of the phase behavior of TAGs we used
our AFM results for mixtures and single components. AFM is shown to be essential for a
correct interpretation of monolayer data.
We obtained the AFM monolayer thickness for the pure systems and mixtures and
we find that it is linear dependent on the average chain length of the TAGs molecules. We
estimated the film compressibility and found that mixed monolayers are slightly easier to
compress ( ) than pure monolayers ( ).
0d
K-10.08 0.01 nNK = ± -10.07 0.01 nNK = ±
Based on the AFM images of LB-monolayers of PPP-SSS and PPP-AAA we conclude
that in these systems incomplete phase separation takes place. The solubility of the shorter
PPP molecules in the “long” (SSS-rich and AAA-rich) phase is significant.
For the mixture SSS-AAA, where the difference in the chain length is two carbon
atoms as for PPP-SSS we did not observe phase separation. The linear dependence of the
monolayer thickness of the composition supports the conclusion that SSS and AAA 0( )d x
106
mix almost ideally. We conclude that, due to the stronger interaction between the longer alkyl
chains, the sensibility for differences in the chain length decreases.
In air the mixtures have the same instability and form crystals on top of the monolayer
as the pure PPP and SSS systems. The thickness of the crystals on top of PPP-SSS and PPP-
AAA corresponds to PPP and SSS crystals in α - like and β - like structures, as in the LB-
films of pure PPP and SSS layers. From our data we cannot deduce the composition of the
crystals that are formed in the second layer. In the mixture SSS-AAA, like on pure AAA,
such crystals were not observed.
References:
[1] Himawan, C., Starov, V.M., Stapley A.G.F., Advances in Colloid and Interface Science
122 (2006) 3-33
[2] Larsson, K., Acta Chem Scand 20 (1966) 2255
[3] Hagemann, J.W. In: Garti, N. and Sato, K., Editors, Crystallization and polymorphism of
fats and fatty acids, Marcel Dekker, New York (1988), p. 9.
[4] Hernqvist L., In: Garti, N. and Sato, K., Editors, Crystallization and polymorhism of fats
and fatty acids, Marcel Dekker, New York (1988), p. 97.
[5] Wesdorp LH, Liquid-Multiple Solid Phase Equilibria in Fats, PhD dissertation, Delft
University of Technology; (1990)
[6] Sato, K., Fett-Lipid 101 (1999) 467
[7] Ghotra, B.S., Dyal, S.D. and Narine, S.S., Food Res Int 35 (2002) 1015
[8] Sato, K. and Kuroda, T., J Am Oil Chem Soc 64 (1987) 124
[9] Kellens, M., Meeussen, W., Riekel, C. and Reynaers, H., Chem Phys Lipids 52 (1990), 79-
98
[10] Kellens, M., Meeussen, W., Gehrke, R. and Reynaers, H., Chem Phys Lipids 58 (1991),
131-144
[11] Kellens, M., Meeussen ,W. and Reynaers, H., J Am Oil Chem Soc 69 (1992) 906.
[12] Desmedt, A., Culot, C., Deroanne, C., Durant, F. and Gibon, V., J Am Oil Chem Soc 67
(1990). 653
[13] Kellens, M., Meeussen, W. and Reynaers, H., Chem Phys Lipids 55 (1990) 163-178
[14] Lutton, E.S., J Am Chem Soc 77 (1955) 2646
107
[15] Kellens, M., Meeussen, W., Hammersley, A. and Reynaers, H., Chem Phys Lipids 58
(1991) 145-158
[16] MacNaughtan, W., Farhat, I.A., Himawan, C., Starov V.M. and Stapley, A.G.F., J Am
Chem Soc 83 (2006) 1-9
[17] Takeuchi, M., Ueno S. and Sato, K., Cryst Growth Des 3 (2003). 369-374
[18] Bursh, T., Larsson, K. and Lundquist, M., Chem. Phys. Lipids 2 (1968) 102-113
[19] Hamilton, J.A., Small, D.M., In Proc. Nat. Acad. Sci. USA 78 (1981) 6878
[20] Hamilton, J.A., Biochem. 28 (1989), 2514-2520
[21] Claesson, P.M., Dedinaite, A., Bergenstahl, B., Campbell B. and Christenson, H.,
Langmuir 13 (1997) 1682-1688
[22] Michalski, M., Brogueira, P., Goncalves da Silva, A. and Saramago, B., Eur. J. Lipid Sci.
Technol. 103 (2001) 677-682
[23] Akita, C., Kawaguchi, T., Kaneko, F., Yamamuro, O., Akita, H., Ono, M. and Suzuki,
M., Journal of Crystal Growth 275 (2005)2187-2193
[24] De Jong, S., Triacylglycerol crystal structures and fatty acid conformations, a theoretical
approach- PhD thesis (1980) University of Utrecht, The Netherlands
108
Summary
Amphiphiles are molecules, which have a hydrophilic head group and a hydrophobic tail.
They form monolayers at air-water interfaces (Langmuir monolayer). These monolayers can
be transferred on solid surfaces by the Langmuir - Blodgett technique (LB layers) and
investigated further by Atomic Force Microscopy. In this thesis we used these techniques to
study the phase behaviour of organic molecules at air-water and air-solid interfaces.
We started our investigation with the simplest amphiphilic molecules - fatty alcohols
(CnH2n+1OH, with even n = 16-24). Their behaviour can be used as a starting point for
understanding other amphiphiles with more complex structure. In Chapter 2 we discuss the
structure of binary mixed LB monolayers of fatty alcohols. The dependence of phase
separation on the difference between the chain lengths of the two components and the surface
pressure is described. Based on our results we conclude that phase separation occurs in
compressed films, if the chain length of the two components differs at least six carbon atoms.
A strong dependence of the domain shape on the surface pressure is observed. At high surface
pressure, 20 35 mN/mπ = − , the domains have tetragonal shapes. This is explained by the fact
that at higher pressures crystalline packing of molecules is favored as compared to disordered
or liquid like packing. The excess Gibbs energy ∆Gex vs. surface pressure and mole fraction is
estimated from Aπ − isotherms. In line with thermodynamic expectation, the tendency of
phase separation increases with increasing ∆Gex. The result that we observe phase separation
already in the range where our thermodynamic measurements indicated is
surprising, since the equilibrium thermodynamics predict phase separation only if
. The stability against phase separation of monolayers of fatty alcohols in non -
equilibrium isobaric conditions apparently is smaller than in equilibrium.
0.1exG R∆ ≅ T
T1exG R∆ ≥
Another interesting research in this area would be to image binary mixed monolayers
of odd fatty alcohols and investigate the conditions (the difference between their chain
lengths) for phase separation.
In the next two Chapters, 3 and 4 we describe the structure and stability of triglyceride
monolayers at air-water and air-solid (mica) interfaces. The triglycerides are typical examples
for materials, whose surface structure is different from the bulk structure. For such materials
different macroscopic properties are expected for surfaces and films as for the bulk. They
adopt a chair or tuning fork conformation in crystals and in bulk solutions, but at air-water
109
interface they rearrange in trident conformation (all hydrocarbon chains pointing in the same
direction). In the trident conformation they behave as amphiphilic molecules at the air-water
interface, though in general they are lipophilic. The idea to study these molecules in 2D
system was based on the previous study done on triglycerides at air-water interface by Bursh
and Larsson (1968). They first proposed the trident conformation for the triglyceride
molecules at an air-water interface. They found that if such a monolayer is compressed
beyond the collapse pressure, some of the molecules leave the monolayer to form new
molecular layers. Bursh and Larsson proposed a trident conformation for the first triglyceride
monolayer and a tuning fork conformation with a packing similar to that in the crystalline
state in the next layers.
By definition a Langmuir monolayer at a given spreading pressure π is
thermodynamically stable if under isobaric conditions at air-water interface it does not change
its structure, i.e. the area of the film remains constant. The pressure at which this happens is
called equilibrium spreading pressure eqπ . At spreading pressures eqπ π> one would expect
that the film area decreases, resulting in the formation of a new structure. The new structure,
depending of the film material, could be e.g. micelles in the subphase or multilayers on top of
the monolayer. Such structures can be formed at a certain pressure if the monolayer is
compressed at a constant rate. 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< .
It is well known that some Langmuir monolayers are unstable at air-water interface at
surface pressures below the collapse pressure. Indeed, one of the surprising results, described
in Chapters 3 and 4, is that triglycerides turned out to be thermodynamic instable at air-water
interface at surface pressures far below the collapse pressure. Under isobaric conditions a
molecular rearrangement process takes place which effectively thickens the film. Using
Atomic Force Microscopy for triglycerides we have shown that this process is growth of 3D
crystals of triglycerides on top of the monolayer for surface pressures eqπ π> . In Chapter 3
we present a new crystal growth model to quantitatively describe this process.
In Chapter 4 this crystal growth model for tristearin is also applied two more
triglycerides – tripalmitin (PPP) and triarachidin (AAA). It was found that the three
investigated triglycerides behave similar. We investigated the influence of the chain length of
triglyceride molecules on the stability of their films on water and mica surfaces. The trident
110
Langmuir – Blodgett monolayers were the less mobile and the crystal phase was the more
stable, the longer the alkyl chains were. The nucleation rate increased with increasing surface
pressure π .The monolayer was compressed and transferred at the sameπ .
In Chapter 5 we describe the phase behaviour of binary mixed LB - monolayers of
triglycerides. We discuss the relation between phase separation and chain length. Incomplete
phase separation was observed for systems with two or more carbon atoms difference of the
chain length of the two components. The solubility of the shorter PPP molecules in the “long”
(SSS-rich and AAA-rich) phase was significant. An interesting result is that we did not
observe phase separation in the mixture SSS-AAA, although the difference in the chain length
is two carbon atoms as for the PPP-SSS mixture. The linear dependence of the monolayer
thickness of the composition supports the conclusion that SSS and AAA mix almost
ideally. The conclusion was that, due to the stronger interaction between the longer alkyl
chains, the sensibility for differences in the chain length decreases.
0( )d x
The study in this thesis illustrates that the Langmuir – Blodgett technique and Atomic
force microscopy are useful tools in the study of phase behavior of organic molecules on
different interfaces. The results could be used as a template for investigation of the phase
behaviour of other kinds of triglycerides (mixed-acid saturated/unsaturated) and their
mixtures in 2D dimension.
I would like to finish this work with a Japanese proverb:
“Beauty is only one layer.”
111
112
Samenvatting
Amphiphielen zijn moleculen met een hydrofiele kop en een hydrofobische staart. Op het
grensvlak tussen lucht en water vormen zij zogenaamde Langmuir monolagen. Deze
monolagen kunnen door middel van de Langmuir-Blodgett (LB) techniek worden
overgebracht op vaste oppervlakken en verder worden onderzocht met atomic force
microscopy (AFM). In dit proefschrift wordt het fase gedrag van organische moleculen aan
het lucht-water en lucht-vaste stof grensvlak onderzocht met behulp van deze technieken.
Dit onderzoek startte met de eenvoudigste amphifilische moleculen - vetachtige
alcoholen (CnH2n+1OH, met even n = 16-24). Hun gedrag kan gebruikt worden als startpunt
voor het begrijpen van het gedrag van andere amphifilische moleculen met complexere
structuur.
In Hoofdstuk 2 wordt de structuur van binaire vermengde LB monolagen van vetachtige
alcoholen besproken. De afhankelijkheid van fasescheiding van het verschil in ketenlengte
van de twee componenten en de oppervlaktedruk is onderzocht. Uit deze resultaten
concluderen we dat fasescheiding optreedt in samengedrukte films indien de ketenlengte van
de twee componenten tenminste zes koolstof atomen verschilt. Een sterke afhankelijkheid van
de vorm van de verschillende domeinen op de oppervlaktedruk is waargenomen. Bij een hoge
oppervlaktedruk, 20 35 mN/mπ = − , hebben de domeinen een tetragonale vorm. Dit wordt
verklaard door het feit dat bij een hogere druk een kristallijne pakking van de moleculen
gunstiger is dat een vloeistofachtige of wanordelijke pakking. De exces Gibbs energie ∆Gex
uitgezet tegen de oppervlaktedruk en molfractie is met behulp van Aπ − isothermen geschat.
In overeenstemming met de verwachting volgens de thermodynamica neemt de neiging tot
fasescheiding toe met toenemende ∆Gex. Het resultaat dat wij fasescheiding waarnemen waar
thermodynamische metingen aangeven dat is nogal verrassend, omdat er
volgens de evenwichts thermodynamica alleen fasescheiding optreedt als . De
stabiliteit ten opzichte van fasescheiding van monolagen van vetachtige alcoholen in niet-
evenwicht isobare omstandigheden is kleiner dan in evenwichtsomstandigheden. Het zou
interessant zijn om te onderzoeken of de condities voor fasescheiding (verschil in ketenlengte)
hetzelfde zijn voor vetachtige alcoholen met een oneven ketenlengte.
0.1exG∆ ≅ RT
T1exG R∆ ≥
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In de volgende twee hoofdstukken, 3 en 4, i de structuur en stabiliteit van triglyceride
monolagen aan lucht-water en lucht-vaste stof (mica) grensvlakken onderzocht. Triglyceriden
zijn typische voorbeelden voor materialen met een oppervlaktestructuur die verschillend is
van de bulkstructuur. Voor zulke materialen worden verschillende macroscopisch
eigenschappen voor oppervlakten en films ten opzichte van de bulk verwacht. Deze
moleculen nemen een stoel of stemvork conformatie aan in kristallen en in oplossingen, maar
aan het lucht-water grensvlak herschikken deze moleculen zich in een drietand conformatie
(alle koolwaterstof ketens wijzen in dezelfde richting). In de drietand conformatie gedragen
zij zich als amphifhiele moleculen aan het lucht-water grensvlak, hoewel deze moleculen in
het algemeen lipofiel zijn. Het idee om deze moleculen in een 2D systeem te onderzoeken is
gebaseerd op een eerdere studie van triglyceriden aan het lucht-water grensvlak door Bursh en
Larsson (1968). Zij stelden de drietand conformatie voor de triglyceride moleculen aan een
lucht-water grensvlak voor en vonden dat indien zo'n monolaag voorbij de zogenaamde
'collapse pressure' wordt samengedrukt, sommige moleculen de monolaag verlaten om nieuwe
lagen te vormen. Bursh en Larsson stelden een drietand conformatie voor voor de eerste
triglyceride monolaag en een stemvork conformatie met een gelijkaardige structuur als in de
kristallijne vorm voor de volgende lagen.
Per definitie is een Langmuir monolaag voor een gegeven oppervlaktedruk π
thermodynamisch stabiel indien onder isobare condities aan het lucht-water grensvlak de
structuur niet verandert, d.w.z. de oppervlakte van de film is constant. De druk waarvoor dit
geldt wordt de evenwicht oppervlaktedruk eqπ genoemd. Bij een lagere druk verwacht men
dat de oppervlakte van de film afneemt, resulterend in een nieuwe structuur. De nieuwe
structuur (die afhangt van het materiaal), zou b.v. micellen in de subphase of multilagen
boven op de monolaag kunnen zijn. Zulke structuren kunnen bij een zekere druk gevormd
worden indien de monolaag bij een constante snelheid wordt samengedrukt. Deze druk wordt
de 'collapse pressure' genoemd. De enige manier om de thermodynamisch stabiliteit te
bepalen is door de monolaag te onderzoeken bij isobare condities voor een oppervlaktedruk
kleiner dan de ‘collapse pressure’, colπ π< .
Het is bekend dat sommige Langmuir monolagen instabiel zijn aan het water-lucht
grensvlak voor oppervlaktedrukken die kleiner zijn dan de ‘collapse pressure’. Een van de
verrassende resultaten (beschreven in hoofdstukken 3 en 4) is dat triglyceriden
thermodynamisch instabiel zijn aan het lucht-water grensvlak voor oppervlaktedrukken die
114
veel kleiner zijn dan de ‘collapse pressure’. Bij isobare condities vindt een moleculair
hershikkingsproces plaats dat resulteert in een dikkere film. Met behulp van AFM metingen
aan triglyceriden hebben we laten zien dat dit proces de groei van 3D kristallen bovenop de
monolaag voorstelt voor oppervlaktedrukken eqπ π> . In hoofstuk 3 wordt een nieuw model
geïntroduceerd voor de kwantitatieve voorspelling van deze kristalgroei.
In Hoofdstuk 4 is dit groeimodel voor tristearin (SSS) ook toegepast op twee andere
triglycerides: tripalmitin (PPP) en triarachidin (AAA). De drie onderzochte triglycerides
vertoonden een gelijkaardig gedrag. De invloed van de ketenlengte van triglyceride moleculen
op de stabiliteit van hun films op water en mica oppervlakten is onderzocht. Voor langere
ketenlengtes waren de drietand Langmuir-Blodgett monolagen het minste mobiel en de
kristalfase was de meest stabiele. De nucleatiesnelheid nam toe met toenemende
oppervlaktedruk. The monolaag werd samengedrukt en overgebracht bij dezelfde druk π .
In Hoofdstuk 5 wordt het fase gedrag van binaire LB monolagen van triglycerides
onderzocht, waaronder de relatie tussen fasescheiding en ketenlengte. Onvolledige
fasescheiding werd waargenomen voor moleculen met twee of meer koolstof atomen verschil.
De oplosbaarheid van de kortere PPP moleculen in de fase met de 'lange' moleculen (rijk aan
SSS en AAA) was aanzienlijk. Een interessant resultaat is dat wij geen fasescheiding in het
mengsel SSS-AAA hebben waargenomen, hoewel het verschil in de ketenlengte twee koolstof
atomen is (net zoals voor het PPP-SSS mengsel). De lineaire afhankelijkheid van de dikte van
de monolaag van het mengsel onderbouwt de conclusie dat SSS en AAA een vrijwel ideaal
mengsel vormen. De conclusie was dat, tengevolge van de sterkere wisselwerking tussen de
langere alkyl ketens, de gevoeligheid voor verschillen in de ketenlengte afnam.
Dit proefschrift illustreert dat de Langmuir-Blodgett en AFM technieken nuttig zijn
voor het bestuderen van het fasegedrag van organische moleculen aan verschillende
grensvlakken. De resultaten zouden als een leidraad voor onderzoek van het fasegedrag van
andere soorten van triglyceriden (vermengde-zuur verzadigd/onverzadigd) en hun mengsels in
2D gebruikt kunnen worden.
Graag beëindig ik dit proefschrift met een Japans gezegde:
'Schoonheid is een enkele laag'.
115
116
List of publications
• A.N. Zdravkova, J.P.J.M. van der Eerden and M.M.E. Snel
Phase behaviour in supported mixed monolayers of alkanols, investigated by AFM
Journal of Crystal Growth, 275 (1-2) (2005) 1029-1033
• Rick van Beek, Leonardus W. Jenneskens, Aneliya N. Zdravkova, Jan P. J. M. van der
Eerden, Cornelis A. van Walree.
Polythiophenes Containing Oligo (oxyethylene) Side Chains as a Thin Film on a ZnSe Single
Crystal Surface, Macromolecular Chemistry and Physics, 206 (10)(2005)1006-1014
• Reza Dabirian, Aneliya N. Zdravkova, Peter Liljeroth, Cornelis A. van Walree, and
Leonardus W.Jenneskens
Mixed Self-Assembled Monolayers of Semirigid Tetrahydro-4H-thiopyran End-Capped
Oligo(cyclohexylidenes), Langmuir, 21 (23) (2005)10497-10503
• Aneliya N. Zdravkova and J.P.J.M. van der Eerden
Structure and dynamics of Langmuir – Blodgett Tristearin films: Atomic Force Microscopy
and theoretical analysis, Journal of Crystal Growth, 293(2) (2006) 528-540
• Aneliya N. Zdravkova and J.P.J.M. van der Eerden
Structure and stability of Triglyceride monolayers on water and mica surfaces,
submitted to Crystal Growth & Design 10/2006
• Aneliya N. Zdravkova and J.P.J.M. van der Eerden
Phase behaviour in binary mixed Langmuir-Blodgett monolayers of Triglycerides
submitted to Crystal Growth 01/2007
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118
Acknowledgements
If someone wants to make a dream come true they need two people, one to give him the
opportunity to develop and prove themselves and another to tell them “You can do it!”
This is why first of all I would like to express my sincere gratitude to my promoter
and supervisor Prof. Dr. Jan van der Eerden for offering me the opportunity and privilege to
work in his group. Jan, thank you very much for your guidance and for everything you did to
make me a scientist. You showed me the beauty of the science and I learned so much from
you. Thank you for being understanding, helpful and patient with me.
The second person I would like to thank is my best friend Katya Ivanova, who told me
“You can do it!” and showed me the way to do it. Katya, thank you for your non - stop
support, in whatever the decisions I have made.
Next I would like to thank to my colleague and friend Thijs Vlugt who was
encouraging me all these four years. Thijs, thank you very much for your help, professional
and personal advices. You made my life in Holland much easier.
My deepest acknowledgements go now to my family, my mother Pavlina, my father
Nikola, my sister Mariana and my niece Niya, whose endless support and love encouraged me
most.
Майко, от цялото си сърце искам да ти благодаря за жертвите и лишенията,
които трябваше да понесеш, за да мога да продължа образованието си и да го завърша с
докторска степен. Мери и Ния, благодаря ви за любовта и подкрепата, без които не бих
могла да се справям с проблемите в живота. Татко, съжалявам че не си между нас, за да
можеш да споделиш с мен радостта от успеха ми. Благодаря ти, че ме научи да бъда
силна!
I would like to thank to my very good friend and first housemate in Holland Marija
Matovic for the support and the fruitful discussions about our work and the life in general.
I would like also to thank to my Bulgarian friends Nikoleta, Ivan, Petar, Boryana, and
Veselka for holding my hand and listening me complaining, when I had problems. Special
thanks to Nikoleta, who was always on line, when I needed her. Petar and Veselka, thank you
for the unforgettable time we spent together in Utrecht. Boryana, thank you for your useful
advices. Ivan, thank you for your warm hospitality and delicious meals.
And last, but not least I would like to thank to my colleagues, especially to Dennis,
Arjan, Floris, Peter Vergeer, Linda and Philipp for the good working atmosphere.
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Curriculum Vitae
Aneliya Zdravkova was born on 10th of December 1973 in Silistra, Bulgaria. In 1991 she
finished the Chemistry class in the Grammar School for Mathematics and Life Science in
Silistra, Bulgaria. She continued her education at Sofia University “St. Kliment Ohridski”,
Sofia, Bulgaria in the Faculty of Chemistry. In 1998 she obtained her Master of Science degree
in Chemistry and Physics. From 1998 until 2001 she was working as logistic controller in a
trade company for pharmaceutical products “Sanita Trading ltd.”, Sofia, Bulgaria. From 2001
until 2002 she worked in the Laboratory of Ultrastructure Research, National Institute for
Physiological Sciences, Okazaki, Japan as research assistant. In 2003 she started her PhD
research in the Condensed Matter and Interfaces group, Utrecht University, The Netherlands
under the supervision of Prof. Dr. Jan van der Eerden. The results of this research are
presented in this thesis.
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