Johannes Kepler Universität Linz
MONOLAYERS BY THE
Bachelor Thesis in PR Biophysik
2 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
2 INTRODUCTION ......................................................................................... 3
2.1 Forming Langmuir monolayers ................................................................................ 3
2.2 From surface tension to surface pressure ............................................................... 3
2.3 Measuring the surface pressure .............................................................................. 5
2.4 Visualizing the surface pressure .............................................................................. 7
2.5 Considerations on the stability of monolayers ..................................................... 10
3 Materials and Methods ........................................................................... 11
3.1 Setup and experimental preparations .................................................................. 11
3.2 Guideline for the use of the Fromherz through ................................................... 13
4 Experiments and Results ......................................................................... 16
4.1 Experiments on different cleaning approaches .................................................... 16
4.2 The platelet: paper or platinum? .......................................................................... 17
4.3 Hysteresis and the relaxation effect of monolayers ............................................. 18
4.4 Studies on the contact angle of the Wilhelmy plate ............................................ 19
4.5 Pressure-area isotherms of phospholipids ........................................................... 21
5 Conclusion & Outlook ............................................................................. 25
6 References .............................................................................................. 26
5 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
2.3 MEASURING THE SURFACE PRESSURE
asically, there are two established approaches in order to measure the surface pressure:
The Langmuir balance makes use of the differential force acting on a float separating pure
water from a film-covered liquid. It should not be discussed in detail here because the instrument
used for the present report is based on the so-called Wilhelmy plate. This is a rectangular plate in
vertical position, touching the subphase on the lower side and connected to a spring on the upper
side. The contact with the liquid leads to a downward force due to the surface tension and this
movement results in a displacement of the spring. This displacement is recognized by a linear
displacement transducer which converts the positional information into an electrical signal. The
measurement principle is schematically depicted in Figure 1 following below.
Figure 1: The Wilhelmy plate. The plate is in direct contact with the liquid, pulling it downward as a result
of the surface tension. Moreover, the plate is connected with a spring whose displacement is finally
recognized by the displacement transducer. Knowing the dimensions of the thin plate (L, w, t) and the
subphase immersion depth h, the total force acting on the plate can be calculated which also depends on the
contact angle θ at the contact zone between plate and liquid. © G. Roberts, Langmuir-Blodgett Films
This method is therefore based on an absolute measurement of the force as a result of the surface
tension acting on a plate partly immersed in the subphase. Various materials are possible for this
plate and among them are glass, mica, quartz and platinum. But in most cases the plate consists of
filter paper due to its simple handling: Every measurement can be started with a new sample of filter
7 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
2.4 VISUALIZING THE SURFACE PRESSURE
s mentioned before, the compression of the monolayer leads to a change of the subphase
surface tension and thus the related surface pressure. Plotting the surface pressure against
the area available for the monolayer gives a characteristic function which conveys a lot of
information about the molecules forming this film. These curves are recorded at constant
temperature and therefore called pressure-area isotherms. Usually the monolayer is compressed at a
very low speed which guarantees pseudo-equilibrium conditions. Real equilibrium values would be
possible by performing a step-by-step movement, but this is in most cases rather unpractical for an
At the beginning of such a measurement, due to the extensive spreading by the volatile solvent,
the amphiphilic molecules are far apart and do not interact. This state is therefore comparable with a
two-dimensional gas. So it takes some time, till the compression of the monolayer actually leads to a
rise in surface pressure. At this point, also called lift-off point, the molecules start to interact
between each other, leading to the interplay of attractive forces between the hydrocarbon chains at
the one hand and repulsive forces between the polar head groups on the other hand. Due to this
confinement the molecules reorganize and the monolayer undergoes several phase transitions
similar to gas, liquid and solid states. At the end of this process the monolayer shows a highly
organized structure with all molecules distinctly aligned .
For these isotherms to become comparable, the plot is performed against the area per molecule
which can be calculated with the assumption that the number of monolayer molecules at the surface
does not change in the time course of an experiment. This is in fact a simplification, as there is some
loss of molecules into the subphase, nevertheless it is a valid approximation. So the area per
molecule is calculated through dividing the trough area by the amount of molecules, finally
expressed in A² or nm² per molecule.
There are some other side effects which are neglected by this calculation. On the one hand it does
not take into account that the surface of the film-covered subphase has some kind of curvature near
the surrounding through walls. Moreover, the filter paper is also covered by the monolayer,
increasing the film area by a small amount .
Nevertheless, these isotherms provide intensive structural information about the monolayer, as
depicted in Figure 2 following below.
8 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Figure 2: Monolayer states. Pressure-area isotherms exhibit a distinct shape that can be related to
different conformations of the monomolecular film. These are the gaseous (G), liquid expanded (LE), liquid
condensed (LC) and solid state (S), this way in an order of increasing density of the molecules. Further
increase of the surface pressure leads to the collapse of the film where multiple layers start to ride on each
other. The occurrence of a first order transition from the liquid expanded to liquid condensed state strongly
depends on the nature of the film forming molecules: So phospholipids tend to show this transition, whereas
fatty acids do not. © Girard-Egrot, Langmuir-Blodgett Technique
An examination of the pressure-area isotherm gives an insight into the structural characteristics of
the monolayer. The gaseous phase at the beginning is followed by a transition to the liquid state
which usually occurs at very low surface pressures and is therefore not resolved by the instrument in
most cases. Further compression leads to the liquid expanded phase (LE) that starts at the lift-off
point and continues with an increase till another transition is reached. This can be observed by
fluorescence microscopy or Brewster angle microscopy which reveals the occurrence of condensed
states . This first-order transition from liquid expanded to liquid condensed (LE-LC) is
characterized by a plateau in the corresponding isotherm, though it does not occur at every observed
monolayer. In fact, there has been intensive discussion on the occurrence of such a plateau region:
Especially single-chain organic compounds like fatty acids often lack the horizontal section which led
to speculations that higher order transitions could occur. However, it is also possible that simple
impurities are responsible for the fact that the plateau is not resolved .
9 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Being in the liquid condensed phase the monolayer now assumes properties of a solid and the
area per molecule is similar to the molecular cross-section which is about 50 A² per molecule for
phospholipid monolayers. The transition occurs gradually and is accompanied by the coexistence of
expanded and condensed domains which can be seen with the use of Brewster angle microscopy .
Further compression finally leads to the collapse of the monolayer: Due to the immense force the
monolayer loses its integrity and the molecules ride on top of each other. The onset of this collapse is
variable and depends on the history of the film, the temperature and the speed of the compression
process. The collapse pressure together with the shape of the pressure-area isotherm provides
extensive information on the stability and properties of the monolayer and can be used to study
phase transitions and conformational transformations .
However, the shape of the pressure-area isotherms is variable and depends on the characteristics
of the monolayer molecules. The interplay of attractive and repulsive forces determines the state of
the monolayer: Van-der-Waals forces between the hydrocarbon chains support the cohesion
between the molecules, whereas repulsive ion-ion interactions between charged headgroups disturb
the integrity of the monolayer. Following the example from Figure 2, fatty acids have only one
hydrocarbon chain, resulting in a dominant gaseous state, while phospholipids have two chains and
show a distinctive liquid expanded and liquid condensed state. In addition to the number of
hydrocarbon chains, the nature and length is important: The existence of a double bond disturbs the
cohesion between the lipid molecules, leading to a more dominant liquid expanded state and a
collapse occurring at a lower surface pressure. In a similar way, the length of the hydrocarbon chain
is decisive: The shorter the chains, the weaker are the van-der-Waals forces which again results in
diminishing solid conformations .
In addition, the pH of the subphase has a decisive influence on the monolayer state: This is
obvious for the ionizable headgroups of some phospholipids, such as the choline group. Hereby, the
pH determines by reversible protonation whether these groups are charged or not. The subsequent
repulsion between charged groups affects the cohesion of the monolayer, shifting it to the liquid
expanded state .
Mixtures of different lipids yield isotherms that are more complicated to interpret. Basically, the
isotherm should be the algebraic sum of the isotherms obtained from the single components.
However, a deviation of this pattern is likely due to attractive or repulsive forces between the
components as well as due to their miscibility .
10 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
2.5 CONSIDERATIONS ON THE STABILITY OF MONOLAYERS
t is important to keep in mind that the monomolecular film is usually not in thermodynamic
equilibrium which can be only maintained by a very slow step-by-step movement of the
barriers. Therefore the monolayer is rather in a metastable state than in a true equilibrium. The so-
called equilibrium surface pressure (ESP) is defined as the pressure generated when a crystalline
sample of the film-forming material is placed on the subphase surface. Following from this, at a
higher pressure there will be a tendency of the monolayer to aggregate into crystals. However, this
equilibrium surface pressure is usually not attained in the time course of an experiment, which
means that monolayers can be held relatively constant at pressures exceeding the ESP.
In fact, the monolayer has to bear a tremendous mechanical stress: For example, a surface
pressure of 100 mN/m acting on a layer of molecules 2.5 nm high is equivalent to a three-
dimensional pressure of 400 atmospheres .
There are various factors having an influence on the stability of the formed monolayer. As
mentioned before, the monolayer forming molecules used for these experiments are biological
phospholipids, which can be readily regarded to be insoluble in the subphase. Therefore the effect of
dissolution of the amphiphilic molecules into the liquid should have a minor influence on the stability
of the monolayer.
In addition to these mechanisms the stability of the monolayer also depends on the subphase
characteristics, especially the presence of multivalent ions and the pH value, as well as the solvent
evaporation time. The stability of the monolayer can be checked by constant-area measurements:
The trough area is reduced to a value corresponding to the monolayer state of interest and
consequently kept constant, while the surface pressure is recorded over time. Another method is to
perform hysteresis experiments: The monolayer is compressed to a defined surface pressure and
relaxed to the original state. After some time another pressure-area isotherm is recorded which is
usually shifted to lower areas in comparison to the former isotherm. Even stable monolayers exhibit
hysteresis characteristics, due to a difference between the aggregation mechanisms of the film
molecules during compression and the relaxation mechanisms during decompression . The
hysteresis effect on monolayers is further discussed in chapter 4.3.
11 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
3 MATERIALS AND METHODS
3.1 SETUP AND EXPERIMENTAL PREPARATIONS
ll pressure-area isotherms were recorded with the use of a circular multicompartment
trough designed by Peter Fromherz. It consists of eight distinct compartments which are
separated by walls slightly lower than the edges. The two barriers are independently movable and
also allow the transport of the monolayer to a different subphase. The trough with a diameter of 19
cm and a total area of 362 cm 2 as well as the barriers are made of PTFE, and the whole assembly is
protected by a Plexiglas cover in order to avoid disturbances by dust and other air pollutants. In
addition, the trough is set on a vibration isolation system, as the stability of the monolayer can be
affected by vibrations. The measurements were carried out under the control of a cooling water
thermostat which guaranteed a temperature of 22 ± 2°C. The temperature in the subphase was
additionally monitored by a digital temperature sensor inserted in a subphase filled compartment
outside of the through area surrounded by the two barriers, so that the monolayer was not affected
by the sensor.
The trough area, as defined by the position of the two barriers, together with the surface pressure
determined by a Wilhelmy plate connected to a linear displacement transducer, were picked up by
an electronic device (Monofilmmeter by Mayer Feintechnik, Göttingen). The Monofilmmeter allowed
a precise control of the electric motor moving the two barriers by setting a defined speed for either
one or both barriers at the same time. An appropriate calibration of both parameters was performed
regularly, following the instructions given in the corresponding manual by P. Fromherz . The
voltage signals acquired by the Monofilmmeter were digitalized by an ADC (National Instruments
USB 6210) which enabled the integration into LabVIEW 2009 software. Thus by creating adapted
virtual instruments a user-defined and software-based visualization of the pressure and area data
was possible: The surface pressure was plotted against the trough area in real-time, while the
collected data was stored in simple text files. This way the pressure and area values could be loaded
into Sigmaplot 11 which was used for the creation of the pressure-area isotherms, as presented in
the following chapters.
The contact angle measurements were performed by using an additional assembly of two LED
lamps for illumination of the filter paper and a webcam connected to the PC. The quality of the
images was optimized with the use of AMCap by switching to a black-and-white mode and adjusting
the brightness and contrast.
12 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
The following figure shows a schematic illustration of the circular trough used for the experiments.
Figure 3: Sketch of the Fromherz trough. The Langmuir trough designed by P. Fromherz is special for its
circular design. In addition, both barriers are movable which makes it possible to move the monomolecular
film to another compartment that may contain a different subphase. The trough has been modified a few
ways: A temperature sensor located in the subphase has been added to achieve a more direct way to control
the temperature. Moreover, two LED-lights as well as a webcam have been assembled in order to perform
the contact angle measurements.
Avoiding contamination by surface active substances dominates all experimental preparations.
First of all, special attendance should be given to the quality of the water used for cleaning as well as
for the subphase. In general, surface active impurities even in the ppb range can cause major
problems, as demonstrated for a concentration as low as 10 -8 M for monolayers of fatty acids on an
aqueous subphase . Considering this requirement to the pureness of water, type 1 ultrapure
Milipore water was used which is deionized by ion-exchange resins and further cleaned with reverse
osmosis to yield 18.2 MOhm∙cm and a TOC value below 10 ppm. However, not only producing, but
also storing the water is critical: On exposure to the atmosphere the water will absorb carbon dioxide
which can significantly influence the measurements . As a consequence, the water taken from the
filtering system was stored in closed glassware bottles and freshly prepared every day.
Still, there are more points to consider concerning the contamination problem. The use of plastic
material is generally disregarded due to concerns about the plasticizers. Therefore, glass ware was
used as far as possible instead, though - to be exact - even glass is not completely inert on the
13 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
contact with water . Glass ware from the dishwasher was additionally rinsed with Milipore water,
as the water used for the washing procedure was not trustable. Moreover, in order to avoid the use
of regular plastic piping, Hamilton syringes were used instead.
All lipids were purchased from Avanti Polar Lipids in the powdered form and stored in portions of
10 mg at a temperature of -80°C. During the preparation of an experiment one of these lipid portions
was dissolved in chloroform to achieve a concentration of 10 mg/ml. As hexane was used as
spreading agent, the chloroform had to be evaporated and the lipids finally dissolved in hexane.
Though hexane is an uncomplicated and easy-to-use spreading agent that is known to evaporate
completely , one can experience problems due to the relatively low solubility of phospholipids in
hexane. As a consequence, different spreading agents have been tried as well, such as mixtures of
hexane with chloroform or alcohols. However, the use of alcohols is critical, though the effects
depend on the lipid .
The subphase was either pure water (18.2 MOhm Milipore) or a Tris-buffer (25 mM Tris, 250 mM
KCl, pH 8), as given in the description of the corresponding measurement. The buffer solution was
prepared with chemicals provided by Sigma-Aldrich in small amounts and stored in closed glass
bottles which were held in a fridge.
3.2 GUIDELINE FOR THE USE OF THE FROMHERZ THROUGH
t was the special task of this bachelor thesis to optimize the handling of the Fromherz through.
The intensive study of adequate literature and - above all - a considerable number of trial &
error experiments on the procedures finally yielded the following step-by-step protocol for an
appropriate use of the trough:
Thorough cleaning of the trough is required: All compartments should be rinsed with EtOH
and Milipore water. More intensive cleaning can be achieved by using a Hellmanex-solution
(2%) which should be left on for at least 45 minutes and then washed off with Milipore water.
Now the required amount of compartments can be filled with the respective subphase.
Considering the area of one compartment (about 45 cm 2 ), filling 3 - 4 compartments is
recommendable for most experiments. The filling is a crucial step as the liquid level should be
neither too high nor to low. To be exact, the level must not be higher than 1 mm above the
outer wall of the compartments.
14 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Eventually the filter paper can be hanged onto the wire of the electro balance. The paper gets
fully wetted and the pressure signal should stay constant now.
Addition: The filter paper can be cleaned in advance by storing it in clean water for a few
hours. The surface has to be sucked off before taking out the filter paper to get rid of surface
The surface of the subphase liquid has to be cleaned as well. This can be done by simple
aspiration, with the use of a very thin pipette tip connected to an appropriate pump. The
barriers should be brought together for a smaller surface.
After this cleaning procedure the surface has to be checked again for being free of
contaminants: While moving the barriers together (at a practical speed, such as 1.2), the
surface pressure should be constantly zero. Deviations of about 0.1 - 0.2 mN/m are still
acceptable, though this is perhaps only achieved after several repetitions of the cleaning
Now the lipid solution* (in hexane or in chloroform) has to be applied on the subphase
surface. This is achieved by using a Hamilton syringe: The tip has to be very near the surface
without touching it so that the drop of the lipid solution merges with the surface. This is
another crucial step which requires sensitivity to avoid collapse of the liquid film.
Addition: The collapse usually happens at the walls separating the compartments. A quick-
and-dirty method to make the best of the experiment is to reconnect the film with the tip of
the syringe by pulling the liquid over the wall.
After waiting for 15 to 20 minutes the solvent should have evaporated and the recording of a
pressure-area isotherm can be started: The lower the speed of the movable barrier, the more
reproducible will be the results. For practical use the value 0.6 seems to be appropriate which
corresponds to 7.2 A²/molecule∙minute.
* The concentration of the lipid solution should be chosen under the following considerations: When
using four compartments, 10 µg of lipid will be a suitable amount. The volume should be large
enough to take it up accurately with the syringe and low enough to avoid film collapse.
15 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Some points should be figured out in detail:
Within this protocol the use of filter paper for the Wilhelmy plate was anticipated. The role of the
Wilhelmy plate material is discussed in detail in chapter 4.2. Moreover, the amount of lipid to be
applied should meet the following considerations: On the one hand the monolayer formed after
spreading the lipid solution should be in the gaseous state, in fact the surface pressure should be
below the lift-off point mentioned in the introductory chapter. With a look on comparative data from
literature, this can be readily calculated with the through area surrounded by the barriers and the
number of lipid molecules, as determined with the molecular mass.
On the other hand practical requirements concerning the application of the lipid solution should be
met. If the volume of the solution is too small, it can’t be taken up accurately with the syringe.
Conversely, if the volume is too large, the drop formed during release by the syringe can cause a
collapse of the subphase surface: The liquid film on the walls separating the compartments is very
thin and can thus collapse very easily. This is also a problem when filling the compartments with the
subphase - a larger subphase volume and subsequently a higher liquid level will reduce the risk of the
mentioned surface collapse, but the subphase will maybe run over the surrounding walls.
Concerning the spreading technique it is also worth mentioning that there exists another method
that is not based on dropping the solution but on dipping the tip a little bit into the subphase and
then releasing the lipids . However, it has been found that preventing contaminations of the
monolayer are much more important for the success of the experiment: The specific spreading
technique is in fact not critical . The choice of the spreading solvent depends on the experimental
requirements, but in general it should evaporate quickly and completely. Moreover, it should not
interact with the monolayer forming material, except of dissolving it efficiently. Finally, it is self-
evident that the density should be below the one of the subphase liquid. Typical spreading materials
include n-hexane, chloroform, benzene, ethyl ether and mixtures with alcohols .
Finally, a few comments should be made concerning the compression speed of the barriers. The
speed has a decisive impact on the slope of the pressure-area isotherms, by influencing the structural
transitions within the monolayer. A fast compression usually leads to an increased occurrence of
condensed lipid domains. Thus, a low barrier speed generally leads to good reproducibility of the
measurements, but on the other hand it is unpractical: An extended compression time results in
more evaporation of the subphase, besides a low speed barely mimics the effective situation in
biological membranes . Considering these facts, a compromise was found by setting the speed to
7.2 A²/molecule∙minute, as mentioned in the protocol above.
16 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
4 EXPERIMENTS AND RESULTS
4.1 EXPERIMENTS ON DIFFERENT CLEANING APPROACHES
ne of the most important aspects when handling a Langmuir trough is proper cleaning.
With respect to this fact, studies on the efficiency of different cleaning procedures were
performed. In addition to various substances for washing the through, the figure below also shows
the influence of the aspiration technique for cleaning the surface of the subphase, as depicted in step
4 of the above protocol.
Figure 4: Experiments on cleaning methods. Pressure-area isotherms have been recorded on a Milipore
water subphase after cleaning with different cleaning solutions: ethanol, acetone and Hellmanex solution
(2% in Milipore water, 90 min. incubation). No monolayer forming material has been added, which means
that the surface pressure should be constantly zero, except for contaminations. Moreover, one of the
isotherms has been recorded after performing the aspiration technique.
The aspiration technique seems to be very important to remove unwanted monolayer-forming
substances, such as dust and other disturbing pollutants originating from the surrounding air. Though
it is itself a possible source of contamination, as the experimentator has to move the capillary tube
by hand over the subphase surface to be cleaned, the positive effects of this technique are
17 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
overwhelming. Alternatively, the tube can be fixed at an appropriate distance of the subphase
surface . Comparing the different cleaning solutions, the Hellmanex solution seems to be the most
effective one. Nevertheless, it should be taken into account that an incubation time of 90 minutes is
not always of practical use. Therefore, for a couple of subsequent measurements the cleaning
procedure with ethanol seems to be the method of choice.
4.2 THE PLATELET: PAPER OR PLATINUM?
he Wilhelmy plate method as used in these experiments can be carried out with different
materials for sensing the surface tension force: The probably most popular ones are
platinum and filter paper. In the course of this work both materials were used for recording pressure-
The Wilhelmy balance generally suffers from a number of disadvantages. The main problem is the
contact angle between the plate and the liquid surface which should be constant during the
measurement. This requires a complete wetting of the plate which is difficult for some materials.
Another point of concern is the position of the plate: It has been shown that the presence of the
plate can perturb the monolayer flow during compression . Therefore it would be ideal to
arrange the plate in the middle of the monolayer and to compress the film symmetrically by both
A direct comparison of the two materials tested here leads to the following considerations: The
platinum flag is chemically inert and does not interact with the monolayer. In contrast, this cannot be
guaranteed when using the filter paper, as it may be partly dissolved in the subphase in the course of
a long ongoing measurement and it perhaps releases surface active substances. The filter paper has
to be prepared before the measurements: The paper has to be cut into right-sized pieces without
contaminating it which requires careful handling. These pieces can be cleaned before by keeping
them in Milipore water for a while and sucking the surface before taking them out. Besides, this
procedure also ensures a complete wetting, as this normally also takes some minutes. Such
preparations are not necessary when using the platinum, as it can be simply cleaned with ethanol
after each measurement. Nevertheless, platinum leads to another problem: As mentioned in the
introduction, the platinum flag has to be roughened to cope with the non-zero contact angle. This
can be done with the use of sandpaper, as performed here .
However, due to ongoing problems concerning the reliability of the results when using platinum,
the experiments described here were performed with filter paper.
18 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
4.3 HYSTERESIS AND THE RELAXATION EFFECT OF MONOLAYERS
sually, only one pressure-area isotherm is recorded after formation of the monolayer.
Expanding the monolayer after the compression does not immediately reverse the
structural changes during the compression. Therefore the isotherms of successive compression-
expansion cycles show a hysteresis effect, shifting the curves to lower areas. However, the changes
are reversible, when the monolayer gets time to recover its structure after expanding: This is known
as relaxation effect of monolayers. In general, the hysteresis depends on the surface pressure at
which the compression is stopped, i.e. the shift to lower areas is more significant, the higher this
pressure is .
Figure 5: Experiments on the relaxation effect of monolayers. The graph shows isotherms of DPhPC on a
Milipore water subphase. The black curve represents the first measurement on the monolayer, while the
others were taken after a certain time lapse, as valued in the legend. In addition to the time lapse the
temperature of the subphase at the beginning of each pressure-area recording is given as well.
Hysteresis is generally a typical effect of phospholipids, due to a variety of reasons: The most
prevalent one is the movement of monolayer molecules into the subphase, but others are the loss of
residual spreading solvent, incomplete spreading of the monolayer (molecules are injected below the
surface and slowly reach the interface), contact angle changes, chemical reactions at the interface
(e.g. with CO2 or O2) and conformational changes of the monolayer molecules .
19 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Above all, the monolayer is very sensitive to ionic impurities which are especially relevant for lipids
extracted from biological material, such as E.coli PLE .
So the hysteresis effect is a key player in all monolayer experiments. Moreover, it might be
interesting to know, how long a monolayer needs for a full relaxation, as this would allow the
recording of a couple of subsequent pressure-area isotherms on the same monolayer. Figure 5 above
shows the results of such an experiment to find out the minimum relaxation time of a given lipid.
A close look at this figure gives thorough information about the hysteresis on DPhPC: The isotherm
recorded after 30 minutes (yellow curve) shows a clear shift to lower areas, as compared with the
first isotherm on the monolayer (black curve). However, the isotherms after 45 minutes (blue curve)
and 60 minutes (green curve) are very similar: Obviously this time lapse is enough for a full relaxation
of DPhPC monolayers. The higher temperature of the subphase at the blue curve seems to make no
clear difference for this very experiment, though this does not allow a general statement.
No comparative literature for the relaxation behavior of DPhPC was found, but for DPPC, which is
structurally similar except of four methyl-groups at the fatty-acid chains, a relaxation time of 30
minutes has been reported .
4.4 STUDIES ON THE CONTACT ANGLE OF THE WILHELMY PLATE
s stated above, the contact angle between the subphase liquid and the filter paper can have
a valuable influence on the detected forces. It should be constant especially during the
compression of the monolayer, because otherwise the calculation of the surface pressure, as
depicted in the introductory chapter, is not valid anymore.
Contact angle changes are also associated with the occurrence of hysteresis effects. So far it has
been elucidated that the head group of the phospholipid plays a decisive role: Choline head-groups
as in DPhPC lead to a nonzero contact angle, whereas other groups such as ethanolamine give nearly
non-zero contact angles .
In order to get more information on this matter, contact angle measurements on DPhPC
monolayers were performed: As mentioned before, photos of the filter paper were taken with a
webcam, while illuminating the scene with two LED lamps. These photos were further processed
with AMCap software, i.e. the black & white mode was turned on and the contrast increased in order
to achieve optimal results.
20 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Figure 6: Contact angle photos. A DPhPC monolayer on Milipore water was formed and photos of the
contact zone between filter paper and subphase surface were taken in different positions of the barriers: just
before starting the compression of the monolayer, at the end and at three positions between. The two
photos above show the plate at the beginning (left) and at the end (right).
Figure 7: The contact angle during monolayer compression. (left) The contact angle between the plate and
the surface of a DPhPC monolayer was obtained at different through areas enclosed by the barriers: at the
beginning and the end of the compression and at three positions between. Each value represents an average
of four contact angles at the same position, attained by repeated measurements under identical conditions.
(right) The contact angle can be used to determine the time after which the solvent has evaporated. The
contact angle was obtained as before by taking photos and analyzing the intersection zone. However, no
compression was made here, but the photos were taken after an increasing period of time. In addition to a
measurement on the DPhPC monolayer, one more was performed after applying a drop of pure hexane. The
red line represents the contact angle at the pure subphase (water).
21 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
The contact angle was then attained by analyzing these photos with Golden Ratio software: The
intersection between the plate and the subphase meniscus was approximated by aligning two lines
whose intersection angle represented the contact angle. In order to obtain statistically relevant data,
these measurements were repeated and the average of the corresponding contact angles was
In figure 7 above we can observe that the contact angle is in fact not constant during the
compression, but varies between 36 and 44 degrees. The contact angle measurements, as done here,
are not very exact, nevertheless this is a significant change of the contact angle. Therefore it is
inevitable to draw the conclusion that the surface pressure calculation, as outlined in the
introductory chapter is rather an approximation than the real situation.
Additionally, the observation of the contact angle can be used to elucidate the influence of the
lipid solvent, as an effect of the reduction of the surface tension. Before beginning the measurement
it should be clear that the solvent has evaporated completely in order to avoid cross-talk on the
surface pressure. Looking at figure 7 we can conclude that it takes about 25 minutes till the solvent
has evaporated completely, so this time span should elapse before the compression is started.
However, it should be noted that this value depends on the volume to be applied, which was in this
case in the range of a few micro liters.
4.5 PRESSURE-AREA ISOTHERMS OF PHOSPHOLIPIDS
n intensive amount of data was collected on pressure-area isotherms of different lipids,
being the central topic of this work. In general, the pressure-area isotherms of analogue
measurements were hard to reproduce, the reason for which is not fully understood. It is possible
that the experimental setup is not optimized to the necessary extent and that the inadequate
handling lead to systematic errors. However, it is known that monolayers of biological lipids are more
difficult to reproduce in comparison to classical experiments on fatty acids. Especially the collapse
phenomenon depends on various factors and is thus sometimes tricky to reproduce .
22 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Figure 9: DPhPC isotherms. (left) Pressure-area isotherms of DPhPC monolayers on a Tris-buffer subphase
were recorded under identical experimental conditions, at a temperature of 23±1°C and a barrier speed of
7.2 A²/molecule∙minute. (right) The analogue isotherm of a DPhPC monolayer on a Milipore water subphase
at 20±0.5°C and a barrier speed of 3 A²/molecule∙minute, taken and edited from .
In figure 9 we can observe that the pressure-area isotherms are not absolutely identical, though
they were recorded under the same experimental conditions. However, as mentioned before the
isotherms of biological lipids are not easily reproducible. Moreover, the similarity to the comparative
isotherm from literature, given in the insert of the illustration, is obvious. They bear widespread
analogy concerning the lift-off point as well as the shape itself: Both have neither a plateau region,
nor a bend in the curve, suggesting the monolayers collapsed directly from the liquid expanded state,
without any occurrence of an intermediating liquid condensed state. Such a behavior is usually
observed at monolayers of unsaturated phospholipids, but this is not unexpected due to the four
methyl groups of DPhPC, which lead to steric hindrance similar to a double bond . Though the
collapse pressures are different, the mean areas of the head group of a lipid molecule, as determined
by the area per molecule at the collapse pressure, come to an agreement (60-70 A²).
In addition to DPhPC, isotherms of E.coli PLE have been recorded. Being an extract of the inner
membrane of E.coli cells, it consists of the three lipids phosphatidylethanolamine (PE),
phosphatidylglycerole (PG) and cardiolipin. Thus the corresponding pressure-area isotherm is
recorded on a mixed monolayer which is characterized by packing states of much higher complexity
compared to homogenous monolayers. This complexity is further increased by the occurrence of
double bonds at the acyl chains of some cardiolipin species . From the biological point of view,
the surface pressure value 30 mN/m is important, as this pressure corresponds to the packing state
in the membrane .
23 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Figure 10: PLE isotherms. (left) Pressure-area isotherms of PLE monolayers on a Tris-buffer subphase were
recorded at a temperature of 23±1°C and a barrier speed of 7.2 A²/molecule∙minute. (right) The analogue
isotherm of a PLE monolayer on a Tris-buffer subphase at 25°C and a barrier speed of 3 A²/molecule∙minute,
taken and edited from .
Again, there are obvious similarities to the isotherm from literature concerning shape and lift-off
point and in addition, the collapse pressures are roughly the same. In this case the differences in the
experimental conditions, i.e. the barrier speed and the subphase temperature had no significant
influence on the corresponding isotherms.
Finally, isotherms of BPS monolayers were recorded, exactly speaking monolayers of SOPS (1-
stearoyl-2-oleoylphosphatidylserine) which are illustrated in Figure 11 following on the next page.
Having a closer look on the structure, a significant difference towards the other phospholipids
presented before can be observed: SOPS contains a double bond in cis-configuration which leads to
steric hindrance upon tight packing in a monolayer. This structural asymmetry has far-reaching
consequences which can be readily illustrated by a comparison of the isotherms recorded on SOPS
monolayers resp. on such of the same phospholipid with saturated acyl-chains.
The isotherms of BPS are generally more difficult to compare with the curve from literature, as the
subphase temperatures are significantly different. This may also be the reason for the fact that there
are relatively few similarities between the isotherms. Beside the collapse pressure there is an
obvious deviation in the shape: The kink in the isotherms of SOPS (Fig. 12a) could indicate a
structural transition from a liquid expanded (LE) to a liquid condensed state (LC). Moreover, the
isotherm of DPPS (Fig. 12c) shows the impact of the cis double bond: Due to the steric hindrance the
collapse pressure of SOPS (48 mN/m in 12b) is essentially lower than the collapse pressure of the
fully saturated DPPS (63 mN/M in 12c). In addition, there is a liquid condensed state (LC) at the DPPS
monolayer (Fig. 12c) that is missing in the SOPS monolayer (Fig. 12b).
24 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
Figure 11: BPS isotherms. The illustration shows isotherms of SOPS monolayers (a, b) and of DPPS
monolayers (c), as well as the structure of SOPS or stearoyloleoylphosphatidylserine, as drawn with the use
of the ChemSketch software. The isotherms of (a) are results of this work, whereas (b) and (c) were taken
from literature . The lipids were spread on a Tris-buffer (a) resp. Milipore water (b, c), at a subphase
temperature of 23±1°C (a) resp. 37°C (b, c) with a barrier speed of 11.4 A²/molecule∙minute (a) resp. 7.2
A²/molecule∙minute (b, c).
25 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
5 CONCLUSION & OUTLOOK
he target of this work was the optimization of the handling of the Fromherz trough and its
practical application to obtain data on phospholipid monolayers. This has been achieved, as
presented by the protocol for the use of the Fromherz trough and the pressure-area isotherms
illustrated in the chapter before. In addition, the impact of the contact angle and the material of the
platelet, as well as the hysteresis effect on monolayers were investigated. However, there is more to
do in order to validate the measurements on the trough concerning accuracy and correctness of the
collected data: Future studies could be focused on pressure-area isotherms of fatty acids, as there is
extensive comparative literature available. Moreover, there are still several ways to further improve
the experimental procedures. For instance, advantages and disadvantages of different spreading
solvent could be analyzed, as well as a variation of the lipid concentration.
Besides, the reproducibility of pressure-area isotherms of phospholipids is still an issue to be
focused on. As mentioned before, there are reports stating that monolayers of biological material
yield poorly reproducible isotherms, but nevertheless it is not clear, whether the variability between
two consecutive measurements under identical experimental conditions is only the result of the
nature of the monolayer forming material. After all, systematic errors could also play a significant
role, leading to non-homogenous data.
Further studies could also investigate and compare different techniques to form the monolayer:
Instead of the classical spreading technique described in this work, monolayers can be established by
fusion of liposomes suspended in the subphase with the air-water interface. These so-called
Schindler membranes can also be formed with proteoliposomes, leading to protein-containing
monolayers. Though a couple of experiments were performed on such differently formed
monolayers, these measurements have to be continued in order to yield representative data.
However, the optimization of the trough’s handling is certainly the most valuable contribution of
this work which forms a trustable foundation for further measurements.
26 CHARACTERIZATION OF PHOSPHOLIPID MONOLAYERS BY THE FROMHERZ TROUGH
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