Brewster Angle Microscopy: Keith J. Stine

Brewster Angle Microscopy
Keith J. Stine
University of MissouriSt. Louis, St. Louis, MO, USA
1 Introduction: The Role of Brewster Angle Microscopy

in Supramolecular Chemistry of Monolayers
1
2 Principles and General Applications of Brewster
Angle Microscopy
2
3 Application of Brewster Angle Microscopy to the
Study of Monolayers of Macrocyclic Compounds
11
Study of Monolayers of Chiral Compounds
20
Study of Monolayers in which Hydrogen-Bond
Complex Formation Occurs at the Water Surface
24
6 Emerging Developments in the Application of
Brewster Angle Microscopy
26
7 Conclusion
27
References
28
INTRODUCTION: THE ROLE OF

BREWSTER ANGLE MICROSCOPY IN
SUPRAMOLECULAR CHEMISTRY OF
MONOLAYERS
The study of monolayers of amphiphilic compounds at the

waterair interface has spanned many decades. Much of
the early work on monolayers has been summarized in
the monograph by Gaines.1 The early work on monolayers
that elucidated the basic thermodynamic behavior of singlechain amphiphiles, lipids, sterols, and other molecules set a
foundation of the understanding of basic monolayer behavior that could be applied as the intense interest in studying
the behavior of supermolecules at the waterair interface
arose over the past two decades. Spreading monolayers at
the waterair interface provides a two-dimensional environment in which the packing and interactions of supermolecules can be studied and in which the mean molecular separation between molecules can be systematically
varied and their interaction with different species present
in the subphase studied. While measurements of surface
pressuremean molecular area ( A) isotherms, surface
potentialmean molecular area isotherms (VA), and
other surface-pressure-related phenomena such as collapse
and relaxation at constant area are necessary to characterize
a monolayer system, the revitalization of monolayer science
that began in the 1980s was driven by the emergence of new
spectroscopic and imaging methods that could be applied
to monolayers in situ at the waterair interface. Amongst
these new methods have been fluorescence microscopy,
infrared reflection absorption spectroscopy (IRRAS), grazing incidence X-ray diffraction (GIXD), and Brewster angle
microscopy (BAM).
The revelation that monolayers exhibited a rich and
varied microstructure of coexisting domains in the micronsto-millimeter size range was first revealed by the application
of fluorescence microscopy to monolayers of phospholipids2, 3 and subsequent application to monolayers of fatty
acids and their esters.4 The fluorescence microscopy technique requires the introduction of a small percentage of a
fluorophore-labeled lipid or amphiphile to provide contrast
between coexisting phases. The orientation of the fluorescent probe has also been exploited successfully in many
cases to image regions of varying amphiphile tilt orientation relative to the interface.5 The method of fluorescence microscopy has limitations with respect to monolayer
studies of supermolecules; for example, it is unlikely that
Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.
This article is 2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc040
Techniques
fluorescent probe lipids would partition in a meaningful way

in a monolayer of a macrocyclic amphiphile. Fluorescence
microscopy does not lend itself to studies of the change in
monolayer morphology upon interaction with compounds
such as guest molecules injected into the subphase. The
method is also not suitable for studies of monolayers formed
by adsorption from the subphase, or of monolayers of
molecules with structures very different from the classical hydrocarbon chain amphiphiles and lipids, or of spread
monolayers of polymers or biomacromolecules. However,
the method is still broadly used for studies of phospholipid monolayers in particular as it is less expensive than
the present state-of-the-art Brewster angle microscopes and
provides superior lateral resolution compared to all except
the latest state-of-the-art BAM instruments.
BAM was introduced in the early 1990s by the groups
of Honig and Meunier.6, 7 The method is applicable to
adsorbed or spread monolayers of any type of surface-active
species. It can be used to image changes in microstructure due to collapse, multilayer formation, or complexation
with species from the subphase beneath the monolayer.
BAM is a broadly useful tool for studying supramolecular
phenomena at the waterair interface. The information provided by BAM is also useful in characterizing monolayers
that will then be transferred to solid supports and become
LangmuirBlodgett films (see LangmuirBlodgett Films,
Techniques). In this chapter, we first present the basic
principles of BAM. The full mathematical derivations for
describing the optical reflectivity observed in BAM are
not presented. It is assumed that the reader will most
likely make use of a commercially available BAM and
thus detailed instrument plans have not been presented.
In this chapter, examples are presented from the application of BAM to the study of monolayers of macrocyclic
compounds, monolayers in which supramolecular complex
formation occurs, monolayers of chiral amphiphiles, and
others. Each general class of compounds studied by BAM
provides a useful insight into how the technique can be
applied in different situations. The chapter does not review
the extensive BAM studies conducted on single-chain fatty
acids, fatty esters, and alcohols related to phase diagram
determination. The chapter also does not review the numerous studies of monolayers of membrane lipids carried out
using BAM. However, examples from these two fields of
study are used to illustrate specific applications of BAM.
The general aspects of monolayer preparation and phase
behavior are not reviewed here in detail and readers with
no background in monolayers should consult other reviews
first. Given that there are many hundreds of papers reporting
use of BAM, referencing is not comprehensive. Some new,
emerging applications of BAM are described and some general insights summarized at the end.
2.1
PRINCIPLES AND GENERAL

APPLICATIONS OF BREWSTER
ANGLE MICROSCOPY
Basic principle of Brewster angle microscopy
The basic principle of BAM is that for p-polarized light

incident on the interface between air and water; there
is a specific angle at which the reflectivity displays a
sharp minimum.8 If the airwater interface was truly
perfect and flat, then at the Brewster angle, the reflectivity
would be zero; however, the presence of capillary waves
and a density transition zone gives the result of a very
low but nonzero reflectivity. The reflectivity detected in
BAM, represented by R = IR /I0 , where I0 is the incident
light intensity and IR is the reflected light intensity, is
typically near 106 in the vicinity of the Brewster angle.
The Brewster angle, B , is determined by the condition
B = tan1 (n1 /n0 ), where n1 is the refractive index of
the subphase and n0 = 1.00 is the refractive index of air.
The refractive index of the aqueous subphase will depend
weakly on temperature, the wavelength of the incident light,
and the presence of other components in the subphase.
Using a reported refractive index9 of 1.33 211 for water at
T = 20 C and = 632.8 nm, one obtains a value of B =
53.10 . Laser beams are collimated well enough to provide
illumination at a precise angle and minimal reflectivity.
Many BAMs use a HeNe laser at = 632.8 nm, but
other wavelengths such as the 514.5 nm line from argon
ion lasers, and others, have also been used.
When an organic monolayer or thin film is present
on the water surface, its effect is to introduce a thin
layer of different refractive index, typically with n 1.45;
this change in the refractive index results in violation
of the Brewster angle condition and an enhancement in
reflectivity. The increase in reflectivity due to the presence
of an organic layer depends upon its thickness, refractive
index, and whether it is isotropic and is described by
a single value of the refractive index or whether the
film is anisotropically ordered and must be described
by a 4 4 dielectric tensor.10 The optical theory behind
BAM is closely associated with that used to describe
the ellipsometry of multilayered thin films on nonmetallic
substrates.11
2.1.1 Reflectivity of the waterair interface

The reflectivity is calculated using the Fresnel reflection
coefficient rp , which is defined as Erp /Eip , where Erp and
Eip are the complex amplitudes of the electric vectors of the
reflected and incident p-polarized light respectively.11 Values may also be calculated for rs = Ers /Eis , the reflection
DOI: 10.1002/9780470661345.smc040
Brewster angle microscopy

coefficient for s-polarized light. The expressions for these
reflection coefficients are given by the following equations
in which 0 is the angle of the incident beam measured from
the normal direction and 1 is the angle of the refracted
beam relative to the normal direction:
tan( 0 1 )
tan( 0 + 1 )
sin( 0 1 )
rs =
sin( 0 + 1 )
rp =
(1)
(2)
The reflectivity Rp = |rp |2 for p-polarized light and Rs =

|rs |2 for s-polarized light. The starting point for the
derivation of (1) and (2) makes use of Maxwells equations
and requires conservation of the electric and magnetic
components of the incident, reflected, and refracted beams.
The angles 0 and 1 are related by Snells law. When
0 + 1 = 90 , meaning that the angle of incidence and
angle of refraction are perpendicular, (1) becomes zero as
the denominator approaches infinity. Using the condition
0 + 1 = 90 together with Snells law, n0 sin 0 =
n1 sin 1 , will result in the condition for the Brewster
angle, tan B = n1 /n0 . Figure 1 shows the variation of
reflectivity with angle for illumination of the waterair
interface by both p-polarized and s-polarized radiation
calculated using (1) and (2).
2.1.2 Reflectivity of an isotropic monolayer

The treatment of reflectivity from a single isotropic layer
on top of a subphase in ambient can be treated by
describing the system in terms of the refractive index of
100
Reflectivity (R)
101
s -polarized
102
103
104
the ambient n0 , the refractive index n1 , and thickness d1 of

the layer, and the refractive index now denoted as n2 for the
subphase. The reflected beam is now the sum of the waves
reflected from the ambient/layer interface and from the
layer/substrate interface.11 A monolayer at the waterair
interface is subject to the thin-layer approximation in that
d , where is the wavelength of the incident light. The
thin-layer approximation is described further in Section 2.9
and is the basis for quantitative applications of BAM to
estimations of film thickness.
2.1.3 Reflectivity of an anisotropic monolayer at the

waterair interface
When significant anisotropy arises due to a structural feature of the monolayer such as long-range tilting of the
molecular orientation or an anisotropic lattice structure,
the monolayer can no longer be described by a single
value of refractive index as the interaction with the incident light now depends on the orientation of the molecules
with respect to the plane of incidence. The consequences for
reflectivity in BAM measurements have been considered in
a number of publications for tilted phases of hydrocarbon
chain bearing amphiphiles such as fatty acids or phospholipids; these are described in more detail in Section 2.5. The
calculations are complicated and are not presented here. The
reflectivity of the p-polarized light varies with the magnitude of the tilt angle and its direction and is calculated in
terms of two refractive index values, no and ne , the ordinary
and extraordinary values, as well as the tilt angle and the
azimuthal angle relative to the plane of the incident light.
Addition of an analyzer to the BAM introduces a strong
dependence of Rp on the analyzer angle that allows for
good contrast in images based on anisotropy, whereas the
variation in reflected intensity without an analyzer due to
anisotropy is much weaker.9 Calculations of how the reflectivity varies with orientation and analyzer angle have been
presented using a 4 4 matrix approach10, 11 ; in this study,
anisotropy is also reported at the glassair interface in a
LangmuirBlodgett film in which the molecules form an
orthorhombic lattice.11
p -polarized
105
106
2.2
20
40
60
General features of the Brewster angle

microscopy experiment
80
Incident angle (q0)
Figure 1 Reflectivity as a function of incident angle for both

s-polarized (blue line) and p-polarized light (red line) as a
function of angle at the waterair interface for a refractive index
of n = 1.332 for water (20 C, = 632.8 nm) and n = 1.000 for
air. The curves are calculated using (1) and (2) in the text together
with Snells law.
The Brewster angle microscope is positioned over the

monolayer trough such that the incident laser beam hits the
part of the water surface where the monolayer is present
at all stages during the experiment. The surface pressure
() and (if used) surface potential (V ) transducers also
need to be positioned in this segment of the trough. The
trough itself may be either commercial or home built; in
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Techniques
either case, the space to accommodate the BAM must be

considered. The BAM and trough should be on a heavy
secure table such as a large balance table for best image
quality and reduction of vibrations; a vibration isolation
table can be used if available. Positioning the trough onto
an XY translation stage allows observation of different
regions of the monolayer, which is of interest as monolayers
are often heterogeneous. Without the ability to laterally
translate either the trough or the BAM, one must rely on
viewing whatever regions of the monolayer drift beneath
the laser spot and this may not be fully representative
of the range of structures present. The refracted beam
will hit the trough bottom, which is generally Teflon and
will scatter light that can contribute to the background
brightness in the image. This refracted beam should be
blocked, and this can be done with a piece of black
glass resting on the bottom of the trough at a small
inclined angle. The alignment of the BAM is critical in
terms of both the angle of incidence and the adjustment
of the polarizer. Most BAMs can adjust the incident
angle with a resolution of 0.001 . The water surface
should be illuminated with p-polarized light oriented in a
plane perpendicular to the surface. The refractive index of
water varies slightly over the temperature range of interest
for most monolayer studies (1040 C). The refractive
index of water for = 632.8 nm has been reported as
n = 1.33 282 at 10 C ( B = 53.12 ) and n = 1.32 972 at
40 C ( B = 53.06 ). The refractive index variation with
subphase composition is also significant if experiments are
carried out on buffers or electrolyte solutions of various
concentrations. Optimization can be made by viewing the
image from just the aqueous subphase and making small
adjustments in the angle of incidence until the lowest
reflectivity is obtained. Recent commercial BAMs carry
out these alignments automatically via software control.
Figure 2 shows the basic components of a Brewster angle
microscope.
BAM is useful for assessing the cleanliness of the
water surface prior to the spreading of a monolayer. If
Incident beam
CCD camera
Analyzer
Polarizer
Subphase
q0
impurities are present, they often appear as fluid streak-like

domains caused by greasy dirt and the water surface appears
as if a monolayer was already present. Dust and particulates
on the water surface show up as brightly reflecting spots
that indicate a need to clean the surface. Repeated surface
aspiration and cleaning of the trough should result in a
featureless water surface under BAM that is then ready
for spreading of the monolayer. A fringe pattern may be
visible due to the inhomogeneous profile of the laser beam.
These observations compliment traditional tests of water
surface purity in which the barrier is run forward to a
high compression ratio, and it is checked if the surface
pressure increases tangibly according to some criterion
such as registering no surface pressure change for a 5 : 1
compression. Running the barrier forward across a clean
water surface should also result in the observation of no
features by BAM.
The images from the BAM experiment can be recorded
and stored as video files in a computer. The setup will
either have a stand-alone video monitor, or the BAM
images can be viewed in real time on the computer
screen depending on what software is available. The
new commercial BAMs come with software for direct
image acquisition and processing. These newer BAMs are
especially powerful compared to older models such as the
BAM-1plus in that they correct for the loss of focus at
the edges of the images caused by imaging at an angle.
In earlier designs, the distortion of the aspect ratio in the
image due to imaging at an angle was corrected by tilting
the CCD chip inside the camera.
A home-built design that removed the problem of the
images being in good focus only along a strip across the
middle of the image was introduced; this required the use
of a specially designed and custom-fabricated objective.12
The incorporation of the objective in a home-built BAM
provided real-time imaging with a resolution of 1 m and
images in focus across the entire image plane. A highly
compact design for a home-built BAM in which the entire
instrument is oriented vertically has also been introduced
as it is especially suitable for positioning in a trough used
for LangmuirBlodgett deposition.13 A low-cost, compact
design constructed from standard optical components has
also been reported.14
Objective
2.3
q1
Figure 2 The basic geometry and components of a Brewster

angle microscopy experiment.
Selection of instrumentation for Brewster

angle microscopy
A number of companies offer commercial BAMs that

can either be coupled with a commercial trough or used
with a home-made trough. It is possible to build a basic
BAM given sufficient technical expertise in optical instrument design; however, most researchers use commercially
DOI: 10.1002/9780470661345.smc040

available BAMs that are now quite sophisticated. The quality of images produced by BAM instruments has improved
significantly for the most recent (and most expensive) models. Nanofilm Technologie GmbH (NFT) made BAM available commercially with the BAM-1 and the BAM-1plus in
the early 1990s. The BAM-1plus corrects for distortion of
the image aspect ratio but not for the image focusing issue
and has a lateral resolution of 4 m. Images reported using
this instrument and similar home-built designs are generally
out of focus on the two ends of the image and are sometimes cropped to show only the central portion which is in
better focus. The BAM-2plus from NFT is a more powerful BAM introduced in the later 1990s. It is superior to the
BAM-1plus in that the lateral resolution is now 2 m. The
instrument also provides images that are in focus within the
entire field of view. The BAM-2plus keeps the images in
focus by scanning the orientation of a mirror so that parallel strips of the image come into focus sequentially and are
then reconstructed. The BAM-1plus and BAM-2plus are
equipped with analyzers for imaging anisotropy in monolayers. A simpler Mini-BAM instrument from NFT without
an analyzer and a wide field of view of 4 mm 6 mm is
also available. A number of more powerful instruments are
now available, such as the nanofilm ultrabam (NFT, now
called Accurion), which provide fully focused images in
real time and are capable of performing quantitative analysis of reflectivity in BAM images. The nanofilm ep3bam
provides high-resolution images and is able to be upgraded
to function as an imaging ellipsometer for use on other substrates. Another current BAM that is available is the Optrel
Bam 3000 from KSV Instruments, which provides 2 m resolution, an adjustable analyzer, and a scanning option for
building focused images. The Optrel and Accurion instruments allow adjustment of the Brewster angle over a wide
range allowing in principle for imaging on surfaces other
than water such as glass slides.
2.4
General features of monolayer phase

behavior observed by Brewster angle
microscopy
The interpretation of BAM images involves the use of

knowledge from fields including the basic phase behavior of monolayers, the range of basic domain forms seen in
monolayer systems, nucleation and domain growth behavior
in monolayers, and binary phase behavior if mixed monolayers are being studied. It is important for the user to
understand how to interpret surface pressurearea ( A)
isotherms that should be measured simultaneously with the
BAM experiment. A keen eye for making and comparing
observations under different monolayer conditions is necessary. While it is often relatively easy to observe some interesting looking images upon starting a monolayer project
using BAM, going beyond qualitative description should

be the goal. While it is not possible to predict monolayer
domain shapes and their behavior from molecular structures, what is seen using BAM generally can be interpreted
using known patterns and principles.
The monolayer is first spread at high molecular area
where the surface pressure is low and then the monolayer is
compressed to record the A isotherm. At such high areas
and very low surface pressures, one generally observes
coexistence of the two-dimensional gas phase (G) with
either a two-dimensional liquid-like phase often referred
to as the liquid-expanded (LE) phase or with a form
of condensed phase, which may be of varying degree
of order from that of a liquid-condensed (LC) phase, as
found in fatty acid and ester monolayers to solid-like
phases found when much stronger intermolecular forces
are present.5 The gas phase is of low molecular density
and is dark under BAM. An LE phase appears as bright
circular islands in a dark background or sometimes as a
two-dimensional foam structure that resembles a network
of cells.12 Upon compression of monolayers with coexisting
gas and LE phases, the fraction of the surface covered by
the gas phase shrinks until a uniform bright field is seen;
ideally, this occurs just as the surface pressure begins to
rise tangibly. In contrast, monolayers showing coexistence
of a gas phase with an LC or solid-like phase appear
differently under BAM. If the second phase is LC or
more solid-like, then generally bright domains are seen that
are not round in shape and can have angular boundaries
with the gas phase. These domains may have anisotropic
ordering (Section 2.5), which can be confirmed by rotating
the analyzer away from 0 and looking for variations in
the reflectivity from domains or regions within domains.
Domains of weakly ordered LC phases often appear bright
and rounded in shape. It is also possible to observe the
coexistence of gas, LE, and LC phases at very low surface
pressures and below a certain temperature as monolayers
possess a triple point and when out of equilibrium the phase
rule can be violated.4, 15
When a solid-like and a gas phase are found coexisting upon spreading, the A isotherm remains flat and
then rises. The gas phase is squeezed out but may persist between domains if they have difficulty in merging.
The merging of solid-like domains upon compression is
not always a smooth process as they are pushed into each
other, sometimes fragment, and reorganize. When the LE
and gas phase are present upon spreading, compression will
first bring the monolayer into the LE phase, which should
appear as an all bright uniform field of view. If there is a
kink or plateau in the surface pressure isotherm, then the
emergence of domains of a condensed phase should become
visible by BAM. The emerging phase will be of higher
reflectivity, and could also potentially possess anisotropic
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Techniques
ordering within domains visible by BAM. If the condensed

phase has a tilted orientation of hydrocarbon chains, then
anisotropy will be generally be visible if the analyzer angle
is set well away from 0 . Under some conditions and especially for more weakly ordered LC phases, the domains
that form may be rounded shapes. In more highly ordered
phases, as found in many of the examples cited in this
chapter, many other domain shapes can be observed. These
are often observed as growth forms and appear as highly
branched dendritic or fractal-like structures. It is possible
that these nonequilibrium growth forms will relax in shape
over time but often they are not observed to do so; for
many monolayers, the equilibrium domain shapes are never
observed. The observation of dendritic growth forms that do
not relax over tens of minutes or longer indicates the formation of a highly ordered phase. Special precautions, such as
the use of enclosed troughs, and against effects including
subphase evaporation or contamination would need to be
taken to carry out observations of monolayers over periods
of many hours. Further compression will generally bring
the monolayer fully into the condensed phase.
In observations of mixed monolayers by BAM, a major
question of interest is the miscibility of the components.
BAM observation of morphologically distinct regions, especially when they resemble what is seen in the pure monolayers of the components, is a sign of immiscibility. When
the components are miscible, generally a uniform morphology of a somewhat intermediate nature is observed.
The phase behavior can be complex, as immiscibility can
be partial or complete and can depend upon temperature,
monolayer composition, and surface pressure. For example,
BAM was used to study mixed monolayers of 7-(2-anthyrl)heptanoic acid (2A7) and myristic acid at a series of mole
ratios and the monolayers of the pure species.16 The A
isotherm of 2A7 was steep and indicated the formation
of a solid-like phase consistent with BAM observation of
domains resembling irregular fragments in coexistence with
the gas phase. The A isotherm of myristic acid showed
a plateau identified as the well-known phase transition from
the LE phase into the coexistence of the LE and LC phase.4
Rounded condensed-phase domains with internal anisotropy
were seen as expected for a tilted LC phase. It was noted
that addition of small amounts of 2A7 to the monolayer
prevented the formation of these domains indicating some
miscibility at low mole fraction of 2A7. Equimolar mixed
monolayers at near-zero surface pressure showed coexistence of foam-like regions characteristic of myristic acid
and regions rich in 2A7, which in contrast appeared darker
as this is a shorter molecule than myristic acid. Upon compression of the equimolar mixed monolayer, alternating
dark and bright stripes formed. Upon collapse, bright bands
formed across the darker stripes, which suggested that the
darker stripes were rich in 2A7.
BAM contrast inversion due to relative thickness changes

in coexisting monolayer regions of a phase-separated
binary mixture was observed during compression of mixed
monolayers of a triaroylbenzene derivative (C8METAB,
see structure in Figure 7) consisting of a 1,3,5-tris(4hydroxy-benzoyl)benzene core ether linked to methyl
octanoate side chains, and methyl stearate, and is shown in
Figure 3.17 Mixed monolayers of C8METAB and methyl
stearate of mole ratio 1 : 2 showed round domains rich in
methyl stearate at higher molecular areas. These domains
were brighter than the surroundings and were internally
anisotropic, as seen in condensed-phase domains of methyl
stearate or similar fatty esters.18 The methyl stearate
domains became darker than the surrounding field as the
compression proceeded. It was concluded that compression
forces the C8METAB molecules to reorient from a flat to a
standing orientation on the water surface now resulting in a
greater reflectivity for the C8METAB regions than for the
methyl stearate domains. Collapse was then visible in the
C8METAB-rich regions, as evidenced by the emergence of
grainy bright pattern.
2.5
Brewster angle microscopy observation of

domain anisotropy
When the refractive index of a monolayer varies with

direction in the plane of the monolayer, anisotropy is
present such as is the case for phases of amphiphiles
with hydrocarbon chains that are tilted. In addition to a
magnitude of tilt, the tilt will have a direction defined by
the azimuthal angle. Fatty acids, esters, and alcohols exhibit
complex phase diagrams with a variety of vertical and tilted
condensed phases, which have been studied extensively and
reviewed.19, 20 Within the domains of a tilted phase, there
can be regions with the same azimuthal angle that are large
enough to be imaged by BAM. When the analyzer of the
BAM is set at an angle other than 0 , regions of different
azimuthal angle will have different reflectivity and there
will be contrast between them in the BAM image. In tilted
condensed phases, there are examples of discontinuous
changes in the azimuthal direction and hence reflectivity
between segments as well as examples of continuous
variation in reflectivity across a segment as the azimuthal
tilt direction gradually varies. There are other reasons, such
as an asymmetric lattice structure, that can give anisotropy
in the refractive index in a monolayer, but anisotropy due to
molecular tilt is by far the most widely studied source and
is also observed in the condensed phases of monolayers of
membrane lipids as well as synthetic amphiphiles.21, 22 The
observations of these domain segments of varied tilt angle
had been achieved using polarized fluorescence microscopy
prior to the introduction of BAM.6
DOI: 10.1002/9780470661345.smc040
(a)
(b)
(c)
(d)
Figure 3 Brewster angle microscopy images of monolayers of mixtures of monolayers of the triaroylbenzene derivative C8METAB
and methyl stearate showing both an example of contrast inversion and also of collapse in a binary mixed monolayer; in this
experiment, the angle of the analyzer is set at 30 . A small X near a domain in panel A locates a condensed-phase domain
showing some internal anisotropy. Reproduced from Ref. 17. Elsevier, 2008, and the caption reads as follows: Brewster angle
micrographs of mixed monolayers of methyl stearate and C8METAB of mole ratio 1 : 2 C8METAB/methyl stearate on water at 295 K
during compression. (A) 69 A 2 per molecule, (B) 53 A 2 per molecule, (C) 37 A 2 per molecule, (D) 22 A 2 per molecule. The scale of the
images is 900 m 700 m.
Monolayers of methyl eicosanoate were examined in

a tilted condensed phase L2 in which the tilt direction
was toward next-nearest neighboring molecules.23 In this
phase, six-armed star defect patterns are seen in which
the domain is divided into six regions, each resembling
one-sixth of a pie shape. Each segment or slice of the pie
has a different reflectivity indicating a different azimuthal
direction. These domains undergo a blooming transition
upon cooling that originates from the center of the domain
and during which the azimuthal directions change but the
segmentation is maintained. An analysis of the reflectivity
variation was presented that depended on nine parameters
including the dielectric constant of water, elements xx and
yy of the molecular dielectric tensor, monolayer thickness,
angle of incidence of the laser beam, the analyzer angle,
and a small tilt of the polarization angle away from the
p-orientation that improved image contrast. These were
processed in a detailed optical model to obtain reflectivity
variations that could be compared with those observed and
used to determine the best fitting values of the monolayer
thickness and tilt angle.
Monolayers of ethyl palmitate and ethyl stearate are
attractive systems in which to study internal domain
anisotropy by BAM.18 Monolayers of these compounds
have two-phase coexistence regions between the LE and LC
phases and the domains of the tilted condensed phase are

round and 100200 m in size.21 In these condensed-phase
domains, the most regular structure that could be observed
was the round domain divided into six equal segments like a
sliced pie with each segment having a different reflectivity,
as seen in Figure 4. Analysis of the reflectivity differences
was consistent with a structure in which the azimuthal direction was parallel to the domain periphery and jumped by
60 at each segment boundary. In these images, the analyzer
angle was set at 60 and, when rotated to 60 , the relative
brightness of the segments in the domain reversed, with the
brightest segment becoming the darkest and vice versa; the
observation of this inversion in contrast confirms the presence of anisotropy. This experiment is a good illustration
of the value of recording BAM images at different analyzer
angles including pairs at positive and negative angles of the
analyzer of the same magnitude.
In these monolayers, not all domains were organized
into neat six segmented pies; others had corner-notched
segments of different brightness or segments with zigzag
boundaries and some domains appeared to have one
orientation within them. Dendritic growth of the condensedphase domains could be observed in monolayers of ethyl
stearate when they were compressed more rapidly, but in
these monolayers the dendrites were transient forms that
DOI: 10.1002/9780470661345.smc040
Techniques
(a)
(b)
(c)
(d)
(e)
(f)
Figure 4 Liquid-condensed-phase domains in monolayers of

ethyl stearate showing anisotropy, the monolayer is in the LE
phase and liquid-condensed phase coexistence region. Reproduced
from Ref. 18. American Chemical Society, 1996, and the figure
caption reads as follows: The most regular texture in ethyl stearate
monolayers is a subdivision of the domain into six parts of uniform
orientation: (a) analyzer angle of about 60 ; (b) analyzer angle
of about 60 ; (c) parallel polarizer and analyzer; (d) crossed
polarizer and analyzer. [In half the domains of this type, the
arrangement of the segments is as in parts a and b. The other
domains of this type are inverted images of those of parts a and b;
(e) analyzer angle of about 60 ; (f) analyzer angle of about 60 .
The bar represents 100 m.
relaxed into circular domains within minutes. These growth

forms were observed to have anisotropic ordering with
different branches having different reflectivity. The contrast in reflectivity due to segments of different orientation
diminished as the magnitude of the tilt angle decreased.
Compression of these monolayers ultimately resulted in
a transition to a vertically oriented phase in which the
anisotropy disappeared. A subsequent study of monolayers
of palmityl acetate included both BAM and GIXD data and
showed a transition within the domains from the azimuthal
tilt directions being oriented perpendicular to parallel to the
segment boundaries.22
Anisotropy within condensed-phase domains can also

be found in which the azimuthal direction of tilt is
varying continuously such that a gradation or pattern
of gradually changing reflectivity is observed within a
domain. Monolayers of the phospholipid dimyristoylphosphatidylethanolamine (DMPE) were found to exhibit cardioid (kidney)-shaped condensed-phase domains within
which the azimuthal direction bended gradually within the
domain.24 Analysis of the variation in the gray levels in the
images with the analyzer set at 60 allowed determination
of the magnitude of the tilt and the azimuthal direction.
The analysis makes use of an expression for the reflectivity as a function of the angle of incidence, angle of the
analyzer, and dielectric anisotropy in the monolayer represented by and a = that was obtained as a
second-order approximation and is less onerous than the
full optical formalism.
A novel extension of BAM useful for the determination of tilt angles in condensed phases was achieved by
adding a photon counter and an autocorrelator to a homebuilt BAM in a method the authors called Brewster angle
autocorrelation spectroscopy.25 The experiment makes use
of the fluctuation in the intensity of the reflected light as
domains of different azimuthal tilt direction drift under
the laser spot when the analyzer is set at an angle of
80 . A derived equation relates the magnitude of the tilt
angle to the limiting value of g2 =< (I < I >)2 > /
< I >2 where the brackets denote time averages of
reflected intensities. The method was applied to determine
the tilt angle variation near a phase transition in octadecanol between tilted and untilted condensed phases. The
obtained variation in the tilt angle with surface pressure as
the monolayer crossed the phase transition agreed very well
with that determined by GIXD experiments.26
2.6
Multilayer formation
BAMs is an ideal method for observing films at the

water surface, which exhibit coexisting regions of discrete
numbers of molecular layers such as monolayer together
with trilayer and thicker. Such a situation is commonly
encountered when studying compressed monolayers of thermotropic liquid crystals (see Self-Organization and SelfAssembly in Liquid-Crystalline Materials, Soft Matter),
which are generally rod-shaped molecules, or bent banana
shapes, or disc shaped in the case of discotic liquid crystals.
The rod-shaped molecules exhibit a plethora of phases in
three dimensions that include the isotropic phase, nematic
phase, and a wide variety of vertically oriented, tilted, or
hexatically ordered smectic phases as a function of decreasing temperature.27 Studies of liquid crystals at the waterair
DOI: 10.1002/9780470661345.smc040
here a range of coexisting multilayer structures was also

seen.30
2.7
Figure 5 BAM image of multilayer regions coexisting in spread

and compressed films of the liquid crystal 8CB. Reproduced
from Ref. 31. American Physical Society, 2007, and the figure
caption reads as follows: Brewster angle microscope image of the
coexistence of 8CB multilayers. The background is 8CB trilayer.
The layer reflectivity increases with thickness, so that different gray
levels correspond to different thicknesses. The rippled variation in
color within one domain is due to variations in illumination. The
black scale bar is 1.0 mm.
interface provide a unique environment in which to observe

orientational ordering and domain dynamics.
One of the most well-known thermotropic liquid crystal
molecules is 4-octyl-4 -cyanobiphenyl (8CB), which has
also been the subject of a number of monolayer studies
using BAM.2831 8CB forms a smectic-A phase in which the
molecules are organized in layers and are of average vertical
orientation, although there is no long-range positional
ordering within a given layer. The formation of layers
is also seen at the water surface for 8CB. The surface
2 per molecule and
pressure of 8CB begins rising near 50 A
then enters a long plateau at 5 mN m1 , which extends
2 per molecule. BAM imaging in the
down to near 10 A
long plateau shows coexisting rounded domains of different
reflectivity,31 as seen in Figure 5.
Inside the plateau region, the film is found to consist of
a monolayer coexisting with domains that are composed of
the monolayer plus an integer number of bilayers above it.
The thicknesses of the different coexisting domains were
examined using quantitative assessment of the reflectivity
within domains, which required calibration of the response
of the gray scale in the images against the incident light
intensity.29 In 8CB, a second rise and second plateau
in surface pressure was seen at very low areas, and
Monolayer collapse
BAM is also well suited for determining when a monolayer

collapses under compression and forms three-dimensional
structures. Collapse during continuous monolayer compression is usually quite obvious by BAM in that distinctly
brighter spots or other shapes such as long bright jagged
or wavy lines appear in the monolayer. The appearance
of such features should coincide with features in the A
isotherm such as a sudden drop in surface pressure followed by a plateau or by a roll over onto a plateau,
which will not always be exactly flat. Such changes should
occur near molecular areas that seem reasonable as possible two-dimensional packing limits as estimated from
the dimensions of the molecular profile. It is possible to
observe the collapse and further three-dimensional aggregation either under slow continuous compression or by
pausing at a surface pressure at which the monolayer is
metastable and observing structures emerge as a function
of time.
A study of the collapse in stearic acid monolayers was
an early application of BAM.32 Stearic acid monolayers
compressed to 30 mN m1 at 20 C on subphases of pH =
2, 3, and 5 exhibited significant surface pressure relaxation
over a period of 150 min. During the surface pressure
relaxation, BAM showed the nucleation and growth of
crystallites whose form depended on the pH, varying from
bright fragments at pH = 2, to structures which clearly
showed internal anisotropy at pH = 3, to smaller dendritic
forms at pH = 5. A subsequent study further examined the
collapse in a series of fatty acid monolayers and also for
methyl arachidate on a variety of subphases.33 The collapse
of stearic acid on a pH 3.0 subphase was examined by
stopping the compression at 40 mN m1 , which is in the
surface pressure spike after which the surface pressure
quickly drops into a plateau near 18 mN m1 . In this case,
BAM showed the nucleation of many small bright dots,
which grew and overlapped if compression was resumed
into the plateau region, as seen in Figure 6.
In contrast, the stearic acid monolayer compressed on a
pH 8.0 subphase containing 104 M CaCl2 showed a surface pressure rising to just over 60 mM m1 and then rolling
over into a plateau without a spike. BAM observation at
63 mN m1 showed chains of many small bright microcrystals. It is important to realize that monolayers at surface
pressures above the equilibrium spreading pressure (ESP)
are thermodynamically metastable and should ultimately
transform to three-dimensional aggregates in equilibrium
with a monolayer whose surface pressure equals the ESP
DOI: 10.1002/9780470661345.smc040
10
Techniques
Surface pressure (mN m1)
60
50
40
(b)
30
20
10
10
(a)
20
BAM enables a number of additional characterizations of a

monolayer (or thin film, in the case of multilayer and other
features) such as the recording of reflectivity isotherms
that can be used to estimate changes in the film thickness
as a function of surface pressure. Quantitative BAM also
enables estimation of differences in film thickness between
regions within an image.
In the simplest approach, the reflectivity R at the Brewster angle is noted to be equal to Cd 2 , where C is a constant and d is the monolayer thickness. A more developed
equation that makes use of the thin film approximation35
is
30
R=
Area (2 mol1)
2
n21 n22 1 +
1+
n22
n21
n22
(3)
(c)
Figure 6 BAM images of a monolayer of stearic acid in

the collapse regime, along with the surface pressure ()mean
molecular area (A) isotherm at 20 C on a pH 3 subphase.
Reproduced from Ref. 33. American Chemical Society, 1996,
and the figure caption reads as follows: Surface pressure/area
isotherm (a) of a stearic acid monolayer on acidified aqueous
subphase (pH 3.0) at 20 C and BAM images of (b) 3D nuclei
(bright dots) observed at a surface pressure of 40 mN m1
within the steep part of the isotherm (i.e., the 3D nucleation occurs
at < c = 50 mN m1 ) and (c) overlap and growth of the 3D
nuclei within the plateau region of the /A isotherm (after the
pressure spike at c ). The bar represents 200 m.
of the molecule at hand for the given subphase conditions.

The possible mechanisms of monolayer collapse, especially
for lipid monolayers, have been reviewed and, in addition
to formation of microcrystals, include ejection of material into the subphase, fracture, and a folding mechanism
in which the layer bulges up and then lays over resembling what would occur on pushing on a rug from two
ends.34
2.8
Quantitative analysis of Brewster angle

microscopy images
While many studies of monolayers using BAM descriptively report the microstructures observed, a more quantitative analysis of the BAM images can yield information on
thickness changes in monolayers and has been referred to as
quantitative BAM. Quantitative BAM can be applied using
a simple optical model for films at the waterair interface
in which there is no or negligible anisotropy within the
film. The measurement requires that a calibration be performed to calibrate gray-scale intensities from pixels in the
image with calculated values of reflectivity, and the conduct
of such a calibration has been reported.3537 Quantitative
In (3), d is the film thickness, is the wavelength of the

laser radiation, n1 is the refractive index of the monolayer,
and n2 is the refractive index of the subphase. The value
of n2 is known and a value or reasonable range of values
for the refractive index of the film material is then used
in the calculation. The next requirement is to calibrate
the gray scale of the CCD camera with reflectivity. In
the calibration procedure, the pure water surface is used.
Camera settings such as shutter time, gain settings, and
the entire optical configuration must be kept unchanged
throughout the calibration and for any experiments that
make use of the calibration. The BAM images are recorded
from the bare water surface as the incident angle is
varied by a few tenths of a degree on either side of
the Brewster angle. The range of variation should not
exceed saturation of the camera response. The gray scale
should be averaged over a region or image. A plot of
gray scale intensity versus incident angle will be parabolic
in shape. The theoretical reflectivity is then calculated
as a function of incident angle using (3). Finally, the
calculated reflectivity values for each incident angle are
plotted versus the gray scale values obtained at those
angles. It will now be possible, subject to the abovestated assumptions, to relate gray-scale intensities from
images with reflectivity and use (3) to estimate the film
thickness.
Quantitative BAM has been applied to studies of lung
surfactant films at the water surface.35 It has been applied
to obtain reflectivity versus surface pressure isotherms for
monolayers of monopalmitin, monoolein, and monolaurin.36 In studies of monolayers of dipalmitoylphosphatidyl
glycerol, the method was used to conclude that a 4 A
increase in thickness occurred during compression across
the LC + LE coexistence region due to a conformational
change.37
DOI: 10.1002/9780470661345.smc040
APPLICATION OF BREWSTER ANGLE

MICROSCOPY TO THE STUDY OF
MONOLAYERS OF MACROCYCLIC
COMPOUNDS
BAM has been applied in studies of amphiphilic derivatives

of macrocycles. As these compounds are capable of forming hostguest complexes, the study of their monolayers
is often motivated by an interest in their transfer as LangmuirBlodgett films for use as sensors or by an interest
in studying their recognition behavior in a membrane-like
environment. The dispersion of amphiphilic macrocycles in
mixed monolayers with fatty acids or lipids has proven to be
of interest. A number of significant issues arise in studies of
amphiphilic macrocycles related to their collapse behavior,
instability toward aggregation, response to recognition of
guest molecules in the subphase, and miscibility behavior
in mixed monolayers that can be at least partly addressed
by BAM. In all the cases, the interpretation of the BAM
images is aided by comparison with the A isotherm data
and considerations of the dimensions of the molecule. The
structures of a selection of the supermolecules mentioned
in this chapter are shown in Figure 7.
3.1
Examples involving monolayers of

amphiphilic calixarenes
There have been a number of studies of calixarene monolayers incorporating the use of BAM addressing a range
of phenomena from behavior and stability of monolayers
of the calixarenes (see Calixarenes in Molecular Recognition, Molecular Recognition) themselves at the water
surface, to their complexation of guest molecules, and
their behavior in mixed monolayers. One of the earliest studies examined the formation of complexes between
para-tertbutylcalix[8]arene and the fullerenes C60 and C70
spread together at the waterair interface.51 The 1 : 1 complexes of the calixarene and fullerene were prepared by
refluxing in benzene and then taking the recovered solid
and dissolving it in chloroform for spreading onto the
water surface presumably as the intact complex. The A
isotherms for the calix[8]arene showed a rise starting near
2 per molecule and then steadily increasing to close
180 A
to 80 mN m1 (which is greater than the surface tension
of water) with no sign of a collapse plateau. The surface pressure of the monolayer of the calix[8]arene + C60
2 per molecule
started to rise at higher areas near 230 A
and started to plateau just above 60 mN m1 . In contrast,
the surface pressure for the calix[8]arene + C70 monolayer started rising at a similar area but during the ascent
shifted to lower areas than seen for the pure calix[8]arene
11
monolayer. BAM observations showed differences amongst

2 per
these three monolayers. The calix[8]arene at 301.6 A
molecule showed bright islands and dark areas indicating
a two-dimensional solid-like phase coexisting with a twodimensional gas phase. Assignment of the phase coexisting
with the darker gas phase in monolayers at higher area and
low surface pressures near zero as solid-like rather than LE
or liquid crystalline can be decided by observing the shape
of the domains. If the boundaries of the bright domains
smoothly vary and especially if any domains are circular or
elliptical, then a LE or possibly liquid-crystalline phase is
present. If the boundaries appear jagged or have sharp turns
or corners, then the phase is assigned as being solid-like.
The domains of solid-like phases do not merge easily upon
compression, while those of the LE or liquid-crystalline
type merge. In this image, the presence of extra bright,
mountain-shaped clusters of molecules was also observable,
and their brightness and bordering by optical interference
patterns, known as Newtons rings, indicates that these are
due to three-dimensional aggregates of the calix[8]arenes.
The calix[8]arene + C60 monolayer was reported to appear
similar at low surface pressure and high surface areas as the
monolayer of the calix[8]arene alone. The monolayer of
the calix[8]arene and C70 showed different features, and
illustrates the important distinction between multilayer formation and anisotropic ordering in a monolayer that can be
made by informed application of BAM. In the BAM image,
regions representing three different intensities of brightness
were seen, and it was concluded that these were monolayer,
bilayer, and trilayer regions. In this case, it is reported that
there is no variation in the brightness of these image regions
as the analyzer is rotated and thus the authors conclude
that they are observing the presence of regions of different
thickness. In this study, a noteworthy dynamic effect is also
reported, In the case of the calix[8]arene + C70 monolayer,
a large region that was fairly uniform suddenly fragmented
like the shattering of a sheet of ice upon a small mechanical
vibration of the trough. This indicates that the monolayer
was of a remarkably brittle, two-dimensional structure. It
was noted that the solid-like domains of these monolayers did not merge easily upon compression but tended to
crash into each other and form some clusters and ultimately
bilayers. These observations indicate that BAM applied to
monolayers should be viewed as more than the static capturing of images at fixed conditions and as a method for
observing dynamic changes in real time.
The behavior of monolayers of a para-tertbutylcalix[8]arene in which the phenolic hydroxyls were ether linked
to 3-hydroxypropionic acid groups was studied using
A and V A isotherms, BAM, and infrared spectroscopy of LangmuirBlodgett films.52 The calix[8]arene
can undergo conformational changes on compression that
alter the nature of the hydrogen bonding present, which
DOI: 10.1002/9780470661345.smc040
12
Techniques
R6
R5
R7
R8
OR2
OR1
R3O
OR4
O
N
H
OCH2CH3
OCH2CH3
P
S
NH2
Calix[4]arene derivatives
OCH3
O
N C16H33
C16H33 N
O
OR
OR
N
H
Chiral crown ether
Diazacrown ether
OR
O
O
CH3O
OR
OCH3 CH3O
HO
O
OH
OCH3
6-position
OH
O
OH
3-position HO
HO
O
OH
O
HO
O
OHOHO
Cryptophane (anti form)
2-position OH
OH
(CH2)n
O
OH
HO
OCH3 OCH3
(CH2)n
(CH2)n
C8METAB
RO
b-cyclodextrin
NH2
O
OH
OH
OH
O OH
OH
OH O
C11H23HN
NHC11H23
2C11H23-melamine
O
OH
Figure 7 Structures of a selection of the molecules described in this chapter. (A) calix[4]arene derivatives: CALIX1 (Ref. 38),
R2 = R4 = H, R1 = R3 = group A, R5 = R6 = R7 = R8 = p-tert-butyl; CALIX 2 (Ref. 39), R1 = R2 = R3 = R4 = n-C12 H25 ,
R5 = R7 = group B, R6 = R8 = H; CALIX3 (Ref. 39), R1 = R3 = n-C12 H25 , R2 = R4 = CH3 , R5 = R7 = group B, R6 = R8 = H;
CALIX4 (Ref. 40): R1 = R3 = group C, R2 = R4 = H, R5 = R6 = R7 = R8 = p-tert-butyl; CALIX5 (Ref. 41), R1 = R2 = R3 =
R4 = H, R5 = R6 = R7 = R8 = dodecanoyl (O=C-(C11 H23 )); CALIX6 (Ref. 41) R1 = R3 = group D, R2 = R4 = H, R5 = R6 =
R7 = R8 = dodecanoyl (O=C-(C11 H23 )); CALIX7 (Ref. 41) R1 = R3 = PO(OEt)2 , R2 = R4 = H, R5 = R6 = R7 = R8 = dodecanoyl
(O=C-(C11 H23 )); CALIX8 (Ref. 42), R1 = R3 = H, R2 = R4 = N-acetyl-pivaloyloxymethyl-6-aminopenicillanic acid, R5 = R6 =
R7 = R8 = p-tert-butyl; CALIX9 (Ref. 42), R1 = R3 = H, R2 = R4 = benzylpenicillin ethyl ester, R5 = R6 = R7 = R8 = p-tertbutyl; CALIX10 (Ref. 42), R1 = R3 = H, R2 = R4 = benzylpenicillin propyl ester, R5 = R6 = R7 = R8 = p-tert-butyl; Cyclodextrin
derivatives: CD1 (Ref. 43): the 2-positions and 3-positions are all ether linked to n-C6 H13 , the 6-positions are all substituted with
NH3 + ; CD2 (Ref. 44), the 2-positions and 3-positions are all ester linked to hexanoyl groups (O=C-C5 H11 ), the 6-positions are all
unmodified; CD3 (Ref. 44), the 6-positions are all modified by tert-butyldimethylsilyl groups, the macrocycle is -cyclodextrin (six
glucose units instead of seven for the -cyclodextrin as shown); CD4 (Ref. 44), the 6-positions are all modified by tert-butyldimethylsilyl
groups; CD5 (Ref. 44), the 6-positions are all modified by tert-butyldimethylsilyl groups, the macrocycle is -cyclodextrin (eight
glucose units instead of seven for the -cyclodextrin as shown); Chiral Crown Ethers (Ref. 45), R = benzyl, p-phenylbenzyl, nC12 H25 , or n-C16 H33 ; Diazacrown Ether (ACE-16 in Ref. 46); C8METAB (Ref. 17), R = (CH2 )7 CO2 CH3 ; Cryptophanes (Ref. 47):
only anti -conformation is shown, n = 3 or n = 5 for anti -cryptophanes, n = 9 or n = 10 for mixed 1 : 1 anti - + syn-cryptophanes;
2C11 H23 -melamine (Refs 4850).
DOI: 10.1002/9780470661345.smc040

can be intramolecular, intermolecular, or directed to water
molecules. This study was carried out using the NFT IElli2000 BAM, which provides especially well-focused
images, as seen in Figure 8.
2 per
The surface pressure rose starting near 320 A
molecule, displayed some changes in slope along its rise,
2 per
and then a plateau starting near 46 mN m1 and 200 A
2
per molecule.
molecule that extended down to near 90 A
Compressionexpansion cycles revealed hysteresis in the
surface pressure. The magnitude of the compressibility
modulus, Cs1 = A(
/A)T was consistent with the
presence of a condensed phase. The BAM observations
were consistent with the earlier assignment51 of the phases
present at low pressure as solid-like and gas. During the
first compression, large, fairly uniform solid domains with
straight edges and some smaller solid domains are seen that
merge on compression, although not easily; some scattered,
but small and brighter features are also seen in the images.
The subsequent expansion shows the bright domains breaking apart and also shows the emergence of many small,
branched domains. During the second compression, these
small, branched domains remain visible and are also visible on a second expansion in the midst of the brighter
islands. Compression to lower areas generated bright striations indicative of monolayer collapse. The emergence of
the smaller, branched domains upon the first expansion
was interpreted as being consistent with infrared spectra
of the monolayers transferred as LangmuirBlodgett films
and molecular mechanics calculations that suggested that
on compression the hydrogen bonding shifted from being
between calixarenes to being between calixarenes and water

molecules, thus resulting in a less cohesive film. Large
difference in the V A isotherms between the first and
second compression also suggested a significant conformational change.
Monolayers of compounds that exhibit hysteresis of surface pressure during compressionexpansion cycles generally also exhibit surface pressure relaxation at fixed area as a
function of time. During surface pressure relaxation, BAM
is often useful for observing morphological changes due to
the structural changes or nucleation and domain growth that
is associated with the surface pressure relaxation. A study
of monolayers of a para-tertbutylcalix[4]arene derivative
1,3 modified on the lower rim hydroxyls with benzylamidoethoxy groups (CALIX1, see Figure 7) was reported
using surface pressure and BAM.38 The monolayers were
studied over a temperature range of 525 C. The surface pressure rise on compression was seen to start at
2 per molecule and was notably linear except at
125128 A
5 C where there was an inflection region near 25 mN m1

indicating a phase transition. The surface pressure plateaus
varied with temperature. The phase transition observed at
5 C manifested itself under BAM as the rapid appearance of many irregular, small, and bright domains. Thus,
in studies using BAM, one should be alert to isotherm
features including small kinks and changes in slope as conditions near it should be especially observant for changes
visible by BAM. The isotherm feature at 5 C also showed
a small maximum and reversal indicating a supersaturation kinetic effect upon compression. Such overshoots in
1st c
1st c
1st c
(i) < 500 2; 0 mN m1
(ii) 410 2; 0 mN m1
(iii) 320 2; 2 mN m1
1st e
2nd c
(vi) 425 2; 0 mN m1
(vii) 330 2; 0 mN m1
13
2nd e
(viii) 330 2; 0 mN m1
1st c
(iv) 275 2; 30 mN m1
1st e
(v) 400 2; 0 mN m1
2nd e
(ix) 340 2; 0 mN m1
(x) 125 2; 46 mN m1
Figure 8 BAM images of monolayers of a para-tert-butyl calix[8]arene derivative described in Ref. 52, showing rigid fractured
ice-sheet-like domains merging during the first compression, hysteresis in the domain morphology on expansion and recompression, and
then collapse. Reproduced from Ref. 52. American Chemical Society, 2005, and the figure caption reads: Brewster angle microscope
(BAM) images of C8A monolayer at the airwater interface during the two successive cycles: first compression (1st c), first expansion
(1st e), second compression (2nd c), second expansion (2nd e), processes and collapse of the film, images iiv, images v and vi, image
vii, images viii and ix, and image x, respectively.
DOI: 10.1002/9780470661345.smc040
14
Techniques
surface pressure in isotherms often precede rapid domain

growth as the two-dimensional supersaturation is relieved.
In this study, experiments were carried out in which the surface pressure relaxation and BAM images were observed
as a function of time over periods up to 800 s. Compres 2 at 20 C corresponding to an initial surface
sion to 104 A
pressure of 25.6 mN m1 showed the emergence of highly
regular dendritic domains after 50 s. The small magnitude
of the surface pressure relaxation suggested that the density
difference between the emerging ordered phase in the dendritic domains and the initial phase was small. Compression
2 , respectively
to lower surface areas of 95.6 and 83.5 A
also showed the emergence and growth of domains of a
lancet-like shape that appeared at later times to indicate
overgrowth of a second layer. It was thus concluded that
during compression this monolayer is far from equilibrium.
The limiting molecular areas were found to be consistent
with a conformation in which the benzylamido groups were
oriented parallel to each other and at an angle to the calixarene cone axis.
Calix[4]arenes modified at the upper rim with two phosphonate groups and on the lower rim hydroxyls with either
four dodecyl chains (CALIX2, see Figure 7) or two dodecyl
chains and two methyl groups (CALIX3, see Figure 7) were
studied as monolayers using BAM and GIXD.39 The monolayers were studied on subphases of pure water, and on
those containing Th(NO3 )4 , Eu (NO3 )3 , CdCl2 , and NaCl at
a series of temperatures from 14 to 39 C. CALIX2 clearly
displayed plateaus in its surface pressure isotherms indicating a first-order phase transition between an LE phase and
a condensed phase. This phase-transition plateau resembled
that seen for single-chain amphiphiles, which is unusual in
the reported studies of monolayers of macrocyclic compounds. The plateau surface pressure was reduced and the
mean molecular area at its onset was increased on the
Th4+ subphase versus pure water. In contrast, CALIX3
did not show a surface pressure plateau and its isotherms
were essentially the same at 15 and 25 C and varied little between the pure water and Th4+ -containing subphases.
BAM in the case of these calixarenes showed the formation
of bright domains only in the two-phase coexistence region
and not immediately upon spreading. CALIX2 showed
the formation of domains described as lancet or sickle
shaped and with an irregular border. The observation of
uniform reflectivity indicated that there was no anisotropy
of molecular orientation within these domains. The domains
of CALIX2 were similar on the NaCl-containing subphase, but were considerably more irregular in shape on
the Cd2+ -, Eu3+ -, and Th4+ -containing subphases. BAM
thus has shown here the effect of cation binding on the
shape of the condensed-phase domains of this compound.
BAM observations of CALIX3 showed roughly rectangular
domains. The observation of domain segments of different reflectivities indicated the presence of anisotropy in the
molecular orientation. The presence of cations in the subphase increased the observance of more rounded domains
with frayed peripheries and sharply defined regions of different reflectivities indicating anisotropic ordering. The filigree structure of the domains in which they resemble
bundles of filaments viewed from the top was interpreted as
indicating the presence of a second molecular layer within
these domains. The GIXD data showed that only CALIX2
formed a two-dimensional lattice and that the lattice parameters were not affected by the presence of Th4+ in the
subphase.
BAM proved useful in evaluating the effectiveness
of using metal complex formation based upon chloride
bridging between Pd(II) centers as a strategy to stabilize monolayers of a 1,3-(distal) p-tert-butylcalix[4]arene
derivative bearing methionine groups amide linked to short
aminoethoxy groups (CALIX4, see Figure 7).40 The two
methionines, tethered to the lower rim of the calix[4]arene
(denoted as L), are capable of forming an intramolecular
complex with Pd(II) with the two sulfur and two nitrogen donor atoms from each methionine coordinated to a
single Pd(II), forming the mononuclear species designated
as PdLCl2 , which required two chloride counterions. Each
of the two methionines can also separately form a complex with Pd(II) through a nitrogen and sulfur donor atom
and with two chloride ions serving as the additional ligands forming the binuclear species designated as Pd2 LCl4 .
Monolayers of these two species were compared with
the Pd2 LCl4 species capable of chloride bridging between
neighboring molecules intended to provide stabilization of
the monolayer. The A isotherms of the calix[4]arene
amphiphile with no Pd(II) denoted as L, and of the PdLCl2
and Pd2 LCl4 species showed rising surface pressure ascend2
ing fairly steeply that gave limiting areas of 162 3 A
2
2
per molecule, and 152 3 A
per
per molecule, 145 3 A
molecule for L, PdLCl2 , and Pd2 LCl4 respectively. These
limiting areas were found to be consistent with the dimensions of the upper rim of the calix[4]arene cone. Compressionexpansion cycles revealed significant hysteresis for all
three species, especially when compressed into the upper
plateau region where collapse occurred. Area relaxation at
constant surface pressures ranging from 33 to 52 mN m1
showed significant decreases in area of as much as 25%
over a 90-min period for the L and PdLCl2 species; in
contrast, the Pd2 LCl4 species showed very limited area
relaxation of at most 5%, indicating that the stabilization
strategy was largely successful. BAM provided complimentary information confirming the success of the stabilization
strategy via chloride ligand bridging between binuclear
complexes. BAM images of L at 0 mN m1 show a twodimensional, hexagonal network pattern on spreading where
DOI: 10.1002/9780470661345.smc040

the brighter walls of the hexagons were of higher molecular
density. Upon compression, first the network merged and
a few bright islands were seen and then, at higher surface
pressure, a pattern of many bright worm-like domains
and darker regions appeared, which was interpreted as representing multilayers or aggregates. The same morphology
was not recovered on expansion for monolayers of L. The
monolayers of PdLCl2 showed regions of lower reflectivity
than those of L with numerous holes. On expansion, PdLCl2
showed formation of a hexagonal network pattern. This
study is a clear example of how hysteresis in the surface
pressure upon a compressionexpansion cycle is generally
correlated with different BAM observations during compression and expansion. The most significant observation
was that for Pd2 LCl4 : the as-spread monolayers displayed
extensive block-like regions of uniform reflectivity that coalesced into a bright uniform film during compression, which
during expansion maintained much of its uniformity except
for the opening of some dark rifts in the monolayer. The
observation of large regions of uniform brightness by BAM,
but with straight borders and corners, is a clear indication
of a cohesive condensed phase, attributed to the chloridebridging effect.
Miscibility studies of calixarenes in mixed monolayers with other species involving use of BAM have been
reported for calixarenes mixed with cholesterol41 and
phospholipids.42, 53 Studies of miscibility in binary mixed
monolayers can be quite complicated as the presence
or absence of phase separation of the components can
be a function of composition, temperature, and surface
pressure. In principle, the mapping of an entire twodimensional binary phase diagram is possible, and as
such would require extensive experimentation. Miscibility studies are often motivated by an interest in the
behavior of the oriented calixarene in a membrane-like
environment or dispersed with a simple amphiphile for
use as a sensor after transfer as a LangmuirBlodgett
film. A study of three amphiphilic calix[4]arenes in
mixed monolayers with cholesterol was reported.41 The
derivatives were para-dodecanoylcalix[4]arene (CALIX5,
see Figure 7), 25,27-bis-diethoxyphosphoryltetradecanoylcalix[4]arene (CALIX6), and 25,27-bis-dihydroxyphosphoryloxytetradodecanoyl-calix[4]arene (CALIX7). These derivatives showed steeply rising A isotherms with limiting
2 per molecule respectively
areas of 120, 115, and 116 A
for CALIX5, CALIX6, and CALIX7. The collapse pressures are reported as 15.2, 23.4, and 35.5 mN m1 respectively for CALIX5, CALIX6, and CALIX7. The collapse pressure did not vary with composition for mixed
monolayers of CALIX5 and CALIX6 with cholesterol
indicating immiscibility of CALIX5 and CALIX6 with
cholesterol. Cholesterol was also immiscible with CALIX7
and partly destabilized the monolayer. BAM images of
15
monolayers of CALIX6 showed smooth curved borders

between the brighter and darker regions at low surface
pressures and a uniform brightness on compression that
indicated formation of a LE-like phase for this monolayer. Examination of mixed films of these three compounds
with cholesterol at 1 : 1 mole ratios showed visual evidence for immiscibility, especially for mixtures of CALIX7
with cholesterol. Monolayers of CALIX5 and CALIX6
mixed with cholesterol showed LE-like domains but with
many small holes. However, in the case of mixtures of
CALIX7 with cholesterol, BAM showed a remarkable
emergence of long fibrillar aggregates during intermediate stages of the compression that were uncharacteristic
of either the pure monolayer of CALIX7 or of cholesterol
monolayers. The miscibility of two of these compounds,
CALIX6 and CALIX7, with the phospholipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidic
acid (DPPA), dipalmitoylphosphatidylserine (DPPS), and
dipalmitoylphosphatidyethanolamine (DPPE) was studied.53 The dependence of the collapse pressure on composition was used to determine that CALIX5 was immiscible with these phospholipids except at mole fractions
either below 20% or above 80%. The collapse pressure
varied proportionately with composition for mixtures of
the phospholipids with CALIX6, and it was concluded
that CALIX6 was miscible with these four phospholipids. BAM images for CALIX5 mixed with DPPA in
a 1 : 1 ratio were reported and showed formation of a
granulated film before compression, different from either
pure monolayer and suggestive of immiscibility. BAM
images at other points during compression or for the
other lipids mixed with CALIX5 were not reported in this
study.
In another study, interest in the miscibility of the calixarene derivative with phospholipid in a mixed monolayer
was motivated by the prospective antibacterial activity of
the para-tertbutylcalix[4]arene derivatives, to which two
penicillin units were appended. 6-Aminopenicillanic acid
was linked by amide bonds to 1,3-bis(O-acetyl)-p-tertbutylcalix[4]arene to form derivative CALIX8 (Figure 7).42
In the second derivative (CALIX9), benzylpenicillin ethyl
ester was ether linked to the 1,3 positions and in the
third derivative (CALIX10) benzylpenicillin propyl ester
was ether linked to the 1,3 positions. Mixed monolayers
of these compounds with DMPE were studied by A
and V A isotherms and by BAM, as seen in Figure 9.
The limiting areas of these compounds were found to be
2 per molecule, respectively. The A
175, 180, and 181 A
isotherms showed the most commonly reported dependence
for amphiphilic macrocycles of near-zero surface pressure at high areas followed by a steady rise and then an
inflection upon entering a collapse plateau. Analysis of
A isotherms measured as a function of composition
DOI: 10.1002/9780470661345.smc040
16
Techniques
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 9 BAM images of mixed monolayers of the three calixarenes described in Ref. 42 with the phospholipid DMPE. The
domains formed in mixed monolayers with DMPE are seen to depend upon the structure of the calixarene derivative (panels GI
below). Reproduced from Ref. 42. American Chemical Society, 2007, and the figure caption reads: BAM images of calixarene,
DMPE, and DMPE/calixarene monolayers. Line AC: pure Calix II; line DF: pure DMPE; line GI: mixed films. (A)
0 mN m1 ;
A = 240 A 2 per molecule; (B)
= 6 mN m1 ; A = 185 .6 A 2 per molecule; (C) collapse point; (D)
0 mN m1 , A = 90 A 2 per
molecule; (E)
= 6 mN m1 ; A = 57 .8 A 2 per molecule; (F) collapse point; (G) DMPE/Calix I; (H) DMPE/Calix II; (I) DMPE/Calix
III. The G, H, and I images were taken at 12, 13, and 14 mN m1 and 110.9, 82.5, and 87.5 A 2 per molecule, respectively. The composition
of all the mixed films was xDMPE = 0 .75 . Scale: the width of the snapshots corresponds to 400 m.
carried out by plotting mean molecular area versus mole

fraction of DMPE at 10, 15, and 20 mN m1 showed positive deviations from ideality. BAM showed that these
three calixarenes showed uniformly bright images once
compressed to rising surface pressures. Observation of a
two-dimensional LE and gas pattern for CALIX9 at high
areas was noted. The observation that these calixarenes
alone form uniform one-phase monolayers along the entire
rise in surface pressure aids interpretation of BAM images
of mixed monolayers, as domain formation in the mixed
monolayer becomes easier to interpret. In the case of mixed
monolayers of DMPE and CALIX8 for xDMPE = 0.75,
domains form that closely resemble those formed by the
pure lipid. In the case of mixed monolayers of CALIX9
or CALIX10 with DMPE, such domains also form but are
considerably smaller, a result indicating a degree of miscibility between the calixarene and DMPE, suggesting that
they would be membrane active.
3.2
Examples involving monolayers of

amphiphilic cyclodextrins
Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucose units joined by -1,4-glycosidic bonds
(see Cyclodextrins: From Nature to Nanotechnology,
Molecular Recognition). The most commonly studied CDs
are -CD composed of six glucose units, -CD composed of seven glucose units, and -CD composed of
eight glucose units. The structure presents a hydrophobic cavity for binding of guests with primary hydroxyl
groups from the 6-positions equal to the number of glucose units presented on the primary face of the open
CD bucket and secondary hydroxyl groups from the
2- and 3-positions equal to twice the number of glucose units presented on the wider secondary face of
the open CD bucket. CDs can form inclusion complexes with a variety of organic guest molecules and thus
are of potential interest for chemical sensor development
and studies of hostguest inclusion complex formation.
DOI: 10.1002/9780470661345.smc040

There have been numerous papers examining the monolayer
behavior of CD derivatives bearing alkyl chains and other
constituents, and many of these studies have employed
BAM.
BAM is highly useful for determining miscibility of components in mixed monolayers and this has been applied to
mixed monolayers of two related CD derivatives.43 Mixed
monolayers of per-6-ammonium-per-6-deoxy-per-O2 ,O3 hexyl--cyclodextrin (CD1, see Figure 7) and the related
derivative per-O2 ,O3 -hexanoyl--cyclodextrin (CD2) were
examined. It was found that CD1 had a higher collapse pressure (
c = 45.3 mN m1 ) than CD2 (
c = 37.4 mN m1 ).
In studies of mixed monolayers, comparisons are often
made of the observed molecular area at a chosen surface pressure in the A isotherms versus that predicted
from the molecular areas of the two pure components at
the chosen surface pressure and assuming ideal mixing
via the equation A12 = x1 A1 + x2 A2 , where x1 and x2 are
the mole fractions of CD1 and CD2 in the monolayer
respectively. Positive deviations from ideality are generally
taken as indicating possible immiscibility between monolayer components. The excess free energy of mixing at a
given temperature can also be calculated using the A
isotherm data on a series of mixtures. In this particular
study, a peak in the deviation of the molecular area from
the ideal value was consistently observed near a mole fraction of compound CD1 of 0.60. BAM was reported to show
phase separation by the emergence of a network-like structure with high-reflectivity cages surrounding regions of
lower reflectivity. It is interesting to note that this was
observed upon decompression, and indicates how BAM can
follow hysteresis-like effects in monolayers where a different microstructure is seen on expansion than is observed
during compression.
Monolayers of the per-6-O-(tert-butyldimethylsilyl)
derivatives of -, -, and -CDs (CD3, CD4, CD5, see
Figure 7) were examined by BAM and A isotherms.44
In this study, BAM provided the key information needed
to understand the problem encountered with reproducibility
of the A isotherms for these monolayers. The isotherms
of these compounds were exhibiting significant variation of
2 molecule in the lift-off area, the mean molec30 A
ular area at which the surface pressure first starts to rise.
The application of BAM demonstrates how surface pressure
isotherms alone cannot reliably indicate correct monolayer
formation. The derivatives were spread from five different
spreading solventschloroform, chloroform/ethanol (4 : 1
v/v), ethanol (which is not a spreading solvent), hexane, and
hexane/isopropanol (7 : 3 v/v). BAM observations showed
the presence of small crystalline aggregates a few microns
in size even at high molecular areas just after spreading
and well above the lift-off area. The formation of these
small crystallites was attributed to supersaturation of the
17
amphiphiles during the solvent evaporation. Variation in

their number for different experiments could account for
the isotherm variability. BAM showed that the aggregates
moved closer together and increased in number upon compression and increased greatly in number upon collapse.
Spreading from the solvent hexane/isopropanol (7 : 3 v/v)
resulted in the desired homogeneous monolayer, which was
observed by BAM to remain homogeneous throughout compression until the collapse pressure was reached. Atomic
force microscopy (AFM) (see Atomic Force Microscopy
(AFM), Techniques) on derivatives transferred onto mica
modified by an initial monolayer of cadmium stearate was
generally consistent with the BAM results, showing less
aggregation in the transferred films originally spread from
hexane/isopropanol. It was determined that the monolayers of the CD derivatives spread from hexane/isopropanol
were more suitable for use in sensor construction due to
more consistent CD orientation and much less prevalence
of aggregates. This study demonstrates the power of BAM
to guide LangmuirBlodgett film development, to assess
reasons for irreproducibility in monolayer behavior, and to
make an optimal selection of spreading solvent.
Studies of unmodified native -cyclodextrin at the
waterair interface alone and together with sodium dodecyl sulfate (SDS) have recently been reported and have
incorporated the use of BAM.54 Pure -cyclodextrin was
found to adsorb significantly and cover most of the interface and could be seen by BAM as a film with many
small gaps, dark areas, and regions of differing reflectivity. The film appeared to be more fluid at 303 K and cyclodextrin concentration of 2.5 mM than it did at 283 K
and a concentration of 35 mM. Using isothermal titration
calorimetry, it was found that -cyclodextrin formed a
complex with SDS of 2 : 1 stoichiometry. BAM observation of the waterair interface for the mixture of 35 mM
-cyclodextrin and 17.5 mM SDS at 286 K showed formation of a rigid continuous film, while upon lowering
the concentrations to 5.0 mM -cyclodextrin and 2.5 mM
SDS the film was still fairly continuous and rigid but displayed some gaps. This study shows how BAM can reveal
new phenomena in studies where a macrocycle is studied
adsorbed at the water surface, and that the inclusion of a
guest to form a stoichiometric complex can alter the properties of the adsorbed monolayer. These studies should be
quite straightforward as the only requirement is the observation of the water surface of the solution at a controlled
temperature. It could also prove of interest to sweep the
surface clean and study the dynamics of the appearance of
the adsorbed film. Measurement of surface pressure versus
time for the adsorbing film could augment the experiment to
assess if thermodynamic equilibrium has been reached. In
this study, the transfer of the -cyclodextrin films onto mica
for subsequent imaging by AFM confirmed the existence of
DOI: 10.1002/9780470661345.smc040
18
Techniques
dimer-like arrangements of -cyclodextrin forming tubular

structures.
BAM is also uniquely suited to study the influence
of the interaction of a macrocycle in the subphase
with a spread monolayer on the monolayer morphology and structure.55 Monolayers of cholesterol, dimyristoylphosphatidycholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), 3/1 DMPC/cholesterol mixtures, and
3/1 DMPG/cholesterol mixtures were examined using surface pressure isotherms, BAM, and polarization-modulation
infrared reflection absorption spectroscopy (PM-IRRAS).
These studies used a BAM-2plus and report making use
of a feature of the I-Elli2000 software that enables easy
reflectivity calibration from the CCD image gray scale and
estimation of film thickness in regions of the image assuming fixed refractive index values for the water and for the
film. This study contains film thickness estimations for different regions reported to have relative errors close to 5%;
the estimate of these thicknesses makes use of the proportionality of the reflectivity to (nd )2 , where n is the film
refractive index and d is the film thickness. Using this quantitative BAM approach, it was found that, after collapse, the
thickcholesterol monolayer showed regions of 18 1 A
ness in coexistence with regions of 54 3 A thickness,

suggesting the formation of trilayer regions upon collapse of
the cholesterol monolayer. The A isotherms of cholesterol monolayers spread on saturated -cyclodextrin solution rose gradually starting from much higher molecular
areas and attained only about half the surface pressure
of cholesterol on water, with dramatic hysteresis seen on
expansion. The BAM images of cholesterol compressed on
the -cyclodextrin solution showed regions of greater and
more variable reflectivity, suggesting thicknesses of 40 2,
While the effect of sub60 3, 80 4, and 100 5 A.
phase -cyclodextrin on the surface pressure isotherms of
the two pure lipids was slight, BAM revealed the presence of -cyclodextrin domains beneath these monolayers at lowmoderate surface pressures. The isotherms of
3/1 DMPC and cholesterol on the -cyclodextrin subphase showed a significant surface pressure arising at much
higher molecular areas and BAM showed bright domains
that persisted until a pressure of 43 mN m1 was reached.
The disappearance of these bright domains upon compression suggested that the -cyclodextrincholesterol complex was being squeezed out of the interface. Similar
behavior was found for the DMPG + cholesterol mixed
monolayers. It is of particular significance that the lipid
DMPC and DMPC/cholesterol mixed monolayers were chosen for these experiments as DMPC and the mixed monolayer are normally homogeneous; therefore, any brighter
domains observed must be due to the interaction with the
-cyclodextrin in the subphase. Using the BAM imaging
results together with PM-IRRAS data and calculations, a
model was presented in which the CDs orient parallel to the

plane of the interface (the axes through the cavity being perpendicular to the interface) in a head-to-head and tail-to-tail
fashion. The association with cholesterol was concluded to
be through interaction with the CD cavity, while that with
phospholipid was through hydrogen bonding to the lipid
head group. The observation by BAM of thicknesses that
was instrumental in arriving
were close to multiples of 20 A
is about twice the length of two CDs
at this model as 20 A
stacked on top of each other.
3.3
Other studies using BAM and amphiphilic

macrocycles
Amphiphilic derivatives of crown ethers (see Crown and

Lariat Ethers, Molecular Recognition) and cryptophanes
(see Cyclotriveratrylene and Cryptophanes, Molecular
Recognition) have been a part of monolayer studies involving the use of BAM and provide additional examples of how
BAM can be applied to supramolecular chemistry in monolayers. Chiral amphiphilic crown ethers (Figure 7)45 were
spread as monolayers and their recognition of the enantiomers of the amino acids valine, alanine, tryptophan, and
phenylglycine introduced into the subphase was evaluated.
BAM showed that the chiral crown ether with two benzyl groups collapsed by forming multilayer islands, while
closely related derivatives bearing alkyl chains instead collapsed via a folding mechanism at the water surface. The
monolayers were spread on acidic subphases containing the
L or D enantiomers of each of the four amino acids of concentration 0.0325 mM. Chiral discrimination effects were
noted by observation of modest but significant differences
in isotherm parameters such as compressibility modulus,
collapse pressure and area, and surface potential. However,
no changes of interest were noted in features observed by
BAM. Thus, it is possible that a monolayer system can
show interesting and quantitatively significant recognition
phenomena of subphase species but no noteworthy changes
under BAM. Larger-scale reorganization of the monolayer
in response to recognition events is needed to see visually
significant changes using BAM.
BAM is an especially useful method for helping in
characterization of the behavior of molecules at the
waterair interface that do not exhibit stable monolayer
behavior but are instead subject to relaxation effects,
which are generally due to slow aggregation. Monolayers of the compound N,N -dihexadecyl-4,13-diaza-18crown-6 (ACE-16, see Figure 7) were examined alone and
in mixtures with palmitic acid; these exhibited significant surface pressure relaxation effects.46 In this study,
BAM is used to distinguish three scenariosdendritic
domain growth during relaxation, monolayer collapse, and
DOI: 10.1002/9780470661345.smc040

separation of components due to immiscibility for the case
of mixed monolayers of ACE-16 and palmitic acid. The
A isotherm for ACE-16 showed a plateau starting at
2 per molecule that extended
30 mN m1 and near 80 A
down to areas much less than any realistic profile of the
2 per molecule for the
molecule that would be close to 43 A
crown ether parallel to the surface and the alkyl chains
perpendicular. When the monolayer was compressed to
32 mN m1 and then held at fixed area, a relaxation of the
surface pressure to 25 mN m1 was observed over a period
of 40 min. BAM showed the growth of dendritic domains
during this period after a brief 2-min induction period; from
their appearance, it was likely that these domains were two
dimensional. In contrast, compression to 50 mN m1 was
followed by relaxation of the surface pressure to close to
40 mN m1 with BAM showing formation of aggregates
assigned as most likely being due to monolayer collapse.
The compression of 1 : 1 mixed monolayers of ACE-16 and
palmitic acid to either 23 or 37 mN1 followed by holding
at fixed area showed significant surface pressure relaxation.
In the case of mixed monolayers of ACE-16 and palmitic
acid, BAM showed the emergence of many small point-like
domains that were assigned as a possible demixing of the
palmitic acid from the ACE-16.
The binding of a cation to a crown ether amphiphile
can significantly alter the domain microstructure observed
by BAM. In the case of monolayers of benzodithia-15crown-5-styryl dye,56 BAM showed large islands on 1 mM
AgClO4 indicating coexistence of a condensed phase with a
gas phase. In contrast, on 1 mM NaCl subphase, the appearance of circular domains of the gas phase surrounded by
brighter regions suggested that in this case the coexistence
was between a LE phase and a gas phase. On pure water,
the domains were more irregular but clearly showed coexistence of a darker two-dimensional gas phase with a brighter
pattern. The assignment of LC phase, as being present at
low surface pressures, is generally done if one sees other
than round or fluid boundaries between the brighter and
darker regions by BAM; if these boundaries appear rounded
and smooth, or if a two-dimensional foam-like arrangement is observed, then the phase is generally assigned
as LE.
Cryptophanes are examples of a molecular structure that
one might not expect to form monolayers at the surface
of water given their roughly spherical profile. However,
their structure possesses well-defined regions that are either
polar or nonpolar; as the upper and lower cyclotriverarylene caps are nonpolar, the two belts of linking ether bonds
are polar and the connecting short alkyl chains are nonpolar. In this study, a series of four cryptophane compounds
(Figure 7) with varying lengths of the alkyl chain linker
(C3, C5, C9, and C10) and pure anti -stereochemistry for
C3 and C5 and mixed anti - and syn-stereochemistry for
19
C9 and C10 were spread on the water surface.47 The A

isotherms measured as compressionexpansion cycles were
reported over two ranges, termed long-range from 250 to
2
2 per molecule and short-range from 250 to 145 A
65 A
per molecule. The long-range isotherms displayed a rise
followed by a plateau and then another rise in surface
pressure. The long-range isotherms showed large hysteresis, while the short-range isotherms showed significantly
less hysteresis. The surface pressure plateau, which resembles that associated with first-order phase transitions with
a coexistence region, was found to represent a range of
gradual formation of three-dimensional aggregates. This
is an important distinction to be aware of when interpreting A isotherms that can be greatly aided by the
use of BAM. While dramatic surface pressure hysteresis
and considerations of molecular dimensions suggested this
conclusion, BAM can definitively confirm whether during a surface pressure plateau one is observing a firstorder phase transition or formation of three-dimensional
aggregates signifying monolayer collapse. In this study, the
C5-cryptophane developed a grainy pattern in the plateau
region, while the C9-cryptophane developed bright domains
of variable sizes that were oriented parallel to the barrier. It
was concluded that the cryptophanes studied only formed
monolayers up to modest surface pressures of 78 mN m1 ,
except 12 mM m1 for the C3 cryptophane. Ellipsometry
measurements, which do not provide images, gave monolayer thicknesses consistent with the dimensions of these
cryptophanes. It is important in monolayer studies to correctly distinguish collapse from actual two-dimensional
phase transitions.
A study of amphiphilic derivatives of a molecular clip
and a molecular tweezer provided an additional example of
how BAM can aid the study of hostguest complexation in
monolayers.57 The molecular clip and tweezer derivatives
had either a 1,4-diacetoxybenzene derivative or a 1,4diacetoxynapthalene spacer. The guest molecule chosen
was 1,2,4,5-tetracyanobenzene (TCNB) and was spread as a
1 : 1 mixture in chloroform with the host molecule. The two
molecular clips and the narrower molecular tweezer showed
fluid-like domains during compression as observed by
BAM, while the wider molecular tweezer derivative showed
solid-like domains with sharper edges. The presence of
TCNB did not influence the A isotherms of the two
molecular clip derivatives. The mean molecular areas from
the A isotherms were consistent with orientation of the
sidewalls of the clips or tweezers perpendicular to the water
surface. Comparison of the BAM images of monolayers
of the narrower molecular tweezer derivative with and
without TCNB showed significant differences. The pure
tweezer at low surface pressure showed shapeless wavy
aggregates, while the complex showed a network of smaller,
bright, fractal-shaped aggregates. At higher pressures, the
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20
Techniques
monolayer of pure tweezer compound showed LE-like

domains with smooth bright bands or smooth boundaries,
while the monolayer of the complex showed smaller
fractal-structured domains. The monolayer of the narrower
tweezer compound complexed with TCNB also showed
anisotropy upon rotation of the analyzer such that contrast
inversion could be observed in a selected small group of
domains. The observation clearly indicates that the domains
of the molecular complex of the tweezer compound with
TCNB have anisotropic orientations.

MONOLAYERS OF CHIRAL
COMPOUNDS
In contrast to the issues of interest in applying BAM

to the study of monolayers of macrocyclic amphiphiles,
studies of chiral monolayers using BAM are primarily
concerned with how molecular chirality is manifested in
terms of the mesoscopic chiral features of condensed-phase
domains (see Chirality, Concepts). Many of the studies
of chiral monolayers focus on chiral discrimination effects,
which may be broadly classified as being either homochiral (interaction between like enantiomers is more favorable
than that between opposite enantiomers) or heterochiral
(interaction between opposite enantiomers is more favorable than that between like enantiomers), by comparing
the domain morphology and isotherm behavior of monolayers of pure enantiomers with those of racemic mixtures.58, 59 Of particular interest in studying monolayers of
racemic mixtures is whether visual evidence for separation of enantiomers in two dimensions can be observed
for the case of homochiral discrimination. In the case
of homochiral discrimination, the A isotherms of the
pure enantiomers appear more condensed than those of
the racemic mixture, while the opposite is observed for the
case of heterochiral discrimination. The A isotherms of
the two pure enantiomers should be equivalent, and this
must be verified in these studies. Monolayers of diastereomers of molecules with more than one chiral center
have also been examined in a smaller number of studies, and these systems can be quite complex. Methods
frequently applied to study these systems in conjunction
with BAM in studies of monolayers of chiral amphiphiles
are IRRAS to assess molecular ordering, and GIXD for
the determination of two-dimensional lattice structures. In
this section, attention is focused on chiral discrimination
in synthetic amphiphiles. Chiral domain formation has
been observed in phospholipid monolayers, dating back
to the pioneering work of McConnell2 using fluorescence
microscopy in the 1980s; many of the earlier studies of

chiral discrimination in monolayers were carried out using
fluorescence microscopy.
In studies of monolayers of chiral amphiphiles with a single chiral center, it has often been the case that the domains
of the individual enantiomers exhibit curved structures with
the sense of the curvature being unique to the handedness of
the chiral center. If such systems have been found to exhibit
homochiral discrimination, as usually assessed by observation of more condensed A isotherm behavior for the pure
enantiomers than for the racemic mixtures, interesting chiral segregation effects can often be observed. These studies
are generally carried out for chain lengths and temperatures for which a clearly defined phase transition from a LE
phase to a two-phase coexistence with the emerging condensed phase is observed. In BAM observation of racemic
monolayers, the simultaneous appearance of domains with
both senses of curvature has been seen. Monolayers of chiral amphiphiles that show heterochiral discrimination do
not show such chiral segregation and show other complex
domain shapes. In the case of homochiral discrimination,
observation of curved domains is not ubiquitous, and in
many cases branched growth forms are observed instead.
BAM studies applied to any supramolecular system containing chiral centers should be cognizant of the possibility
of observing domains with chiral features. Such curvature
can be explained on the basis of anisotropy of the variable
known as the line tension, which is the two-dimensional
equivalent of surface tension, and exists at the interface
between the condensed-phase domains and the surrounding
LE phase. A long-range twisting of the molecular orientation of tilted chiral molecules in the condensed phase can
also explain the bending of domains. Many of the systems
studied have been amphiphilic amino acid derivatives, and
amideamide hydrogen bonding promoting that long-range
ordering in these monolayers is a major force resulting in
the formation of large curved domains.
One of the earliest studies on the use of BAM examined
monolayers of the racemic mixture and of the pure L- and
60
D-enantiomers of N-dodecylgluconamide.
This system
presented A isotherms at 10 C that were more condensed for the racemic mixture than for the pure enantiomers, which were equivalent; however, at 25 C, the
opposite was true, although the isotherms crossed at a certain point. Significant surface pressure relaxation at fixed
area was observed, which differed in extent between the
enantiomers and the racemic mixture and was much greater
at 25 C than at 10 C, as expected on the basis of the
short chain length. BAM observation during compression
of either of the pure enantiomers at 25 C showed extensive growth of two-dimensional dendritic domains. Differences could not be found between the dendritic forms
of the two enantiomers. The monolayers of the racemic
DOI: 10.1002/9780470661345.smc040

mixture gave an indistinct grainy pattern on compression.
The domains seen in monolayers of these pure enantiomers
were nonequilibrium growth forms that did not manifest
chiral differences. Chiral segregation was not observed in
the racemic mixture, at least on the length scales probed
by BAM. It must be kept in mind in BAM studies that
structures on length scales below the lateral resolution will
not be seen by the method. Chiral single-chain molecules
capable of very strong lateral hydrogen bonding are more
likely to exhibit this sort of extensive dendritic growth on
compression.
In a following study, monolayers of the enantiomers
of N-dodecylmannonamide and of the racemic mixture were examined and very different results were
obtained than those found for the enantiomers of Ndodecylgluconamide.61 In this case, the surface pressure
isotherms clearly show homochiral discrimination. BAM
observation of the monolayers of the pure enantiomers
showed formation of feather-like dendrites with curved
main growth axes and with curved side branches. The side
arms were observed to curve exclusively counterclockwise
for the L-enantiomer and clockwise for the D-enantiomer.
(a)
21
BAM allowed observation of the growth of these curved

side arms in real time. BAM observation of the monolayers of the racemic mixture showed dendritic growth
with the side arms curved in either direction in equal
proportions.
Chiral discrimination effects have been observed using
BAM in monolayers of a range of single-chain Nacylamino acid derivatives,62 some of which had previously been imaged using fluorescence microscopy. The
domain morphologies observed by BAM and by fluorescence microscopy were in agreement. BAM observation
of monolayers of N-stearoylserine methyl ester (SSME)
enantiomers showed distinct curved domains resembling
spirals that curved clockwise for the D-enantiomer and
counterclockwise for the L-enantiomer, as seen in Figure 10.
Racemic monolayers of SSME showed a number of
twinned domains showing both senses of curvature as
well as larger flower-like domains with curved segments growing around their peripheries either in a clockwise or counterclockwise sense. These observations were
taken as evidence for chiral segregation in the racemic
monolayer.
(b)
(c)
Figure 10 Images of domains of monolayers of the D-enantiomer, L-enantiomer, and racemic mixture of N-stearoylserine methyl
ester clearing showing chiral discrimination effects. The domains of the enantiomers display unique curvature, and the domain of the
racemic mixture shows features with both senses of curvature and hence evidence for chiral segregation. Reproduced from Ref. 62.
American Chemical Society, 2003, and the figure caption reads as follows: Chiral discrimination of the condensed-phase domains
of N-stearoyl serine methyl ester monolayers spread on pH 3 water. (a) D-enantiomer (b) L-enantiomer (c) and (d) 1 : 1 DL racemate.
Image size 80 80 m.
DOI: 10.1002/9780470661345.smc040
22
Techniques
The A isotherms for these monolayers indicated

homochiral discrimination. Similar curved spiral domains
were seen in monolayers of N-palmitoylaspartic acid. Very
striking, curved, condensed-phase domains were observed
by BAM in monolayers of N--palmitoylthreonine, curving
with opposite sense for the two pure enantiomers and
showing twin-like structures with one arm curving in each
sense for the racemic mixture paper.63
Further studies of chirality-related effects using BAM
pointed out the complexities involved in considering how
the structure of the observed domains relates to the molecular chirality but is also strongly and often dominantly
affected by domain nucleation and growth rates.64 The presence of Zn2+ in the subphase was found to have dramatic
effects. Monolayers of N-hexadecanoyl-L-alanine spread at
298 K showed large islands of the condensed phase upon
spreading that resembled floating ice packs. These experiments were carried out with a 4 mm 6 mm field of view
(provided by the NFT Mini-BAM instrument); some of
these domains were a few millimeter wide. The phases upon
spreading were the condensed phase and the gas phase, and
the A isotherm did not exhibit a plateau at this temperature. Upon compression, the condensed-phase domains
covered more of the surface and the gas was squeezed out
as they merged, in accordance with the thermodynamic
lever rule for two-phase coexistence. The domains seen
in these monolayers were sensitive to temperature and a
change from 298 to 303 K altered the morphology entirely
such that the condensed phase appeared as hook shapes that
were chiral and whose two arms defined axes of dendritic
growth as the compression continued. This observation
underscores the need for temperature control during monolayer experiments and BAM observations especially since
the A isotherm was essentially the same at these two
temperatures. Monolayers of N-hexadecanoyl-D,L-alanine
showed a plateau in the A isotherm and the condensedphase domains that formed were highly branched fractal
shapes and could be as large as 10 mm in size, as seen in
Figure 11.
Visual evidence of chiral phase separation could not be
found in this racemic monolayer despite the differences in
the A isotherms and IRRAS data showing a homochiral
60
(mN m1)
50
40
30
E
20
A-C
10
0
0.0
0.2
0.4
0.6
A (nm2 mol1)
0.8
(a)
(b)
(c)
(d)
(e)
Figure 11 Reproduced from Ref. 64. American Chemical Society, 2005, and the figure caption reads as follows:
/A isotherm
and corresponding BAM images (AE) of N-hexadecanoyl DL-alanine methyl ester on a pure aqueous subphase (pH 6, T = 298 K). The
images were recorded at the points indicated on the
/A plot. They represent an area of W H = 6 mm 4 mm.
DOI: 10.1002/9780470661345.smc040

preference based upon greater ordering of the hydrocarbon chains. This illustrates that the domains seen here are
nonequilibrium growth forms and have grown so rapidly
that the two-dimensional diffusion required for opposing
enantiomers to form separate domains was not possible.
The behavior of N-hexadecanoyl-L-alanine on a subphase
containing Zn2+ was strikingly different as domains of the
condensed phase showed chiral S-shaped domains, torusshaped domains, and seahorse-shaped domains. Monolayers of N-hexadecanoyl-L-alanine methyl ester and of Nhexadecanoyl-D,L-alanine methyl ester were also studied
and the condensed-phase domains observed were primarily
dendritic growth forms.
A consideration often overlooked in BAM studies is the
possible influence of the compression rate on the domain
structures. In the case of N-acylamino acid monolayers,
the associations due to amideamide hydrogen bonding
are very strong and promote rapid domain growth and
also make it unlikely that relaxation to an equilibrium
domain shape can occur on any realistic experimental
timeframe. Domain shape relaxation kinetics are noted to
be dependent on the strength of intermolecular forces; for
example, dendritic condensed-phase domains formed in a
phospholipid monolayer required 5 h to relax to equilibrium
shapes and, for the phospholipid DMPE, compression rates
2 per molecule per minute were needed
as slow as 0.2 A
to observe equilibrium domain shapes.21 Examination of
the variation of domain structure with time after their
formation or with compression rates are not commonly
reported; however, it is advisable to consider examining
these variables when carrying out BAM experiments.
The significance of the amideamide hydrogen bonding in monolayers of the amino acid derivative Nstearoylvaline was assessed by comparing the behavior
of monolayers of N-stearoyl-L-valine with N-stearoylN-methyl-L-valine.65 While dendritic growth forms were
observed by BAM upon compression for monolayers of
N-stearoyl-L-valine, methylation of the amide nitrogen that
removes the prospects for amideamide hydrogen bonding
resulted in the observation of condensed-phase domains that
were irregular, mostly rounded shapes.
In the case of monolayers of the compound N-tetradecyl ,-dihydroxy-pentanoic acid amide (TDHPAA), chiral discrimination effects in the domain structures were observed
by BAM.62 The small, spear-like crystals showed a longer
and shorter branch at one end and were seen to be mirror
images of each other when the two enantiomers were compared. The racemic mixture formed a symmetric, almost
straight and narrow crystallite for its condensed phase.
Monolayers of the R-enantiomer of 3-palmitoyl-snglycerol were found to behave differently than monolayers
of 3-palmitoyl-rac-glycerol under BAM observation,
23
although in a more subtle manner that required observation of domain anisotropy making use of the analyzer
of a BAM.66 The condensed-phase domains of the Renantiomer were almost round and were divided neatly
into seven segments that appeared with different reflectivities, indicating that the azimuthal tilt direction is different
in each pie-piece-shaped segment. The molecules within
these domains were all tilted by the same angle and the
tilt was directly radially; however, the azimuthal direction of the tilt direction jumped discontinuously at the
borders between the domain segments. At 20 C, the Renantiomer and the racemic mixture behaved identically
under BAM observation and also exhibited identical surface pressure isotherms. At a lower temperature of 5 C,
a phase transition was observed for the racemic mixture
that was not observed for the R-enantiomer in that BAM
showed changes in the azimuthal orientations upon compression inside the racemic domains. A kink in the surface
pressure isotherm for the racemic compound was seen that
was not seen in the isotherm of the enantiomer. GIXD
data found that the enantiomer was ordered in an oblique
lattice, while that for the racemic mixture was rectangular. Observing this difference in domain behavior and
detecting the phase transition using BAM required attention to the effect of rotating the analyzer. A derivative of
1-stearoyl-rac-glycerol bearing a hydroxyl group on the 12position showed very different behavior and BAM images
of monolayers of this compound at 6 C showed domains
with several large curved arms that were sometimes closed
into rings and were of either sense of curvature.67 The tendency to form curved domains diminished with increasing
temperature. Thus, it is important to conduct studies at a
series of temperatures as condensed-phase domain structures can often vary significantly.
Monolayers of ethyl 4-fluoro-2,3-dihydroxystearate diastereomers were examined recently and provide an example
of a more complex chiral system as these molecules contain three chiral centers.68 It was not possible to produce all of the possible diastereomers in pure form in
this case. Four synthetic products were studied as monolayers: (a) a 69 : 31 ratio mixture of the (R,R,S)/(R,R,R)
enantiomers referred to as RDIA, (b) a 67 : 33 ratio mixture of the (S,S,R)/(S,S,S) enantiomers referred to as SDIA,
(c) a 60 : 40 ratio mixture of the two enantiomers pairs
(R,R,S)/(S,S,R) and (R,R,R)/(S,S,S) referred to as RAC,
and (d) a pure enantiomer whose absolute configuration
was not determined referred to as ENAN. Surface pressure isotherms at 20 C showed that the ENAN compound
gave a highly condensed isotherm, while RAC was the
most expanded and showed signs of a phase transition near
15 mN m1 . Monolayers of SDIA and RDIA were intermediate in their behavior and fairly close to each other
in surface pressure. BAM showed different morphologies
DOI: 10.1002/9780470661345.smc040
24
Techniques
for all four of these monolayer systems. The RAC monolayer formed rounded domains with fringes. Both SDIA
and RDIA monolayers formed long, curved needle structures. The domains of ENAN were much smaller, elongated bead shapes and were present upon spreading. Their
appearance immediately on spreading was consistent with
the surface pressure isotherm, which suggested finding
the gas and condensed phases at low pressure. Additional
information was obtained from tapping mode AFM study
of these monolayers transferred as LangmuirBlodgett
films and from molecular modeling. The structural differences between systems are a challenge to unravel, but
BAM can certainly provide some directly relevant information concerning two-dimensional organization at the water
surface.
5.1

MONOLAYERS IN WHICH
HYDROGEN-BOND COMPLEX
FORMATION OCCURS AT THE
WATER SURFACE
Molecular recognition by melamine
amphiphiles
Amphiphilic derivatives of melamine of the form 2,4-di(nalklyamino)-1,3,5-triazine (2Cn H2n+1 -melamine) present
three hydrogen-bond donors and two hydrogen-bond acceptors when spread at the waterair interface. The recognition of small soluble species with complimentary hydrogen bonding by these melamine amphiphiles has been
studied in monolayers using surface pressure isotherms
BAM and GIXD.48, 6971 Binding to both faces of the
melamine by the recognized molecule could result in formation of linear hydrogen-bonding networks at the waterair
interface. The recognition of thymine by monolayers of
2C11 H23 -melamine (Figure 7) was examined.69 Monolayers of C11 H23 -melamine alone showed surface pressure
isotherms with a plateau, indicating a first-order phase transition from a LE phase into a coexistence with a condensed
phase. The monolayers were studied over the temperature
range 10.231.9 C, and the surface pressure of the plateau
increased regularly with temperature. The monolayers collapsed at the end of the plateau at 31.9 C. The observation
of the first-order phase transition leads to the expectation
of observing well-defined, condensed-phase domains by
BAM in the coexistence region and such was the case in
this study. In the case of amphiphiles with a head group
and hydrocarbon chain structure, the temperature range
for observing the first-order phase-transition plateau can

be adjusted into the experimentally accessible temperature range by adjusting the hydrocarbon chain lengths with
longer chain lengths, generally increasing the temperature at
which the transition plateaus will be observed. The authors
previously studied monolayers of 2C10 H21 -melamine and
2C12 H25 -melamine alone using surface pressure isotherms,
equation of state calculations, BAM and GIXD.71 For
monolayers of 2C11 H23 -melamine alone, BAM showed the
appearance of numerous compact domains of similar size
in the coexistence regions that grew larger under compression. The monolayers of 2C11 H23 -melamine were then
studied on subphases containing thymine (0.05 or 0.10 mM)
and striking differences were observed. The surface pressure isotherms depended upon compression rates such that,
the slower the rate, the greater was the reduction in the
pressure of the plateau, which could be lost altogether at
a low enough compression rate. Monolayers of 2C11 H23 melamine on thymine subphases are not in equilibrium
2
under compression except at the slowest used rate of 1 A
per molecule per minute. This indicates that time is required
for the completion of the recognition of the dissolved
thymine by the 2C11 H23 -melamine monolayer. BAM observation of 2C11 H23 -melamine on 0.1 mM thymine subphase
2 per molecule per minute at 27 C to
and compressed at 1 A
2
per molecule over a 30-min period showed the forma80 A
tion of large, bright, symmetric, dumbbell-shaped domains
as large as 400 m. The interiors of these domains had a
subtle texture of smoothly varying brightness that indicated
the presence of anisotropy most likely associated with a
small molecular tilt varying in direction within the domains.
The formation of domains on the 2C11 H23 -melamine could
also be followed by stopping compressions carried out at
slightly higher rates and then following surface pressure
relaxation and BAM observation. Compression stopped at
13.5 mN m1 , showing formation of narrower dumbbells
during relaxation, while stopping at 15.6 mN m1 showed
formation of many small irregularly branched domains that
merged into a network. GIXD indicated a centered rectangular lattice with a small molecular tilt toward next
nearest neighbors. The same lattice was seen on the aqueous thymine subphase but with different dimensions. These
studies show the promise of BAM for studying recognition
processes with hydrogen bonding between the amphiphile
and a subphase species. It is also clear that such a system
can display very rich behavior as function of the physical
and chemical parameters and that attention must be paid to
questions of appropriate compression rates and approaches
to equilibrium. The BAM observations can clearly show
dramatic differences in domain structures due to molecular
recognition at the water surface.
A further study compared the recognition of uracil, which
can hydrogen-bond to one face of the 2C11 H23 -melamine
DOI: 10.1002/9780470661345.smc040

molecules, with the recognition of barbituric acid, which
can bind to both faces by 2C11 H23 -melamine monolayers.48
The presence of 0.5 mM uracil in the subphase had a similar
effect as that seen in the study using thymine in that its
complete recognition by the melamine amphiphile resulted
in disappearance of the surface pressure plateau and the
formation of very different domain structures, as seen in
Figure 12.
On the uracil-containing subphases, many round domains
were seen with a gradually varying inner texture, indicating a varying molecular tilt direction within the domains.
Over time, the number of domains decreased and the larger
domains expanded in size significantly at the expense of
the smaller ones, a process generally known as Ostwald
ripening and seen in many situations involving nucleation
and growth of domains. The isotherms of the 2C11 H23 melamine monolayers were shifted to lower areas on a
0.01 mM barbituric acid subphase, indicating the formation of a more condensed structure. BAM observation
of 2C11 H23 -melamine monolayers on barbituric acid containing subphase showed the formation of large, homogeneously bright regions that could merge and become
uniform. This observation was viewed as being consistent
with formation of an extensive hydrogen-bonded network
between melamine and barbituric acid. As an interesting
reminder of how modest changes in molecular structure can
result in completely different domain structures, 2C11 H23 melamine was modified to 2C12 H25 -O-(CH2 )3 -melamine.
On aqueous uracil subphases, the domains formed for this
modified melamine amphiphile showed a distinctly varying
inner texture indicating greater anisotropy. Their appearance became more frayed at the peripheries upon increasing the uracil concentration from 3 to 5 mM. On 0.1 mM
aqueous barbituric acid subphases, this modified melamine
amphiphile showed starfish-like domains with an irregular
number and size of arms in which each arm appeared with
a different brightness, indicating different azimuthal orientations within each arm. The weakening of the hydrogenbonding network was cited as a possible reason for these
different observations. A subsequent study developed a
kinetic model of the recognition process that could fit the
surface pressure relaxation data.70
5.2
Highly ordered monolayers formed by

adsorption from the subphase
A particularly strong advantage of the BAM method is that

it can be used to directly image monolayers formed by
adsorption from the subphase. Phase transitions as a function of surface coverage, related to the subphase concentration, adsorption time, and temperature can be observed
for such adsorbed monolayers. In one series of studies, the
25
(a)
(b)
(c)
Figure 12 Brewster angle microscopy images of the condensedphase domains of the melamine amphiphile from the studies
described in Section 5.1 shown on different subphases illustrating
the effect of molecular recognition by lateral hydrogen bonding on domain structure. Reproduced from Ref. 70. American
Chemical Society, 2005, and the figure caption reads as follows: BAM images of characteristic condensed-phase domains
of C11H23-melamine monolayers spread on water (a), 0.5 mM
uracil subphase (b), and 0.1 mM thymine subphase (c). Compression rate per molecule, 0.04 nm2 min 1 ; T, 25 C; image size,
400 m 400 m.
DOI: 10.1002/9780470661345.smc040
26
Techniques
behavior of adsorbed monolayers of molecules containing

amide groups and thus capable of strong lateral hydrogen
bonding was examined by BAM and surface pressure measurements. The molecule N-dodecyl- -hydroxybutyric acid
(DHBAA) was investigated, both as adsorbed and spread
monolayers.49 The chain length here is chosen to be long
enough to allow formation of spread monolayers but short
enough to allow sufficient solubility such that solutions can
be prepared from which adsorbed monolayer formation can
be studied. BAM observation of the adsorption process was
carried out by sweeping the surface of the DHBAA solution
clean and then measuring the change in surface pressure as
a function of time for close to 1 h while simultaneously
observing the interface with BAM. The measurements were
carried out at a series of temperatures from 5 to 30 C
and at 10 C and adsorption was followed at a series of
DHBAA concentrations from 0.01 to 0.01 mM. The measurement of surface pressure versus time during adsorption
at 10 C showed a sharp inflection point near 16 mN m1
at all five concentrations studied, and the time required to
observe the inflection decreased with increasing concentration. These observations defined a critical surface pressure
c and critical time tc for the transition; at tc , the surface
pressure that had been rising steadily abruptly changed to
increasing with a very gradual slope. The transition was not
observed above 15 C. At 15 C, BAM showed the formation of highly ordered condensed-phase domains with four
growth directions that grew in size until covering most of
the field of view. In this experiment, measurement of the
integrated BAM reflectivity as a function of time was performed. The reflectivity remained flat for about 10 min after
the surface pressure inflection and then steadily climbed,
although with up and down fluctuations. After the induction
time following the phase transition, there were condensedphase domains sufficient in size and number to increase the
reflectivity. The integrated reflectivity represents the light
reflected from the entire illuminated spot on the water surface. As the monolayer domains are slowly drifting about
on the water surface, their exact numbers under the laser
spot will fluctuate with time, and this accounts for the somewhat jagged appearance of the increase in reflectivity versus
time. DHBAA was then used to form spread monolayers
and A isotherms were measured by compression. Given
the short chain length of the species, high compression
rates were used to avoid dissolution. In the compression
isotherm, surface pressure plateaus were observed, indicating a first-order phase transition and a two-phase coexistence region. The surface pressure at onset of the phase
transition in the spread monolayers increased with temperature, the molecular area at onset decreased, and the width
of the plateau region also decreased, signifying the coexistence of two phases. The onset surface pressures for spread
monolayers were close to those for the surface pressures
observed at the inflection points in the adsorbed monolayers. The morphology of the condensed-phase domains
formed upon compression of the spread monolayers was
similar in characteristic features to that seen formed in the
adsorbed monolayers.
A related study compared spread and adsorbed monolayers of the molecules N-( -hydroxypropyl)tridecanoic acid
amide (HTRAA) and N-( -hydroxypropyl)tetradecanoic
acid amide (HTEAA).50 HTEAA is water insoluble and
more suitable for GIXD. This study included the application of GIXD, which revealed an oblique lattice structure independent of whether the monolayers were formed
by spreading or adsorption. The dendritic appearance of
the condensed-phase domains under BAM was attributed
to the strong hydrogen bonding present between these
molecules. These studies suggest that the prospects for
studying adsorbed monolayers of supermolecules and their
complexes directly as adsorbed monolayers should be considered and that BAM is well suited for this purpose.
6.1
EMERGING DEVELOPMENTS IN THE

APPLICATION OF BREWSTER
ANGLE MICROSCOPY
Time-resolved Brewster angle microscopy
A recent extension of BAM involves equipping the setup

with a pulsed Nd-YAG laser that can deliver a 5 ns pulse
of 355 nm wavelength and 60 mJ energy radiation to the
water surface from a direction perpendicular to the water
surface.72, 73 The same spot as subjected to the pulse is
then observed by BAM using a 532 nm laser illumination
at the Brewster angle using a CCD camera that can record
an image every 40 ms and with a 1 ms shutter time. This
pulse and then probe (image) version of BAM was applied
to monolayers of a spiropyran derivative that underwent
a conversion to a merocyanine form upon illumination
but relaxed thermally back to the spiropyran form.73 The
technique made observing a number of photochemically
induced changes possible using BAM. Collapsed aggregates
of the spiropyran form were observed to transiently spread
into larger, thinner domains of the merocyanine form. The
merocyanine form has a more polar head group and a
different alkyl chain orientation that makes it able to spread
better on the water surface. The spreading was transient due
to conversion back to the spiropyran over a few hundred
microseconds. A transient wave structure induced by the
laser pulse was observed and attributed to jumping the
monolayer from a gas phase for the spiropyran to an LE and
gas state. The modification of BAM to include pulsed laser
illumination followed by high-speed imaging should find
DOI: 10.1002/9780470661345.smc040

application to additional photophysical and photochemical
studies in monolayers.
6.2
Light-scattering microscopy coupled with

The method of light-scattering microscopy (LSM) was

introduced and can provide information that is new and
complimentary to that provided by BAM. The LSM
method has not yet been widely applied to monolayers of
supramolecular compounds and seems well suited to provide information especially for those systems subject to
aggregation effects. LSM is an application in which the
BAM setup is modified by adding an intensified CCD camera perpendicular to the water surface and a microscope
objective that can collect light that is scattered normal to
the surface, while the reflected light is detected and imaged
by BAM. The microscope and camera perpendicular could
also be used for fluorescence microcopy together with BAM
and LSM to give a powerful combination of techniques.
Particles or aggregates of 100 nm or greater in size should
be able to be imaged by LSM using the same low-power
lasers such as a 10 mW HeNe laser as used for BAM
illumination. This method could be applied to a number of
problems, and revealed some new, remarkable monolayer
phenomena.73
LSM was applied to observe the formation of biominerals beneath phospholipid monolayers. The formation of
calcium oxalate crystallites underneath phospholipid monolayers in the LE + LC region was readily observed.
Streptavidin crystals growing beneath monolayers of the
synthetic lipid 1,2-dioleyl-rac-glycero-3-(8-(3,6-dioxy)
octyl-1-amino-diacetic acid (referred to as Cu-DO-IDA)
were simultaneously imaged using BAM and LSM. The
protein streptavidin coordinates to this lipid through two
histidines, forming a complex with a Cu2+ ion from the
subphase that also complexes to the lipid head group. LSM
was able to discern vacancies within these two-dimensional
streptavidin crystals that grew beneath the Cu-DO-IDA
monolayers. LSM revealed a remarkable topographic instability previously unknown for phospholipid monolayers.
LSM images of monolayers of DPPC compressed through
the coexistence region of the LE and LC phases and into
the LC phase showed corrugated patterns of scattering originating around where the boundaries of the LC domains
had been located.74 These monolayers were transferred to
mica for AFM analysis in a tapping mode, and regions that
represented budding, a bulging of lipid up away from the
surface, were found. These buds proliferated rapidly during
collapse but, in the LC phase below collapse, represented
a small fraction of <1% of the phospholipid present. The
LSM method seems to be of high interest for applying to
27
supramolecular systems, such as the amphiphilic macrocycle systems, subject to much speculation about formation
of three-dimensional aggregates under various conditions
of their study on the water surface.
The LSM method was used to follow a unique solitonlike phenomenon in observations of monolayers of amphiphilic bistable rotaxanes.75 LSM showed 10-m-wide
bright spots moving in linear trajectories across the water
surface. These features moved perpendicular to the compression direction at speeds of 50350 m s1 . The features disappeared if the compression was slowed to <3 mm
min1 . Resuming compression resulted in the features reappearing at the same points and starting to move again. The
features were found to display the behavior expected for
solitary traveling waves (solitons). The observation of the
solitons required the presence of the cyclobis(paraquat-pphenylene) ring component and was not associated with
the moving of the ring component between two sites on
the chain. It was speculated that the solitons were associated with defects in the monolayer. This study indicates
that monolayers of supramolecular species can give rise to
unexpected phenomena discovered when monolayers are
observed using new methods probing different physical
aspects of their microstructure.
CONCLUSION
BAM imaging of monolayers of supramolecular systems

can reveal many valuable insights into molecular ordering
at interfaces. In some systems, the BAM images are quite
striking and remarkable, and fascinating domain structures
are revealed. In other systems, one may only see rather
mundane features, but even in these cases insight is
provided into the molecular ordering at the surface at
least to the extent that it manifests itself in micron-sized
structures. Some of the key points to keep in mind when
applying BAM to monolayers in supramolecular chemistry
are as follows:
1.
2.
It is important to use the BAM images together

with the A isotherms and careful consideration is
required of molecular dimensions to judge whether the
compound at hand is spreading as a true monolayer or
if it is displaying aggregation or dissolution behavior
throughout the experiment; in such cases, applying
monolayer-based interpretations to the system may face
significant uncertainty in interpretation.
It is important to consider whether the domains
observed are growth forms or are closer to being
equilibrium domain shapes. An appreciation of the
significance of observing dendritic or fractal shapes
DOI: 10.1002/9780470661345.smc040
28
3.
4.
5.
Techniques
signifying the appearance of growth forms versus various more compact shapes is important. Experiments
in which the compression rate is varied or in which
domains are observed over periods of time after stopping compression may yield surprising observations.
In studies of mixed monolayers, examination of a series
of compositions is important along with the variation
in the temperature. The assignment of miscibility or
immiscibility in mixed monolayers using BAM can be
subtle.
Observation of anisotropy within domains is an important indication of molecular ordering and thus monolayers should be viewed both with the analyzer at 0
and other angles to check for anisotropic ordering.
Experiments in which supermolecules and their guests
are simply dissolved in the subphase and allowed to
form adsorbed monolayers can be performed using
BAM and can yield interesting results. This removes
the need to carry out synthetic modification with
hydrocarbon chains, and can at least constitute an
additional approach to studying the system.
The most recent BAM instruments make the application of quantitative BAM and thus estimation of relative
thickness changes in regions of a BAM image accessible to
those with these instruments. The quantitative BAM method
can provide insight into the stability/instability of supermolecules at the waterair interface, formation of multilayer structures, height differences between domains, and
thickness changes due to conformation changes or due to
binding of large enough guest species from the subphase.
Application of quantitative BAM should be considered as
a means to making studies of supermolecules provide more
insight. The more detailed analysis of BAM images with
anisotropy, although difficult, should also be fruitful for
future studies. The image quality has improved, although
at present the most capable instruments are not available
to many labs. As new supramolecular systems of interest
emerge, undoubtedly study of their assembly in two dimensions will become of interest in many cases and BAM will
be a major technique for examining such monolayers at the
waterair interface.
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DOI: 10.1002/9780470661345.smc040

Brewster Angle Microscopy: Keith J. Stine

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Brewster Angle Microscopy

1 Introduction: The Role of Brewster Angle Microscopy

INTRODUCTION: THE ROLE OF

The study of monolayers of amphiphilic compounds at the

fluorescent probe lipids would partition in a meaningful way

PRINCIPLES AND GENERAL

The basic principle of BAM is that for p-polarized light

2.1.1 Reflectivity of the waterair interface

Brewster angle microscopy

The reflectivity Rp = |rp |2 for p-polarized light and Rs =

2.1.2 Reflectivity of an isotropic monolayer

the ambient n0 , the refractive index n1 , and thickness d1 of

2.1.3 Reflectivity of an anisotropic monolayer at the

General features of the Brewster angle

Incident angle (q0)

Figure 1 Reflectivity as a function of incident angle for both

The Brewster angle microscope is positioned over the

either case, the space to accommodate the BAM must be

impurities are present, they often appear as fluid streak-like

Figure 2 The basic geometry and components of a Brewster

Selection of instrumentation for Brewster

A number of companies offer commercial BAMs that

Brewster angle microscopy

General features of monolayer phase

The interpretation of BAM images involves the use of

using BAM, going beyond qualitative description should

ordering within domains visible by BAM. If the condensed

BAM contrast inversion due to relative thickness changes

Brewster angle microscopy observation of

When the refractive index of a monolayer varies with

Brewster angle microscopy

Monolayers of methyl eicosanoate were examined in

phases and the domains of the tilted condensed phase are

Figure 4 Liquid-condensed-phase domains in monolayers of

relaxed into circular domains within minutes. These growth

Anisotropy within condensed-phase domains can also

BAMs is an ideal method for observing films at the

Brewster angle microscopy

here a range of coexisting multilayer structures was also

Figure 5 BAM image of multilayer regions coexisting in spread

interface provide a unique environment in which to observe

BAM is also well suited for determining when a monolayer

Surface pressure (mN m1)

BAM enables a number of additional characterizations of a

Figure 6 BAM images of a monolayer of stearic acid in

of the molecule at hand for the given subphase conditions.

Quantitative analysis of Brewster angle

In (3), d is the film thickness, is the wavelength of the

Brewster angle microscopy

APPLICATION OF BREWSTER ANGLE

BAM has been applied in studies of amphiphilic derivatives

Examples involving monolayers of

monolayer. BAM observations showed differences amongst

Chiral crown ether

Cryptophane (anti form)

Brewster angle microscopy

between calixarenes to being between calixarenes and water

5 C where there was an inflection region near 25 mN m1