Competitive Adsorption of Proteins and Low-Molecular-Weight Surfactants Computer Simulation and Microscopic Imaging

Advances in Colloid and Interface Science 107 (2004) 2749
Competitive adsorption of proteins and low-molecular-weight surfactants:

computer simulation and microscopic imaging
Luis A. Pugnalonia, Eric Dickinsona,*, Rammile Ettelaiea, Alan R. Mackieb, Peter J. Wildeb
a
Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK

Food Materials Science Division, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney NR4 7UA, UK
Abstract
Proteins and low-molecular-weight (LMW) surfactants are used in the food industry as emulsifying (and foaming) ingredients
and as stabilizers. These attributes are related to their ability to adsorb at fluidfluid (and gasfluid) interfaces lowering the
interfacial (and surface) tension of liquids. Hence, the study of the properties of adsorbed layers of these molecules can be
expected to lead to a better understanding of their effect on food products. Direct proof of the validity of mesoscopic models of
systems of proteins and LMW surfactants can only be achieved by quantitative theoretical predictions being tested against both
macroscopic and mesoscopic experiments. Computer simulation constitutes one of the few available tools to predict mathematically
the behaviour of models of realistic complexity. Furthermore, experimental techniques such as atomic force microscopy (AFM)
now allow high resolution imaging of these systems, providing the mesoscopic scale measurements to compare with the
simulations. In this review, we bring together a number of related findings that have been generated at this mesoscopic level over
the past few years. A useful simple model consisting of spherical particles interacting via bonded and unbonded forces is
described, and the derived computer simulation results are compared against those from the imaging experiments. Special attention
is paid to the adsorption of binary mixtures of proteins, mixtures of LMW surfactants, and also proteinqsurfactant mixed systems.
We believe that further development of these mathematically well-defined physical models is necessary in order to achieve a
proper understanding of the key physicochemical processes involved.
2003 Elsevier B.V. All rights reserved.
Keywords: Proteins; Surfactants; Competitive adsorption; Emulsion and foam stability; Atomic force microscopy; Brownian dynamics simulation;
Phase separation
Contents
1. Introduction .........................................................................................................................................
1.1. The background .............................................................................................................................
1.2. Computer simulation ......................................................................................................................
1.3. A simple model for proteins and surfactants at interfaces ...................................................................
1.4. Microscopy at interfaces .................................................................................................................
1.5. Phase separation at interfaces ..........................................................................................................
2. Adsorption of one-component systems ....................................................................................................
2.1. Adsorption of LMW surfactants ......................................................................................................
2.2. Adsorption of globular proteins .......................................................................................................
3. Competitive adsorption of mixed LMW surfactants .................................................................................
4. Competitive adsorption of mixed proteins ...............................................................................................
5. Competitive adsorption of proteins and LMW surfactants .........................................................................
*Corresponding author. Tel.: q44-113-3432956; fax: q44-113-3432982.
E-mail address: e.dickinson@leeds.ac.uk (E. Dickinson).
0001-8686/04/$ - see front matter 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.cis.2003.08.003
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L.A. Pugnaloni et al. / Advances in Colloid and Interface Science 107 (2004) 2749
6. Concluding remarks .............................................................................................................................. 47

Acknowledgements ................................................................................................................................... 48
References ............................................................................................................................................... 48
1. Introduction
1.1. The background
Proteins and low-molecular-weight (LMW) surfactants are key components of many foodstuffs w1x. Some
dairy products, for example ice cream, contain both
proteins and LMW surfactants in their formulation. Both
types of molecules can adsorb at fluid interfaces, reducing the interfacial tension and so facilitating the formation of emulsions and foams and providing stability to
droplets and bubbles. However, their molecular properties are very different w2x.
LMW surfactants are small molecules each consisting
of a hydrophilic head group and one or several hydrophobic tails. When such molecules reach an airwater
or oilwater interface, they tend to adsorb by arranging
the hydrophobic tails within the non-aqueous phase and
the hydrophilic head in the water phase (see Fig. 1a).
LMW surfactants are very mobile and they are particularly efficient at reducing the interfacial tension. As a
result, they rapidly coat the newly created oilwater and
airwater interface during emulsification and foaming.
Proteins are high-molecular-weight molecules each
consisting of a chain of amino acids. As there are polar,
non-polar and ionic amino acids, proteins contain a
mixture of hydrophilic and hydrophobic groups. In
aqueous solution, a protein molecule will tend to fold
Fig. 1. Schematic representation of surface-active species. (a) Protein

and surfactant molecules. (b) Representation of the proteins and surfactants as particles according to the model presented in this paper.
in a coil-like structure in order to expose the most

hydrophilic groups to the water and hide the most
hydrophobic segments in the centre of the coil (see Fig.
1a). However, when a protein molecule reaches an air
water or oilwater interface, the molecule will partially
unfold orientating its hydrophobic groups towards the
non-aqueous phase (Fig. 1a). Proteins are very slow at
diffusing and adsorbing as compared with LMW surfactants; and they do not normally lower the interfacial
tension so efficiently. However, proteins form thick
protective layers at the surface of oil droplets and gas
bubbles which, under appropriate conditions, can prevent
coalescence after an emulsion or foam has been formed
thereby conferring long-term stability to the system.
When a mixture of LMW surfactants or a mixture of
proteins or a surfactantqprotein mixed system is
exposed to an interface, the different species compete
to adsorb and lower the interfacial tension w3x. During
the equilibration of the interfacethis may take from
seconds in the case of pure LMW surfactants to up to
several hours for protein-containing systemsthe molecules adsorb and desorb dynamically. If one of the
components of the mixture is presented to the interface
first, then the second component will tend to replace the
adsorbed molecules of the first type partially or totally
depending on the relative surface-activity of the species
and their mutual cross interaction. Interestingly, in some
cases, the mixture does not adsorb homogeneously over
the surface. On the contrary, interfacial regions rich in
one or the other species are present either during the
equilibration process or in metastable states.
It is a common practice in the study of such systems
(and in general of most physicochemical systems) to
generate a mesoscopic model of the molecules involved
in order to explain the behaviour of the system in a
qualitative fashion. These kinds of models, which are
often not very precisely defined, take into account a few
essential characteristics of the molecules under study
such as molecular weight, flexibility, hydrophiliclipophilic balance, presence of reactive groups, and charge
distributioninstead of the detailed chemical structure.
This provides a useful way of elucidating the important
factors affecting a particular phenomenon. However, the
majority of these models are expressed in a qualitative
language during the interpretation of experimental
results, but if these types of models are amenable to be
expressed in a physicomathematical language describing quantitatively the molecules and their interactions,
the calculation of the behaviour of an ensemble of
molecules can be made by solving the basic equations
of motion through the use of a suitable computer

simulation algorithm w4,5x.
Atomic force microscopy (AFM) is a powerful experimental tool to probe very small length scales on a
surface w6x, hence the usefulness of this technique to
inspect the structure of adsorbed protein films at interfaces w7x. It is particularly interesting that with AFM
we are able to reach resolutions almost down to molecular length scales. Therefore, any mesoscopic model can
be assessed at the ultimate level of description, i.e. down
to the small-scale structures formed by the molecules
themselves. Additionally, fluorescence microscopy provides a method to distinguish different species in a
protein mixture w8x. This can be particularly useful, for
example, if one is interested in studying the homogeneity
of the layer formed at the interface.
In this paper, we review a number of experiments and
computer simulations of model systems that have been
carried out in recent years. We pay special attention to
the description of the structure of the interface. We
describe a general but simple mesoscopic model for
adsorbing proteins and surfactants, and several examples
of computer simulation results on the competitive
adsorption of these types of molecules are shown.
Atomic force microscopy and fluorescence microscopy
experiments are presented and compared with the simulation results. Additionally, we present some rheological properties of adsorbed protein layers that can be
extracted from the simulations.
1.2. Computer simulation
In this section we give a concise overview of the
most common computer simulation techniques relevant
to this topic. We recommend the reader to refer to the
book by Allen and Tildesley w5x for a thorough introduction to the subject.
Every physicochemical system comprises a number
of basic interacting entities. Depending on the intended
level of description, these entities can be atoms, molecules or molecular complexes. We give the name particle to any of these basic entities. A theoretical model
for these particles consists in a mathematical definition
of the way they interact with each other and with
external forces. When such a model is complex, as is
usually the case when dealing with complex fluids, the
physical equations that describe the behaviour of a
system composed of these model particles are unattainable analytically. In a few cases, further approximations
can be used to simplify the equations. Mainly, however,
a numerical solution of the set of fundamental physical
equations is normally the practical alternative. This
numerical approach is referred to as computer simulation. The two basic techniques for performing computer
simulations consist in either solving the equations of
motion of the involved entities (particles) that form the
29
systemthis is termed molecular dynamics (MD) simulationor in generating ensembles of representative

configurations of the system at randomwhat is called
Monte Carlo (MC) simulation.
It is important to mention here that there exist other
numerical techniques to study the properties of different
molecular models. Self-consistent field theory for example, is a very useful technique to study the equilibrium
properties of molecular systems (see for example Ref.
w912x). However, such types of studies are not computer simulations in the sense that the fundamental
physical equations are solved indirectly. In self-consistent field theory, the unknown function to be calculated
numerically is the density distribution of molecular
segments at thermodynamic equilibrium rather than the
positions andyor velocities of each segment.
In a classical MD simulation, Newtons equations are
solved for a set of particles. The computation involves
the solution of a system of N coupled differential
equations (one for each particle) of the form
mi
d2rit.
sFiextwyrit.z~q8Fi,jwyrit.,rjt.z~,
x
dt
(1)
j/i
where mi is the mass of particle i, ri(t) is its position at

time t, and Fext
and Fi,j are the forces applied on i by
i
any external field and by particle j, respectively. Of
course, solution of such a system of differential equations requires initial conditions for the positions of the
particles ri(ts0) and their velocities vi(ts0). Then,
any appropriate finite difference technique can be used
to solve the equations numerically and so obtain the
position ri(t) and velocities vi(t) of the particles at
discrete intervals of time Dt. Once the trajectories of the
particles have been calculated for a long enough period
of timemillions of units of Dt, typicallythe macroscopic properties (specific heat, viscosity, etc.) and
mesoscopic properties (structure factor, clustering, etc.)
can be extracted through the appropriate time averages.
Classical MC simulation relies on the ensemble statistical theory. According to this theory, an average over
all possible configurations of the system is equivalent
to an average over the configurations that the system
visits during its time evolution, as long as it is in
thermodynamic equilibrium. Therefore, a representative
set of MC configurations can be generated at random
and then used to obtain mesoscopic and macroscopic
averages. The simulations carried out under the MC
scheme are normally simpler and more efficient than
the ones based on MD. However, since the real trajectories of the particles are not calculated, no direct
dynamic information can be extracted from MC simulations. Thus, only equilibrium properties of the systems
of interest can be studied via MC simulation. Unfortunately, many of the most interesting properties of real
surface-active molecules are related to their slow dynam-
30
ics, which evolves over periods of hours, days or even

weeks, without reaching proper thermodynamic
equilibrium.
There is also a third, more coarse-grained simulation
approach, namely Brownian dynamics (BD). In studying
systems of surface-active molecules (and complex fluids
in general), a mixture of solvent molecules (typically
water) and surface-active molecules (proteins or LMW
surfactants) has to be simulated. Normally, such solutions contain a few surface-active molecule for every
tens of million of water molecules. Therefore, it is clear
that the simulation of a few hundred surface-active
molecules by the MD or MC approach would involve
the calculation of trajectories or configurations of a
prohibitively large number of solvent molecules. However, if we are not actually interested in the behaviour
of the solvent, it is more sensible to avoid such a waste
of computing resources by focusing just on the surfaceactive species alone, and including the solvent contribution as an effective external perturbation. This is the
basis of Brownian dynamics.
In a BD simulation, as in MD, the equations of
motion of the particles are solved numerically. However,
no solvent particles are included explicitly. Instead,
every other particle is subjected to two external forces
that mimic the effect of the solvent on them. These two
forces are the drag force and the random buffeting that
generates the Brownian motion of the solute. In other
words, BD is concerned with the numerical solution of
the Langevin equation. For an isolated spherical particle
the drag force is taken as y3p h s vi, where h is the
solvent viscosity, s is the particle diameter, and vi is
the particle velocity. The random buffeting is simulated
through a random Gaussian distribution with zero mean
and variance 2kBTy(3p h s), which is consistent with
the fluctuationdissipation theorem and leads to Einsteins relation for the diffusion coefficient wD0skBTy
(3p h s)x. Consequently, we have to solve a system of
N coupled differential equationsone for each surfaceactive particleof the form
2
mi
d rit.
sFiextwyrit.z~q8Fi,jwyrit.,rjt.z~
x
dt
j/i
y3phs
drit.
dt
R
i
qFRi t..
expects that the presence of any container wall should

affect significantly the behaviour of the entire simulated
system. A way to lessen this effect is to use periodic
boundaries conditions. That is, we allow particles on
one side of the system to interact with particles on the
opposite side, as if copies of the virtual container were
placed all around the system to create borders of the
same nature as the system itself. Within the same
scheme, a particle that crosses the boundaries of the
system (say by diffusion) is introduced through the
opposite edge to create a smooth motion across the
border. Although this computational trick is very useful
and efficient, it cannot eliminate other small-size effects:
for example, in the calculation of long-ranged particle
particle forces or in the analysis of density fluctuations
larger than the basic simulation system size. In practice,
the only way of ensuring that size effects do not
influence a particular result is to perform independent
simulations with different system sizes and then extrapolate the results to infinitely big systems.
It is appropriate also here to mention briefly the nonequilibrium simulation techniques used to analyse the
response of a system to an external perturbation w5,13x.
It is the case that the linear response of the system to
small perturbations can be obtained from an equilibrium
MD or BD simulation through the fluctuationdissipation theorem w5x. However, non-equilibrium simulation
techniques require less computer effort to obtain a
similar degree of accuracy, and they also allow us to
study the response of the system to large perturbations,
where the response is likely to be highly non-linear.
The response of the system to an external perturbation
can be measured through the interparticle stress tensor
s. For a pairwise-additive interaction it is given by the
Kirkwood formula w14x
sabsrkBTdaby
1 N Ny1
raijFbij,
V 88
j)i is1
(3)
where V is the volume of the system. Here, a and b

indicate the different Cartesian components of the stress
tensor s, the interparticle distance rij, and the interparticle force Fij, respectively. Normally, the macroscopic
rheological quantities of interest are related to the
symmetric part of s, i.e.,
(2)
Here, F t. represents the random force. Again, as in

MD simulation, finite difference algorithms are used to
solve these equations numerically.
An important point has to be made with respect to all
types of computer simulation. Because the systems
studied are invariably much smaller than real systems,
an unrealistically large proportion of particles are close
to the edges of the system boundaries. Therefore, one
1
sabs sabqsba..
2
(4)
Shear flow can be imposed on a simulated ensemble

of particles in much the same fashion as for a real
system in the laboratory. The most widely used method
involves the creation of a shear flow profile by the
addition of a position and time-dependent external force
quantified by the affine strain gxy(t) applied on the xy-
plane (or any other appropriate plane). The periodic

boundary conditions have then to be modified to account
for a linear flow profile across the infinite replicas w15x.
For a sine wave input at frequency fsv y2p, we have
(5)
gxyt.sg0sinvt.,
and the time domain response is
sxyt.
g0
31
approach has been developed by Wijmans and Dickinson

w18,19x. The model is a straightforward variation of a
previous one designed to describe interacting protein
systems in the bulk (i.e. far from interfaces) w20x. In its
simplest version, the model consists of two types of
spherical particles (of diameters s1 and s2) interacting
via a steeply repulsive spherical core potential:
B s E36
sG9v. sinv t.qG0v.cosv t.,
(6)
where G9(v) and G0(v) are the storage and loss moduli,
respectively. These can be extracted by integrating over
a sufficiently large integral number of shear cycles n as
v
G9v.s
npg0
2np
v
G0v.s
npg0
sxyt.sinvt.dt,
(7)
0
2np
sxyt.cosvt.dt.
(8)
Several initial cycles of the oscillation have to be

discarded from the analysis so as to ensure that only the
steady state response to the perturbation is accounted
for.
The dilatational rheology of a system can be extracted
in a completely analogous fashion to the shear rheology
w16,17x. In this case, the system is subjected to a small
oscillatory change in volume. This is achieved by
creating an extensional flow profile on application of a
position- and time-dependent external force quantified
by the dilatational strain-rate. Again, the periodic boundary conditions have to be adapted in order to account
for a volume-changing system. After an integral number
of dilatational cycles, the dilatational storage modulus
9(v) and loss modulus 0(v) can be extracted through
equations analogous to Eqs. (7) and (8). Additionally,
we can investigate large deformation-dilatational rheology by compressing (expanding) the system at a constant strain-rate down to (up to) few times the original
size and analysing the response of the system though
the stress tensor w18x.
It is worth mentioning here that in what follows we
concentrate on interfacial systems. Therefore, the rheological properties discussed above should be adapted to
interfacial shear and interfacial dilatational quantities by
using the appropriate interfacial stress tensor. A detailed
discussion and application of interfacial shear and dilatational rheology in computer simulation can be found
in Ref. w17x.
1.3. A simple model for proteins and surfactants at
interfaces
In order to study the competitive adsorption of different surface-active components, a simple simulation
fcrij.sC
F ,
(9)
D rij G
where ss1y2 siqsj., si and sj are the diameters of

the interacting particles, rijsri yrj is the centre-tocentre distance of the particle pair, and is an energy
parameter that is set equal to kBT. As usual, kB is the
Boltzmann constant and T is the absolute temperature.
All quantities are then expressed in units of , s1 and
s1ym1 y for energy, length and time, respectively, with
s1 and m1 being the diameter and mass of the particles
of type 1.
To mimic adsorption, each particle of type a (as1
or 2) interacts with an external potential, acting in the
z-direction:
fas zi.s
S B
E36
s1
C s qw.yBz y s E F
T
TC
D
E36
s1
C s qw.qBz y s E F
2 GG
B
sa E a
Cz y
FFz
i
c
D
2 G
2 GG
s1
B
E36
sa E
s1qw.y z y F
D
V D
2 GG
C a
c
s1
E36 B
sa E a
Cz y
F)z
D i 2 G c
C s qw.qBz y s E F
C a
c
2 GG
(10)
This potential has a square-well-like shape with one

infinite wall and one finite wall. The infinite wall
prevents particles from escaping to the phase in which
they are not solubletypically air or oil in the case of
proteins. Conversely, the finite wall allows for interchange of particles between the interface and the phase
where they dissolvetypically an aqueous solution in
an experimental setup. It is important to note that some
surfactants can also dissolve in a non-aqueous medium.
The adaptation of Eq. (10) to account for oil-soluble
surfactants is straightforward but will not be considered
here. The parameter w in Eq. (10) defines a minimum
width for the potential well, and it is usually set to
0.05s1. Then, zca should be greater than w. The parameter zac defines simultaneously the total width of the
potential well and the particle adsorption energy for
each type, namely Eaads'fas zac .yfas 0. w18x. A particle
i of type a is said to be adsorbed if ziFzca. Notice that
the potential representing the presence of the interface
is positioned in such a way that particles adsorb by
touching the interface, which is at zs0. Therefore,
particles of different sizes adsorbed at the interface will
32
not have their centres on the same z-plane, as exemplified in Fig. 1b.
In order to account for the intermolecular crosslinking behaviour of some proteins, the adsorbing particles can also interact through flexible bonds (see Fig.
1b). Nodes are created on the nominal surface of the
spherical particles (si y2 from the centre). The bond
interaction acts along the straight line that joins the
corresponding nodes, and it depends on the node-tonode distance bij only:
0
bijFb1
T
T
w
zz
q8fcwyrijt.z~qfBwybijt.z~qfab
UByrijt.~|,
x
Bb
D
Bb
BC
D
yb1 E2
F
b0 G
ij
(13)
where b identifies the corresponding type for particle j.

The two terms on the right hand side of Eq. (13)
correspond, respectively, to the two first terms on the
right hand side of either Eq. (1) or Eq. (2). The torque
around the centre of particle i is
tit.s8ybj==bjfBwybijt.z~.
|
(14)
b1-bijFbmax .
(11)
yb1 E2
F bij)bmax
b0 G
max
We note that, as these forces do not need to operate

in purely radial directions, they can give rise to torques
acting on each particle. Consequently, a rotational contribution to the equation of motion has to be applied in
addition to Eq. (1) or Eq. (2).
The interparticle bonds can be created or destroyed
through different protocols. Normally, a bond would be
created with the given probability Pab
B when two particlesone of type a and one of type bapproach within
a distance b1. Initially, nodes are created on the line that
joins the particle centres. After bond formation, the
nodes that define the ends of the bond are fixed within
the corresponding particle reference system. That is, the
nodes remain fixed at the initial position on the surface
of each particle. If the particle moves such that the
length of a bond exceeds bmax, the bond is deemed to
be broken. By setting bmaxs`, irreversible (unbreakable) bonds can be simulated.
In some cases, to allow for various kinds of repulsive
and attractive interactions, an additional non-binding
force between the particles can be introduced. The
simplest case is dictated by the interaction pair potential
B rabyr E
c
ij
Tab
F
UBC ab
U
ab
D
G
r
ys
fUBrij.sT
c
V0
j/i
fBbij.sUBC
Fit.sy=riwxfsawyzit.z~
rijFrab
c
rij)rcab
(12)
where rab
defines the range of the interaction, ab
c
UB
accounts for its strength, and again ss1y2 (saqsb);
sa and sb are the diameters of the interacting particles.
The indices a and b indicate the type of particle i and
j, respectively.
Adding together all the above contributions, the force
acting on particle i of type a at time t is given by
This last sum runs over all the bonds of particle i, where
bj is the position of node j with respect to the centre of
the particle.
The system is not homogeneous in the z-direction
since the interfacial external potential depends on the zcoordinate of the particles. Therefore, the periodic
boundary conditions can be applied in the x- and ydirections only. Particles cannot escape in the negative
z-direction (upwards in Fig. 1b), as they are trapped by
the infinite wall of the interface, but they can move
away (and become lost) in the positive z-direction
(downwards in Fig. 1b). In order to avoid this effect,
which would otherwise not be present if periodic boundary conditions were applicable in the z-direction, an
additional wall needs to be added at the given zposition. One way of achieving that is by introducing
an external potential of the form:
B
sa E36
F ,
D zwyzi G
fawzi.sC
(15)
where zw is the position of the restricting wall. In this

way particles initially placed between zs0 and zszw
can only move within this range in the z-direction.
The properties of the present model can, in principle,
be studied by means of MD, MC or BD. However, the
first two techniques would require, in principle, the
introduction of an extra species in the system to account
for the solvent. Therefore, BD simulations are normally
more efficient, as they introduce the solvent effect by
just assuming a bulk solvent viscosity, h. In this case,
the unit of time is generally changed to a more convenient scale: this is the average time taken for a particle
to diffuse a distance equal to its radius in an infinitely
diluted system, i.e.
trs
sy2.2
6D0
ps3h
.
8kBT
(16)
The model defined so far consists of a number of
parameters that characterize the system. These parameters are: the sizes of the particles s1 and s2, the
corresponding adsorption energies E1ads and E2ads (determined by z1c and z2c , respectively), the maximum length
of the bonds bmax, the strength of the bonds defined by
Byb02, the equilibrium length of the bonds b1, the
22
12
reaction probabilities P11
B , PB and PB , the strengths of
11
22
the long-range forces defined by UB, UB
and 12
UB and,
finally, the ranges of the non-bonded forces
22
12
r11
c , rc and rc . By selecting different sets of parameters,
we are able to model various sorts of systems like binary
mixtures of proteins, binary mixtures of LMW surfactants or proteinqLMW surfactant mixtures. In later
sections, we show how this can be done and how results
from the simulated systems compare to some recent
experimental results from AFM and fluorescence
microscopy.
It is worth emphasising that the model presented in
this section does not consider the internal structure and
flexibility of the molecules. This is particularly inadequate for very flexible protein molecules, as the model
cannot account for the unfolding of the molecule upon
adsorption. However, some globular proteins such as blactoglobulin may be reasonably well represented by the
model at mesoscopic resolution, since unfolding takes
place over long time scales, especially with strongly
interacting proteins. A molecular dynamics simulation
of the entire structure of a single b-casein molecule
adsorbing to an oilwater interface over a time period
of 1 ns has been carried out recently w21x. Naturally, the
computational cost of simulating an entire protein film
using this scheme for experimentally significant time
scales is prohibitive for todays computer resources.
1.4. Microscopy at interfaces
As has already been stated, interfacial films formed
by the adsorption of surface-active species to a gas
liquid (or liquidliquid) interface can be simulated by
BD. In order to make a comparison between the patterns
formed by these simulations and experimental systems
we require methods of probing similar length scales.
This is complicated by the fact that the film sits at a
liquid boundary, making it inherently unstable. The
problem of imaging such interfacial films can be
approached in two different ways. Either the sample can
be viewed non-invasively in-situ using optical techniques or the interfacial film can be transferred, unchanged, onto a solid support where it becomes amenable to
imaging at high resolution by probe microscopy. Both
approaches have advantages and disadvantages, which
make them useful under different circumstances.
The foremost probe microscopy is atomic force
microscopy (AFM), which uses a sharpened silicon
nitride cantilever to scan over the surface of the sample.
In its normal mode of operation the deflection of the
33
cantilever is maintained at a predetermined value as the

scan progresses in order to keep the forces acting
between tip and sample constant. By monitoring the
movements of the scanner that are required to maintain
constant deflection of the cantilever, the sample topography is revealed as a three-dimensional image. AFM
has the advantage over electron microscopy of being
able to operate in liquids at ambient temperatures and
pressures, and so it is an ideal instrument for biological
studies. Its resolution is limited not by diffraction but
by the sharpness of the tip. In practice, AFM can
routinely achieve molecular resolution and in ideal
circumstances sub-molecular resolution w22x. However,
in order to obtain these high levels of resolution, the
sample needs to be mounted on an atomically flat solid
substrate such as mica. This is achieved by a technique
known as LangmuirBlodgett (LB) transfer where the
solid support is dipped, at low speed through the
interface into the sub-phase, and then removed. The
transfer of the interfacial film is controlled by the surface
properties of the substrate. With a hydrophilic substrate
the contact angle between the liquid and the substrate
dictates that the film will only transfer on the upward
stroke, removing a single section of interfacial film.
Although the film may be altered during the transfer,
comparisons with Brewster angle microscopy w23x and
specially designed in-situ AFM experiments w24x have
shown the same behaviour and morphology as those
using the LB transfer technique. Despite the high spatial
resolution, because the technique is essentially topographical, it cannot be used to distinguish between
molecules with only small differences. Most protein
molecules for example look very similar to one another
in AFM images. In Section 5, we give some examples
of AFM images of LB films drawn from fluidfluid
interfaces where mixtures of proteins and LMW surfactants were adsorbed competitively.
Optical techniques that can interrogate an interfacial
film in-situ such as Brewster angle microscopy (BAM)
suffer from limited resolution (typically 1 mm). Despite
the limited spatial resolution of BAM it can be extremely
sensitive to surface composition. The two requirements
for obtaining good BAM images are phase regions of
sufficient size, typically tens of microns, and significant
variation of the optical properties between the two
phases. For example, one common use of BAM is to
look at the complex interfacial phase behaviour of lipids
w25x. In such systems small changes in the orientation
(tilt) of the molecule between liquid-expanded and
liquid-condensed phases are readily apparent if the
different domains are large enough. The limited spatial
resolution means that this technique is not suited to
looking at the formation of structures in protein films
or mixed proteinqLMW surfactant films unless (or
until) the phase separated regions become sufficiently
large. BAM is certainly not suitable for looking at phase
34
separation in mixed protein systems because of the

similarity in optical properties.
Although neither of the two techniques discussed
above can distinguish between different proteins, the use
of a combination of LB transfer to a transparent solid
substrate and fluorescence microscopy can be used to
provide images with a resolution of a few hundred
nanometreswhich is the diffraction limit for optical
methods. In this method proteins are covalently linked
to various fluorescent probes. The labelled protein solutions are then used to form an interfacial film that is
transferred to a solid support for imaging. An additional
disadvantage of this approach is the effect the fluorescent moiety might impart to the functionality of the
protein. Examples from two groups who have used
variations on this technique are given in Section 4.
1.5. Phase separation at interfaces
When surface-active molecules adsorb at an interface
they achieve a higher local concentration than in the
corresponding bulk solution. Consequently, the pair
interactions between the molecules become more important at the interface in determining the overall behaviour
of a mixture of surface-active components than they do
in solution. One of the most interesting features of more
concentrated mixtures is the greater likelihood of phase
separation. It seems relevant, therefore, to speculate on
situations under which surface phase separation might
occur. In the following, we discuss the possible cases
that may arise in this respect during the adsorption of
binary mixtures of surface-active molecules.
A crucial factor in determining the possible occurrence of phase separation at an interface relates to the
time scale over which the interface equilibrates with its
corresponding bulk solution. For highly non-soluble
surfactants or high-molecular-weight proteins, present on
surfaces, such processes can be particularly slow w26x.
Thus, it is easy to encounter situations where the
duration of an experiment is far shorter than the necessary equilibration times. In such cases, the overall
composition of the interface remains virtually constant
during the experiment, having initially been set by the
amount of each surface-active species that is introduced
onto the interface. For this very slow exchange dynamics, then, the interfacial region may be considered as a
separate phase isolated from the bulk. Associated with
such an interface, covered by a binary mixture of two
different surface active molecules, is an excess free
energy per unit area f s(G1, G2), where G1 and G2 denote
the coverage of each species in the mix. It must be
noted that in general, should phase separation occur at
a certain range of values of G1 and G2, then f s(G1, G2)
is not experimentally accessible in this range. Rather,
its behaviour has to be inferred, albeit qualitatively, from
a suitable theoretical model; a situation not dissimilar
to bulk systems. Outside this range, however, information regarding f s(G1, G2) is obtained from experimental
data which, for example, might involve surface pressure
and interfacial tension measurements. The phase separation behaviour at the interface is dictated by the form
of f s(G1, G2). In particular, if the matrix of second
derivatives (f s yGiGj) (Hessian matrix) is positive
definite everywhere, then the interface remains stable
against fluctuations in composition at all values of
coverage and will show no tendency for surface phase
separation. For an interface to begin to exhibit such
behaviour, the Hessian matrix must cease to be positive
definite at least over a certain range of G1 and G2. In
this range of values of surface coverage, the mechanism
of phase separation is spinodal decomposition, while
outside the range phase separation can occur through a
nucleation and growth process.
From a more physical point of view, conditions that
lead to the convex form of f s(G1, G2) and the onset
of phase separation at airwater interfaces, are only
realised if certain degree of unfavourable interactions
are present between the two surface-active species or
between these and the water molecules. While the
former set of interactions can arise in certain circumstances, the latter are highly unlikely. Even for highly
insoluble surfactants at the interfaces, it is the polar or
ionic hydrophilic sections of the molecules that remain
in the aqueous phase. Such sections have a strong
preference to be in contact with the solvent molecules
and in the absence of the hydrophobic parts would, by
themselves, be soluble in water. Thus, for amphiphilic
molecules at airwater interfaces, the only remaining
interaction that can realistically lead to phase separation
behaviour is that existing between the two surface-active
species. This in turn is dominated by the lateral interactions between the hydrophobic parts of the two different sets of molecules. For high-molecular-weight
synthetic co-polymers, consisting of large hydrophilic
and hydrophobic blocks, the required unfavourable interactions are relatively easy to obtain. Small incompatibilities between different monomers, comprising the
hydrophobic parts of the two species, become far more
prominent because of the substantial size of the interacting blocks concerned, as well as the relatively higher
concentration of the molecules on the interface. Achieving the same level of unfavourable interactions between
LMW surfactant species is more difficult and requires
the hydrophobic parts of the two sets of molecules to
be chemically very different. An interesting example of
such a system, which has received some attention in the
literature (see for example Ref. w27x), involves binary
mixtures of fluorocarbonhydrocarbon-based insoluble
surfactants placed on airwater interfaces.
In contrast to synthetic block co-polymers, proteins
are made from a variety of hydrophilic and hydrophobic
amino acids that tend to be more uniformly distributed
along the length of the chains. In other words, such

molecules rarely possess large blocks, which happen to
consist almost entirely of the same strand of amino acid
monomers. Furthermore, the same variety of amino acid
groups is to be found in different protein species. Thus,
even if a certain degree of incompatibility between
different hydrophobic amino acid types exists, the structure and composition of proteins, as discussed above,
makes the possibility of large enough direct unfavourable lateral interactions between such molecules unlikely.
At first sight then, the above discussion suggests that
mixtures of different proteins should not exhibit interfacial phase separation behaviour, at least not in the
strict thermodynamic sense. Any observed patterns
resembling onset of phase separation are to be interpreted as transients, which should decay out over sufficiently
long periods of time. However, in a previous publication
w51x, we have argued that this picture might not always
be true. Protein molecules often contain groups, which
can associate or even form inter-molecular bonds. An
extreme and rather well studied case w28x involves
proteins which contain thiol groups (SH), as say with
b-lactoglobulin. In bulk solutions, such groups are
hidden from each other within the interior of the
molecules. However, on the airwater interface, blactoglobulin molecules unfold and expose these reactive
groups, thus giving rise to the possibility of formation
of covalent bonds between the chains. Though in the
case of covalent bonds these bonds tend to be irreversible, in other circumstances weaker bonds (e.g. hydrogen
bonding) might form, which can break and reform. It
has been speculated w51x that the presence of such bonds
between one set of proteins, but not the other, can
provide an effective attraction, which can overcome the
entropy of mixing, causing formation of separate phases
on the interface. Certainly, examples of such behaviour
have been found in bulk solutions for water soluble
polymers. For example, incorporation of a few small
hydrophobic groups, comprising no more than 2% wyw
of the polymer to polyethylene glycol (PEG) chains, is
known to cause phase separation in solutions consisting
of mixtures of such modified and identical non-modified
PEG molecules w29x. Both resulting phases have been
reported to be clear solutions, with roughly the same
amount of PEG present in each. However, the viscosities
of the two phases are found to be five orders of
magnitude different w54x. This is thought to be due to
the presence of predominately modified PEG in one
phase and that of non-modified chains in the other. The
hydrophobic groups on the modified chains are known
to associate reversibly w30x in water, forming spanning
networks that lead to the observed high viscosity. It has
been shown theoretically that such association results in
a net attraction between modified PEG molecules sufficient to explain the observed behaviour of such mixtures
w54x. As discussed in the later section, following the
35
presentation of a number of simulation results, it is

proposed that a similar mechanism can operate between
mixtures of two different proteins residing on interfaces.
However, an important point to emphasise is that such
association or bonds have to be transient to allow
sufficient mobility on the interface. Permanent bonds
lead to a system that becomes kinetically trapped,
arresting the subsequent phase separation dynamics.
While bonds have to be weak enough to be reversible,
they have to nevertheless have sufficient strength to lead
to enough effective attraction to induce phase separation.
An important question is, therefore, whether these two
opposing requirements can simultaneously be met in
real or even in model simulated systems. We shall
attempt to provide some clues to the answer in Section
4.
Our discussion so far has been entirely concerned
with systems where the dynamic of exchange between
the bulk and the interface is assumed to be very slow.
It is also useful to briefly consider the opposite case
where the bulk solution and the interface are at equilibrium. For such systems, just as before, there is an excess
free energy per unit area f s(G1, G2), arising from the
presence of the interface. However, unlike the situation
discussed above, the overall composition of the interfacial layer is no longer constrained. Since molecules of
both species can freely exchange with the bulk, it is
appropriate now to consider the interfacial thermodynamic potential (per unit area), i.e.
AsG1,G2.sfsG1,G2.ymb1G1ymb2G2
(17)
where mbi is the chemical potential of the species i in

the bulk solution. The composition of the interface is
determined by the values of G1 and G2, which happen
to minimise the function defined in Eq. (17). At this
lowest value, As is also equal to the interfacial tension
for the interface w31x. In most circumstances one expects
a single global minimum for the function in Eq. (17).
Nevertheless, one might argue that for some particular
set of values of bulk concentration of the two surfaceactive species, As(G1, G2) might develop two minima,
at different interfacial compositions, which just happen
to have the same minimum value. In principle then, two
such phases can co-exist on the interface. In practice,
however, achieving this situation is highly unlikely. First
of all, the situation will only arise at some precisely
related values of bulk concentration of the two species.
Controlling the amount of two sets of molecules to such
a degree would be difficult. Even if such a situation
could be realised, there are no constraints on the overall
composition of the interface. Therefore, in time, one of
the phases will tend to dominate at the expense of the
other. This will be determined by the kinetic of adsorption of the two species and the initial conditions of the
experiment.
36
Our motivation, in discussing these cases involving

free exchange of surface-active species with the bulk, is
to emphasise the importance of irreversible adsorption
as a prerequisite to having true phase separation processes at the surface. We finish this section by drawing
attention to a situation intermediate between the two
cases discussed above, where one of the surface-active
species is irreversibly adsorbed, whilst the other is in
equilibrium with the bulk solution. This is an interesting
possibility that can arise when a high-molecular-weight
protein is very slowly displaced by relatively soluble
LMW surfactant molecules, adsorbing from bulk. Once
again the simulation results (presented in Section 5) can
provide some clues as to the likelihood of phase separation behaviour of such systems.
2. Adsorption of one-component systems
To understand the simulations of the competitive
adsorption of proteins and LMW surfactants we have
first to consider the behaviour of each separate type of
molecule adsorbing to an interface. Although no AFM
imaging can be done for pure surfactant layersmainly
because of the high mobility of these moleculessome
images have been obtained for b-lactoglobulin adsorbed
at oilwater interfaces w3x. Furthermore, interfacial rheological properties of adsorbed proteins have been investigated extensively in the laboratory w32,33x and also in
computer simulations w18,17x. In the following two
sections we describe separately the main features of
simulated adsorption of pure LMW surfactants and pure
proteins.
2.1. Adsorption of LMW surfactants
When an oilwater or airwater interface is presented
to a LMW surfactant dissolved in one of the phases, its
amphiphilic character drives a surface adsorption process. Although there is an entropy loss (mainly due to
the loss of a degree of freedom in the direction perpendicular to the interface), the orientation of the surfactant
molecules at the interface (with the hydrophobic tail
protruding into the hydrophobic phase and the hydrophilic group into water) reduces the free energy of the
system as a whole. There is a binding energy per
molecule of, say, a few kBT. Therefore, surfactant molecules tend to accommodate themselves preferentially at
the interface thereby reducing the interfacial tension g.
Surfactant molecules are relatively small and so they
diffuse rapidly to and from the interface. After a short
period of time a dynamical equilibrium state is reached,
where the number of molecules adsorbing at the interface at any time equals the number of molecules
desorbing back into the bulk phase, driven by thermal
motion. Following equilibration, the concentration of
the adsorbate molecules at the interface is constant and
Fig. 2. Adsorption of spheres to an initially clean interface for various

adsorption energies form BD simulations. (a) Occupied area fraction
of the interface fa as a function of reduced time tytr. The bold line
corresponds to the theoretical prediction for a diffusion-limited
adsorption. (b) Adsorption isotherms, i.e. fa vs. bulk volume fraction
fb at equilibrium. The continuous lines are fitted according to the
Volmer isotherm with Ks35, 250 and 1100 for Eadss6.00 (h), 8.94
(n), and 13.29 (s), respectively.
so is the interfacial tension g. Dynamical interfacial

tension measurements w35x often provide useful information regarding the equilibration process in such
systems.
The concentration of highly idealized spherical adsorbate species at the interface, obtained by BD, is shown
in Fig. 2 as a function of time and bulk concentration
fb. The surface concentration is expressed as area
fractionthe fraction of the interfacial area covered by
the adsorbate molecules. For N adsorbed spherical molecules of diameter s at an interface of area A, the area
fraction is fa'p Ns2 y(4A). The simulation corresponds to the model described in Section 1.3, but with
just a single type of particle used in order to simulate
pure surfactant adsorption. In this case, the only particleparticle interaction involved is the repulsive spherical core potential fc(rij). Particles were initially
randomly distributed in bulk. In Fig. 2a, the time
37
Fig. 3. Interfacial structure of a close-packed protein monolayer from simulation (a) and AFM (b). The AFM image corresponds to a blactoglobulin LangmuirBlodgett film at the oilwater interface (100=100 nm).
evolution of the concentration at the interface is displayed for various adsorption energies Eads. For high
values of Eadswhere every molecule that collides with
the interface is irreversibly adsorbed, and so the
process is strictly diffusion-limitedthe initial part of
the curves agrees with experiment w34x and diffusion
theory w35x in that the area fraction fa increases as
yD0typ.
In Fig. 2b, a set of adsorption isotherms is

shown w18x. The area fraction is plotted against the bulk
volume fraction of the particles. The fitted curves
correspond to Volmer isotherms w36x,
w
z
fa
fa
exp
x
|sKfb,
2
a
ps2y4yfa
y ps y4yf ~
(18)
with K an adjustable constant. As expected, for a given

bulk volume fraction, the equilibrium area fraction at
the interface increases for increasing adsorption energy.
Equally, for a given adsorption energy, the equilibrium
area fraction increases with increasing bulk
concentration.
We should point out here that our model surfactant
molecules are not strictly amphiphilic in the sense that
they do not associate into micelles, and they interact
with each other only through a repulsive core potential.
The surfactant attributes captured by our model are the
small molecular size and the tendency to adsorb reversibly at the interface through the moderately low adsorption energy per molecule.
2.2. Adsorption of globular proteins
Proteins, like LMW surfactants, can also adsorb to
interfaces lowering the interfacial tension. Since the
molecular mass is typically 100 times that of LMW
surfactants, they diffuse more slowly to the interface.
Protein chains are normally highly folded in solution

with the hydrophobic amino acids tending to reside in
the centre of the globuleaway from waterwhile the
hydrophilic amino acids tend to be at the globule surface.
When a protein adsorbate reaches an oilwater or air
water interface, it partially unfolds exposing its hydrophobic groups to the non-aqueous phase. In that way
the protein reduces the surface free energy of the system,
and the binding energy per molecule is much higher
than for surfactant adsorbates. Therefore, proteins are
also surface-active molecules, although LMW surfactants can typically reduce the interfacial tension to a
larger extent. This is due to the simpler structure and
smaller size of LMW surfactants, which allows them to
be arranged more efficiently at the interface. (In Section
3, we will show how important this size effect can be
for mixed systems.)
Many globular proteins can also form chemical and
physical linkages with other protein molecules. When
proteins adsorb, they reach higher local concentrations
than in the bulk, and so the proteinprotein interactions
play a more important role. Moreover, when they unfold,
a large number of chemical groups on the protein
molecule are exposed to other neighbouring molecules.
As a consequence, the formation of a proteinprotein
linkage is a very common event in globular protein
films. Indeed, many of these films behave like twodimensional gels, held together by a cross-linked network of bonds, some strong (permanent) and some
weak (transient).
In Fig. 3, we show the interfacial structure of a
simulated layer of protein-like spherical particles
adsorbed at an interface (a) together with a AFM image
of a real b-lactoglobulin monolayer adsorbed at the oil
water interface (b). As in the previous section, particles
of a single species are used in simulating the model
using a BD algorithm. However, this time, the adsorbate
38
spheres are allowed to interact via the formation of

bonds wsee Eq. (11)x in addition to the core repulsive
potential. The particles adsorb to the interface with a
high adsorption energy Eadss44.0, and the bond parameters are: Bs1.0, b1s1.0, b0s0.1 and bmaxs0.3.
Bonds are created with probability PBs1.0 when a pair
of particles approaches to within b1, and destroyed when
the bond length exceeds bmax. However, as proteins are
much more reactive at the interface than in bulk solution,
no bond is created unless both approaching particles are
fully adsorbed. All the adsorbate spheres (4000 in total)
are initially placed at random in the simulation box
(LxsLys40, Lzs6). Soon after the simulation starts,
the spheres pack the interface and develop the networklike structure shown in Fig. 3a. As we can see, this
structure compares rather well (in terms of the lengthscales of heterogeneities) with the experimental one,
obtained using AFM imaging of b-lactoglobulin at the
oilwater interface (Fig. 3b).
Detailed studies of the rheological properties of these
types of simulated films have been carried out w18,17x.
However, definitive comparisons with experimental
results are still awaited (for a review on experimental
techniques see Ref. w37x). The small-deformation shear
rheology of a monolayer of bond-forming spheres shows
similar characteristics to the shear rheology of an analogous bulk system. The main difference is that the stress
time correlation function, defined as Cst.s
ab0.s
abt.MyrkBT., decays more slowly in the
NNs
two-dimensional case, perhaps not surprisingly given
the more restricted topology of a two-dimensional network. In Fig. 4, we show the stress time correlation
function (a), and the storage and loss moduli as a
function of the frequency of the applied strain (b). The
results correspond to a system with Ns1000 particles
irreversibly adsorbed (i.e. Eadss`, which corresponds
to zcs`). After adsorption (fas0.31), particles
formed bonds with PBs10y3, assuming bond parameters Bs1.0, b1s1.0, b0s0.1 and bmaxs1.0. Once a
stable structure had been formed, the bonding probability
was set to zero and the rheological properties of the
interface determined.
The slow decay of the stress time correlation function
(Fig. 4a), as compared with a similar three-dimensional
system, indicates that the interfacial viscositywhich
corresponds to zero frequency shearis rather high.
This is to be expected since at the interface the particle
concentration is normally much higher than in bulk
solution, and as already mentioned, the particles have
also a more restricted mobility in the two-dimensional
space of the interface. As far as the storage and loss
moduli are concerned (Fig. 3b), the same qualitative
features are seen in the interfacial and bulk gel-like
protein systems. Frequency-dependent storage and loss
moduli have been measured w38,39x, but no direct
Fig. 4. Interfacial shear rheology of a cross-linked monolayer of particles (reproduced from Ref. w17x). (a) Normalised stress time correlation function cs(t)ycs (0) for a two-dimensional system (the area
fraction is fas0.31) and a three-dimensional system (the volume
fraction is fbs0.05) as a function of reduced time tytr. (b) Storage
and loss moduli G9(d) and G0(j) vs. (reduced) shear frequency
ftr. The maximum applied strain is g0s0.05, which is in the linear
regime.
quantitative comparison with simulated data is possible

because of the somewhat arbitrary choice of the parameters in the simulation model.
The small-deformation dilatational rheology of this
same system has been also studied w17x. In Fig. 5a, we
show the storage and loss dilatational moduli based on
isotropic expansion and compression of the interface.
Again, the gel-like behaviour of the film is clear since
the storage modulus builds up with increasing frequency
while the loss modulus vanishes at relatively low frequencies. This information can be obtained experimentally by using, for example, a pendant-bubble dynamic
tensiometer where a protein-coated bubble is subjected
to volume oscillations that induce surface area changes
(see for example Ref. w40x).
In Fig. 5b, the large-deformation surface dilatational
rheology of a simulated protein film is represented as
stress vs. time w18x. The model film (Ns1000 particles
adsorbed with fas0.63 and bond parameters Bs1.0,
b1s0.1, b0s0.1 and bmaxs`) is compressed in the x-
Fig. 5. Interfacial dilatational rheology of a cross-linked monolayer

of particles. (a) Storage and loss dilatational moduli 9(d) and 0(j)
vs. dilatational frequency ftr for the same system as in Fig. 4 (reproduced from Ref. w17x). The maximum relative increase of the interfacial area (isotropic compression and expansion) during the
oscillation was 0.05. (b) Interfacial stress response sxx (normal component of the stress tensor parallel to the direction of compression
and expansion) of a system with area fraction fas0.63 (reproduced
from Ref. w18x). From t *s0 to t *s0.5 the interface is compressed
(up to 50%) and it is re-expanded between t *s0.5 and t *s1.0.
Curves ac correspond to adsorption energies Eadss2.66, 6.00 and
8.94 kBT, respectively. The strain-rate is 0.283ty1
r . Curve d corresponds to the same system as b, but for a strain-rate of 0.0283ty1
r .
direction up to roughly half of its initial surface area

and re-expanded back to its original size. The compression and expansion at constant strain-rate is achieved
by rescaling the position of the particles so that the
length of the simulation box in the x-direction varies
with time as Lxt.sLx,0exp t.. In addition, the positions
of the particles are rescaled in the z-direction accordingly
to maintain the system at constant volume. The time
scale in Fig. 5b has been adjusted so that t *s0.5
corresponds to the time at which the maximum compression is reached and t *s1.0 corresponds to the time
at which the system is returned back to its original
shape. Therefore, results corresponding to different
strain-rates are presented with different time scales.
Curves ac are for different adsorption energies, and
curve d is the same adsorption energy as b but at a
lower strain-rate. As we can see, the stress becomes
negative during the compression phase (i.e. the interfacial tension decreases), and it is greater in absolute
39
value for the systems with higher particle adsorption

energies. This is what we expect: particles need to be
removed from the interface in order to relax the film
stress, and this is harder to achieve with strongly
adsorbing particles. The minimum of the curves is
associated with the point at which many of the particles
start to desorb to release interfacial pressure and an
orogenic mechanism has been suggested to produce
this process w7x. Comparing curves b and d in Fig. 5b,
it becomes clear that a low strain-rate allows the protein
film to rearrange its internal structure at the interface
more efficiently leading to a lower stress in absolute
terms. Recently developed instruments w41x might be
able to produce experimental results in situations comparable to those studied here by computer simulation.
To conclude this section, we should emphasise that
these simulations on large-deformation dilatational rheology were carried out with irreversible proteinprotein
bonds (bmaxs`) and no bonds were allowed to form
during the compressionexpansion process. Therefore,
the topology of the bond network was restricted to
remain unaltered during the deformation process. In Fig.
6, we show a compressed and a re-expanded protein
film where bonds were allowed to break (bmaxs1.0s)
and new bonds were allowed to form (PBs10y3). After
re-expansion, the film presents cracks due to the bonds
broken during the rearrangement of the internal structure
of the film. The restrictions imposed by the newly
formed bonds make the film protrude into the bulk after
re-expansion because many of the particles cannot then
re-adsorb. This is in contrast with the previous simulations where the film re-adsorbed fully after expansion
w18x.
3. Competitive adsorption of mixed LMW surfactants
When a mixture of two different LMW surfactants
(or any mixture of surface-active species) is allowed to
adsorb at an interface, the two kinds of molecules have
to compete for space: hence, the name competitive
adsorption. Naively, we might expect the adsorbate
with the larger value of Eads to dominate the interface if
the mixture bulk concentration ratio is approximately
1:1. For species with equal binding capacity for the
surface, it would be expected that the one present at
higher bulk concentration would predominate at the
interface. However, the moleculemolecule interactions
also play an important role in determining the final
composition at the interface.
There is, however, another equally important factor
regardless of inter-molecular interactionsaffecting the
composition of the mixture at the interface: the size
ratio of the species. It has been shown w42x by MD
simulation that small spheres can displace bigger ones
from an interface even if their adsorption energies are
identical. This effect is explained from the thermodyn-
40
Fig. 6. Snapshot of a compressed interfacial film after a large number of extra bonds have been formed (a), and the same system after reexpansion (b). In each case a top-view and a side-view are given. Reproduced from Ref. w18x.
amical point of view, by the BraunLe Chateliers

principle applied to surfaces w43x. In fact, this can also
be the case for small spheres having lower adsorption
energy than the big spheres. In Fig. 7, we show some
simulated data illustrating this behaviour. Using the
model described in Section 1.3, we explore the displacement of big spheres by smaller ones. One thousand
particles of type 1 (with unit diameter) are initially
placed at the interface. Then, twice this number of
smaller particles (diameter 0.3, 0.5, 0.7 or 0.9) is added
to the bulk, beneath the interface. The particles interact
with each other only via the repulsive core interaction
potential, and both species have the same adsorption
energy (E1adssE2adss6.0kBT). Fig. 7 shows the timedependence of the number of particles of type 1 as they
are displaced by particles of type 2 during the equilibration process. Results for various sizes of type 2 particles
are compared. As we can see, smaller particles promote
a more efficient displacement. This is because the
number density at the interface can be greatly enhanced
by adsorbing several small particles in the space required
for adsorbing a big particle. As the adsorption energy is
identical for both species, the more particles of any type
that the interface contains, the lower is the free energy
of the system. The actual number of small adsorbates
that can be placed in the same space as a big adsorbate
grows as the square of the diameter ratio. Therefore, if
the size ratio is large enough, even small adsorbates
with low adsorption energies (LMW surfactants) can
efficiently displace big particles that have a high affinity
for the interface (even proteins). This effect is further
enhanced by the non-ideal entropy of mixing of nonequally-sized molecules as demonstrated by LucassenReynders w44x by recognising that the total Gibbs free
energy of the adsorbed mixture is expressed more
appropriately as a function of the area fraction rather
than the mole fraction of the molecules. Of course, the
equilibrium adsorbed amount of type 1 particles ultimately depends not only on the size ratio, but also on
the bulk concentration of both species, and also the
intermolecular interactions. We do not attempt here to
Fig. 7. Displacement of a monolayer of adsorbed spheres by smaller

particles. Number of adsorbed big spheres Nb as a function of time.
The 1000 initially adsorbed spheres (adsorption energy 6.0 kBT) are
of unit diameter. Displacement is produced by 2000 small spheres (of
same adsorption energy as big ones) of diameter 0.3 (e), 0.5 (h),
0.7 (n), and 0.9 (s).
analyse the thermodynamic properties of the model since

we are more interested in the non-equilibrium properties
of the initial stages of the competitive adsorption.
We conclude this section with a comment on the
likelihood of lateral phase separation in this kind of
system. Surfactants are in general small molecules and
their adsorption energies are of the order of just a few
kBT w37x. Therefore, surfactant molecules adsorb and
desorb from liquidliquid interfaces in a relatively rapid
manner, so promoting a constant exchange of particles
with the bulk phase. As we have discussed in Section
1.5, a mixture of adsorbates with such a property is
unlikely to form different co-existing phases at the
interface even if their cross interaction favours demixing.
4. Competitive adsorption of mixed proteins
The competitive adsorption of sticky hard spheres has
been studied in the framework of the PercusYevick
integral equation theory, both in general w45x and in
relation to protein mixtures w46x. The model system w47x
consists of a binary mixture of particles interacting
through a hard sphere particleparticle potential and a
sticky surfaceparticle potential. It has been shown w46x
that for binary mixture of equally sized adsorbing hard
spheres with no extra interactions, the isotherms calculated from the PercusYevick theory reproduce qualitatively the experimental results obtained for the
competitive adsorption of as1-caseinyb-casein mixtures
in oil-in-water emulsions. It is worth mentioning that
this type of integral equation theory applies properly
just to systems at equilibrium. Fortunately, mixtures of
the disordered proteins as1-casein and b-casein come to
equilibrium fairly rapidlyunlike most globular proteinsand so the theoretical assumption is realistic in
this case.
In relation to the kinetics of the competitive adsorption of proteins, there has been a significant amount of
work done on the diffusion equation and its relationship
to surface tension measurements (for a review see Ref.
w35x). As far as the sticky surface model is concerned,
the assumption that the proteinprotein interaction is
well represented by a hard sphere potential may be
reasonably reliable only at low or moderate ionic
strength where the protein molecules do not aggregate
as a result of electrostatic screening of the attractive
interactions. Unfortunately, theoretical studies that
include proteinprotein stickiness in addition to proteinsurface stickiness w48x have not provided a simple
expression for the isotherms.
Recently, there have been experimental reports w49,50x
of high-resolution microscopic images of mixed protein
films. Binary mixtures of proteins labelled with different
fluorescent dyes have been adsorbed from bulk aqueous
solution to the airwater interface. After transferring
41
each adsorbed film onto an appropriate substrate, separate images of each species could be recorded by
illuminating the sample with different wavelengths of
light. The results of some of these experiments appear
to be contradictory, however, as phase separation of the
components after ageing has been seen in some cases
w8,49x, but not in others w50x. In any case, it is clear
that the viscoelastic character of the films kinetically
limits the molecular mobility at the interface and hence
any thermodynamically driven structural rearrangements. As a result, lateral phase separation, if it occurs,
may typically take place only after several days of
surface ageing. However, when strong intermolecular
interactions are present, the system can be kinetically
trapped, so that interfacial phase separation cannot occur
at all during normal experimental time-scales.
In order to model the dynamic behaviour of this type
of system, some over-damped BD simulations have been
performed w51x. The assembly contains 2000 particles
of two types (i.e. 4000 particles altogether). Particles
were initially distributed at random in a prism-like box
of sides Lx=Ly=Lz (40=40=6). The value of the
parameter zc in the interfacial potential, Eq. (2), was set
to 0.15s. This implies an adsorption energy of 44.0kBT
for both kinds of particles. As the simulation evolves,
the individual particles reaching the interface are trapped
by the strong external force field and so they remain
adsorbed essentially irreversibly (no spontaneous
desorption over the simulation time-scale). However,
they can readily move in the plane of the interface. The
strong adsorption energy here is essential in order to
avoid complications of desorption during the simulation,
and hence to set the composition of the interface once
it is packed with particles. Otherwise, phase separation
is not to be expected, as pointed out previously (Section
1.5).
The model particles of type 1 can interact through
flexible bonds as described by Eq. (11). In order to
mimic the effect of the interfacial unfolding behaviour
of real proteins w33x, we allow these particles to form
bonds only when they are located at the interface
reflecting the fact that macromolecular reorganization at
the interface generally enhances greatly the probability
of reacting groups becoming exposed to neighbouring
proteins molecules.
In Fig. 8, the simulated structure of the interface is
illustrated by representing type 1 particles as light
spheres and type 2 particles as dark spheres. In the set
of top-view pictures (a)(c), various different types of
interactions have been adopted in addition to the repulsive hard-core spherical potential. While pictures (a)
(c) include the adsorbed particles only, picture (d)
shows a side view of the entire system (displaying both
adsorbed and non-adsorbed particles) corresponding to
(c). Picture (a) refers to a system where the type 1
particles can form elastic bonds once they reach the
42
Fig. 8. Two-dimensional patterns formed by mixtures of model spherical particles as they adsorb to a planar interface. Parts of the replicas of the
central simulation box are also displayed. Dark particles do not form bonds. In (a), light particles form irreversible bonds. In (b), light particles
form reversible bonds. In (c), neither dark nor light particles form bonds but an effective repulsion has been introduced between unlike particles.
A three-dimensional profile of the system in (c) is also shown (d). Reproduced from Ref. w51x.
interface. These bonds are irreversible (i.e. bmaxs`)

and moderately inflexible (bs3.0kBT)a case that
might for instance represent the situation where intermolecular covalent disulfide bonds are formed between
adsorbed b-lactoglobulin molecules w52x. After a short
time, corresponding to the time necessary to saturate the
interface with particles, the interfacial structure becomes
trapped in a particular configuration, like the one shown
in Fig. 8a. It now no longer evolves with time. The
bond-forming type 1 particles build a two-dimensional
network, which resembles a gel-like structure with pores
containing the type 2 particles. Although regions rich in
one or other of the species can be seen in picture (a),
these are of the order of a few molecules in size. A
reduction in free energy would be achieved if the system
were to become phase-separated. However, the rigidity
of the interfacial network traps the system kinetically,
preventing the structure from becoming more heterogeneous. This picture (a) seems to agree rather well with
fluorescence microscopy experiments on b-lactoglobulinyb-casein w50x where a perfectly mixed system was
observed after three days of ageing. In Fig. 9a, we
present a fluorescence image of a mixed b-casein (light
colour)qb-lactoglobulin (dark colour) film after 2 days
of ageing showing no evidence of surface phase

separation.
In picture (b) of Fig. 8 we see the result of a similar
kind of simulation to the one displayed in picture (a),
but, in this new example, the bonds were allowed to
break and reform (bmax50.3s), and they were also
more flexible (bs1.0kBT), corresponding to less
strong, physical association, as opposed to chemical
bonds in (a). Because of these changes, the more mobile
type 1 particles at the surface can now rearrange more
efficiently and it is clear from picture (b) that the system
is indeed phase separating. In the initial stages, the
system builds a network similar to the one shown in
(a). However, the reversible character of the bonding
allows the surface structure to rearrange, leading to more
compact type 1 clusters that become increasingly disconnected. The ageing of this system would be expected
eventually to lead to a proper macroscopic phaseseparated structurebut the simulations become prohibitively expensive at these later stages because the
diffusion of a big type 1 cluster through a dense type 2
environment is inevitably very slow. This type of scenario is normally referred to as transient gelation, and
it has been well documented in bulk systems w53x. Based
43
Fig. 9. Fluorescence images of mixed protein films adsorbed to airwater interfaces. (a) b-casein (light colour)yb-lactoglobulin (dark colour)
after 2 days of ageing (reproduced from Ref. w50x). The image is 63=48 mm and it presents a homogeneous grey texture. (b) a-lactalbumin
(light colour)yb-casein (dark colour) after 4 days of ageing (reprinted with permission from Ref. w49x Copyright 2001 American Chemical
Society). The image is 1200 mm across and it shows an a-lactalbumin-rich oval region.
on the experimental evidence, phase separation of this

character might well be encountered in soy 11Sybcasein, a-lactalbuminyb-casein w49x, and bovine serum
albuminyb-casein mixtures w8x. It has also been demonstrated elsewhere w54,55x that phase separation
between two very similar polymeric species can occur
in the bulk if the molecules of one of the species can
self-associate. In Fig. 9b is a fluorescence image w49x
of a mixed a-lactalbumin (light colour)qb-casein (dark
colour) film after 4 days of ageing showing an alactalbumin-rich oval region. Interestingly, these types
of interfacial phase-separated structures do not appear
for this system when investigated at low bulk concentrations w49x.
An example of a simulated layer of two non-bondforming species is shown in picture (c) of Fig. 8. In
this case, we have switched off the formation of bonds
between type 1 particles, but instead have included a
repulsive interaction between the unlike species (12
UBs
2.0kBT and rc12s1.2s). Additionally, the simulation box
is enlarged in the z-direction (40=40=18) to reduce
the bulk volume fraction of the particles and so to avoid
possible bulk immiscibility. We find that the interfacial
region phase separates very quickly through what seems
to be the spinodal decomposition mechanism. This effect
is seen soon after the interface is packed with particles,
in sharp contrast to the case depicted in picture (b), for
which much longer runs were required in order to
observe the nucleation of clusters of type 1 particles.
As we have discussed in Section 1.5, rapid phase
separation is commonly seen in binary polymer mixtures
rather than in protein mixtures since much higher
degrees of incompatibility are normally achieved in the
former case. Fig. 10 shows an experimental example of
a phase-separating polymer mixture at the airwater
interface w56x.
By studying experimental adsorption isotherms,
incompatibility in interfacial mixing of some pairs of
proteins has been inferred w57x. That is, the actual
adsorption isotherm for a mixture of proteins can be
compared against the theoretical Langmuir isotherm for

a non-interacting mixture. Then, the extent of the deviation can be used to provide a measure of the degree of
cross interaction present in the system. This measure
has been suggested w57x as being an indicator of the
degree of (im)miscibility of the system. However, this
might not actually be the case. For example, if the
proteins of the unlike species cross-link strongly to each
other at the surface, they could form a very well mixed
system w58x, even though the strong cross interaction
would lead to a large deviation of the actual adsorption
isotherm from that corresponding to a non-interacting
mixture. A further complicating fact is that true thermodynamic equilibrium might be extremely hard to
achieve even after long periods of ageing in these highly
Fig. 10. Fluorescence scanning near-field optical micrograph for a

phase-separated poly(octadecyl methacrylate)ypoly(isobutyl methacrylate) binary mixture at the airwater interface (reprinted with permission from Ref. w56x Copyright 2001 American Chemical Society).
The light coloured areas correspond to the poly(octadecyl methacrylate) molecules.
44
concentrated and highly interacting protein mixtures.

Therefore, the incipient phase separation may not actually be seen in some experiments, because the particular
preparation procedure of the system leads to a kinetically
trapped configuration, as discussed above.
5. Competitive adsorption of proteins and LMW
surfactants
When a solution containing protein and LMW surfactant is adsorbed, the surfactant is likely to prevail at the
interface, after equilibration, if both species are present
at high enough bulk concentrations w37,52,59x. This is
due to the fact that LMW surfactants are much smaller
in size than proteins, and so they can reduce the
interfacial tension more efficiently by adsorbing a much
larger number of molecules within the same surface area
(see the discussion in Section 3 about the relative
particle size effect on competitive adsorption of LMW
surfactants). However, during the adsorption of the two
species, the protein molecules do partially adsorb at the
interface in the very initial stages of the process w60x1.
Soon after, however, an increasing number of small
surfactant molecules adsorb at the interface and displace
the protein molecules, eventually removing all of the
initial protein film. A similar sort of behaviour occurs
if the surfactant is introduced into the sub-phase after
the protein film has developed at the interface.
It is important to mention that there exists an additional mechanism for the displacement of a protein film
by surfactant molecules. Water-soluble surfactant molecules can bind to protein molecules both by electrostatic
interaction (if the surfactant is ionic) and by hydrophobic interaction involving their hydrophobic tails and
hydrophobic groups of the protein w35,37x. If the hydrophiliclipophilic balance is larger for the proteinsurfactant complex than for the protein molecule on its
own, a solubilization mechanism will facilitate the displacement of some of the protein from the monolayer
on the interface w52x.
Although many experiments have been devised to
study the properties of protein films being displaced by
LMW surfactants, it is only recently that the first images
of these films have been obtained by means of the
atomic force microscope w7x. The outcome of these
experiments has been very valuable, providing evidence
against the common assumption in many theoretical
1
Although the diffusion coefficient is lower for proteins, they
occupy a larger area when they reach the interface. Indeed, during
the very initial stages of adsorption the surface concentration increases
with time as G(t)s2 c0 (D0 typ)1y2, with c0 the molar bulk
concentration w35x. Therefore the area fraction occupied by the
particles increases as fa(t)sp G(t) s2 y4;s3y2. Hence, the bigger
the particles are the more surface they initially cover at the interface
for a given bulk concentration. This effect is very much enhanced by
unfolding in the case of proteins, since they can diffuse faster in the
folded configuration and occupy a larger area after unfolding.
approaches (see for example Ref. w35x), namely that the

preferential adsorption of the surfactant occurs in a
homogeneous way throughout the interface. The main
structural features of the displacement of milk proteins
by LMW surfactant have been identified in this work
w7x. In Fig. 11, we show AFM images of b-casein
(pictures ac) and b-lactoglobulin (picture d) films
displaced by the non-ionic surfactant Tween20. After
the protein film has formed, small pools of surfactant
develop in the defects of the protein monolayer (picture
a). Then, these surfactant domains grow with time and
with increasing surfactant bulk concentration, increasing
the interfacial pressure and compressing the protein film
(picture b). Beyond a critical surface pressure the protein
film starts buckling into the bulk phase and consequently
increasing the film thickness. As surfactant molecules
continue adsorbing, the surfactant domains start to coalesce and parts of the protein film detach from the
interface (picture c). Finally, a continuous surfactant
phase develops at the interface leaving just a few
disconnected protein areas adsorbed, which eventually
also detach from the interface.
The compression mechanism that promotes the local
thickening of the protein layer has led to the description
of the competitive displacement process as orogenic
displacement by analogy with the geological process of
mountain formation. The regions of the interface covered
with protein behave like a protein film subjected to
mechanical compression (see Section 2.2) under the
influence of the growing surfactant domains.
Fig. 11d corresponds to an intermediate state of the
displacement process for a b-lactoglobulinyTween20
mixture. From this AFM image, we see that although
the general process described above applies to most
proteins, individual protein films can show very different
structures. Whereas the disordered protein b-casein
allows the formation of nearly circular surfactant
domains, the globular protein b-lactoglobulin develops
very irregular gaps. The main reason for the irregular
shaped domains is the gel-like structure of the blactoglobulin films created by a network of disulfide
bonds w52x, which can support local stresses, leading to
a fracture-like disruption of the film. The structures
encountered depend not only on the type of protein
(disordered or globular, bond-forming or weakly-interacting), but also on the type of surfactant (ionic or nonionic, water-soluble or oil-soluble) w61x and the nature
of the fluid interface (airwater or oilwater) w3x. In
Fig. 12, for example, we can note the different structures
obtained during displacement of b-casein by Tween20
at the airwater and oilwater interfaces. It has been
suggested w3x that the b-casein network may be stronger
at the oilwater interface than at the airwater interface,
which makes it more likely to present fracture-type
disruptions as the surfactant domains grow at the oil
water interface. The study of the displacement of mixed
45
Fig. 11. Atomic force micrographs of spread protein films displaced by adsorbing surfactant Tween20: (a)(c) b-casein films for increasing
surface pressure (PsgH2Oyg) as the surfactant adsorbs; (d) b-lactoglobulin at intermediate displacement. (a) 1.6=1.6 mm, Ps15.9 mNym.
(b) 6.4=6.4 mm, Ps16.7 mNym. (c) 6.4=6.4 mm, Ps19.2 mNym. (d) 3.2=3.2 mm, Ps21.8 mNym. Similar results were obtained when
the protein was co-adsorbed with the surfactant instead of spread on the water surface. Reproduced from Ref. w7x.
protein films by surfactants has shown w50x structures

intermediate between those obtained with the corresponding pure protein films.
An attempt has been made to study these proteinq

LMW surfactant systems by computer simulation
w19,62x using the general model presented in Section
Fig. 12. Atomic force micrographs of spread b-casein films displaced by adsorbing Tween20: (a) b-casein film at the airwater interface; 6.4=6.4
mm, Ps19.2 mNym (reproduced from Ref. w7x); (b) b-casein film at the oilwater interface; 6.4=6.4 mm, Ps30.5 mNym (reproduced from
Ref. w3x).
46
Fig. 13. Interfacial structure of a simulated protein film displaced by surfactant as seen from below (the bulk phase). The big light spheres
represent the protein-like molecules and the small dark spheres represent the surfactant. The images are labelled in chronological order from the
insertion of the surfactant beneath the interface (a) to the intermediate stages of the displacement (d). Only those surfactant molecules adsorbed
directly at the interface are displayed. By contrast, all the protein molecules are drawn, and so many of them are actually protruding into the bulk
solution. The central simulation box plus a part of the replicas are shown to give a better idea of the spatial texture of the system.
1.3. In Fig. 13, we show a typical result from a BD

simulation. The model system consists of two types of
particles: protein-like particles represented as large light
spheres (type 1) and the surfactant-like particles represented as small dark spheres (type 2). Proteins are
modelled as bond-forming particles (Bs1.0kBT, b0s
0.1s1, b1s0.1s1, bmaxs0.4s1 and PB11s1.0) with a
relatively low adsorption energy (E1adss2.66kBT). Surfactants, in contrast, are modelled as small particles
(s2s0.5s1) that do not form bonds and have a relatively high adsorption energy (E2adss29.5kBT). As
already mentioned, real surfactant molecules normally
have lower adsorption energies than proteins, and the
main reason for their great effectiveness in reducing the
interfacial tension is the more efficient coverage of the
interface due to their smaller size. However, in a
simulation, the use of very small surfactant molecules
is prohibitively expensive in terms of computational
resources. The smaller the surfactants the more molecules we need to introduce in the system in order fill
the space between the big protein molecules. Using a
higher adsorption energy for the simulated surfactant
molecules is a pragmatic way to overcome the problem
of using not such small surfactant molecules but still

making them more efficient in competition for the
interface. It is worth mentioning that we compare the
results of these simulations with experiments on blactoglobulinqTween20 mixtures. In this case, where
the surfactant molecules are reasonably big, the diameter
ratio between the two species is approximately 3 if we
assume size is proportional to the cube root of molecular
weight. Therefore, the diameter ratio of 2 used in the
simulations is not too unrealistic with respect to this
particular system.
The simulation of the competitive displacement of
globular protein by surfactant is carried out as follow.
Firstly, 1000 type 1 particles (the proteins) are placed
at the interface (fa1s0.64) and they are allowed to
cross-link to form a gel-like film. Then, after equilibration, 2000 type 2 particles (the surfactants) are introduced beneath the interface. The type 2 particles are
constrained by a reflecting wall located at z53.5s1. In
this way we avoid the problem of the surfactant diffusing
away from the interface and diluting the bulk solution.
In Fig. 13, we can see how the surfactant molecules
adsorb in the gaps between the proteins. Then, these
47
Fig. 14. Comparison of a simulated protein film partially displaced by surfactant with a high resolution AFM image. (a) Simulated protein film,
where surfactant molecules adsorbed into the film gaps are not displayed. (b) AFM image of a b-lactoglobulin film displaced by Tween20. The
image is 0.8=0.8 mm.
gaps expand as an increasing number of surfactant

molecules adsorb. The growing surfactant domains compress the protein-rich regions of the interface. As a
consequence, surfactant molecules find it increasingly
difficult to adsorb in the progressively smaller gaps of
the protein-rich regions, therefore, increasing the adsorption at the already existing surfactant pools. It is clear
from Fig. 13 that proteinprotein bonds are broken up
throughout this process. Some simulations performed
with irreversible bonds w19,62x showed similar structures
at the interface, but in those cases the protein-rich
regions were actually connected underneath by parts of
the displaced protein network.
The type 1 particles in Fig. 13 can be regarded either
as protein molecules or as small aggregates of such
proteins. In particular, due to the gel-like structure that
the bonds induce in the simulated model film, we can
expect the simulation to agree better with experiments
on bond-forming globular proteins such as b-lactoglobulin. Furthermore, since no extra interactions have been
introduced between the molecules apart from the core
potential and the bonds, a non-ionic surfactant like
Tween20 should be well represented by the type 2
particles. In Fig. 14, we present an image obtained from
the simulation (for clarity only the protein-like molecules have been displayed) along with a high-resolution
AFM image of a b-lactoglobulin film displaced by
Tween20. The similarity of the two images is rather
impressive, particularly considering the simplicity of the
model. However, initial attempts to compare quantitatively the structures in the experiments with those in the
simulations w63x have been less successful. The main
reason for this is the fact that the simulation represents
length scales that are typically an order of magnitude
smaller than the best that can be resolved by AFM. In
spite of this, some of the main qualitative features, such
as the increase in thickness of the protein film during
competitive adsorption, and the fracture-like growth of

the surfactant domains are well reproduced by the
simulations.
6. Concluding remarks
We have shown in this review that the combination
of imaging experiments and computer simulation based
on simple models has brought about important new
understanding of the mechanisms involved in the competitive adsorption of mixtures of different adsorbates.
Both experiments and simulations show that the displacement of protein layers by LMW surfactants normally occurs in an inhomogeneous way throughout the
interfacewith the LMW surfactant forming separated
domains. This type of situation is not typically considered in many existing theoretical treatments of competitive adsorption, such as integral equation theories w45x,
thermodynamic models w35x and kinetic models w34,35x.
Regarding the competitive adsorption of protein
mixtures, it is clear that complex interactions between
molecules can lead to different scenarios as far as the
structure of the adsorbed layer is concerned. Whereas
mixtures containing a species that can form covalent
bonds (b-lactoglobulin) upon unfolding seem to rapidly
reach homogeneous kinetically trapped structures, others
present more fluid-like and well-separated phases. The
cause of these different kinds of behaviour, which lies
in the degree of reversible (irreversible) character of the
proteinprotein association, has been clarified by means
of the computer simulation of the model proteins.
In this study of the adsorption of different surfaceactive species, simple mesoscopic models of the components and their interactions have been introduced in
order to attempt to explain various experimental results.
These models are mesoscopic in the sense that no
detailed information of the entire molecular structure
48
(atom-by-atom) is considered. Clearly, further development of theoretical models for these types of adsorbate
is needed to allow for surfactant micellization and
proteinsurfactant complexation. However, we believe
that the model adsorbates should still remain mesoscopic
in character. The reason for this is two-fold. Firstly, the
way we want to describe these generic systems is by
using handy uncomplicated models of the molecules. At
this stage, it seems worth putting these types of mesoscopic model to the test, rather than incorporating highly
detailed chemical structures that might hide the key
features that determine the generic behaviour of the
systems under study. Secondly, the computer resources
required to simulate over a long time-scale even just
one protein molecule at the level of its atomic structure,
not to mention an entire adsorbed protein layer, is at
present unavailable to us, and is likely to remain so for
several years to come.
Acknowledgments
This research was supported by the BBSRC (UK).
Computing was done on the Leeds Grid Node 1 facility,
funded under the 2001 HEFCE Science Research Investment Fund initiative at the University of Leeds, which
is one of the partners in the White Rose Grid project.
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Competitive Adsorption of Proteins and Low-Molecular-Weight Surfactants Computer Simulation and Microscopic Imaging

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Advances in Colloid and Interface Science 107 (2004) 2749

Competitive adsorption of proteins and low-molecular-weight surfactants:

Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK

6. Concluding remarks .............................................................................................................................. 47

Fig. 1. Schematic representation of surface-active species. (a) Protein

in a coil-like structure in order to expose the most

of motion through the use of a suitable computer

systemthis is termed molecular dynamics (MD) simulationor in generating ensembles of representative

where mi is the mass of particle i, ri(t) is its position at

ics, which evolves over periods of hours, days or even

expects that the presence of any container wall should

where V is the volume of the system. Here, a and b

Here, F t. represents the random force. Again, as in

Shear flow can be imposed on a simulated ensemble

plane (or any other appropriate plane). The periodic

approach has been developed by Wijmans and Dickinson

sG9v. sinv t.qG0v.cosv t.,

Several initial cycles of the oscillation have to be

where ss1y2 siqsj., si and sj are the diameters of

This potential has a square-well-like shape with one

where b identifies the corresponding type for particle j.

We note that, as these forces do not need to operate

where zw is the position of the restricting wall. In this

The model defined so far consists of a number of

cantilever is maintained at a predetermined value as the

separation in mixed protein systems because of the

along the length of the chains. In other words, such

presentation of a number of simulation results, it is

where mbi is the chemical potential of the species i in

Our motivation, in discussing these cases involving

Fig. 2. Adsorption of spheres to an initially clean interface for various

so is the interfacial tension g. Dynamical interfacial

In Fig. 2b, a set of adsorption isotherms is

with K an adjustable constant. As expected, for a given

Protein chains are normally highly folded in solution

spheres are allowed to interact via the formation of

quantitative comparison with simulated data is possible

Fig. 5. Interfacial dilatational rheology of a cross-linked monolayer

direction up to roughly half of its initial surface area

value for the systems with higher particle adsorption

amical point of view, by the BraunLe Chateliers

Fig. 7. Displacement of a monolayer of adsorbed spheres by smaller

analyse the thermodynamic properties of the model since

interface. These bonds are irreversible (i.e. bmaxs`)

of ageing showing no evidence of surface phase

on the experimental evidence, phase separation of this

compared against the theoretical Langmuir isotherm for

Fig. 10. Fluorescence scanning near-field optical micrograph for a

concentrated and highly interacting protein mixtures.

approaches (see for example Ref. w35x), namely that the

protein films by surfactants has shown w50x structures

An attempt has been made to study these proteinq

1.3. In Fig. 13, we show a typical result from a BD

of using not such small surfactant molecules but still

gaps expand as an increasing number of surfactant

competitive adsorption, and the fracture-like growth of

w19x C.M. Wijmans, E. Dickinson, Langmuir 15 (1999) 8344.

w61x A.P. Gunning, Encyclopedia of Surface and Colloid Science,

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