Engelhardt BSA SFG Foams Langmuir 2012 001

Article
pubs.acs.org/Langmuir

Protein Adsorption at the Electrified Air−Water Interface:
Implications on Foam Stability
Kathrin Engelhardt,† Armin Rumpel,†,‡ Johannes Walter,† Jannika Dombrowski,§ Ulrich Kulozik,§
Björn Braunschweig,† and Wolfgang Peukert*,†,‡
†

Institute of Particle Technology (LFG), University of Erlangen-Nuremberg, Cauerstrasse 4, 91058 Erlangen, Germany
Erlangen Graduate School in Advanced Optical Technologies (SAOT), University of Erlangen-Nuremberg, Paul-Gordan-Strasse 6,
91052 Erlangen, Germany
§
Chair for Food Process Engineering and Dairy Technology, Research Center for Nutrition and Food Sciences (ZIEL) − Department
Technology, Technische Universität München, Freising-Weihenstephan, Germany
‡

ABSTRACT: The surface chemistry of ions, water molecules,
and proteins as well as their ability to form stable networks in
foams can influence and control macroscopic properties such
as taste and texture of dairy products considerably. Despite the

significant relevance of protein adsorption at liquid interfaces,
a molecular level understanding on the arrangement of
proteins at interfaces and their interactions has been elusive.
Therefore, we have addressed the adsorption of the model
protein bovine serum albumin (BSA) at the air−water
interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the
composition and average orientation of molecules at interfaces, while complementary information on the thickness of the
adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified
interface, pH dependent charging, and electric field-induced polar ordering of interfacial H2O and BSA. Varying the bulk pH of
protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in
vibrational bands of interfacial H2O are simultaneously observed. These observations have allowed us to determine the isoelectric
point of BSA directly at the electrolyte−air interface for the first time. BSA covered air−water interfaces with a pH near the
isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of
the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of
BSA at water surfaces and macroscopic properties such as the stability of protein foams.
In order to reveal the interactions of proteins at interfaces,
information on the interfacial composition, for example, the
arrangement of ions and water molecules in the adjacent
electrolyte subphase, possible protein unfolding processes, and
the formation of single or multilayers, is imperative.10−13 The

physical and chemical stability of proteins is influenced by
different factors such as temperature, chemical composition of
the electrolyte, and the pH of the bulk electrolyte.14 While it
seems to be accepted that structural rearrangement of proteins
due to the adsorption to the interface can occur,15−18 the extent
of unfolding or surface aggregation and a possible reversibility
of this process are still a matter of considerable debate.9,19,20
The lack of molecular level information is mainly due to a
lack of suitable experimental techniques that can actually reveal
both composition as well as conformation of protein adlayers
and other interfacial molecules such as H2O. In previous
studies, it was already shown that sum-frequency generation
(SFG) is a powerful optical probe for the investigation of

INTRODUCTION
Foams are materials of particular importance since they are
applied in a broad range of applications such as metal foams for
lightweight structures,1 polymer foams for thermal insulation or
foams in food products, just to mention a few. Although the
chemical composition of these foams is largely different, they

share common similarities for foam formation and stabilization.
The latter is controlled by adsorption processes at the interface
between the gaseous and the surrounding solid or liquid phase.
Therefore, it is of great importance to understand foam
stabilization processes at interfaces in order to design advanced
materials with tunable properties.2 Stabilization of protein
foams is dominated by a molecular layer at the interface which
can be controlled experimentally.3−6 For that reason, protein
foams represent a model system for mechanistic studies of
foams and investigations on the origin that causes a liquid to
foam. Hence, hierarchical studies of the relationship between
molecular structure and interactions, interface design and
macroscopic properties, has become an important part of
current research in this field.7−9 A detailed molecular level
understanding of the surface chemistry of proteins at liquid
interfaces has, however, not been established.

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© 2012 American Chemical Society

Received: April 3, 2012
Revised: April 23, 2012
Published: April 24, 2012
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protein interfaces.21−26 Wang et al.27 reported the pH
dependent charging of bovine serum albumin (BSA) at the
air−liquid interface where the influence of pH on the SFG
spectra in the region of methyl and OH stretching vibrations
was attributed to a charge reversal of interfacial BSA. As the
surface charge of the proteins is affected by the electrolyte pH,
it leads to a strong electric field and, consequently, to the
formation of an electric double layer at the protein surface. The

local electric field of proteins and its effects on the surrounding
electrolyte layer is minimized at a pH where the net charge of
the protein with bound ions is zero.28 Obviously, this point is of
great scientific interest as it is protein specific and is a function
of the amino acid sequence at the protein surface. Although the
isoelectric point of proteins in the bulk electrolyte can be
determined by zeta potential measurements, the conditions at
electrolyte interfaces can be dramatically different from those in
the bulk. The concentrations of proteins, ions, and water
molecules as well as their lateral interactions may be modified
significantly at an interface. Consequently, it is a priori not clear
if the isoelectric points of proteins at interfaces and in bulk
electrolytes are identical.
In order to reveal the intriguing relationship between pH
dependent charging of a protein, protein adsorption, interfacial
molecular structure, and macroscopic properties such as foam
stability, we have studied the model protein BSA at the
electrified air−water interface with broadband SFG, ellipsometry, and a macroscopic analysis of the foam stability. This
hierarchical approach has enabled us to determine the
interfacial isoelectric point as well as the structure of BSA

layers adsorbed to the electrolyte−air interface. New
information on the link between microscopic properties of
BSA at interfaces and macroscopic properties such as the
stability of foams from BSA solutions is provided.

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rapid acquisitions of experimental data compared to the conventional
nulling technique.29 For each experiment, 15 μM BSA sample solution
was poured into a Petri dish with a diameter of 10 cm and was allowed
to equilibrate for about 30 min. Angle scans between 51° and 55°
versus the surface normal were performed with a step width of 0.5°. In
order to ensure reproducibility, at least six measurements were
recorded and averaged for every pH value. Angle-resolved data from
ellipsometry were fitted under the assumption of a three layer model
with refractive indices of 1.33, 1.40,12 and 1.00 for the electrolyte
subphase, the protein layer, and air, respectively. In general two
parameters are unknown in this three layer system: the thickness of the
adsorbed protein layer and the corresponding refractive index. Due to
the fact that these parameters cannot be determined independently,

one of them, for example, the refractive index, has to be chosen as a
fixed input parameter for all model calculations. Since the refractive
index of BSA at an interface is unknown, the latter assumption causes a
systematic error of the layer thickness that depends on the deviation of
the assumed refractive index from its actual value. However, since we
only compare relative changes in the layer thickness as a function of
the electrolyte pH, interpretations in this respect are not impaired.
Vibrational Sum-Frequency Generation (SFG). SFG is a
second-order nonlinear optical technique30 where two laser beams
are overlapped temporally and spatially at the interface of interest and
generate photons with the sum frequency of the two impinging laser
beams. One laser has a frequency ωVIS in the visible region (800 nm),
and the other laser is tunable in the infrared region with frequencies
ωIR. The intensity of sum-frequency output I(ω) depends on the
intensities of the impinging laser beams IVIS and IIR as well as on the
(2)
nonresonant χ(2)
NR and resonant χk parts of the second-order nonlinear
susceptibility χ(2) as follows:
2

(2)
I(ω) ∝ χNR
+

∑ χk(2)
k

IVISIIR

with

χk(2) =

Ak exp(iφk )
ω k − ω + i Γk
(1)

depends on the amplitude Ak =
The resonant contribution χ(2)
k

N⟨αkμk⟩, the relative phase φk, the resonance frequency ωk, and the
bandwidth Γk of the vibrational mode k. The amplitude Ak is a
function of the molecular number density N at the interface and an
orientational average of the Raman polarizability αk and the dynamic
dipole moment μk. The latter dependence of Ak on the orientation of
molecules at interfaces has far reaching consequences: SFG is not
allowed in materials with centrosymmetry or isotropic materials
without long-range order, that is, liquids and gases when the positions
of molecules are averaged over time. At interfaces, the bulk symmetry
is necessarily broken and nonzero components of χ(2) solely from the
interface exist and give rise to surface sensitive SFG. A perfectly polarordered adlayer results in the highest amplitude and SFG intensity,
while a layer with identical coverage, but randomly oriented interfacial
molecules, has negligible SFG intensity. Hence, SFG combines the
advantages of optical techniques with intrinsic surface/symmetry
sensitivity and is a very powerful and highly versatile spectroscopic tool
for studies of surfaces and interfaces. However, most studies of
proteins at the air−water interface were limited to the methyl and
water stretching region27,31−34 which yields information about the
interfacial water structure but not about the protein amide I band
around 1650 cm−1. For our SFG measurements a home built

broadband SFG setup was applied, as described elsewhere.35 The setup
enables us to tune the IR frequency and record the SFG intensity for
IR frequencies which are within the bandwidth of a broadband IR
pulse (200 cm−1). All spectra were recorded with s-polarized sumfrequency, s-polarized visible and p-polarized IR beams (ssp). The
presented spectra were normalized to a reference spectrum of a
polycrystalline Au sample that was previously subjected to oxygen
plasma. The 15 μM BSA samples were poured in a Petri dish, and SFG
spectra were collected. Each spectrum was measured by scanning the
broadband IR beam with a step width of 130 cm−1 and total
acquisition times of 7 and 8 min for 2800−3800 and 1300−1800 cm−1
spectral regions, respectively.

EXPERIMENTAL SECTION

Sample Preparation. BSA (essentially acid free) was purchased
from Sigma Aldrich (A7030) and was used as-received. BSA solutions
(15 μM for spectroscopic measurements, 150 μM for zeta potential
measurements) were prepared by dissolving the dry protein in
ultrapure water (18.2 MΩ cm−1; total oxidizable carbon < 10 ppb).
The pH was adjusted by adding either HCl (Merck; Suprapur grade)

or NaOH (99.99%; Alfa Aesar) and measured with an InLab Micro
Pro pH electrode (Mettler Toledo). In order to remove possible
contaminations, the necessary glassware was soaked in a mixture of
concentrated sulfuric acid (98%; analytical grade) and NOCHROMIX
for at least 24 h, thoroughly rinsed with ultrapure water, and
subsequently cleaned in boiling ultrapure water. All measurements
were performed at a temperature of 24 °C.
Zeta Potential Measurements. Zeta potentials were measured
with a commercial Zetasizer Nano ZS instrument (Malvern Instruments). The pH of 150 μM BSA aqueous solutions was adjusted by
adding either acid or base. The samples were filtered using 0.2 μm
cellulose acetate filters (VWR 514-0060) and thoroughly cleaned glass
syringes before transferring them into the cuvette. For every data
point, at least four measurements with four different cuvettes were
performed. For good and reproducible zeta potential measurements, a
minimum BSA concentration of approximately 150 μM is needed. For
that reason, 10-fold higher BSA concentrations have been chosen for
measurements of the bulk zeta potential compared to ellipsometry and
SFG measurements at air−water interfaces.
Ellipsometry. The thickness of adsorbed protein layers was
determined with a phase modulated ellipsometer (Picometer
Ellipsometer; Beaglehole Instruments) that was operated with a
wavelength of 632.8 nm. Phase modulated ellipsometry offers the
possibility to record data near or at the Brewster angle (∼55°) of the
studied system which increases the sensitivity considerably and enables
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Foam Preparation and Characterization. Foaming experiments
were carried out with a commercial dynamic foam analyzer DFA 100
(Krüss GmbH, Germany). Foams were produced in a glass column of
0.25 m length and 0.04 m thickness by a stream of air that was
introduced into the protein dilutions through a porous glass filter
(pore size: 9−16 μm) with a constant flow rate of 5 mL/s.
Experiments were performed in triplicates. According to Glaser et
al.,36 stability and capacity are key parameters which can be used to
characterize macroscopic foam properties. The foam capacity can be
determined by the volume increase due to the foaming of the initial
BSA dilution with a volume Vi of 40 mL. In our experiments the foam
capacity is given by the maximum (foam) volume (Vf) that is reached
after 10 s of gas flow through the BSA dilution. Foam capacity [%] =
(Vf − Vi)/Vi × 100. Subsequent to the formation, foam degradation
given by the Foam stability [%] = (Vt=300s/Vt=0) × 100 was monitored
for an additional 300 s.

behavior of surface adsorbed BSA layers in the following
sections.
Ellipsometry. The use of ellipsometry enables us to
investigate the pH effects on the thickness of adsorbed BSA
layers and to estimate the interfacial number density N of BSA.
In Figure 2, the thickness of BSA layers adsorbed to the air−

RESULTS
Determination of the Bulk Zeta Potential. The zeta
potential is often used as stability parameter in colloidal
chemistry whereby a potential larger than |30 mV| leads to
stable suspensions.37 In contrast to this, the zeta potential of
proteins is determined to be mostly lower than |40 mV|28,38 and
is often used to determine the isoelectric point.39 The
isoelectric point is defined as the point where the zeta potential
is equal to zero. At the isoelectric point, the protein carries no
net charge, while there is an excess of positive or negative
charge for pH values higher and lower than the pH of the
isoelectric point, respectively. Previous studies of the BSA’s
isoelectric point have shown that its exact determination is
impaired by the applied experimental method and the
background electrolyte. For that reason it is not surprising
that isoelectric points between pH 4.7 and 5.6 have been
reported.28
Figure 1 shows the zeta potential of BSA in a bulk solution as
a function of the electrolyte pH. From a close inspection of

■

Figure 2. Thickness of adsorbed BSA layers at the air−water interface
as a function of the electrolyte pH. As explained in the text, the
thickness was determined by ellipsometry under the assumption of a
simple water−BSA−air layer model. The dashed line is a guide to the
eye.

water interface is presented as a function of electrolyte pH. For
acidic conditions, the thickness first increases with increasing
pH, reaches a pronounced maximum at pH ∼5.5, and decreases
subsequently for higher pH values. Obviously, pH values near
the bulk isoelectric point (see previous section) lead to much
thicker BSA layers as compared to more acidic or alkaline
conditions.
Assignment of Vibrational Bands in SFG Spectra. To
gain further insight in the molecular structure of the interface,
vibrational SFG spectra were recorded in the entire spectral
region of 1000−3700 cm−1. To the best of our knowledge these
are the first vibrational SFG spectra of a protein that were
measured in such a broad spectral region. This approach allows
us to identify vibrational fingerprints of BSA and interfacial
water molecules and to select specific regions of interest where
changes with the electrolyte pH are most pronounced.
Figure 3 shows vibrational SFG spectra of BSA adsorbed to
the air−water interface for pH values of 8.2 and 4.3. A close
comparison of the spectra reveals that they are dominated by

Figure 1. Zeta potential of BSA as a function of the electrolyte pH.
Lines are a guide to the eye.

Figure 1, the pH of the isoelectric point of BSA can be
determined to 5.2 ± 0.1. Although the isoelectric point of BSA
can be established in the bulk electrolyte, it is not a priori
known whether the isoelectric points of bulk and surface
regions are identical. In fact, the small electric charge of BSA
near or at the isoelectric points leads to very small repulsion
between individual proteins and therefore to the tendency of
agglomeration and possibly to precipitation of BSA. Consequently, it is not clear if and how stable BSA layers can
actually form at the air−water interface under these conditions.
To study this effect further, we will show the pH dependent

Figure 3. Vibrational SFG spectra of BSA at air−water interfaces for
pH 8.2 and 4.3. Solid lines are fits to the experimental data as
explained in the text. Details to the spectral regions I−III can be found
in the text.
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CH aromatic bending vibrations.58 Since substantial changes as
a function of the bulk pH are observed for CH, OH, amide I,
and carboxylate vibrations (Figure 3), we have focused further
pH dependent studies to the latter vibrations. An overview of
the SFG active BSA resonances relevant to the further
discussion and their attribution to specific molecular vibrations
can be found in Table 1.

vibrational bands in unique spectral regions where substantial
changes as a function of the electrolyte pH can be observed:
(I) Functional groups of adsorbed BSA give rise to strong
vibrational bands at ∼2875 and ∼2936 cm−1 that are
attributable to CH3 symmetric stretching vibrations and to
the CH3 Fermi resonance, respectively. Furthermore, CH3
asymmetric, symmetric CH2, and aromatic CH stretching
vibrations give rise to bands at 2964, 2850, and 3060 cm−1,
respectively.27,31−33 Strong bands between 3100 and 3800 cm−1
originate from OH stretching vibrations of interfacial water
molecules.34,40,41
(II) In a second spectral region, additional vibrational bands
of interfacial BSA at ∼1654 and 1410 cm−1 are observed and
can be attributed to amide I and to carboxylate (R−COO−)
symmetric stretching vibrations, respectively.42,43 The amide I
band originates from R−CO carbonyl vibrations of
molecular groups in the protein backbone44−48 where they
can form hydrogen bonds to neighboring amide groups49,50
(Figure 4).

Table 1. Assignment of Vibrational Bands in SFG Spectra of
BSA Adsorbed to the Air−Water Interfacea
band

[cm−1]

ref

amide III
COO− (ss)
amide II
amide I
CH2 (ss)
CH3 (ss)
CH3 (F)
arom. CH
OH (ss)
OH (ss)

1250
1410
1550
1650
2850
2875
2936
3060
3200
3450

57
43
50
48, 53
31−33
31−33
31−33
31−33
40, 41
40, 41

a

(F), (ss), and (as) stand for Fermi resonance, symmetric, and
antisymmetric stretching vibrations, respectively.

pH Dependence of the Interfacial Molecular Structure. In order to reveal changes in the interfacial molecular
structure, SFG spectra of BSA adsorbed to the air−water
interface were recorded and analyzed for pH values of 3−10.
Figure 5 shows representative SFG spectra in this pH range.
OH, CH, and amide I bands have strong pH dependencies. In
particular, for the OH stretching bands of interfacial water
molecules, we observe dramatic changes in the SFG intensity
(Figure 5b).
At a pH of 5.5, the SFG intensity of the H2O bands is
negligible, but increases substantially for lower and higher pH
values than 5.5. Similar but less pronounced behavior is
observed for the SFG intensity in the CH stretching region.
Compared to the latter bands, BSA amide I and carboxylate
modes show much weaker changes in SFG intensity with the
pH (Figure 5a). In order to analyze changes of SFG bands as a
function of pH, we have fitted our spectra with model functions
according to eq 1 and determined the amplitude of the
vibrational bands in our SFG spectra. We have used the
amplitude Ak, the resonance frequencies ωk, and the nonresonant contribution χNR(2) as adjustable parameters in our
fitting procedures. Here, the overview spectra in Figure 3 are
extremely helpful, since the nonresonant contribution and its
influence on the dispersion of the vibrational resonances can be
easily estimated.
In Figure 6a, b, and c, the results of our fitting procedures are
presented for the amplitudes of amide I, carboxylate (R−
COO−), and OH vibrations, respectively. The pH dependence
of the three amplitudes shows a local minimum around pH 5.
While the amplitudes of amide I and carboxylate bands have a
narrow minimum at pH 5 and are only slightly smaller for more
alkaline than for acidic pH values, the amplitude of OH
vibrations is also minimal at pH 5, but varies in a much broader
pH range. Nearly all carboxyl groups with a pK of 4.3 from the
amino acid side chains of BSA59 are deprotonated in the
studied pH range and exist as carboxylates. This causes the
amplitudes of the R−COO− vibration to stay nearly constant.
However, since the density of these groups at the interface

Figure 4. Schematic representation of amide groups and their local
orientation in a protein with an α-helix secondary structure.

The strength of the hydrogen bonds influences the frequency
of the amide I band greatly and is, therefore, strongly
dependent on the secondary structure of the protein. In
previous IR studies, changes in the position of this band were
often referred to denaturation, unfolding, or aggregation
processes.51,52 Since BSA consists mainly of an α-helical
structure, unfolding would lead to a blue shift in the amide I
frequency. As was previously shown, the spectral frequencies of
amide I vibrations can be attributed to different secondary
structures42,53 and the kinetics of conformational changes was
also resolved with SFG.54
At this point, it should be noted that the so-called amide II
band at ∼1550 cm−1 that can be observed with linear IR
spectroscopy50,55 contributes only weakly to the SFG intensity.
Prerequisite for an SFG active mode is both Raman as well as
IR activity. Since the amide II mode does lead to weak Raman
resonances only,56 weak resonant SFG contributions from this
band are also likely. Furthermore, the amide II band is a
combination of C−N stretch and N−H bending vibrations and
has a dynamic dipole moment that is perpendicular to the
dipole moment of the amide I band (Figure 4). As a result, the
intensity of this band is additionally weakened due to weaker
excitations with p polarized IR light.
(III) In the spectral region below 1400 cm−1, a band
centered at ∼1250 cm−1 is observed and has been attributed to
amide III vibrations.56,57 Three weak bands at ∼1140, ∼1070,
and ∼1020 cm−1 are also observed in our spectra, which we
relate to −C−O− stretching modes, N−H deformation, and
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Figure 5. Vibrational SFG spectra of BSA at air−water interfaces as a
function of the electrolyte pH: (a) Symmetric carboxylate (R-COO−)
and amide I bands and (b) CH and OH stretching bands as explained
in the text (Table 1). The pH was as indicated in the figure. Solid lines
are fits to the experimental data according to eq 1. The color scale
represents schematically the interfacial charge density as a function of
the pH.

Figure 6. pH dependence of the amide I (a) and carboxylate R−
COO− (b) vibrational amplitude in arbitrary units. (c) Amplitude of
OH stretching vibrations at 3200 cm−1 (black solid square) and 3400
cm−1 (red solid triangle). Dotted Lines are guide to the eye.

increases (Figure 2), the observed change in SFG amplitude is a
signature of a decrease in interfacial polar order that will be
discussed in more detail in the following section.
The vibrational band of interfacial H2O shows a dramatic
decrease in amplitude for pH 3−5 with a subsequent dramatic
increase of almost 1 order of magnitude (Figure 6c). However,
not only the amplitudes of the vibrational bands change, but
there is also a notable change in the polarity of the band at
∼3060 cm−1 due to aromatic CH stretching vibrations. At pH <
5, this band appears as a positive going feature, while for pH >
5 a negative going feature is observed (Figure 5b). These
changes are not directly related to the ∼3060 cm−1 band but to
a change in the orientation of the interfacial water molecules.
The phases of the broad OH stretching bands are rotated by
180°, and according to eq 1 the spectral interference of H2O
and CH bands is altered. The change in the average orientation
of the interfacial water molecules points to a charge reversal of
the interface and, consequently, to a reversal of the electric field
that causes the polar ordering of H2O at the interface. This
result confirms previous SFG studies of BSA adsorption by
Wang et al.27

DISCUSSION
We will now compare the observed pH effects of adsorbed BSA
adlayers from ellipsometry with SFG measurements and the
zeta potential of BSA in the bulk electrolyte. It is obvious that
the bulk isoelectric point of BSA at pH 5.2 with a zeta potential
equal to zero (Figure 1) clearly corresponds to a minimum in
SFG amplitudes of the BSA and interfacial H2O related bands
(Figure 6), but to a maximum in the thickness of adsorbed BSA
adlayers (Figure 2). In fact, our ellipsometry results indicate the
presence of multilayers for pH values near the bulk isoelectric
point and are in agreement with previous neutron reflection
studies of BSA at air−water interfaces. In their report, Lu et al.
have estimated the adsorbate thickness to approximately 7 nm
and a reduced thickness of approximately 4 nm for pH values
higher or lower than the bulk isoelectric point.11 Modeling the
geometric dimensions of BSA by a simple incompressible
ellipsoid, thicknesses of ∼4 nm in Figure 2 point to a
monolayer of adsorbed BSA proteins with their long axis
oriented parallel to the interface.12 Threefold thicker layers at a
pH near 5 are also observed (Figure 2) and suggest the
formation of multilayers. Although we observe a seemingly

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can be rationalized in terms of hydrophilic−hydrophobic
interactions, where the hydrophobic parts of the BSA proteins
tend to protrude into the gas phase. In contrast to the protein
layer, the interfacial water molecules are highly disordered and
lead to negligible SFG amplitudes (Figure 6c).
Having established the interfacial molecular structure, we can
now deduce (to some extent) macroscopic properties such as
foam stability and foam capacity. Prerequisite for good foam
formation is a fast diffusion of the proteins to the interface,
where they can build a viscoelastic adsorbed layer around the
gas bubbles and prevent destabilization of the foam lamella.62
As we have shown, at the interfacial isoelectric point of BSA, a
network of agglomerated proteins is formed and held together
by weak attractive forces, while at a pH more alkaline or more
acidic than the isoelectric point BSA forms monolayers with
repulsive interactions. It is therefore likely that producing BSA
foams at the isoelectric point will lead to foams with higher
stability.
We have tested this hypothesis with macroscopic foams from
BSA dilutions. The results for foam capacity, which describes
the ability of a protein solution to enclose air, and foam stability
measurements are shown in Figure 7a and b, respectively. Both

opposite behavior of SFG amplitudes and adsorbate thickness,
it can be concluded that the isoelectric points of bulk and
interfacial BSA proteins are identical. pH values at the
isoelectric point of the interface lead to a highly disordered
electrolyte subphase and BSA (multi)layers with a low degree
of order:
We recall that the SFG amplitude Ak is dependent on both
the number density of interfacial molecules and their molecular
order. In the present case, the contribution of the number
density to the SFG amplitudes is only minor otherwise the
amplitude should reach a maximum at a pH where the
thickness of the BSA adlayer is also maximal. Since we observe
the opposite, signals in SFG spectra must be dominated by the
interfacial molecular order. At a pH near the bulk isoelectric
point, the net charge of BSA proteins is negligible and a
macroscopic electric field at the interface is absent. As the pH
increases or decreases with respect to the isoelectric point of
BSA, the charge density and, consequently, the electric field of
the interface increase. As a result, polar ordering of interfacial
water molecules and BSA is induced by the interfacial electric
field. Further support for this hypothesis comes from SFG
experiments at electrified oxide interfaces where electric fieldinduced polar ordering of interfacial water molecules was
shown.40,41,60,61
Observations of field induced polar ordering in vibrational
SFG are, consequently, directly related to the strength of the
interfacial electric field and allow an estimate of the isoelectric
point of the interface. At this point, it should be noted that
electrokinetic zeta potential measurements and SFG spectroscopy actually probe different physical properties which,
however, have their physical origin both in the charge
distribution at the surface. The zeta potential refers to the
electric potential at the hydrodynamic shear (or slipping) plane.
The slipping plane separates ions (if present) and solvent
molecules which travel with the migrating protein from those in
the diffuse layer that do not travel with the charged protein in
an external electric field. In contrast, SFG probes the average
orientation of molecules within the interfacial electric field.
Since the amide I band originates from molecular groups in the
interior of the protein (see above), pH effects on polar ordering
must be related to the net charge of the protein surface which is
also determined by the charge of possible bound counterions in
the adjacent Stern layer. The physical origins of polar ordering
as seen in SFG and in the zeta potential are, thus, identical. The
remarkable resemblance of amide I, carboxylate (R−COO−),
and OH amplitudes in Figure 6 also signifies a similar origin for
the latter bands.
For pH values near the isoelectric point, BSA adsorption is
controlled by a gain in entropy and the formation of attractive
noncovalent interactions such as van der Waals forces and
hydrogen bonds of the BSA hydration shell. The situation is
different for pH values of 6.7 where electrostatic
forces dominate and lead to a polar ordered BSA monolayer. In
order to establish multiple BSA layers at the interface, the
lateral interactions between individual BSA proteins have to be
attractive. In fact, the absence of a strong electric charge at the
BSA surface, as it is suggested by the zeta potential of BSA in
the bulk, leads to weak repulsive electrostatic interactions and
consequently to an agglomerated BSA adlayer. At the isoelectric
point, the orientation of adsorbed BSA is, however, not
completely random since the nonzero SFG amplitudes of amide
I and carboxylate vibrations indicate that to some extent a
preferential orientation of BSA is maintained. This observation

Figure 7. Foam capacity (a) and stability (b) as a function of pH.
Lines are a guide to the eye.

the foam capacity and stability show a clear maximum around
pH 5 (Figure 7). Obviously, a network of agglomerated protein
multilayers can encapsulate the air and prevent the foam from
drainage much more efficiently than ordered protein
monolayers with repulsive interactions.

SUMMARY AND CONCLUSION
New information on molecular processes at interfaces and
macroscopic phenomena of soft matter is provided. For that
purpose, we have addressed the surface chemistry of the protein
bovine serum albumin (BSA) at the air−water interface and the
effect of pH. Combining ellipsometry and broadband sumfrequency generation (SFG) has allowed us to reveal the
molecular composition and molecular order of BSA adlayers
and the electrolyte subphase in unprecedented detail. pH
dependent charging of BSA leads to the formation of electrified
interfaces and to polar ordering of interfacial BSA and H2O.
Using the latter as a measure of the electric field at the interface,

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(12) McClellan, S. J.; Franses, E. I. Effect of concentration and
denaturation on adsorption and surface tension of bovine serum
albumin. Colloids Surf., B 2003, 28, 63−75.
(13) Stocco, A.; Drenckhan, W.; Rio, E.; Langevin, D.; Binks, B. P.
Particle-stabilised foams: an interfacial study. Soft Matter 2009, 5,
2215−2222.
(14) Wang, W. Instability, stabilization, and formulation of liquid
protein pharmaceuticals. Int. J. Pharm. 1999, 185, 129−188.
(15) Douillard, R. Kinetics of lysozyme adsorption at the air-buffer
interface. Thin Solid Films 1997, 292, 169−172.
(16) Miller, R.; Fainerman, V. B.; Makievski, A. V.; Krägel, J.;
Grigoriev, D. O.; Kazakov, V. N.; Sinyachenko, O. V. Dynamics of
protein and mixed protein/surfactant adsorption layers at the water/
fluid interface. Adv. Colloid Interface Sci. 2000, 86, 39−82.
(17) Tripp, B. C.; Magda, J. J.; Andrade, J. D. Adsorption of Globular
Proteins at the Air/Water Interface as Measured via Dynamic Surface
Tension: Concentration Dependence, Mass-Transfer Considerations,
and Adsorption Kinetics. J. Colloid Interface Sci. 1995, 173, 16−27.
(18) Tronin, A.; Dubrovsky, T.; Dubrovskaya, S.; Radicchi, G.;
Nicolini, C. Role of Protein Unfolding in Monolayer Formation on
Air-Water Interface. Langmuir 1996, 12, 3272−3275.
(19) Felsovalyi, F.; Mangiagalli, P.; Bureau, C.; Kumar, S.; Banta, S.
Reversibility of the Adsorption of Lysozyme on Silica. Langmuir 2011,
27, 11873−11882.
(20) Desfougères, Y.; Saint-Jalmes, A.; Salonen, A.; Viés, V.; Beaufils,
S.; Pezennec, S.; Desbat, B.; Lechevalier, V.; Nau, F. Strong
Improvement of Interfacial Properties Can Result from Slight
Structural Modifications of Proteins: The Case of Native and DryHeated Lysozyme. Langmuir 2011, 27, 14947−14957.
(21) Kim, G.; Gurau, M.; Kim, J.; Cremer, P. S. Investigations of
Lysozyme Adsorption at the Air/Water and Quartz/Water Interfaces
by Vibrational Sum Frequency Spectroscopy. Langmuir 2002, 18,
2807−2811.
(22) Fick, J.; Wolfram, T.; Belz, F.; Roke, S. Surface-Specific
Interaction of the Extracellular Domain of Protein L1 with
Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers. Langmuir 2009, 26, 1051−1056.
(23) Kim, J.; Somorjai, G. A. Molecular Packing of Lysozyme,
Fibrinogen, and Bovine Serum Albumin on Hydrophilic and
Hydrophobic Surfaces Studied by Infrared−Visible Sum Frequency
Generation and Fluorescence Microscopy. J. Am. Chem. Soc. 2003,
125, 3150−3158.
(24) Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z. Detection of chiral
sum frequency generation vibrational spectra of proteins and peptides
at interfaces in situ. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4978−4983.
(25) Thennarasu, S.; Huang, R.; Lee, D.-K.; Yang, P.; Maloy, L.;
Chen, Z.; Ramamoorthy, A. Limiting an Antimicrobial Peptide to the
Lipid−Water Interface Enhances Its Bacterial Membrane Selectivity: A
Case Study of MSI-367. Biochemistry 2010, 49, 10595−10605.
(26) Nguyen, K. T.; Soong, R.; lm, S.-C.; Waskell, L.; Ramamoorthy,
A.; Chen, Z. Probing the Spontaneous Membrane Insertion of a TailAnchored Membrane Protein by Sum Frequency Generation Spectroscopy. J. Am. Chem. Soc. 2010, 132, 15112−15115.
(27) Wang, J.; Buck, S. M.; Chen, Z. Sum frequency generation
vibrational spectroscopy studies on protein adsorption. J. Phys. Chem.
B 2002, 106, 11666−11672.
(28) Salis, A.; Boström, M.; Medda, L.; Cugia, F.; Barse, B.; Parsons,
D. F.; Ninham, B. W.; Monduzzi, M. Measurements and Theoretical
Interpretation of Points of Zero Charge/Potential of BSA Protein.
Langmuir 2011, 27, 11597−11604.
(29) Tompkins, H. G.; Irene, E. A. Handbook of ellipsometry; William
Andrew Pub; Springer: Norwich, NY, Heidelberg, Germany, 2005.
(30) Shen, Y. R. The principles of nonlinear optics; John Wiley & Sons:
New York,1984.
(31) Wang, J.; Buck, S. M.; Chen, Z. The effect of surface coverage
on conformation changes of bovine serum albumin molecules at the
air-solution interface detected by sum frequency generation vibrational
spectroscopy. Analyst 2003, 128, 773−778.

we have deduced the isoelectric point of BSA at air−water
interfaces to pH ∼ 5, which is close to that of bulk BSA. The
molecular level information presented in this study can explain
the high foam stability around pH 5, which was determined in
additional experiments: Around pH 5, disordered multilayers
are present at the interface and form an agglomerated network
of BSA proteins that can be used to form macroscopic foams
with excellent stability. Here, BSA agglomerates stabilize gas
bubbles at the ubiquitous air−water interface extremely
efficiently and prevent the foam from drainage. For pH values
smaller or larger than the isoelectric point, BSA monolayers
with repulsive interactions are formed and lead to a decreased
stability of BSA foams.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: W.Peukert@lfg.uni-erlangen.de.
Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS
The authors gratefully acknowledge funding of the Erlangen
Graduate School in Advanced Optical Technologies (SAOT)
and by the German National Science Foundation (DFG)
through the Leibniz program and project PE427/21-1. B.B. is
grateful for support by the Alexander von Humboldt
foundation and a Feodor Lynen fellowship. J.D. is grateful for
support by the German Ministry of Economics and Technology
(via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn), project AiF 17124 N.

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REFERENCES

(1) Lefebvre, L.-P.; Banhart, J.; Dunand, D. C. Porous Metals and
Metallic Foams: Current Status and Recent Developments. Adv. Eng.
Mater. 2008, 10, 775−787.
(2) Fameau, A.-L.; Saint-Jalmes, A.; Cousin, F.; Houinsou-Houssou,
B.; Novales, B.; Navailles, L.; Nallet, F.; Gaillard, C.; Boué, F.; Douliez,
J.-P. Smart Foams: Switching Reversibly between Ultrastable and
Unstable Foams. Angew. Chem. 2011, 123, 8414−8419.
(3) Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M.
Understanding foods as soft materials. Nat. Mater. 2005, 4, 729−740.
(4) Schramm, L. L. Emulsions, foams, and suspensions: Fundamental
and applications; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim,
2005.
(5) Townsend, A.-A.; Nakai, S. Relationships Between Hydrophobicity and Foaming Characteristics of Food Proteins. J. Food Sci.
1983, 48, 588−594.
(6) Foegeding, E. A.; Luck, P. J.; Davis, J. P. Factors determining the
physical properties of protein foams. Food Hydrocolloids 2006, 20,
284−292.
(7) Tolstoguzov, V. Foods As Dispersed Systems. Thermodynamic
aspects of composition-property relationships in formulated food. J.
Therm. Anal. Calorim. 2000, 61, 397−409.
(8) Goff, H. D., Vega, C. Structure-engineering of ice-cream and
foam-based foods. In Understanding and controlling the microstructure of
complex foods; McClements, D. J., Ed.; CRC Press; Woodhead Pub.:
Cambridge, 2007.
(9) Wierenga, P. A.; Gruppen, H. New views on foams from protein
solutions. Curr. Opin. Colloid Interface Sci. 2010, 15, 365−373.
(10) Miller, R.; Fainerman, V. B.; Wüstneck, R.; Krägel, J.; Trukhin,
D. V. Characterisation of the initial period of protein adsorption by
dynamic surface tension measurements using different drop
techniques. Colloids Surf., A 1998, 131, 225−230.
(11) Lu, J. R.; Su, T. J.; Penfold, J. Adsorption of Serum Albumins at
the Air/Water Interface. Langmuir 1999, 15, 6975−6983.
7786

dx.doi.org/10.1021/la301368v | Langmuir 2012, 28, 7780−7787

Langmuir

Article

(32) Chen, X.; Flores, S. C.; Lim, S.-M.; Zhang, Y.; Yang, T.; Kherb,
J.; Cremer, P. S. Specific Anion Effects on Water Structure Adjacent to
Protein Monolayers. Langmuir 2010, 26, 16447−16454.
(33) Chen, X.; Yang, T.; Kataoka, S.; Cremer, P. S. Specific Ion
Effects on Interfacial Water Structure near Macromolecules. J. Am.
Chem. Soc. 2007, 129, 12272−12279.
(34) Kim, J.; Cremer, P. S. Elucidating Changes in Interfacial Water
Structure upon Protein Adsorption. ChemPhysChem 2001, 2, 543−
546.
(35) Rumpel, A.; Novak, M.; Walter, J.; Braunschweig, B.; Halik, M.;
Peukert, W. Tuning the molecular order of C60 functionalized
phosphonic acid monolayers. Langmuir 2011, 27, 15016−15023.
(36) Glaser, L. A.; Paulson, A. T.; Speers, R. A.; Yada, R. Y.;
Rousseau, D. Foaming behavior of mixed bovine serum albumin−
protamine systems. Food Hydrocolloids 2007, 21, 495−506.
(37) Heurtault, B.; Saulnier, P.; Pech, B.; Proust, J.-E.; Benoit, J.-P.
Physico-chemical stability of colloidal lipid particles. Biomaterials 2003,
24, 4283−4300.
(38) Anema, S. G.; Klostermeyer, H. ζ-Potentials of casein micelles
from reconstituted skim milk heated at 120 °C. Int. Dairy J. 1996, 6,
673−687.
(39) Jachimska, B.; Wasilewska, M.; Adamczyk, Z. Characterization
of Globular Protein Solutions by Dynamic Light Scattering, Electrophoretic Mobility, and Viscosity Measurements. Langmuir 2008, 24,
6866−6872.
(40) Richmond, G. L. Molecular Bonding and Interactions at
Aqueous Surfaces as Probed by Vibrational Sum Frequency Spectroscopy. Chem. Rev. 2002, 102, 2693−2724.
(41) Shen, Y. R.; Ostroverkhov, V. Sum-Frequency Vibrational
Spectroscopy on Water Interfaces: Polar Orientation of Water
Molecules at Interfaces. Chem. Rev. 2006, 106, 1140−1154.
(42) Wang, J.; Even, M. A.; Chen, X.; Schmaier, A. H.; Waite, J. H.;
Chen, Z. Detection of Amide I Signals of Interfacial Proteins in Situ
Using SFG. J. Am. Chem. Soc. 2003, 125, 9914−9915.
(43) Braunschweig, B.; Mukherjee, P.; Kutz, R. B.; Wieckowski, A.;
Dlott, D. D. Sum-frequency generation of acetate adsorption on Au
and Pt surfaces: Molecular structure effects. J. Chem. Phys. 2010, 133,
234702−234708.
(44) Byler, D. M.; Susi, H. Examination of the secondary structure of
proteins by deconvolved FTIR spectra. Biopolymers 1986, 25, 469−
487.
(45) Wang, J.; Chen, X.; Clarke, M. L.; Chen, Z. Vibrational
Spectroscopic Studies on Fibrinogen Adsorption at Polystyrene/
Protein Solution Interfaces: Hydrophobic Side Chain and Secondary
Structure Changes. J. Phys. Chem. B 2006, 110, 5017−5024.
(46) Clarke, M. L.; Wang, J.; Chen, Z. Conformational Changes of
Fibrinogen after Adsorption. J. Phys. Chem. B 2005, 109, 22027−
22035.
(47) Yang, P.; Ramamoorthy, A.; Chen, Z. Membrane Orientation of
MSI-78 Measured by Sum Frequency Generation Vibrational
Spectroscopy. Langmuir 2011, 27, 7760−7767.
(48) Wang, J.; Clarke, M. L.; Chen, X.; Even, M. A.; Johnson, W. C.;
Chen, Z. Molecular studies on protein conformations at polymer/
liquid interfaces using sum frequency generation vibrational spectroscopy. Surf. Sci. 2005, 587, 1−11.
(49) Knoesen, A.; Pakalnis, S.; Wang, M.; Wise, W.; Lee, N.; Frank,
C. Sum-frequency spectroscopy and imaging of aligned helical
polypeptides. IEEE J. Sel. Top. Quantum Electron. 2004, 10, 1154−
1163.
(50) Barth, A.; Zscherp, C. What vibrations tell about proteins. Q.
Rev. Biophys. 2002, 35, 369−430.
(51) Murayama, K.; Tomida, M. Heat-Induced Secondary Structure
and Conformation Change of Bovine Serum Albumin Investigated by
Fourier Transform Infrared Spectroscopy. Biochem. 2004, 43, 11526−
11532.
(52) Shanmugam, G.; Polavarapu, P. L. Vibrational circular dichroism
spectra of protein films: thermal denaturation of bovine serum
albumin. Biophys. Chem. 2004, 111, 73−77.

(53) Liu, Y.; Jasensky, J.; Chen, Z. Molecular Interactions of Proteins
and Peptides at Interfaces Studied by Sum Frequency Generation
Vibrational Spectroscopy. Langmuir 2012, 28, 2113−2121.
(54) Fu, L.; Ma, G.; Yan, E. C. Y. In Situ Misfolding of Human Islet
Amyloid Polypeptide at Interfaces Probed by Vibrational Sum
Frequency Generation. J. Am. Chem. Soc. 2010, 132, 5405−5412.
(55) Haris, P. I.; Severcan, F. FTIR spectroscopic characterization of
protein structure in aqueous and non-aqueous media. J. Mol. Catal. B:
Enzymatic 1999, 7, 207−221.
(56) Socrates, G. Infrared and Raman characteristic group frequencies:
Tables and charts, 3rd ed.; John Wiley & Sons: Chichester [u.a.], 2004.
(57) Lin, V. J. C.; Koenig, J. L. Raman studies of bovine serum
albumin. Biopolymers 1976, 15, 203−218.
(58) Yu, N.; Jo, B. H. Comparison of protein structure in crystals and
in solution by laser Raman scattering: I. Lysozyme. Arch. Biochem.
Biophys. 1973, 156, 469−474.
(59) Peters, T. All about albumin: Biochemistry, genetics, and medical
applications; Academic Press: San Diego, 1996.
(60) Sung, J.; Zhang, L.; Tian, C.; Shen, Y. R.; Waychunas, G. A.
Effect of pH on the Water/α-Al2O3 (1102) Interface Structure Studied
by Sum-Frequency Vibrational Spectroscopy. J. Phys. Chem. C 2011,
115, 13887−13893.
(61) Braunschweig, B.; Eissner, S.; Daum, W. Molecular Structure of
a Mineral/Water Interface: Effects of Surface NanoRoughness of αAl2O3 (0001). J. Phys. Chem. C 2008, 112, 1751−1754.
(62) Kinsella, J.; Morr, C. V. Milk proteins: Physicochemical and
functional properties. Crit. Rev. Food Sci. Nutr. 1984, 21, 197−262.

7787

dx.doi.org/10.1021/la301368v | Langmuir 2012, 28, 7780−7787