SDS Mixtures

Chapter 7

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Simultaneous versus Sequential Adsorption of β-Casein/SDS Mixtures. Comparison of Water/Air and Water/Hexane Interfaces A. Dan,1 G. Gochev,1 Cs. Kotsmar,2 J. K. Ferri,3 A. Javadi,1 M. Karbaschi,1,4 J. Krägel,1 R. Wüstneck,1 and R. Miller*,1 1Max-Planck

Institute of Colloids & Interfaces, Potsdam/Golm, Germany 2University of California at Berkeley, Berkeley, California 94720-1462, U.S.A. 3Department of Chemical Engineering, Lafayette College, Easton, Pennsylvania 18042, U.S.A. 4Sharif University of Technology, Teheran, Iran *E-mail: [email protected]

This chapter is dedicated to the surface properties of mixed protein/surfactant adsorption layers, formed by two different experimental approaches, i.e. by sequential and simultaneous adsorption, respectively. A special modification of a drop profile analysis tensiometer, consisting of a coaxial double capillary, provides a unique protocol for studies of mixed surface layers formed by sequential adsorption of the individual components in addition to the traditional simultaneous adsorption from their mixed solution. A CFD simulation allowed to optimize the drop exchange process performed with the special double capillary arrangement. The experiments show that properties of sequentially formed layers differ significantly from those formed simultaneously, which can be explained by the different nature and structure of the complexes formed at the two different locations. The nature of the interface, water/air or water/oil, influences strongly the adsorption behavior of the protein molecules and consequently the mixed layers due to their different degree of polarity and hydrophobicity. Washing out experiments are performed in order to support the proposed

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

mechanism of the protein displacement process from mixed surface layers, i.e., to check how many protein molecules are left in the adsorption layer. Based on the experimental studies of the milk protein β-casein (βCS) mixed with the anionic surfactant sodium dodecyl sulfate (SDS) at water/air and water/hexane interfaces, the results are discussed according to different mechanisms describing the different location of interaction.


1. Introduction The stability of many products in the field of food, cosmetic, pharmaceuticals is controlled by mixed adsorption layers of proteins and low molecular weight surfactants (1). While neither single protein nor the surfactants are suitable, their mixtures at optimum mixing ratios are able to provide the right properties to the products, which is essentially true for foams and emulsions. It is generally accepted that in mixed protein/surfactant solutions complexes are formed. According to Kotsmar et al. (2) the main interaction forces between protein and surfactant molecules are of electrostatic and hydrophobic nature. Depending on the pH and the mixing ratio, the resulting complexes are more or less surface active as compared to the protein alone. By adding a non-ionic surfactant to a protein solution, the surface activity of the resulting complex is gradually decreased. Consequently, the protein would be step by step displaced from the interface by free surfactant molecules (3). For ionic surfactants, the situation is much more complicated. At small amounts the ionic surfactant binds to the protein molecules via electrostatic interactions (4) and they proceed until the available charges in the protein molecules are compensated by the surfactant ions. The resulting complexes show an increase in surface activity as compared to the original protein. With decreasing protein/surfactant mixing ratios, the hydrophobic interaction becomes more important, thus making the complex more hydrophilic and hence less surface active. A competitive adsorption between the hydrophilized protein/surfactant complexes and free surfactant molecules sets in. Depending on the total concentration and the pH in some systems even a precipitation is observed, caused by an aggregation of the most hydrophobic complexes. The described phenomena are observed for different proteins, such as β-casein, β-lactoglobulin, lysozyme, at different pH and ionic strength and also at the water/air and water/oil interfaces. The possibility for the protein to interact directly with the molecules of the oil phase is also changing the conformation of the adsorbed complexes. It is observed that the proteins are much stronger adsorbed at a water/oil interface, the adsorbed amount is larger and also the resulting adsorption layers are thicker (5). As it was shown recently, the structure and properties of the complexes are different when formed in the bulk of the solution or at the interface, respectively, which can be probed by performing experiments with different adsorption routes. In processes of a simultaneous adsorption, the proteins und surfactants are mixed 154 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


in the solution bulk and the complexes are formed in the bulk before they adsorb at the interface. These complexes may eventually change their conformation there. In a sequential adsorption protocol, the components are allowed to adsorb one after another (3). Thus, proteins can be adsorbed first from a protein solution of a given concentration. Then, the protein is removed from the bulk phase and surfactant is added. The surfactant molecules also diffuse to the interface and adsorb there. They can either penetrate into the existing protein adsorption layer, or interact with the proteins and form surface complexes. At higher surfactant concentration, the proteins are displaced into the bulk phase by the surfactant molecules and the adsorption layer is mainly covered by the free surfactant molecules. The structures of these complexes are different at the water/air or water/hexane interface, and depend on the place where they are formed. The target of this manuscript is to present the experiment protocols essentially for the sequential adsorption route, and to demonstrate the extent of proteins replaced from the interface due to the addition of surfactant. Due to the interaction with the oil phase the conformation of protein molecules at the water/oil interface is different. Adsorption experiments with the two types of protocols, simultaneous and sequential adsorption, show clearly that the properties of the complexes formed in the solution bulk differ from those formed at the interface. The present state of the art is that the community of interfacial dynamics and 2D rheology assumes that the adsorption dynamics is closely linked to the process of foam or emulsion formation. Regarding the stability of foams and emulsions, there is not yet a clear view due to the complexity of stabilizing mechanism. Obviously, the 2D rheology has impact on this however we cannot give velar relationships now. Until recently, there were no reliable experiments available and we are now in the stage of accumulating knowledge in order to sooner or later provide clear mechanisms, i.e. relationships between dilations and shear rheology of interfacial layers and the stabilizing mechanisms of foams and emulsions.

2. Theoretical Background There is a well established thermodynamic theory for describing the formation of adsorption layers from protein or mixed protein/surfactant solutions. However, these models do not allow appreciating yet the peculiarities arising from different adsorption routes. Therefore, we give only a very brief summary of the adsorption models here, in order to present the state of the art. For the adsorption of proteins alone at a liquid interface, the following basic equation of state was derived in (6):

where Π is the surface pressure, R the gas law constant, T the absolute temperature, is the intermolecular interaction parameter, ω0 is the molar area of the solvent, which is taken equal to the area occupied by an adsorbed segment of the protein molecule. The total adsorption of proteins in all n possible 155 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

states

allows to determine the total surface coverage by proteins


. The index P stands for protein. For each state j of the adsorbed protein we obtain a respective adsorption isotherm,

Here cP is the protein bulk concentration and bPj is the equilibrium adsorption constant for the protein in the jth state. The model given by Eqs. (1) and (2) describes the evolution of states of protein molecules with increasing adsorption or surface pressure. This model agrees in many details very well with experimental results (2–5). The first thermodynamic model for adsorption layers formed from mixed solutions was presented in 2004 (7). This model was further refined and allows a semi-quantitative description of the adsorption of proteins mixed with ionic as well as non-ionic surfactants. The main equation of this model reads

The parameter is the only one additional to those characterizing the single components (subscripts S and P refer to parameters characteristic for the individual surfactant and protein) and describes the interaction between protein and surfactant molecules. Small differences between ω0 and ωS can be accounted for by introducing

For a protein molecule adsorbed in the state of minimum molar area ω1=ωmin, and the surfactant, respectively, the adsorption isotherms have the following form:

This set of equations was used in (2) to describe different mixed protein/ surfactant adsorption layers. We will, however, not use it here, as the model cannot distinguish between the specific properties between layers built up on a sequential or simultaneous adsorption route. 156 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


3. Experimental Protocol for Sequential and Simultaneous Adsorption Routes The basis of the experimental setup used in the present studies is a classical drop profile analysis tensiometer (PAT1, SINTERFACE Technologies, Berlin, Germany). The image of a droplet formed at a capillary tip is acquired via a video camera and the extracted profile fitted by the Gauss-Laplace equation in order to determine the surface tension value (8). A special equipment of the PAT1, a coaxial double capillary, is used for the bulk exchange experiments, as it was first described by Wege et al. (9). The details of the setup were explained recently in (10, 11). In brief, a drop of a solution of concentration C1 is formed using syringe 1. The drop bulk is then exchanged by injecting a second solution of concentration C2, via syringe 2 (cf. Figure 1). Regarding the experimental protocols and technical data for monitoring and control of the droplet size, a pulse-like flow with different injection volume dV and waiting time dt between two pulses are applied via syringe 2 into the drop. The software of PAT1 allows to keep the drop volume VD or area AD constant via a feedback control algorithm using syringe 1. During the exchange process, the concentration of the compound in the drop, i.e. C1(t) evolves continuously from the initial C1 to the final concentration C2 within several seconds, minutes or even hour, depending on the liquid exchange rate (12). The surface tension values are measured simultaneously from the drop profile via the PAT standard software. The great advantage of the double capillary technique for the investigation of interfacial properties is based on the assumption that the material exchange between the bulk and interface is governed by an adsorption/desorption mechanism. Clearly the diffusion and convection transport mechanisms support the adsorption process via exchanging the bulk. However, during the exchange process the sub-layer should not be disturbed by forced convection or turbulences, otherwise the adsorbed layer cannot be formed correctly. For high liquid flow rates and a position of the inner capillary tip deeper inside the drop, the probability of this problem is higher. On the other hand, for low injection rates far from the drop apex, a very weak convective flow can correspond to a slow exchange process that could never lead to a complete bulk exchange during a reasonable experimental time. Therefore, optimized operational conditions for the liquid exchange should be applied in order to establish a complete bulk exchange. Mixed adsorption layers composed of protein and surfactant molecules can be formed via two different experimental strategies. As noted by several authors, the mixing protocol and equilibration time also for classic mixing can largely affect the adsorption. The classical way is when the protein and surfactant molecules adsorb simultaneously from a mixed solution. Alternatively, the individual components are adsorbed one after another, i.e., in a sequential way (13). The interfacial properties of the mixed layers formed in these two different ways can differ significantly, depending on the location where the protein/surfactant complexes are formed. The complex formation in the simultaneous adsorption route happens in the solution bulk, while in the process of sequential adsorption the surfactants form complexes with the pre-adsorbed proteins at the interface 157 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


only. The formation of both types of mixed adsorption layers was studied earlier by the drop profile analysis tensiometer PAT-1, specially equipped with a coaxial double capillary (3, 9, 13–15). The general setup and measuring principle were described in detail in (8) while Ferri at al. (12) described all up to date known fields of application of the double capillary/double dosing arrangement first proposed by Wege et al. (9) for drop bulk exchange processes. Using this arrangement we can easily perform an ‘in-situ’ subphase exchange in a single pendent drop without disturbing the surface layer, providing the required experimental protocol for performing a sequential adsorption of two individual components besides the traditional simultaneous adsorption route.

Figure 1. Schematic of the PAT1 tensiometer with a double dosing system and a coaxial double capillary (a); schematic of a bulk exchange with a protein drop, subsequent washing off the bulk with pure buffer and then injection of a surfactant solution (b). In Scheme 1 we show the timeline of mixed protein/surfactant adsorption layers formed in a sequential adsorption experiment. First, a droplet is formed via the outer capillary with a pure protein solution (in buffer at a fixed pH), which documents the adsorption kinetics up to the equilibrium reached in the plateau region (stage-I). 158 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


At a sufficiently high protein concentration the interface is completely covered by adsorbed protein molecules. Once the proteins reach the interface, the hydrophobic parts are directed towards the air or oil phase, while hydrophilic parts remain in the aqueous bulk phase. Depending on the available time and space, this can lead to an unfolding of the molecular structure and consequently, increases the free energy of adsorption due to the larger attachment of proteins at the interface (18). During the subsequent first bulk exchange experiment with the pure buffer solution the protein molecules are washed off from the drop bulk, while keeping the drop volume and hence the drop surface area constant (stage-II). As we can see, there is no remarkable desorption of protein molecules from the interface during this exchange, which is confirmed by an almost negligible increase in surface tension.

Scheme 1. Experimental protocol for mixed βCS/SDS layers formed via sequential adsorption made with a coaxial double capillary to measure dynamic surface tensions: I – pre-adsorption of βCS until equilibrium, II – bulk exchange against a pure buffer solution, i.e. replacement of proteins from the drop bulk, III – bulk exchange with SDS solution, i.e. modification of the surface structure by forming protein/surfactant complexes at the interfaces, IV – final bulk exchange against a buffer solution (washing out). 1,2,3,4 – Drop oscillations as a possible additional test of the surface layer composition (not discussed here)

The result of this exchange is a drop with a protein covered surface but containing no protein molecule in the drop bulk. In the second bulk exchange process, now we inject the surfactant solution into the drop through the inner capillary, replacing the buffer solution (stage-III). The protein/surfactant complexes are formed at the interface, which leads to modification of the pre-adsorbed protein layer. A decrease in surface tension is observed during 159 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


this bulk exchange process; the absolute values of the new surface tension plateaus depend on the surfactant concentration of the injected solution. The final bulk exchange (washing out experiment) is performed again with a pure buffer solution (stage-IV). This washing-off removes any molecules from the drop bulk. We can also assume that all the surfactant molecules, freely adsorbed and bound to the protein molecules at the interface, are removed provided sufficient time is available to reach the respective local equilibrium for the surfactants. However, the protein molecules still in the adsorbed state after the sequential adsorption stay in the interfacial layer even after this washing off experiments and hence, it provides a qualitative estimation of the remaining amount of proteins. This experimental approach cannot be applied to systems where the complexes precipitate. This will affect the drop stability and hence the results. For the case of simultaneous adsorption, the experimental protocol is shown in Scheme 2. The mixed solution of protein and surfactant forms a droplet at the outer capillary and the protein/surfactant complexes formed already in the bulk adsorb with free surfactant molecules in a competitive manner at the drop surface (stage-I). After this competitive adsorption, when the adsorption kinetics reached equilibrium, a washing off experiment is performed against a pure buffer solution (stage-II) to understand the replacement of adsorbed complexes. Note, the low frequency harmonic oscillations of the drop surface area are performed after each stage of the experiments to obtain the dilational viscoelastic properties of the adsorption layers as a qualitative measure of the surface composition.

Scheme 2. Experimental protocol for simultaneously formed mixed βCS/SDS adsorption layers performed with a coaxial double capillary to measure dynamic surface tensions: I – adsorption from a mixed solution of βCS and SDS until equilibrium, II – bulk exchange with a pure buffer solution (washing off). 1,2 – periodic drop oscillations (not further analyzed here) 160 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

4. Fluid Dynamics of the Drop Exchange


As discussed above, the bulk exchange via the double capillary is a new experimental tool. However, a verification of its efficiency is required in order to know if the drop bulk is really perfectly mixed. Then, the simplest description of the evolution of the bulk concentration is given by (10):

Here = VD/RE is the residence time of the liquid in the drop, C1,∞ is the concentration of bulk at the beginning and C2,∞ is the concentration of the species at the final stage (i.e. C1,∞ = 0).

Experimental studies via the injection of a suitable dye solution and the related computational fluid dynamic (CFD) simulation results show that the mentioned assumptions are not a priori true (11). For a high injection flow rate of a liquid with a density higher than the droplet bulk this assumption is acceptable after a certain exchange time, e.g. t= 4 s, see Figure 2. However, for low injection rate conditions the inhomogeneous exchange process shown in Figure 3 is very far from a complete mixing. Another important issue is that during the exchange process the quality of drop profile has to be very high as it is used for determining the surface tension.

Figure 2. Dye exchange experimental snapshots (top) and CFD simulation of dye concentration contours (bottom) for high injection rate conditions dt=0.1 s and dV=0.1 mm3 (11). 161 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2 shows that for high injection rates, after starting the exchange process, a significant drop shaking and instability due to hydrodynamic and vibration effects appear. Hence, there are difficulties for the online surface tension monitoring. These observed instabilities can also cause strong disturbances of the adsorbed layer and must therefore be avoided. For any inhomogeneous exchange conditions a surfactant-rich region could come close to the drop surface and then creates a Marangoni convection (see for example Figure 4, at about t=25 s and 30 s). Marangoni convection can enhance the distribution of surface active components in the drop and also boosts mixing in the bulk (11, 16).

Figure 3. Dye exchange experimental snapshots (top) and CFD simulation of dye concentration contours (bottom) for low injection rate conditions dt=0.33 s and dV=0.033 mm3 (10).

For the injected solution with a density lower than that in the drop bulk, for example just due to a difference in concentration of the solute or temperature, the exchange process can be influenced significantly (Figure 5). The injected flow may return back to the outer capillary without reaching drop center. The easiest ways to overcome this problem is to increase the injection rate within the suitable limits, or protrusion of the inner capillary tip deeper into the droplet center. In Figure 5 we see these two options for improving the drop bulk exchange. 162 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 4. Surface tension measurements during drop exchange (starting around t=10 s) for high (♦ dt=0.1 s, dV=0.1 mm3) and low (● dt=0.33 s, dV=0.033 mm3) injection rates, Marangoni onset appears at about 25 and 30 s, respectively.

Figure 5. Dye exchange experimental snapshots (top) for dye solution 0.3 mg/ml + 1.5% Ethanol (density slightly lower than water), for dt=0.1s and dV=0.1 mm3 (top row) and for dt=0.1 s, dV=0.2 mm3 (central row), and for dt=0.1 s, dV=0.1 mm3 with deep immersed inner capillary (bottom row). 163 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


For the current setup in absence of surface activity effects and thus no Marangoni convection, the evolution of drop surface concentration (Cs) respect to the inlet dye concentration (Cinlet) are predicted via CFD simulation of the corresponding Navier Stokes equations presented in Figure 6. This figure shows the effect of increasing injection rate (dV) on the normalized drop surface concentration (Cs/Cinlet) at constant injection interval time dt. For the studied case and conditions (dt=0.1 s), the medium injection rate 0.067 mm3 looks optimal, as the lower injection rate needs a very long time for getting close to the complete exchange and the higher injection rates cause drop shaking problem.

Figure 6. Normalized drop surface concentration (Cs) respect to the inlet concentration (Cinlet) during drop water exchange by a dye solution 0.3 mg/ml for dt=0.1 s and different injection rates dV=0.033 (1), 0.067 (2) and 0.1 mm3/pulse (3), respectively.

5. Adsorption Isotherm of Frequently Studied Proteins The surface behaviour of an aqueous protein solution depends on the native characteristics of the protein macromolecule such as chemical and spatial structure, molecular weight and net charge. Figure 7 shows experimental surface pressure isotherms, obtained by drop/bubble profile analysis tensiometry, for the globular proteins β-lactoglobulin (BLG), lysozyme, bovin serum albumin (BSA) and human serum albumin (HSA) and the random coil protein β-casein (BCS). For the different proteins, the onset of measurable π-values varies within a wide concentration region and the shapes of the π-c curves are quite different. The runs for BSA, HSA and BCS virtually follow the same trend of an initial steep increase and subsequent formation of a plateau region, while those for BLG and lysozyme 164 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


show continuous increase of the surface pressure with increasing concentration in the studied region. It is evident from Figure 7 that BSA and HSA behave rather similarly since the molecular structure of these albumins is analogous, i.e. their amino acids sequences coincide by about 80%. Comparison between the adsorption kinetics data for BSA, BCS, HSA and BLG obtained with and without forced convection in the solution sub-phase leads to the conclusion that the adsorption of proteins at the water/air interface is thermodynamically reversible, in contrast to the kinetic irreversibility of the process as discussed in (17, 18). In contrast to the pure protein system, the addition of a low molecular weight surfactant to the solution provokes replacement of the protein from the surface (19, 20). The mechanism of this process comprises both modification of the surface activity of the protein due to formation of less adsorbing complexes and competitive adsorption by the surfactant molecules, and is discussed in details below.

Figure 7. Surface pressure vs. protein bulk concentration plots for BLG, redrawn from (16), Lysozyme, redrawn from (22), BCS, new data, BSA, redrawn from (23), HSA, redrawn from (24). The interfacial properties of proteins are strongly affected by the type of the hydrophobic fluid (gas or oil) which is in contact with the solution. Figure 8 shows experimental surface pressure isotherms, again obtained by the most reliable drop/bubble profile analysis tensiometry, for BLG and BCS at water/air (w/a) and water/hexane (w/h) interface. One clearly distinguishes between the curves 165 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


for the different interfaces as those for w/h interface are shifted to lower protein concentrations. As discussed in the literature, e.g. in (16, 21), such shift of the adsorption isotherm is related to the stronger affinity of the hydrophobic residues of the protein for the oil phase. Theoretical processing of the experimental data for BLG by the model mentioned above shows smaller ω-values at the water/hexane interface, which indicates a more compact layer of the protein that has a different conformation in contact with the oil phase (16, 18). As discussed below, the elucidation of the interfacial properties of protein/surfactant solutions are based on these findings.

Figure 8. Surface pressure vs. protein bulk concentration plots for BLG, redrawn from (16, 18) and BCS, new data, at water/air and water/hexane interface.

6. Exchange Dynamics at Water/Air and Water/Hexane Interfaces In order to analyze under which condition a pre-adsorbed protein can be more or less displaced from a liquid interface by competing surfactant molecules, we followed the two strategies detailed above, i.e. injected surfactant solutions of various concentration and then replaced this solution with a pure buffer solution in order to probe how much protein is left at the surface after the final washing. 166 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 9 shows the adsorption dynamics curves for mixed βCS/SDS layers formed after the second bulk exchange with different SDS concentration at W/A and W/H interfaces.

Figure 9. Surface tension dynamics during the drop exchange against different [SDS] obtained for sequential adsorption experiments at W/A (A) and W/H (B) interfaces; the concentration of βCS is kept constant to 10-6 mol/dm3.

One can see in both cases, the higher the SDS concentration, the faster is the surface tension decrease and the lower is the final surface tension plateau. It suggests that mixed layers are formed by the interaction between protein and surfactant only at the interface and at higher surfactant concentration an increasing amount of protein molecules is displaced from the surface layer by the surfactant molecules due to stronger competition, i.e., the surface layer is dominated by the adsorbed surfactant molecules. In this case the situation is that a protein/surfactant complex in the bulk of the droplet and the interface has free adsorbed surfactant molecules in addition to protein/surfactant complexes combined to different extents. Note, the dynamic curves for both interfaces W/A (Figure 9A) and W/H (Figure 9B) certainly show two humps before the tension values reach a plateau, which become more pronounced at higher SDS concentration. This is probably due to the convection pattern inside the drop, i.e. the flow field of the inflowing liquid does not involve all the liquid inside the drop immediately, as we can understand from the discussion of the hydrodynamics above. A quantitative understanding still requires further investigations. 167 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 10. Surface tension dynamics during final drop exchange (washing out) experiments measured after sequential adsorption from an initial βCS solution and subsequently from SDS solutions of different concentrations at W/A (A) and W/H (B) interfaces; the concentration of βCS is kept constant to 10-6 mol/dm3.

The curves shown in Figure 10 represent the corresponding final bulk exchange (washing out experiments) of the previously injected SDS of different concentrations against a pure buffer solution. In contrast to protein, only surfactants molecules are reversibly adsorbed and thus desorb completely as described in a recent report (9). This means that within the experimental time all protein molecules which had not been displaced from the drop surface by the surfactants further stay in the surface layer after this washing out experiment due to their high free energy of adsorption (18). One can see for both W/A (Figure 10A) and W/H (Figure 10B) interfaces, at lower SDS concentrations there are no significant changes in the final surface tension plateaus with increasing surfactant concentrations and the absolute values are close to that of pure βCS solution. The addition of the anionic surfactant SDS to the pre-adsorbed proteins leads to a complex formation first via strong electrostatic interaction resulting in an increased surface activity of the complexes and hence, they cannot be displaced from the surface layer by any co-adsorbing free surfactant molecules. Any hydrophobic interaction is of secondary importance in the complex formation at these low surfactant concentrations. Consequently, almost all the adsorbed protein molecules are left in the surface layer after washing off. At higher surfactant 168 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


concentrations, however, the hydrophobic binding of SDS becomes increasingly significant. As a consequence the complexes become hydrophilized once all the charge sites of the proteins have been compensated by the electrostatically bound SDS. The resulting complexes become less surface active and can easily desorb into the solution bulk, leading to a decreasing presence of remaining proteins after washing off. This results in a higher surface tension plateau after the washing out experiment. The results presented in Figure 11 show the desorption dynamics of mixed surface layers formed after simultaneous adsorption (the dynamic adsorption curves are not shown here) at different SDS concentrations. Similar to the sequential adsorption experiments, the desorption of SDS molecules from the mixed surface layer results in an almost negligible change in the surface tension plateaus at low surfactant concentrations for both W/A (Figure 11A) and W/H (Figure 11B) interfaces and the absolute values increase with increasing SDS concentration at higher concentrations. At low surfactant concentration, the surface structure does not depend on the location where the proteins and surfactant interact and neither the conformation of the protein.

Figure 11. Surface tension dynamics during drop-bulk exchange experiments against pure buffer solution measured after simultaneous adsorption from a mixed solution of βCS/SDS for different used [SDS] at W/A (A) and W/H (B) interfaces; the concentration of βCS is kept constant to 10-6 mol/dm3. 169 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Once the complexes formed in the bulk reach the interface, they adopt a conformation that the surface activity of the adsorbed complexes are almost identical to the complexes only formed in the surface layer. However, at higher SDS concentration, the dynamics of desorption from the surface layers formed from mixed solutions deviate significantly, which can be explained only by the different composition and structure of the adsorbed complexes. This can be better understood in the isotherms shown in Figure 12, representing the state of remaining proteins after the final washing off. For both W/A and W/H interfaces, the exchange dynamics obtained from sequential and simultaneous adsorption experiments follow the similar trend. However, there are some definite differences between the behaviors of such mixed adsorption layers formed at the W/A interface and those at the W/H interface, i.e., dependency on used surfactant concentrations and the composition of the mixed adsorption layers must be different. Indeed, the complexes formed at the W/H interface are more stable obviously due to different conformation and consequently, deep penetration of the hydrophobic segments of the protein molecules into the organic subphase (24, 25). It suggests that the amount of hydrophobically bound SDS complexes should be lower at the W/H interface. Compared to the W/A interface, the behavior of the mixed layers at the W/H interface is governed by the properties of the complexation rather than by the amount adsorbed competing with the free surfactants.

Figure 12. Isotherms for washing off experiments after sequential (5,4) and simultaneous (□,○) adsorption at W/A (A) and W/H (B) interfaces. 170 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


7. Comparison of the Two Routes at Water/Air and Water/Hexane Interfaces The mixed protein/surfactant layers formed via two different routes show different equilibrium surface properties, which can be caused obviously only by the location where the proteins and surfactants meet each other – in the bulk as it occurs in the simultaneous adsorption or in the surface layer during sequential adsorption. In order to compare the results, the isotherms shown in Figure 13 have been constructed from the equilibrium surface tension values for the mixed βCS/SDS layers formed via sequential and simultaneous adsorption experiments after stage-III and stage-I, respectively, and illustrated along with the isotherms of pure SDS. The patterns of the isotherms for the two different interfaces are similar.

Figure 13. Adsorption isotherms of pure SDS at W/A (black ■) and W/H (red ●) interfaces, and its mixed layers with βCS built-up via sequential (5,4) and simultaneous (□,○) adsorption at W/A (5,□) and W/H (4,○) interfaces. The symbol a, b, c and d are used to show the region-wise comparison of two different isotherms; the horizontal lines indicate the equilibrium surface tension values of 10-6 mol/dm3 βCS solution at W/A (black solid) and W/H (red dash) interfaces. 171 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The tensiometric profiles evidence formation of a mixed layer (Figure 13, open symbols). All start from a lower surface tension value, corresponding to the value of the βCS solutions in absence of SDS (horizontal lines). It has been found that there are surface tension maxima in the simultaneous adsorption isotherms in the region ‘bcd’ in Figure 13. In contrast, the adsorption isotherms obtained for the sequential adsorption route do not show such maxima. At this stage of interaction the complexes formed in the bulk aggregate and form larger structures and precipitate. This corresponds to a partial depletion of adsorbing species and hence to an increase in the measured tensions. In contrast, in sequential adsorption, an aggregation of the complexes formed only at the interface is not feasible obviously due to different conformation of the pre-adsorbed protein molecules. There is actually no excess of more and more hydrophobic complexes available to form such superstructured aggregates. This remarkable difference is clear evidence that the way how mixed protein/surfactant layers are formed has a decisive role on their equilibrium surface properties. A quantitative analysis is not possible here as the respective theoretical models do not exist. Therefore, modification of the available theoretical models (18, 24, 26, 27) is needed in order to quantify the characteristic features of the mixed layer owing to phenomenological differences in the two different ways of their formation. Despite this remarkable difference, the simultaneously and sequentially formed mixed layers of βCS and the non-ionic surfactant C12DMPO (dodecyl dimethyl phosphine oxide) showed similar equilibrium surface properties, as it was observed by Kotsmar et al. in a recent study (3). The similarity in the behavior is attributed to the type of surfactant used (non-ionic surfactant in pH 7), i.e. the interaction is only of hydrophobic nature and happens between the hydrophobic groups in the protein and surfactant molecules. In contrast, for the anionic surfactant SDS and βCS, the complex formation is considered to be driven first by electrostatic and only then mainly by hydrophobic interactions. Thus, one can suggest that the behavior of the resulting protein/surfactant complexes differ in their structure and composition. In Figure 12, the isotherms for washing off after sequential and simultaneous adsorption at the two interfaces are illustrated. The results shown here are corresponding only to higher SDS concentration, as there is no significant difference in the isotherms for the two different adsorption routes. A steep increase of the surface tension values suggests an increasing amount of displaced proteins from the surface layer by surfactant molecules. At the W/A interface, after washing off in the sequential adsorption experiment, the surface tension reaches much higher values in comparison to those obtained in the simultaneous adsorption case (Figure 5A). The accessibility of the hydrophobic parts of the pre-adsorbed proteins is more favorable by the surfactants to form less surface active complexes, which can be easily displaced from the surface layer by the surfactant molecules. This leads to a decreasing presence of the protein/surfactant complexes at the interface after sequential adsorption. In the other case, an increased number of surfactants are consumed in the formation of associated complexes in the bulk solution that restricts the adsorption of free surfactant molecules at the interface. The amount of freely adsorbed surfactants is essentially 172 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


less and, consequently, the surface is covered by an increased amount of adsorbed complexes after simultaneous adsorption. At the W/H interface, the picture is totally opposite, i.e., in simultaneous adsorption after washing off, the surface tension increases much earlier than in a sequential adsorption (Figure 12B). As discussed before, the complexes formed at the W/H interface are more stable and cannot desorb easily into the bulk. This suggests an increasing presence of the adsorbed complexes at the interface after sequential adsorption. In contrast, in simultaneous adsorption, the surfactants adsorb strongly with high adsorption energy at the W/H interface and consequently, an increasing amount of complexes desorb from the surface layer after simultaneous adsorption.

8. Conclusions and Outlook As was shown above, there are significant differences in the interfacial behavior of mixed protein/surfactant complexes adsorbed at the water/air and water/hexane interfaces. Moreover, the location at which these complexes are formed is essential for their properties in the interfacial layer. For mixtures of β-CS with different surfactants we have demonstrated that there are two main types of interaction, a hydrophobic and for ionic surfactants also an electrostatic interaction. The cartoon (cf. Scheme 3) shows schematically that the surface activity of the formed complexes decreases gradually with the amount of added surfactant. This leads to a progressive replacement of protein molecules from the interface. This mechanism was confirmed by the experimental findings shown in Figs. 9 to 13. For mixtures of β-CS and ionic surfactants we postulated a primary electrostatic interaction, resulting in an increased surface activity of the complex. Further addition of surfactant molecules leads to a subsequent hydrophobic interaction with the complexes. As a consequence, the charge of the complexes is reversed and the surface activity decreases step by step until their complete replacement from the interface by free surfactants. This is schematically shown in Scheme 4. Note, the mentioned mechanisms are valid for both discussed interfaces. When we compare the amount of protein replaced from the interface by surfactants, we can see that due to the interaction with the molecules of the oil phase, the proteins are stronger adsorbed at the water/oil interface and, therefore, a larger amount remains there. Hence, the conformational changes of the proteins, caused by a direct interaction between surfactants and proteins in the solution bulk, as it is the case for simultaneous adsorption processes are such, that the complexes cannot be anchored at the water/oil interface as strongly as it is possible for the protein alone.

173 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Scheme 3. Illustration of protein displacement from the surface layer by the surfactant molecules in sequential adsorption: A – adsorption of pure protein, B – penetration of surfactant molecules into the preformed protein layer, C – more and more surfactants binding to the protein backbone, D – denaturation of protein from the interface; redrawn from (13)



Scheme 4. Protein/surfactant complex formation in the solution bulk and their competitive adsorption with the free surfactant molecules at the interface: A – binding of SDS by initial electrostatic and subsequent hydrophobic interaction, B – change in conformation of βCS to accommodate more surfactant molecules, C – self-association of the complexes; redrawn from (13)



In sequential adsorption, one of the possible mechanisms for the displacement of protein from the surface layer was proposed in the light of ‘orogenic displacement’ (28–32) and it occurs in several steps as depicted in Scheme 3A-D. The proteins first adsorb at the interface by changing its conformation and tightly attach to the surface (Scheme 3A). Then, surfactant molecules gradually penetrate into the pre-adsorbed protein layer, adsorb and modify the surface structure by forming protein/surfactant complexes at the interface (Scheme 3B-C). The denaturation of the protein molecules continues until the protein network loses its rigidity and is able to desorb into the droplet bulk (Scheme 3D). However, again the surfactant would first have to hydrophilize the patches formed after a break-off of a preformed protein layer network to make them wetted by the aqueous solution. Thus, the protein/surfactant mixed layer formation must be at least driven by the interplay between an orogenic mechanism and hydrophilization of the protein layer (patches) with the surfactants. In contrast, for simultaneous adsorption, this mechanism cannot be applied, as the adsorbing species are the protein/surfactant complexes and available free surfactant molecules, which simply adsorb in a competitive manner. Future work will have to focus on a quantitative understanding of the described differences in the adsorption behavior for different interfaces. We can expect that also the polarity of the oil will have a significant influence, however, there are only results for pure protein solutions available in literature so far. Moreover, the pH of the solution influences strongly the number of net charges in the protein, hence, the surface activity and also the amount of ionic molecules bound to each protein molecules. These peculiarities will have to be reflected by future theoretical models as well. As it was addressed recently (33), a single set of model parameters should be able to describe all interfacial properties in an acceptable quality, including the adsorption kinetics, the equilibrium state and the dilational visco-elasticity. Although the shear visco-elasticity cannot be linked quantitatively to the adsorption layer properties, it has at least to be in a qualitative agreement with the picture obtained by the other experimental techniques. A throughout understanding of mixed protein/surfactant adsorption layers at liquid interfaces is yet pending.

Acknowledgments The work was financially supported by projects of the DFG (Mi418/20-1) and the European Space Agency (PASTA).

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