DoTAB Layers

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Adsorption and Dilational Rheology of Mixed β‑Casein/DoTAB Layers Formed by Sequential and Simultaneous Adsorption at the Water/ Hexane Interface Abhijit Dan,†,* Rainer Wüstneck,† Jürgen Krag̈ el,† Eugene V. Aksenenko,‡ Valentin B. Fainerman,§ and Reinhard Miller† †

Max-Planck Institute of Colloids & Interfaces, Potsdam/Golm, Germany Institute of Colloid Chemistry and Chemistry of Water, 03680 Kiev, Ukraine § Donetsk Medical University, 16 Ilych Avenue, Donetsk 83003, Ukraine ‡

S Supporting Information *

ABSTRACT: The interfacial behavior of β-casein (βCS) has been investigated in presence of the cationic surfactant dodecyl trimethyl ammonium bromide (DoTAB) at the water/hexane interface and compared to that obtained for the water/air interface. The used experimental technique is a drop profile analysis tensiometer specially equipped with a coaxial double capillary, which allows investigation of sequential adsorption of individual components besides the traditional simultaneous adsorption of two species. This method also provides the dilational rheological measurements based on low frequency harmonic drop oscillations. The tensiometric results show that the equilibrium states of the mixed βCS/DoTAB layers built up on the two different routes do not differ significantly, that is, the general compositions of the mixed layers are similar. However, the results of dilational rheology for the two adsorption strategies are remarkably different indicating different dynamic characteristics of the adsorbed layers. These findings suggest that the respective mixed layers are more proteinlike if they are formed via sequential adsorption and more surfactant-like after simultaneous adsorption. In contrast to the W/A interface, at the W/H interface proteins remain at the interface once adsorbed and cannot be displaced just by competitive adsorption of surfactants.

1. INTRODUCTION Protein/surfactant mixed systems have attracted much interest due to their frequent occurrence in many areas, such as pharmaceutics, cosmetics and food processing.1−5 Beside the complex structures and biological functions of proteins, a variety of new issues arising from the interaction with surfactants have to be considered to understand the particular behavior in various applications. Proteins can interact with the surfactant molecules in the bulk and at the interface in different ways, which results in complexes of different surface activity leading to different adsorption dynamics and surface rheological properties.6,7 Studies of interfacial properties of mixed protein/ surfactant adsorbed layers are useful for understanding their effect on the emulsion or foam stability.1,8−10 Increasing the amount of surfactant in the mixtures affects the complexation process and adsorption layer composition, as free surfactant molecules can gradually displace the protein molecules from the surface layers due to a competitive adsorption mechanism10−15 or a more complex one called orogenic displacement.16−20 The behavior of mixed adsorbed layers at the water/ oil interface obviously differs significantly from that at the water/air interface due to their specific interaction between the © 2013 American Chemical Society

hydrophobic segments of adsorbed molecules and the organic solvent.21−23 Indeed, different orientation, molecular areas, or unfolding of proteins have to be taken into account at the water/oil interface as it has been discussed for example by Chipot et al.24 The description of the interfacial phenomena at the water/oil interface involving these compounds is quite complex and still not fully understood. Mixed protein/surfactant layers can be built up via two different experimental strategies − sequential adsorption and simultaneous adsorption. The method of formation can significantly influence the equilibrium properties of the surface layer. A sequential adsorption is possible using a special setup consisting of a coaxial capillary driven by a double dosing system, as it was first proposed by Wege et al.25,26 This arrangement allows for in situ internal subphase exchange in a single pendent drop. This is a unique protocol for studies of mixed surface layers formed by a subsequent adsorption of the protein followed by surfactant. The double capillary idea was Received: November 23, 2012 Revised: January 23, 2013 Published: January 24, 2013 2233

dx.doi.org/10.1021/la304664x | Langmuir 2013, 29, 2233−2241

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Scheme 1. Timeline and Probable Equilibrium States for Sequential and Simultaneous Experiments Performed with a Coaxial Double Capillary to Measure the Dynamic Surface Tension for the Mixed βCS/DoTAB Layer

layers. For example βCS/C12DMPO (dodecyl dimethyl phosphine oxide)54 and βCS/SDS (sodium dodecyl sulfate)55 have been investigated in the recent years focusing on the difference of the interfacial behavior of the mixed adsorbed layers resulting from two different pathways, that is, simultaneous adsorption from mixed solution and sequential adsorption of the individual components. Hence, DoTAB was chosen in the present study as a cationic surfactant with the same alkyl chain length like SDS and C12DMPO. Note, so far only the adsorption layer formation process for mixed βCS/ SDS layers formed in two different routes has been investigated at the water/oil interface.55 The evaluation of the observed effects of these different pathways of mixed layer formation gives additional insight in the knowledge of protein/surfactant systems, which is a topic of general concern, and, therefore, we want to further explore this topic. In this present article, we have investigated the interfacial properties of mixed βCS/DoTAB adsorption layers built up in two different pathways at the W/H interface and discuss the differences to those of the W/A interface. In particular, the illustration has been made for the consecutive stages of the adsorption of the protein/surfactant complex describing in detail the peculiarities of the simultaneous and sequential adsorption. The measurements performed are dynamic interfacial tension as well as dilational rheology, obtained using drop profile analysis tensiometer specially equipped with a coaxial double capillary. Complementary measurements of surface dilational rheology are added to correlate the proposed mechanisms drawn from tensiometry data.

practiced in several experimental approaches, such as for desorption studies,27 penetration experiments,28 washing out studies,29 or even for multilayer formation.30 It should be noted that a sequential adsorption of different components does not lead to an equilibrium state of the mixed adsorbed layer within a realistic time scale due to the high activation energy of the required protein desorption process.10 β-Casein (βCS) is one of the most frequently used proteins,31 the adsorption dynamics, thermodynamics and rheological properties of which have been extensively investigated.32−34 βCS contains relatively hydrophobic molecular parts, which consist of a high number of not interacting proline peptides, and it does not contain disulfide bridges. As a result, compared with typical globular proteins, βCS is classified as an intrinsically unstructured (random coil) protein35 with no characteristic denaturation temperature, although the unfolded state of βCS is less chaotic and randomlike than widely believed. βCS behaves like a block-copolymer and has a strong tendency of self-assembling into a micelle-like structure.36−38 The molecular rearrangement of the protein during adsorption was described recently33 and its structural analysis was carried out with numerous methods and illustrated by various theoretical models, however, mainly at the water/air interface.39−44 The hydrophobic/hydrophilic distribution of the residues in the peptide sequences rather than the length of the peptides seem to be relevant to the adsorption of βCS at the water/oil interface.45 The interfacial properties of the cationic surfactant DoTAB was also the target of many investigations using different methods both at water/air7,46−48 and water/oil49 interfaces. The dynamics of adsorption of βCS in presence of different surfactants have been studied frequently during the past decade, however, mostly via traditional simultaneous adsorption.23,50−53 There are limited studies on the sequentially formed mixed

2. EXPERIMENTAL SECTION The milk protein βCS (minimum 90% pure) with a molecular weight of 24 kDa and an isoelectric pH 5.2 was obtained from Sigma-Aldrich Chemical Co. (Germany) and used without further purification. 2234


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DoTAB (MW = 308.35 g/mol) was purchased from Fluka (Switzerland) and used in the concentration range between 10−6 mol/dm3 to 10−2 mol/dm3. All solutions were prepared in 10 mM phosphate buffer (pH 7, Na2HPO4/NaH2PO4 purchased from Fluka, assay >99%) using Ultrapure Milli-Q water with a resistivity of 18.2 MΩ. Hexane was purchased from Fluka (Switzerland); it was distilled and purified with aluminum oxide. Hexane was chosen because it is a largely unreactive nonpolar solvent (boiling point ≈ 70 °C), and its purification is very simple. The interfacial tension of water and Na2HPO4/NaH2PO4 buffer were 72.5 and 49 mM/m at W/A and W/ H interfaces, respectively. In the βCS/DoTAB mixtures, the concentration of βCS was kept constant at 10−6 mol/dm3. All measurements were performed at room temperature of 22 °C. For the simultaneous adsorption, the respective protein and surfactant solutions were mixed and 30 min allowed for protein/surfactant complex formation before the measurement. The drop profile analysis tensiometer PAT-1, equipped with a special double dosing system (SINTERFACE Technology, Berlin, Germany)25,26,54,55 was used to determine the interfacial tension and dilational rheological parameters of the surface layers. The general setup and the measuring principles were described elsewhere.56 The rheological parameters were calculated via Fourier transform from the interfacial tension variation as a response to the generated harmonic area oscillations of the drop surface at low frequencies of 0.005, 0.01, 0.02, 0.04, and 0.1 Hz. A used setup consisting of two syringes (50 μL syringe from ILS, Germany) connected to a coaxial double capillary provides the possibility of an exchange of the drop volume without disturbing its surface layer during the experiments. The operation of drop exchange is based on a simple master−slave principle: one syringe, connected to the inner capillary pumps small quantities of liquid into the drop, whereas the second syringe, connected to the outer capillary, controls a constant drop size, that is, sucks excess liquid out of the drop. Thus, new liquid comes into the drop through the inner glass capillary of 1 mm diameter and simultaneously, and the excess solution leaves the drop through the outer PEEK capillary of 2 mm diameter. This process also creates some convection/mixing inside the drop. Thus, this instrumental arrangement allows performing adsorption experiments of different components sequentially one after another at the same interface.

(stage-I). Once the protein reaches the interface, it adopts a conformation that the hydrophobic parts expose toward the air or oil phase, whereas the hydrophilic parts are attracted inward to the aqueous bulk phase. This leads to an unfolding of the protein molecules, which strengthens their attachment at the interface and consequently increases their adsorption energy.29 The proteins in the drop bulk are washed out by the first bulk exchange experiment against pure buffer solution, whereas keeping the drop volume constant (stage-II). This subphase exchange does not affect the adsorbed protein at the interface significantly, which is confirmed by an almost negligible increase in surface tension. The result of this exchange is a drop covered by a protein layer consisting of no protein molecules in the drop volume. The second bulk exchange is performed against a surfactant solution at different concentrations replacing the pure buffer solution from the drop bulk (stage-III). Protein/surfactant complexes are formed at the interface and this leads to a modified surface structure. The higher the surfactant concentration is, the lower is the new surface tension plateau for the mixed adsorption layer. The final exchange of the drop bulk is made again with a pure buffer solution, which replaces any molecules from the solution bulk. Also, the freely adsorbed surfactant molecules and those bound to the protein are removed from the drop surface and bulk (stage-IV). In contrast to proteins, we can assume that surfactant molecules adsorb reversibly and desorbed completely as reported in a recent study.27 For simultaneous adsorption, a drop of the mixed protein/ surfactant solution is formed with the outer capillary and kept constant until an equilibrium state is reached (stage-I). In this case, the complexes already formed in the bulk adsorb simultaneously with free surfactant molecules in a competitive manner. A washing off experiment is performed with pure buffer solution to understand if the location of interaction has an impact on the nature and structure of the adsorbed layers (stage-II). Note, after each stage for both experimental protocols, low frequency drop oscillations are performed in order to determine the dilational rheological parameters as a possible additional information on the surface layer composition. Figure 1 shows the dynamic surface tension obtained by the second bulk exchange with different DoTAB concentrations for sequential adsorption at the W/H interface. A decrease in the final surface tension plateau is observed with increasing surfactant concentration indicating an increasing number of complexes and/or surfactant molecules in the surface layer. Similar to the observed results for βCS/SDS mixed systems,55 the dynamic curves show some humps before reaching the plateau, which become more pronounced at higher DoTAB concentrations. The appearance of these humps is probably an experimental artifact and caused by the inflow pattern of injected surfactant or pure buffer solution. Once the liquid of different concentration reaches directly the drop surface, a temporary surface tension gradient and consequently a Marangoni flow may set in and leads to these unexpected changes in the total surface tension. A more accurate analysis requires further investigation, for which additional experiments as well as CFD simulations are under way. The curves presented in Figure 2 show the corresponding final bulk exchange (washing out) of the previously injected DoTAB molecules by a pure buffer solution. Figure 3 represents the kinetics of desorption measured by the dynamic surface tension after simultaneous adsorption, whereas the corresponding

4. RESULTS AND DISCUSSION Interfacial Adsorption. The adsorption behavior of the random coil protein βCS at the W/H interface is very different as compared to the W/A interface due to their different interfacial conformations that allows parts of the molecule to penetrate deeper into the oil phase leading consequently to a thicker adsorption layer. For the adsorption of DoTAB at W/A interface, a smaller molar area is required by the DoTAB molecules when compared to SDS.23,46 At the W/H interface, there is an additional dispersive interaction between the hexane molecules and the hydrocarbon chains of DoTAB molecules, which results in the surrounding of each adsorbed surfactant molecule by hexane molecules. This increases the distance between the adsorbed DoTAB molecules in comparison to that at W/A interface. Kotsmar et al.46 found a larger negative value of the interaction constant (as) for DoTAB molecules at the W/H interface. Mixed protein/surfactant layers were studied via two different experimental approaches − sequential adsorption and simultaneous adsorption. Scheme 1 shows the two timelines of sequential and simultaneous adsorption experimental protocols. A detailed description of the sequential adsorption protocol consisting of four different stages has been discussed recently.54,55,57 In brief, a droplet of the 10−6 mol/ dm3 βCS solution is formed with the outer capillary and the surface tension registered until the equilibrium state is reached 2235


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Figure 3. Dynamic surface tension measured during final drop-bulk exchange process (washing out) after simultaneous adsorption from a mixed solution of βCS/DoTAB with varying [DoTAB] at W/H interface; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

Figure 1. Dynamic surface tension measured in sequential adsorption experiments during the drop-bulk exchange against a DoTAB solution of different concentration at W/H interface; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

observed difference is certainly the result of the different applied experimental protocols. It should be taken in mind that convection due to the bulk exchange process supports the adsorption or desorption rate. For sequential adsorption experiments, after the second bulk exchange process, the inner capillary tube is filled with the surfactant solution, however, during the third bulk exchange it is filled with pure buffer solution. Hence, mixing of surfactant and buffer solutions inside the capillary tube a dilute surfactant solution comes into the droplet through the inner capillary and takes part in the convection process. As the exchange process proceeds, the incoming solution becomes more and more diluted and, at the end, the exchange process is continued with the pure buffer solution. As a whole, the liquid flow through the inner capillary during the third bulk exchange corresponds to a slow washing process that could delay a complete bulk exchange during a respective period of time. However, for simultaneous adsorption experiments the bulk exchange starts with pure buffer solution and the process ends up much earlier in comparison to that in the sequential adsorption strategy. In Figure 4, the equilibrium surface tension profiles of sequentially and simultaneously formed βCS/DoTAB interfacial layers as functions of the DoTAB concentration after stage-III and stage-I respectively are presented together with the isotherm of pure DoTAB. One can see that there is no significant difference in the isotherms for the respective mixed layers builds up in two different ways. The isotherms start from lower surface tension values when compared to the pure DoTAB isotherm as expected due to the surface activity of βCS (horizontal lines). The decrease of interfacial tension with increasing DoTAB concentration suggests the formation of mixed layers in which the surfactant and protein molecules are adsorbed together at the interface, partly in form of complexes. Whereas the isotherms show a quite similar pattern at both the W/A (part a of Figure 4) and W/H (part b of Figure 4) interfaces, at the W/A interface they meet with the pure DoTAB isotherm fairly early before reaching its CMC. This suggests that βCS is at least partly displaced from the W/A interface already before reaching the CMC.

Figure 2. Dynamic surface tension measured during final drop-bulk exchange (washing out) process after sequential adsorption from an initial βCS solution and subsequently from DoTAB solutions of different concentration at W/H interface; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

adsorption kinetic curves are given in Figure 1S of the Supporting Information. In both cases, the final surface tension values remain almost constant and close to that of pure βCS. This allows us to conclude that the DoTAB molecules cannot displace the protein molecules from the surface layers due to increased hydrophobicity of the protein/surfactant complexes in the studied concentration range, that is, almost all adsorbed protein molecules stay at the surface after washing off. One can see in Figures 2 and 3 that the kinetics of desorption takes a relatively long time for the sequentially formed mixed layer, that is, the processes last about 4000 s, whereas for the simultaneous adsorption it is by orders of magnitude faster. However, in both cases the exchange processes were continuous and the solutions were injected with the same rate until a constant surface tension value was reached. The 2236


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Figure 4. Surface tension isotherm of DoTAB, equilibrium surface tension values for mixed βCS/DoTAB layers measured for the sequential adsorption experiments after the drop-bulk exchange with different [DoTAB], and for simultaneous adsorption experiments at W/A (a) [taken from our earlier report, ref 57] and W/H (b) interface; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

Figure 5. Equilibrium surface tension measured after the drop-bulk exchange processes with different [DoTAB] and subsequently with pure buffer solution (washing out) after sequential adsorption experiments and after simultaneous adsorption experiments at water/air (a) and water/hexane (b) interfaces; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

We can summarize from part b of Figure 5 that the simultaneous adsorption route leads to the same amount of adsorbed protein as the sequential protocol. At pH 7, the net charge of βCS is negative and the oppositely charged DoTAB favors to form complex of higher surface activity than the original protein via strong electrostatic interaction, whereas the hydrophobic interaction is of secondary importance. So, one can expect a greater degree of protein adsorption from a mixed solution in simultaneous experiments relative to the case of subsequent adsorption of protein first and then followed by surfactant. However, the competitive adsorption of free surfactants in simultaneous adsorption balances the increased surface activity of the protein complexes formed in the bulk solution and accounts for the fact that the amount of adsorbed protein is the same as in case of sequential adsorption. At the W/A interface (part a of Figure 5), the sequential and simultaneous adsorption isotherms increase slightly at higher DoTAB concentrations, whereas this increase is slightly more pronounced in the sequential adsorption route when compared to the sequential adsorption. The dispersive interaction between the methyl groups of the neighboring DoTAB

From the equilibrium surface tension values obtained from sequential and simultaneous adsorption experiments after the final washing off step, the isotherms in Figure 5 can be constructed representing the state of remaining proteins in the surface layer. The isotherms for the W/H interface are almost identical (part b of Figure 5) and show no change with DoTAB concentration. This would allow us to conclude that the method of mixed layer formation does not much influence the surface composition. Nevertheless, the surface tension values are almost similar to that of adsorbed βCS in absence of surfactant. At the W/H interface, the resulting complexes are stabilized due to their deep penetration into the organic subphase. The additional dispersive forces between hexane and hydrocarbon chains of DoTAB result in a larger distance between the adsorbed surfactant molecules at the W/H interface (larger molar area). This has also impact on the mixed layer composition, which leads to a relatively decreasing presence of freely adsorbed DoTAB molecules at the interface and consequently the adsorbed proteins cannot be displaced by the surfactant molecules. 2237


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molecules causes an increasing presence of freely adsorbed surfactants in the mixed layers at the W/A interface. This leads to the progressive replacement of the proteins from the interface at higher DoTAB concentrations. In case of sequential adsorption, due to accessibility of the hydrophobic segments of the preadsorbed proteins, increasingly hydrophilic complexes are still formed via hydrophobic interactions once all charge sites of the proteins have been saturated at high DoTAB concentrations. These increasingly hydrophilic complexes become less surface active and can easily desorb into the bulk resulting in a decreasing presence of adsorbed complexes after sequential adsorption. However, the proteins are compact in the bulk, the hydrophobic segments are buried in the core, and, hence, the complexes formed in the bulk are less surface active. This suggests that the amount desorbed from the surface layer would be more after simultaneous adsorption. Contrary to the cationic surfactant DoTAB, anionic surfactants, such as SDS, hydrophilize the βCS at a certain surfactant concentration after compensation of the positive charged sites of the protein molecules resulting in a decreased surface activity of the complexes.50,55 As a consequence, a significant amount of protein (complex) is desorbed from the interface during the washing off experiment, which is not observed with DoTAB. In sequential adsorption experiments at the W/H interface, the hydrophobic parts of the preadsorbed proteins are fixed in the oil phase, but still there are some hydrophobic segments in the aqueous bulk phase to which the SDS molecules can bind hydrophobically. The accessibility of the hydrophobic parts in the aqueous bulk phase can be explained by the fact that hydrophilic parts are displaced from the proximal region and protrude into the aqueous subphase thus forming tails and loops. Therefore, SDS can displace the protein molecules from the W/H interface even in case of sequential adsorption. Indeed, this displacement is more effective for simultaneous adsorption, which correlates to higher surface activity of the protein complexes as compared to the case of sequential adsorption. Thus, in contrast to the βCS/DoTAB system, the mixed βCS/SDS adsorption layers formed in two different ways show different amounts of remaining proteins after the washing off steps. Note, an adsorption study of βCS mixed with nonionic surfactant, C12 dimethyl phosphine oxide (C12DMPO) at the W/H interface is unrealistic as the this surfactant is highly soluble in the oil phase, so that even those molecules bound to the proteins may transfer into the oil. Interfacial Dilational Rheology. The viscoelasticity in terms of Er and Ei measured for a 10−6 mol/dm3 βCS solution and pure DoTAB solutions at different concentrations and perturbation frequencies at the W/H interface had been discussed recently.23 Parts a and b of Figure 6 show the measured viscoelastic modulus as a function of DoTAB concentration for mixed βCS/DoTAB layers built up via sequential and simultaneous adsorption, respectively. Whereas the general course of the obtained dependencies is the same for all measured frequencies, there are two main peculiarities in the results to be discussed in more detail. At first, for the sequential adsorption route we have almost no frequency effect, but we see a local minimum/maximum in the DoTAB concentration range between 10−4 to 10−3 mol/L (part a of Figure 6. In contrast, for the simultaneous adsorption route, there is a significant effect of the perturbation frequency (part b of Figure 6). The absence of frequency dependence typically points to the fact that no significant amounts of surfactant is available in

Figure 6. Dilational viscoelastic moduli of the mixed βCS/DoTAB layers formed via sequential (a) and simultaneous (b) adsorption plotted as a function of [DoTAB] at different oscillation frequencies at W/H interface; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

the interfacial layer to relax as response to the harmonic area compressions and expansions. On the contrary, the remarkable differences in the elasticity |E| shown in part b of Figure 6 tell us that the interfacial layer built via a simultaneous adsorption contains surfactant molecules that are able to adsorb and desorb with a measurable effect on the interfacial tension. We yet have to find the explanation of the minimum/ maximum in the |E|(c) for the sequential adsorption route. In Figure 7, we show again the dependencies for both adsorption routes at a fixed frequency of 0.01 Hz (at other frequencies the picture is equivalent) before and after the final washing off process. What we can unambiguously see is that all surfactant has been washed off, independent of coadsorbed at the interface or bound to the βCS via electric or hydrophobic forces, because the elasticity values are identical to the value of the pure protein layer and independent of the DoTAB concentration added before. The viscoelastic modulus for both adsorption routes starts from the corresponding values of pure βCS and decrease with increasing DoTAB concentration. This continuous decrease is presumably due to destruction of a rigid structure of protein, followed by the formation of βCS/ DoTAB complexes in the surface layer. Note, any change in the elasticity value can be explained by two effects: the relaxation of bound surfactant, which are released upon expansion and rebound upon compression of the interfacial layer, and the 2238


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Scheme 2. Illustration of Mixed βCS/DoTAB Layers Representing the Surface Structures Formed at Different [DoTAB] after Sequential and Simultaneous Adsorptions at the W/H Interfacesa

Figure 7. Dilational viscoelastic moduli as a function of [DoTAB] for sequentially (solid and hollow red squares) and simultaneously (solid and hollow blue triangles) formed mixed βCS/DoTAB layers before (solid red square and solid blue triangle) and after (hollow red square and hollow blue triangle) washing off experiment at 0.01 Hz oscillation frequencies at W/H interface; all experiments were performed at a fixed βCS concentration of 10−6 mol/dm3.

relaxation of single segments of adsorbed βCS at the interface. The intermediate extreme for the sequential adsorption route can be explained by a first larger compaction of the protein molecules at the interface due to increased charge density produced by hydrophobic interaction of surfactant chains with the protein or with electrostatically prebound surfactants (i.e., hydrophilization). With the further increase in surfactant concentration, free surfactant starts coadsorbing so that a larger exchange of matter sets in and hence the elasticity decreases. In the end, at sufficiently high DoTAB concentration the surfactant dominates the interfacial layer properties. Note, however, this does not mean that the protein has been removed from the interface, but it has just been subject to conformational changes giving space to the surfactant to coadsorb at the interface. It seems even possible that parts of the protein, originally hydrophobic, become hydrophobized via electrostatic binding of DoTAB molecules and protrude into the hexane phase. In case of simultaneous adsorption, an increasing presence of free DoTAB molecules hinders the elasticity response of the protein/surfactant complexes at the interface, and hence the course of |E|(c) does not show any maximum. In contrast, in the sequential adsorption route, the preadsorbed protein occupies the interface and establishes an optimum conformation so that any added surfactant cannot easily enter the interface to coadsorb or to provide changes in the electric surface charge density. Hence, the interfacial layer structure is changing in a way similar to the situation for the simultaneous adsorption route beyond the local elasticity maximum. The continuous elasticity decreased via protein compaction is caused by an increasing relaxation between different compaction states. The two situations are summarized in Scheme 2.

(i) − Pre-adsorbed protein layer, (ii) − compensation of charges makes the layer more flexible and consequently, decreases molar area of βCS, (iii) − surface layer becomes more rigid due to accumulation of more charges caused by hydrophobic interaction, (iv) − increased exchange of matter DoTAB, (v) − the complexes formed in the bulk are more hydrophobic, (vi) − similar mixed layer but reached at lower DoTAB bulk concentration, therefore, the layer is frequency dependence. a

feature is that the protein molecules can penetrate into the oil phase and a stronger anchoring of these molecules at the W/H interface is the consequence. The tensiometry results suggest that the two adsorption strategies for the formation of mixed βCS/DoTAB layers lead to a rather similar adsorption behavior at this water/oil interface. However, there is a striking difference from the W/A interface, where the addition of a cationic or anionic surfactant displaced part of the protein from the interface. Although the surface tension isotherms for the mixed adsorption layers indicate that the increased addition of DoTAB changes their properties from a pure protein to a pure surfactant behavior, the wash off experiments demonstrate unambiguously that almost no protein was replaced. The addition of DoTAB leads only to changes in the structure of the mixed adsorption layers such that the protein molecules penetrate deeper into the oil phase and take also a more compact conformation in the interfacial layer. This creates sufficient space for the surfactant molecules to compete for the interface.

5. CONCLUSIONS A comparative study of adsorption and rheological properties of mixed βCS/DoTAB layers formed via sequential and simultaneous adsorption at the W/H interface has been performed for different interfacial compositions. The observed behavior is different from that at the W/A interface. The main 2239


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Whereas the tensiometry data do not clearly show differences in the composition of the mixed layers depending on the mixing rations, the dilational rheology provides undoubtedly evidence that the structure and in particular the dynamics of the adsorbed layers depend on the adsorption route. In the simultaneous adsorption route, protein/surfactant complexes are formed in the solution bulk and lead to a respective conformation with hydrophilic segments pointing to the aqueous phase and decrease their surface activity. In contrast, the sequential adsorption route allows the protein to preadsorb at the W/H interface in a way that more of the hydrophobic parts are already buried in the oil and cannot be further decorated by surfactants via hydrophobic interaction.

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ASSOCIATED CONTENT

S Supporting Information *

Dynamic surface tension for simultaneously formed mixed βCS/DoTAB adsorption layer. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was financially supported by projects of the DFG (Mi418/20-1), the European Space Agency (FASES MAP AO99-052, PASTA), and the COST actions CM1101 and MP1106.

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REFERENCES

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