J. Phys. Chem. B 2008, 112, 5381-5392
5381
Effect of r-Lactalbumin on Aerosol-OT Phase Structures in Oil/Water Mixtures Jun Y. Kim and Stephanie R. Dungan* Department of Food Science and Technology, Department of Chemical Engineering and Materials Science, UniVersity of California, DaVis, One Shields AVenue, DaVis, California 95616 ReceiVed: NoVember 27, 2007; In Final Form: February 7, 2008
The ability of water-soluble, globular proteins to tune surfactant/oil/water self-assemblies has potential for the formation of biocompatible microemulsions and also plays a role in protein function at biological interfaces. In this work, we examined the effect of the protein R-lactalbumin on Aerosol-OT (AOT) phase structures in equivolume mixtures of oil and 0.1 M brine. In this pseudo-ternary system, surfactants are free to move to either oil or water phase to adopt phase structures close to the spontaneous curvature of the surfactants. Using small-angle X-ray scattering, we observed that addition of this protein changed the spontaneous curvature of the surfactant monolayer substantially. In the absence of protein, AOT adopted a negative spontaneous curvature to form spherical w/o microemulsion droplets. When less than 1 wt % of R-lactalbumin was added into the system, the w/o droplets became nonspherical and larger in volume, corresponding to an increase in water uptake into the droplets. As the protein-to-surfactant ratio increased, protein, surfactant, and oil increasingly partitioned toward the aqueous phase. There the protein triggered the formation of o/w microemulsions with a positive spontaneous curvature. These protein-containing structures exhibited significant interparticle attraction. We also compared the influence of two oil types, isooctane and cyclohexane, on the protein/surfactant interactions. We propose that the more negative natural curvature of the AOT/cyclohexane monolayer in the absence of protein prevented protein incorporation within organic phase structures and consequently pushed the system self-assembly toward aqueous aggregate formation.
Introduction In the present study, we investigated the ability of watersoluble proteins to interact with and alter surfactant monolayers. Such interactions can cause changes to interfacial curvature, an effect that is drawing increasing attention in the biomedical literature.1 Here we probe such effects by studying how they are manifested in changes to the behavior of self-assembled surfactant solutions, which can respond to protein/surfactant interactions and changes in curvature energies. There have been a number of investigations of protein-surfactant interactions within a single solution phase. In aqueous solvents, proteins can cooperatively bind surfactant micelles, with this association often thought to lead to a “necklace” structure containing an unfolded protein chain.2-7 However, other studies have shown more complex surfactant phase changes upon adding protein to water, and have proposed a compact protein structure within the protein/surfactant self-assemblies.8 In organic solvents, proteins can be hosted by water-in-oil microemulsion solutions with a consequent perturbation to the size of individual, proteinfilled droplets.9-14 We have become interested in a somewhat different phenomenon: the effect of protein on surfactant self-assembly in two-phase systems consisting of both oil and water.15-20 Selfassembly is more unconstrained in such two-phase mixtures, and more far-reaching changes in phase morphology can potentially be observed. Water-in-oil microemulsion droplets can change their size by taking up or releasing water from the coexisting aqueous phase, while oil-in-water droplets can do the same with the adjacent oil phase. Surfactant can also partition * Corresponding author. Tel.: +1-530-752-5447. Fax: +1-530-7524759. E-mail:
[email protected].
to either solvent to adopt negatively (curved toward water) or positively (curved away from water) curved structures. We recently reported on interesting shifts in the phase behavior of the anionic surfactant AOT in organic/aqueous mixtures, induced by the globular protein R-lactalbumin (RLA).18,20 In the absence of protein, AOT is known to form spherical, water-in-oil (w/o) microemulsion droplets in equivolume mixtures of oil and brine.21 Addition of less than 1% of protein to the mixture boosts considerably the amount of water solubilized in those w/o droplets, and induces the formation of aqueous phase surfactant aggregates that contain solubilized oil. Rohloff et al.18 showed that these compositional changes were significantly influenced by the protein concentration, as well as the total AOT concentration in the system. An interesting feature of these results with RLA/AOT mixtures is the apparent lack of a strong attractive force driving their interaction. Substantial protein-surfactant association is generally attributed to strong attractive interactions, created by opposite charges on the protein and surfactant, or by a large hydrophobic domain within a membrane protein. In contrast, R-lactalbumin is a highly water-soluble, globular protein with a low isoelectric point of ∼4.2-4.4. It thus has the same net negative charge at neutral pH as the AOT headgroup.22 The quite extensive changes in phase behavior observed upon addition of protein most likely signal changes in phase structures in the organic phase, and formation of new structures in the aqueous phase. However, previous research did not determine the detailed nature of the phase structures found with protein. In this paper, we describe studies using small-angle X-ray scattering (SAXS) to investigate the size and shape of the nanosized aggregates of AOT, and the effect of protein-tosurfactant ratio on these phase structures. We investigated
10.1021/jp7112413 CCC: $40.75 © 2008 American Chemical Society Published on Web 04/24/2008
5382 J. Phys. Chem. B, Vol. 112, No. 17, 2008 equivolume mixtures of oil with aqueous solutions containing 0.1 M NaCl, and varied both the surfactant and protein concentrations independently. Ultimately, understanding the role of proteins in modifying self-assembled structures may lead to the use of proteins as versatile components in formulating biocompatible microemulsions or other nanostructured fluids. Our results will also provide insights into the behavior of protein/ surfactant combinations in products containing oil and water, (e.g., foods, cosmetics, pharmaceuticals, and cleaning products), in multiphase reactions involving enzymes, and in biological structures such as lipoproteins and membranes. Materials and Methods Materials. Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) of 99% purity and the Ca2+-depleted form of bovine R-lactalbumin (type III) with 85% purity were obtained from Sigma (St. Louis, MO). The protein contained less than 0.3 mol of Ca2+ per mole. Spectroscopy grade isooctane and cyclohexane and ACS grade sodium chloride (NaCl) were purchased from Fisher Scientific (Fair Lawn, NJ). Water for sample preparation was distilled and deionized (18 MΩ) using a Barnstead filter (Dubuque, IW). For quantitative 1H NMR measurements, 3-trimethylsilylpropionate-d4 (TMSP, Cambridge Isotope Laboratories) was used as an internal standard. Dimethyl sulfoxide-d6 (DMSO) purchased from Sigma was used as a solvent for NMR experiments. All chemicals were used without further purification. Sample Preparation and Composition Analysis. Equal volumes of RLA in 0.1 M NaCl aqueous solution and AOT in organic solvent were mixed in a sealed container and allowed to reach equilibrium at 25 ( 0.2 °C.18 The establishment of equilibrium in these samples was determined by following the change of the sample appearance and the amount of water solubilized in the organic phase over time, until no further changes were observed (typically 24 h). Overall concentrations of protein or salt in these mixtures are presented using an aqueous basis (i.e., g or mol of solute per L water in the overall system), while overall AOT concentrations in the mixtures are given as moles of AOT per L organic solvent in the overall mixture. On a total weight basis these dilute mixtures contained 0.3 wt % salt, various AOT concentrations between 0.6 and 5 wt %, and various protein concentrations between 0 and 0.4 wt %, After the system reached equilibrium, the organic and aqueous phases were separated and analyzed for each component. RLA in each phase was quantified using UV absorbance at 280 nm.18 Karl Fisher titration was used to measure the amount of water solubilized in the organic phase. The amount of isooctane or cyclohexane solubilized in the aqueous phase was measured using 1H NMR18 as was AOT in both the organic and aqueous phases. For the quantification of isooctane, the peak at 1.1 ppm was used, and a peak at 1.4 ppm was used for cyclohexane. For AOT, the peaks at 1.2 and 3.9 ppm were used for the aqueous or organic phase concentrations, respectively. Quantification of water and surfactant in the organic phase allowed us to calculate wo ) [H2O]/[AOT], the molar ratio of water to surfactant in the w/o microemulsion. Small-Angle X-ray Scattering Measurements. SAXS experiments were performed using a Bruker AXS NanoSTAR instrument in the Material Science Central Facilities at the University of California, Davis. Ni-filtered Cu KR radiation (λ ) 1.54 Å) was generated at 40kV and 35mA, and a pinhole collimator with a 0.3 mm diameter was used. The SAXS intensity was recorded as a function of the magnitude of the scattering vector q ) 4π sin θ/λ with 2θ defined as the scattering
Kim and Dungan angle in the region 0.01 Å-1 < q < 0.2 Å-1. Organic phase data was collected over 1-4 h, while data for the aqueous phases was twice collected over 8 h and averaged. In general, signalto-noise ratios are lower when performing X-ray scattering in aqueous solvents. Samples were contained in glass capillaries with outer diameter 1.0 mm and wall thickness 0.01 mm (Hampton Research, Aliso Viejo, CA), and sealed with beeswax. All the measurements were performed at 23 ( 1 °C. The scattering intensity was detected in a two-dimensional plane and then averaged in the radial direction. The scattering intensity from the sample was corrected by subtracting the scattering intensity of a solvent-filled capillary using appropriate measured transmission coefficients. The absolute calibration was performed using the scattering intensity of a water-filled capillary at the same experimental conditions. Analysis of Scattering Data. By the assumption that particle size and orientation are not correlated with the position of the particle,23 the scattering intensity of a sample can be modeled as
I(q) ) npP(q)S(q) )
{
np〈|F(q)|2〉 1 +
|〈F(q)〉|2 〈|F(q)|2〉
(Sh(q) - 1)
}
(1)
where np is the number density of particles. P(q) is a form factor describing the average shape and size of the particles in the sample, and the structure factor S(q) accounts for the effects of interparticle interactions. Only a few analytical expressions are available for the structure factor in systems with nonspherical particles or nonuniform sizes or shapes. We followed Kotlarchyk’s procedure23 and calculated an effective structure factor Sh(q) by treating our particles as monodisperse spheres with the same volume as the average volume of the actual particles. The effect of polydispersity and anisotropy of the particles is then considered in the ratio |〈F(q)〉|2/〈|F(q)|2〉 in eq 1. Microemulsion droplets are coated with surfactant molecules, which consist of head and tail groups. The electron density of the tail groups is similar to that of an organic phase, while the head groups usually have a higher electron density than either organic or aqueous phase solvents.24 Thus, the microemulsion droplet can often be represented with a core and shell model, with electron density F0 in the continuous phase and densities F2 and F1, respectively, of the core and shell. We found in many cases that addition of protein to the surfactant solution created microemulsion droplets that were significantly nonspherical. The single particle form factor of a core-shell ellipsoid model is given by
F(q,t) ) (F2 - F1)V2φ(u2) + (F1 - F0)V1φ(u1)
(2a)
u2 ) qr xν2(1 - t2) + t2 and u1 ) q(r + d)
x(νrr ++dd) (1 - t ) + t for oblate (2b) 2
2
2
u2 ) qr x(1 - t2) + ν2t2 and u1 ) q(r + d)
x(1 - t ) + (νrr ++dd) t for prolate (2c) 2
2
2
Here φ(u) ) 3(sin u - u cos u)/u3, t is the cosine of the angle of particle orientation, d is the thickness of the shell, r is the short radius, and ν is the aspect ratio of the particle. In eq 2a, V1 is the total volume of the particle and V2 is the volume of
R-Lactalbumin on Aerosol-OT Phase Structures
J. Phys. Chem. B, Vol. 112, No. 17, 2008 5383
Figure 1. SAXS data for the differential scattering cross section from the organic phases of AOT/isooctane/0.1 M brine (protein free) mixtures at various values of [AOT]total. To separate the curves for easier viewing, each data set for I(q) was multiplied by 5n, n ) 0-5.
by changes in the counterion dissociation. The opposite procedure (fix F1 and fit d) was found to produce better results for the aqueous phase. The other fit parameters in our form factor analysis were the mean radius jr, aspect ratio ν, and the polydispersity index, p. Scattering intensities from the aqueous phase were quite weak, as is generally the case for aqueous phase studies. Consequently, the intensity in the high q region was sensitive to small errors in the background subtraction, leading to unphysical values for the shell properties. To correct for this error, we included for these aqueous data an offset parameter, b, that was added to the right side of eq 1 to account for background scattering levels. O/w microemulsion droplets with charged surfactants often experience repulsive electrostatic interactions over their interparticle distance x, which are described by a screened Coulomb potential within a continuous medium with a uniform dielectric constant , in combination with a hard-sphere repulsion27
{
-k(x-1) /x, U(x) ) Ae ∞,
Figure 2. The core radius jr (O) of w/o microemulsion droplets from fits to the SAXS data, and the volume fraction φwater of water (0) in the w/o microemulsion phase as a function of [AOT]total.
the core. For ν ) 1, eq 2 reduces to the form factor for a single spherical particle with a radius r as given by Guinier and Fournet.25 Whether spherical or nonspherical, the particles are assumed to have some polydispersity in size. To calculate the form factor for polydisperse particles, we need to average the single particle form factor for an sphere/ellipsoid over the size and orientation of the particle as
P(q) ) 〈|F(q)|2〉 )
∫0∞ ∫01 |F(q,r,t)|2 dt f (r)dr; f(r) )
1 z + 1 z+1 z re z! jr
( )
z+1
jr
r
(3)
Here f(r) is a Schultz distribution function with jr being the average core short radius and z the distribution width parameter. A polydispersity index is given by
p ) xjr2 - 〈r2〉 / jr ) 1/xz + 1
(4)
In fitting our SAXS data, the electron density of 0.1 M brine, isooctane, and cyclohexane were fixed at 0.336, 0.241, and 0.267 e/Å3, respectively, where e is the electronic charge. It is well known that changes in the shell thickness and the shell electron density have similar effects on the scattering curve in systems with either polydisperse droplet sizes or ellipsoidal shapes.26 For the organic phase structures, we set the shell thickness of the surfactant heads to be 4.5 Å, and fit the scattering curve by changing the shell electron density. Changes in shell electron density can be explained by varying extents of the solvent and protein penetration into the surfactant head group region and
xg1 x 0.05 Å-1 reflects scattering from the thin surfactant shell surrounding the droplet, as incorporated theoretically in a core-shell model for the form factor with a higher electron density in the shell. The shell corresponds to the region of the AOT head groups, whose lengths are fixed to
4.5 Å. We could then fit the experimental data with a value of 0.53 e/Å3 for the shell electron density. This value is slightly smaller than the reported electron density of the AOT head groups,12 likely due to the penetration of solvent between the AOT head groups. We found that scattering in the low q region, which is sensitive to interdroplet interactions, was well fit using eq 5 for the structure factor, if we set A ) 0. This indicates that the w/o microemulsion droplets interact weakly through a hard sphere potential. Similar results for interdroplet interactions within w/o microemulsions formed with dilute anionic surfactants are found in the literature.23 Results from these fits are given with composition measurements in Table 1. Karl Fischer titration measurements on systems with AOT concentrations between 0.005 and 0.2 M indicated that the amount of water dissolved in the microemulsion phase linearly increased with the total concentration of AOT in the system. Figure 1 shows SAXS data measured for these six different AOT concentrations, with structural parameters obtained from the model fits (solid curves) discussed above (Table 1). These fits indicated that the size of the w/o droplets increased as the AOT concentration increased until ∼0.1 M, as indicated by the shift in the “dent” in the curves to lower q values, and remained approximately constant at higher concentrations. The effect of AOT concentration on droplet core size obtained from the SAXS data and water solubilization is shown in Figure 2. A similar size dependency of w/o microemulsion droplets on the AOT concentration was observed previously using photon correlation spectroscopy.42 At higher AOT concentrations, the radius (and the molar ratio of water-to-surfactant) becomes constant, as expected;19,42 in this region, larger amounts of water in the organic phase are accommodated within an increased number of droplets, whose size remains the same. For the sample with 0.2 M AOT, the SAXS curve could not be fit with a structure factor of hard-sphere interactions alone, and the Yukawa tail structure factor (5) was used to fit the data, with parameters A ) 4kBT and k ) 0.27. Here kBT is the Boltzmann energy. The aqueous concentration of AOT increased slightly as the total AOT concentration increased (Table 1). However, no isooctane was detected in any aqueous phase sample and there was no significant scattering from these phases. These results indicate that, in the absence of protein, AOT did not form nanosized aggregates in the brine and simply dissolved in the aqueous phase as a monomer. The measured concentration of this monomer is similar to that observed by other groups in an excess aqueous phase in equilibrium with oil.39 A value of 8 × 10-4 M was set as the critical micelle concentration in this twophase system for the calculation of the Hayter-Penfold structure factor in the aqueous phase in the presence of the protein.28 Protein-Containing Mixtures: rLA/AOT/Isooctane/0.1 M brine. We prepared samples at different protein and AOT concentrations, and analyzed the structures formed in the resulting equilibrated organic and aqueous phases. The scattering
R-Lactalbumin on Aerosol-OT Phase Structures
Figure 3. SAXS data for the differential scattering cross section of phase structures in mixtures of 0.025 M AOT/isooctane/0.1 M brine and with 0, 4, and 7 g protein/L water. Data obtained from the (a) organic or (b) aqueous phase. Data are fit to the scattering models using Hayter-Penfold (‚‚‚‚‚‚), Yukawa (s), or fractal (- - -) structure factors.
Figure 4. SAXS data for the differential scattering cross section of phase structures in mixtures of 0.1 M AOT/isooctane/0.1 M brine, and with 0, 4, and 7 g protein/L water. Data obtained from the (a) organic or (b) aqueous phase. Data are fit to the scattering models using Hayter-Penfold (‚‚‚‚‚‚), Yukawa (s), or fractal (- - -) structure factors. To separate the curves for easier viewing, each data set for I(q) in part (a) was multiplied by 5n, n ) 0-2.
curves from the organic phase in the presence of protein are shown in part a of Figures 3-5. In general, the scattering intensities for q > 0.08 Å-1 from systems with 4 and 7 g/L protein look very similar to those from the system without protein. This indicates that the added protein does not change the shell properties of the droplets significantly. However, curves from the protein-containing systems have higher scattering
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Figure 5. SAXS data for the differential scattering cross section of phase structures in mixtures of 0.2 M AOT/isooctane/0.1 M brine and with 0, 4, and 7 g protein/L water. Data obtained from the (a) organic or (b) aqueous phase. Data are fit to the scattering models using Hayter-Penfold (‚‚‚‚‚‚), Yukawa (s), or fractal (- - -) structure factors. To separate the curves for easier viewing, each data set for I(q) in part (a) was multiplied by 5n, n ) 0-2.
intensities in the low q region, especially at the two higher AOT concentrations. (The behavior at [AOT]total ) 0.025 M is more complex). The polydisperse core-shell model based on a spherical shape could not fit this low q region without unrealistically low values of the total volume fraction φtotal. Instead, the scattering from the droplets in these proteincontaining microemulsions could be well fit with the polydisperse core-shell model with a prolate shape, in which we allowed the aspect ratio ν to vary. A deterministic identifiability analysis28,43 of the model indicates that there is a finite set of alternative values of p and ν that fit the scattering curves, as also reported in other small angle scattering studies.35 For example, the scattering intensity from the system with 4 g/L protein and [AOT] ) 0.1 M can be fit with 0.27 and 2.3 for the polydispersity and the aspect ratio, respectively; it can also be fit with p ) 0.28 and ν ) 2.0. We therefore chose the set of the parameters with the value of the polydispersity closest to the value of the system in the absence of protein. Making the opposite choice, allowing the polydispersity to grow while the aspect ratio decreases, would change the two parameters by less than 25%, according to our analysis. Thus, our choice did not substantially affect our conclusions about the droplet structure, especially because a slightly smaller aspect ratio is compensated for by a larger distribution of sizes, indicating that the protein’s addition is enhancing the range of volumes of the now nonspherical drops. In addition, as is expected for a system with some polydispersity,26 it was in most cases not possible to distinguish between the goodness of fit of the prolate and oblate models. In the results that follow, the curves were fit using a prolate form factor, unless otherwise noted. Results from the fits to the SAXS data are shown along with results of compositional measurements in Table 2a. Using the
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Kim and Dungan
TABLE 2: Influence of [AOT]total and [rLA]total on Equilibrium Composition Data and SAXS Results in Equivolume Mixtures of Isooctane (oil) and 0.1 M Brine a. Organic Phase Results SAXSa
composition [RLA]total [g/L]
φwater
[AOT]org [M]
[RLA]org [g/L]
φtotal
jr [Å]
ν
p
0.04 0.02 0.01
48 39 66
1.0 1.7 1.0
0.25 0.24 0.28
0.14 0.16 0.15
62 66 63
1.0 2.1 2.3
0.25 0.27 0.27
0.21 0.21 0.22
66 62 61
1.0 1.7 2.0
0.25 0.27 0.29
[AOT]total ) 0.025 M 0 4 7
0.017 0.010 0.0043
0.025 0.017 0.0088
0 4 7
0.079 0.086 0.086
0.095 0.088 0.083
0 4 7
0.15 0.16 0.17
0.18 0.17 0.17
0.18 0.07 [AOT]total ) 0.1 M 1.47 1.50 [AOT]total ) 0.2 M 2.49 3.52
b. Aqueous Phase Results: Composition and Structure SAXSb
composition [RLA]total [g/L]
[oil]aq [M]
[AOT]aq [M]
0 4 7
0.000 0.015 0.027
0.0004 0.0091 0.017
0 4 7
0.000 0.0076 0.020
0.0008 0.0064 0.015
0 4 7
0.000 0.0032 0.0086
0.0013 0.0034 0.0082
[RLA]aq [g/L]
φtotal
jr [Å]
ν
p
0.0002 0.0087 0.016
c 12 13
c 2.7 3.0
c 0.20 0.20
0.0004 0.0055 0.013
c c 13
c c 2.5
c c 0.20
0.0006 0.0027 0.0071
c c 15
c c 1.5
c c 0.20
[AOT]total ) 0.025 M 3.84 6.83 [AOT]total ) 0.1 M 2.54 5.72 [AOT]total ) 0.2 M 1.25 3.56
c. Aqueous Phase Results: Interactions Yukawa [RLA]total [g/L]
d [Å]
A [kBT]
fractal σ/2 [Å]
k
d [Å]
D
ξ [Å]
σ/2 [Å]
4.4 4.6
1.8 2
40 50
29 33
2
50
30
2.5
50
25
4 7
4.9 5.2
-10 -5
[AOT]total ) 0.025 M 3 29 3 34
7
5.5
-20
7
[AOT]total ) 0.1 M 31
4.7
-30
[AOT]total ) 0.2 M 5 27
4.7
7
6.4
a
A core-shell prolate model with 4.5 Å shell thickness was used for the SAXS model fit. density 0.53 e/Å3 was used for the SAXS model fit. cScattering too weak to analyze.
prolate model, we found that the aspect ratio of the w/o microemulsion droplets generally increased from unity (spherical) in the absence of protein to approximately two as the overall protein concentration in the system increased. The short core radius did not change substantially with the addition of protein, except at 0.025 M AOT. Such changes in droplet dimensions with protein addition were accompanied by an increase in the amount of water solubilized in the organic phase, especially at the two higher AOT concentrations. On the other hand, increasing protein concentration always caused a decrease in AOT concentration in the organic phase, as surfactant instead partially partitioned toward the aqueous phase. Thus, overall we observed an increase in water solubilization at the same time as a decrease in AOT concentration, together causing a boost in the molar ratio of water-to-surfactant ratio (wo) in the w/o microemulsion as protein was added. The organic phase concentration of protein also exhibited a complex dependence on overall protein and AOT concentration. The added protein
bA
core-shell oblate model with shell electron
had little effect on the inter-droplet interactions in the w/o microemulsion. With the addition of RLA, the aqueous phase concentrations of all three components, isooctane, AOT, and RLA, increased directly with overall protein concentration (Table 2b), and significant scattering intensities from the aqueous phase were detected (part b of Figures 3-5). After adjusting the background intensity, the scattering curves from the protein-containing aqueous phase were fit using the models discussed above. Scattering for q > 0.05 Å-1 is most sensitive to the particle size and shape, and we found that a form factor for the coreshell oblate model fit the data well. Regardless of how we treated the interparticle interactions through the structure factor, we obtained similar values for the size and shape, corresponding to an aspect ratio of 2-3 and a core short radius of 12-15 Å, as shown in Table 2b. The polydispersity values of 0.2 were smaller than those of w/o microemulsion droplets in the organic phase. Contrary to the organic phase structures, the type of the
R-Lactalbumin on Aerosol-OT Phase Structures ellipsoid (oblate versus prolate) for the aqueous structures could be distinguished: the quality of the curve fit, as quantified by the reduced χ2r parameter, differed by an order of magnitude for the two shapes. The ability to identify the ellipsoid type for this case is probably due to the lower polydispersity in these aqueous structures. The scattering data increased steeply in intensity as q decreased in the small q range (q < 0.05 Å-1), signaling the presence of attractive interparticle interactions. For this reason, the Hayter-Penfold model for the structure factor did not capture this region effectively (part b of Figures 3-5). Both the Yukawa potential and the fractal model, in contrast, fit the data well with fit parameters given in Table 2c. The fractal dimension D ≈ 2 indicates that any clusters that form are rather compactly packed, but their overall cluster size would have to be quite small, as indicated by the small correlation lengths obtained. In the aqueous fits, the shell electron density was fixed to 0.53 e/Å3, and we allowed the shell thickness to vary. Fits of the intensity in the low q region required a somewhat larger shell thickness than that found for the w/o microemulsion droplets, especially for the Yukawa tail structure factor. 1H NMR measurements of the aqueous phase composition indicated that appreciable quantities of isooctane were present in the solution in the presence of protein (Table 2b). Its concentration increased as the aqueous concentration of AOT increased, which in turn was boosted by the increased protein concentration in the aqueous phase. Thus, results from SAXS and composition measurements on the aqueous phase indicated that the addition of RLA induced self-assembly of AOT in the aqueous phase to form structures like o/w microemulsion droplets containing some isooctane. In this picture, the AOT head groups form the shell of the oblate droplets with the AOT tails and isooctane in the droplet core. As shown in Table 2b, as the overall protein concentration in the system increased, the aqueous concentrations of all three components more than doubled. This increase in the amount of aqueous material was accompanied by an increase of the core radius and aspect ratio of the o/w microemulsion droplets, according to scattering for the system with 0.025 M AOT overall. The results discussed above exhibited a significant dependence on the surfactant concentration. Although increases in aspect ratio and drop volume of the organic structures were observed with increasing protein at the two higher surfactant concentrations (Table 2a), the samples at 0.025 M AOT exhibited a more complex behavior. These shifts appear to be driven by the stronger partitioning of surfactant and protein to the aqueous phase as the surfactant concentration decreased. As overall [AOT] decreased and material in the aqueous phase increased, we observed a concomitant increase in the SAXS scattering from the aqueous phase. At 0.2 M AOT, we could only characterize phase structure scattering at the highest protein concentration, at 0.1 M AOT the aqueous scattering was also just barely detectable in some samples at 4 g/L protein, and at 0.025 M AOT scattering could be analyzed with both protein concentrations. The aspect ratio of the aqueous aggregates also appeared to be larger at the lowest [AOT] (Table 2b). We noted that even for protein-containing samples with 4 g/L RLA, compositional measurements suggested that aggregates were forming in the aqueous phase, even though X-ray scattering data was generally too weak to be measured there. This is a consequence of the low sensitivity of X-ray scattering measurements in aqueous solvents. Effect of Oil: RLA/0.1 M AOT/Cyclohexane/0.1 M brine. The influence of oil type on the AOT phase structure in the
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Figure 6. SAXS data for the differential scattering cross section of phase structures in mixtures of 0.1 M AOT/cyclohexane/0.1 M brine and with 0, 4, and 7 g protein/L water. Data obtained from the (a) organic or (b) aqueous phase. Data are fit to the scattering models using Hayter-Penfold (‚‚‚‚‚‚), Yukawa (s), or fractal (- - -) structure factors. To separate the curves for easier viewing, each data set for I(q) in part (a) was multiplied by 5n, n ) 0-2.
presence of RLA was examined by substituting isooctane with cyclohexane at an overall AOT concentration of 0.1 M. In such systems in the absence of protein, most of the AOT was found in the organic cyclohexane phase, where it formed spherical w/o microemulsion droplets, as was the case with isooctane (Figure 6a). However, in cyclohexane, AOT formed much smaller w/o droplets, with SAXS data yielding a core radius of 33 Å: this is about one-half the value of droplets in the isooctane system at the same AOT concentration. These results are given in Table 3a. It appears that the smaller molecular size of cyclohexane allowed it to penetrate into the layer of the AOT tails, causing the formation of smaller w/o microemulsion droplets.16,44 The smaller value for the shell electron density used in the model fits for the cyclohexane system (0.52 e/Å3), compared to that in isooctane, is also consistent with this explanation. When 4 g/L of protein was added, less than 4% of protein was found in the organic phase (Table 3a), which is approximately one-tenth the amount seen in the isooctane solutions. Increasing the protein concentration from 4 to 7 g/L had little effect on the organic protein concentration. In the presence of these small amounts of RLA, AOT maintained spherically shaped w/o droplets. The amounts of AOT and water solubilized in the organic phase decreased as the overall protein concentration increased, with more of these compounds moving into the aqueous phase where most of the protein was found (Table 3b). In the system with 7 g/L overall of RLA, there was enough scattering intensity in the aqueous phase to be analyzed (Figure 6b). Fits to the SAXS data given in Table 3b indicate that AOT in the presence of protein formed spherical o/w microemulsion droplets with a core radius of 26 Å. Evidence of attractive interactions was even stronger in the studies with cyclohexane than for isooctane-containing mixtures, and the scattering intensities could again be fit with an attractive Yukawa model
5388 J. Phys. Chem. B, Vol. 112, No. 17, 2008
Kim and Dungan
TABLE 3: Influence of [AOT]total and [rLA]total on Equilibrium Composition Data and SAXS Results in Equivolume Mixtures of Cyclohexane (oil) and 0.1 M Brine a. Organic Phase Results SAXSa
composition [RLA]total [g/L] 0 4 7
φwater 0.039 0.037 0.036
[AOT]org [M]
[RLA]org [g/L]
0.079 0.074 0.072
φtotal 0.11 0.10 0.09
0.14 0.13
jr [Å] 33 31 35
ν
p
1.0 1.0 1.0
0.24 0.24 0.24
b. Aqueous Phase Results: Composition and Structure SAXSb
composition [RLA]total [g/L] 0 4 7
[oil]aq [M] 0.000 0.0061 0.015
[AOT]aq [M]
[RLA]aq [g/L]
φtotal
3.85 6.64
0.0003 0.0061 0.011
0.0005 0.0070 0.013
jr [Å]
ν
p
c c 26
c c 1.0
c c 0.22
c. Aqueous Phase Results: Interactions Yukawa
fractal
[RLA]total [g/L]
d [Å]
A [kBT]
k
σ/2 [Å]
d [Å]
D
ξ [Å]
7
7.0
-50
12.5
34
5.4
2
80
σ/2 [Å] 32
a A core-shell prolate model with 4.5 Å shell thickness was used for the SAXS model fit. bA core-shell oblate model with shell electron density 0.52 e/Å3 was used for the SAXS model fit. cScattering too weak to analyze.
(A < 0) or a fractal model (Figure 6b). Fits to eq 6 gave again a fractal dimension D ≈ 2, but here the correlation length ξ was larger than the values in Table 2c, suggesting a larger cluster size for the cyclohexane-containing system. The Yukawa tail model also fit reasonably with a strongly attractive value for A ) -50kBT over a very short range. However, the Yukawa model for S(q) also yielded a rather large value for the shell thickness. Discussion Our small-angle X-ray scattering results are consistent with previous findings18,20 that substantial changes in the selfassembly of AOT, oil and water are caused by the presence of the protein R-lactalbumin. Analysis of our SAXS data showed that shifts in AOT phase behavior were accompanied by changes in phase structure morphology that are induced by the protein. Such overall shifts in phase structures upon addition of protein have not previously been reported and could indicate a particular ability of R-lactalbumin to interact with and modify surfactant monolayers. On the other hand, it is possible that other proteins could have similar effects but simply have not been studied under conditions where such changes would be evident. Within the extensive literature on protein/surfactant interactions, most studies have focused on low protein concentrations, generally in single-phase systems, where nanostructures are less likely to alter their structures. A few observations of enhanced water solubilization or phase separation upon protein addition have been reported,15,16,45,46 but the associated phase structures have yet to be investigated in detail. Thus, whether R-lactalbumin’s impact on surfactant self-assembly is typical of protein behavior in general remains an open question. A surprising aspect of the protein effects we have observed is that they take place at pH values well above the isoelectric point, where both the protein and surfactant have a net negative charge. Under such conditions segregative behavior might be expected, as is generally observed with mixtures of negative polyelectrolytes and anionic amphiphiles.47 It is for this reason that most studies of proteins with anionic surfactants investigate conditions where the protein is positively charged. In such cases of opposite charge, strong attraction between the molecules leads to precipitation at higher protein-to-surfactant ratios.15,46 The
presence of some electrostatic repulsion in the R-lactalbumin/ AOT system, on the other hand, may act to balance attractive interactions in such a way as to create the more interesting phase shifts we observe here. The nature of the attractive part of this balance is still unknown, with likely possibilities including hydrophobic effects and/or AOT attraction to localized positive residues on the protein.20 Rohloff et al.18 demonstrated that in AOT/isooctane/brine mixtures at equilibrium, the amount of water solubilized in the organic phase increased with increasing fraction of protein in the mixture. Results in Table 2a indicate that this additional water was taken up by microemulsion droplets that became nonspherical in the presence of protein, and contained larger core volumes because of their larger aspect ratios. The result was w/o microemulsion droplets with larger hydrodynamic radii, accounting for the lower diffusion coefficients measured previously by dynamic light scattering.20 This shift in phase behavior is made more complex by the fact that changes in water solubilization occurred alongside a decrease in surfactant concentration in the organic phase, also induced by the presence of protein. To gain more insight into these changes, we used our SAXS and compositional data to estimate component ratios and droplet properties in the two phases with results given in Tables 4 and 5. As the amount of total RLA added into the system increased, the partitioning of the protein shifted toward the aqueous phase. AOT likewise moved to the aqueous phase, to yield an approximately constant AOT/RLA ratio in the aqueous aggregates at all concentrations (Tables 4b and 5b). These results are consistent with the trend seen by Rohloff et al.18 in which protein, surfactant, and oil moved increasingly into the aqueous solvent as the overall RLA concentration increased. The remaining protein in the organic phase tended to increase the molar ratio of water to surfactant in that phase (wo) by enhancing the aspect ratio and core volume of the droplets, yielding a generally positive correlation between protein/AOT ratio in the organic and wo (Table 4a and Figure 7). These changes are induced with just 1-2 proteins contained per droplet in the w/o microemulsion. Figure 7 presents a comparison of the dimensions and volumes of the w/o droplets in the absence and presence of
R-Lactalbumin on Aerosol-OT Phase Structures
J. Phys. Chem. B, Vol. 112, No. 17, 2008 5389
TABLE 4: Influence of Protein and Surfactant Concentration on Component Ratios, Droplet Concentration, and Droplet Properties in Equivolume Mixtures of Isooctane and 0.1 M Brine a. Organic Phase [AOT]total [M]
[RLA]total [g/L]
0.025 0.025 0.025 0.1 0.1 0.1 0.2 0.2 0.2
0 4 7 0 4 7 0 4 7
[AOT]total [M]
[RLA]total [g/L]
0.025 0.025 0.1 0.1 0.2 0.2
4 7 4 7 4 7
# RLA per droplet
[droplet]org× 10-16 [#/cm3]
[RLA]org/ [AOT]org
3.1 2.0 0.3 6.7 2.8 2.9 10 8.0 7.2
7.4 × 10-4 5.8 × 10-4 1.2 × 10-3 1.3 × 10-3 1.0 × 10-3 1.5 × 10-3
0.4 1.1 2.2 2.1 1.3 2.1
b. Aqueous Phase
a
υcore [Å3]
[droplet]aq×10-16 [#/cm3]
# RLA per droplet
5.9 × 104 9.3 × 104 a 6.4 × 104 a 3.6 ×1 04
8-9 10-11 a 11-12 a 9-11
2 3 a 2 a ∼1.5
[AOT]aq/ [RLA]aq
[isooctane]aq/ [AOT]aq 1.6 1.6 1.2 1.3 0.9 1.1
34 36 36 37 40 33
Scattering too weak to analyze.
TABLE 5: Influence of Protein and Surfactant Concentration on Component Ratios, Droplet Concentration, and Droplet Properties in Equivolume Mixtures of Cyclohexane and 0.1 M Brine with 0.1 mol AOT Per L Cyclohexane Overall a. Organic Phase [RLA]total [g/L]
[RLA]org/ [AOT]org
[droplet]org× 10-16 [#/cm3]
# RLA per droplet
0 4 7
1.3 × 10-4 1.2 × 10-4
22 25 17
