J. Phys. Chem. 1994,98, 369-376
369
Characterization of a Nonionic Surfactant Reversed Micellar System for Enzyme Catalysis Claire F. Komives,' Daniel E. Osborne, and Alan J. Russell' Department of Chemical Engineering and Center for Biotechnology and Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261 Received: May 27, 1993; In Final Form: October 18, 1993"
Reversed micelles have merited attention as a medium for carrying out enzyme-catalyzed reactions because of their interesting properties as hosts for biocatalysis. In order to understand the function of enzymes in these systems, it is necessary to know certain fundamental physicochemical properties of the micelles and how they interact. To date, the majority of characterization studies have been on ionic surfactant mixtures, in spite of the generally accepted notion that these systems can have deleterious effects on solubilized enzymes. Poly(oxyethylene) sorbitan trioleate (Tween 8 5 ) is a nonionic surfactant which forms reversed micelles in hexane using isopropyl alcohol or ethylene glycol as a cosurfactant. Tween 85 is completely biodegradable and can solubilize larger volumes of water that many other ionic or nonionic surfactants. Characterization studies on reversed micelles of Tween 85 are presented in this paper, including dynamic light scattering, viscometry, and conductivity. Micelle sizes have been determined at different water contents, along with intermicellar interactions.
1. Introduction
Reversed micellar enzymology has become a popular route to carrying out a wide variety of reactions in an environment that is predominantly nonaqueous. The enzyme resides in finely dispersed water pools, which areencapsulated by surfactant, within a nonpolar solvent. The interest in using micelles as hosts for enzymes lies primarily in the advantages of the continuousorganic phase. Many industrially important reactions are limited to organic media because of solubility. Solutes in the organic phase of the micelle system are driven into the water pools as they are converted to product by the enzyme, and the products, in turn, partition back into the organic bulk phase. Reversed micelles to date have found a number of applications, including some commercial uses. The history and progress of the work can be found in a number of recent reviews.14 The surfactant most often used for these studies is anionic bis(2-ethylhexy1)sodium sulfosuccinate(AOT). Several studies have shown,however, that AOT can promote rapid degradation of encapsulated protein~.~-8 A characteristic feature of reactions in reversed micellar media with catalysts, such as enzymes, which reside in the water pools, is kinetic limitations due to unfavorable partitioning of organicsoluble substrates. As a result, an understanding of the structural makeup of the micelles is useful in order to describe these limitations. Knowledge of the volumes of the continuous oil and dispersed water phases, as well as the width of the surfactant layer can provide insight to the partitioning behavior of the substrate and, hence, the potential kinetic limitation of reaction rates. While extensive efforts to elucidate structural features of ionic micelle systems have been carried out, relatively few studies with nonionic detergents have been performed. Recent work has explored Brij surfactants (poly(oxyethy1ene) alkyl ether, CiEj)9-14 and ethoxylated nonylphenol (Triton X-100).15J6CiEjsurfactants are popular because they are available in highly pure form and it is possible to select the length of the nonpolar (Ci, alkyl chains) and polar (Ej, ethylene oxide chains) portions of the chain to create a system with known shape and size of the micelles. Further, no cosurfactant is required for solubilizing water in the alkane continuous phase. A principle assumption for many characterization experiments is that the micelles are spherical and do not depend on micelle-micelle interactions for stability, t Current address: ETH Hhggerberg HPT,Zurich, Switzerland. *Abstract published in Advance ACS A6srracrs. December IS, 1993.
which has been demonstrated under certain conditions with nonionic surfactants.9 Poly(oxyethy1ene)sorbitan trioleate (Tween 8 5 ) is a nonionic, organic soluble surfactant that has some interesting features for reversed micellar enzymology. Previous work has demonstrated the utility of Tween 85 micelles for protein extraction and activity.I7-l9 In these studies, it was shown that Tween 85 micelles can solubilizelarger volumes of water and protein using isopropyl alcohol as cosurfactant than other detergents, such as AOT20and Brij.13 Tween 85 doesnot havedeleteriouseffectson thestructure, function, and stability of either cytochrome c or subtilisin. Recently, the solubilizationand growth of whole cells in reversed micelles composed of Tween 85 and Span, a related surfactant, was dem0n~trated.I~ In addition, Tween 85 ( ~ $ 3 5per gallon) is biodegradable and has been successfully tested for use as an additive in fertilizers.*' To date, characterization work on micelles of Tween 85 has not been reported. The high molecular weight (1.84 kD) as well as molecular structure and shape (See Figure 1) (a sugar moiety and four polyoxyethylene chains in the head group) provides an unusual foundation for a micelle monomer. One can expect the characterization of such a system to be both interesting and challenging. Indeed, to arrive at the knowledge of micelle size, one must also understand the role of micellar interactions present in Tween 85 micelles. Given the unusual features of Tween 85 and the need for cosurfactant with this amphiphile, one must investigate micelle structure directly, rather than by inference from data obtained with different surfactants. We have chosen to study the degradation of organophosphorus pesticides with organophosphorus hydrolase as a model enzymecatalyzed reaction for our system.5 The enzyme catalyzes the degradation of pesticides such as Paraoxon and Parathion, as well as nerve gases such as Soman and Dimebu, which have low water solubility, to products which can be metabolized by bacteria found in soil. The solubilities of the pesticide substrates for organophosphorus hydrolase are significantly enhanced in the micelle system. Measurement of the diameter of the reversed micelles can be done routinely by a number of techniques. The method most often described is that of dynamic light scattering (DLS) or photon correlation spectroscopy, which gives a measure of the hydrodynamic radius from the translational diffusion coefficient. The polydispersity of the system can also be obtained from the measured intensity fluctuations,which are a result of the Brownian
0022-365419412098-0369$04.50/0 0 1994 American Chemical Society
Komives et al.
370 The Journal of Physical Chemistry, Vol. 98, No. I , 1994 2. Materials and Reagents
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Spectrophotometricgrade isopropylalcohol and ethyleneglycol were obtained from Mallinckrodt Chemical Co. (Ann Arbor, MI); pnitrophenol(98%) was obtained from Aldrich Chemical Co. (Milwaukee, WI); hexane and Tween 85 are from Sigma Chemical Co. (St. Louis, MO); paraoxon was either purchased from Sigma (stated 90%pure) or synthesized from 4-nitrophenyl phosphorodichloridate(Fluka Chemical Co., Ronkonkoma, NY) and ethanol (punctilious, dehydrated), according to the method of Steurbauet et al.,27and purified on a silica gel column with chloroforom (SpectrophotometricGrade, Mallinckrodt Chemical Co.). IH NMR and mass spectrometryof the purified substrates confirmed their structures and showed purity of approximately 98%. Water used in the experiments was distilled and deionized (Milli-Q, Millipore Inc., Bedford, MA) and Trizma-base was purchased from Fisher (Pittsburgh, PA).
0
3. Metbods: Dynamic Light Scattering
;
Figure 1. Structure of Tween 85. Hatched balls and dotted balls represent
carbon and oxygen atoms, respectively. Hydrogen atoms are not shown for simplicity. The length of the three extended ethylene oxide chains may vary, with the total number of ethylene oxide groups being approximately 20.
motion of the micelles. In a polydisperse system, the observed diffusion coefficient can yield a measure of the average micelle size. A description of the general theory of DLS follows, as well as a discussion of micelle interactions, which were apparent in all of the experimental techniques used. Viscosity measurementscan also lead to a deeper understanding of micelle systems. The volume of the aqueous pool, as well as the length of the surfactant tail can be determined using viscometry, which when combined with the total size calculated from DLS measurementsyields the aggregation number, fi, which is the number of surfactant molecules surrounding a micelle.t2 From measurements of intrinsic viscosity, the extent of micelle interactions can be determined from the Huggins coefficient.9 Another important factor which can govern reaction rates in reversed micellar systems is the micelle exchange rate.23~2~ However, this continuous process of collision and stabilization, which results in the exchange of micelle contents, is limiting only for restrictions with substrates that are predominantly water soluble.2s The pesticides used in our studies are predominantly soluble in the organic continuous phase and Tween 85 layer. Typically, exchange rates are measured by time-resolved fluorescence using water-soluble probe molecules.12J6J7 In the system we describe, these studies were complicated with Tween 85 reversed micelles because of significant fluorescence of the surfactant itself, which obliterates the fluorescenceof the probe molecules. Nevertheless, onecan assume that the exchange rates of Tween 85 micelles are not rate limiting, since Fletcher and Horsup found exchange rates for three polyoxyethylene ether reversed micelle systems ranging from lo9 L mol-' s-1 for in hexane to 1Olo L mol-' s-I for CsE3 in decane.12 Thus, for the Tween 85 system, it is reasonable that the enzyme-catalyzed reaction rates are limited by substrate partitioning. In this paper, the sizes of Tween 85 reversed micelles are reported at various water contents, as well as a description of the intermicellarinteractions, which play a role in the stability of the micelle system. The data generated are useful to enable a deeper understanding of the extensive kinetic studies performed with organophosphorus hydrolase in this micelle system.5
Dynamic light scatteringcan be used to determinethe diffusion coefficient of particles in suspension from fluctuations of light intensity detected at a particular angle from the incident radiation through a sample. The intensity autocorrelation function, C(7), describes these fluctuations: where Iis the intensity of scattered light, q is the scatteringvector, t is time, and 7 the delay time. The scattering vector is a simple function of system parameters: q = (4?m/Ao) sin(t9/2)
with n the refractive index of the solution, &, the incident wavelength, and 8 thescatteringangle. C(7)can be approximated as a sum of exponentials in the case of light scattered from a suspension of polydisperse particles.29 The distribution function of particlesizescan be obtained from the intensity autocorrelation function by the method of c u m ~ l a n t s . ~If~ the micelles are spherical, the hydrodynamic radius, r ~can , be found from the Stokes-Einstein relation
(3) where ke is the Boltzmann constant, Tis the absolute temperature, and 7 is the viscosity of the continuous phase. The intensity autocorrelation function can be defined in terms of static and dynamic structure factors, which consider direct and hydrodynamic interactions. At low concentrations, these interactions have less impact on particle diffusion. The diffusion coefficient, which can be measured with DLS, is a direct function of micelle volume fraction, 4, and can be written
Dc = Do(l + A#
+ O(#*)]
(4) where Dois the diffusion coefficient in the absence of interactions, at infinite dilution, A is a combination of the hydrodynamic virial coefficient, A,, and a frictional term, At,
A=x,+A, 0 represents an order of 42. The terms for 49, 4', etc. are not shown for simplicity. Various models have been used to predict A, A,, and Xf. The effects of thermodynamic interactions among the particles in short range have been explored in a model by Batchelor.30 In short, attractive interactions between the particles will result in a negative value for A, which becomes more negative with increasing attractions. In fact, if the attractionsare strong enough, Dc may be less than Do. It should be noted that the equations described above are valid for low 4 (4 < 0.03).
The Journal of Physical Chemistry, Vol. 98, No. I , 1994 371
Reversed Micellar System for Enzyme Catalysis Dynamiclight scatteringis used todetermine the hydrodynamic diffusion coefficient of micelles. Because particle interactions affect the diffusion coefficient in a manner that is likely distinct from their effect on micelle size, it is necessary to evaluate the diffusion coefficient in the limit of infinite dilution of micelles. From eq 4, a plot of apparent diffusion coefficient plotted with micelle volume fraction can yield Dor as well as A. The micelle volume fraction can be estimated from the volume of surfactant and water added to the solution. This method is clearly an estimation because it neglects volume changes due to mixing and the formation of micelles, but it is reasonable for the purposes of the calculation. The micelle volume fraction is varied by diluting the solution with thecontinuousphase. Theobjectiveof thedilution procedure is to maintain a constant water/surfactant ratio of the droplets. It is important to consider that in reversed micelles, unlike normal micelles, the critical microemulsion concentration (cpc) at which micelles begin to form is on the order of millimoles per liter. This value also represents the concentration of surfactant monomers in a micelle solution. As the DLS experiments must be carried out at low concentrations, the continuous phase solvent should contain the surfactant at the cpc so as not to require removal of surfactant from the micelles to maintain the solutioneq~ilibrium.~ The cpc can be found most easily by comparing the maximum soluble water in a micelle solution with the surfactant concentration. This method yields the cpc measured at the phase boundary, and assumes that the droplet composition within the single-phase microemulsion domain is the same. Once the cpc is known, the water content of the micelles, W,, can be expressed as
w,= [Tween 851[HPI - [Tween 85],,]
(6)
The Wl of a dilution series of micelles is constant. 3.1. Micelle Preparation. Solutionsof micelles were prepared in the following manner: Tween 85 and isopropyl alcohol were mixed in apprcpriate concentrationsand hexane and buffer were added. Water content of the solutions was checked with KarlFisher Titration (Fisher CoulomaticTitrimeter Model 447, Fisher Scientific, Pittsburgh, PA). Samples 10 pL in volume were injected into the titrator, which uses an electrochemicalmethod to determine the micrograms of water in the injected sample. 3.2. Dynunic Ligbt Scattering and Conductivity. Dynamic light scattering was carried out using a Wyatt Technology Dawn F fixed multiangle unit (Wyatt Technology, Inc., Santa Barbara, CA). The light source is a helium cadmium laser operating at 441.6 nm with power stabilization (Liconix model 4210N Laser, Liconix 5OSA power stabilizer, Liconix, Inc., CA). A Langley Ford autocorrelator was used to process the signal (Model 1096, LFI, Amherst MA). Micelles were prepared as described above and filtered (Gelman acrodiscs, teflon, 0.45-pm pore size). Refractive indices were measured for all solutions with a Bausch and Lomb refractometer. Viscosity of the continuous phase was taken as 0.0027 P. For all experiments,the scattering angle was 3 5 O . Conductivity was measured with a YSI Model 35 conductance meter. We found the diffusion coefficients at infinite dilution by extrapolating plots of measured diffusion coefficients versus micelle volume fraction. The micelle volume fractions were calculated from the volume of Tween 85 (10 wt %) in initial solution, density = 1.055 g/mL) and water (determined with Karl Fisher Titration) with isopropyl alcohol (8 v 96). The cpc was subtracted from the surfactant concentration to approximate the concentration of surfactant employed at the interface of micelles. The solution was diluted with a solution of hexane containing isopropyl alcohol (8 v %) and Tween 85 at the cpc (10.5 mM, 24.5 "C). The dilution curves were fit to eq 4 using nonlinear regression with the Marquardt algorithm (Kaleida-
graph). The conductivity of each solution was measured to ensure that no marked changes in micelle structure were taking place across the range of micelle volume fractions studied.
4. Viscosity Theory and Equipment Measurement of viscosity can give the volume of the aqueous po01.~ A correlationwhich relates thevolume fractionof solution taken up by a solute (4) to the specific viscosity of the solution ( v , ~ was ) developed empirically by Guth and S i ~ n h (eq a ~ 7).The ~ qsp= 2.54
+ 14.1rf~~
specific viscosity is a function of the solution ( (q,lv) viscosities
(7) ~ ~ and 1 ~ solvent )
= (lIsoln/9solJ - 1 This correlation was an extension of the Einstein equation %p
= 2.54
(9) which is true for ideal solutions of noninteracting spheres. The extended relation was tested by Day et al. for thecharacterization of reversed micelles with I$ values up to about 0.10. From the measured micelle volume and volume fraction, the number of surfactant molecules per micelle, or aggregation number, It, can be found 9,
4=
N[surf.] V, It
where N is Avagadro's number and [surf.] is the concentration of surfactant. Viscosity can also be used to determine micelle shape and the extent of micelle interaction~.~,33 Specific viscosity is related to the concentrationof suspended particles, and, at infinite dilution, they are proportional,as expressed by eq 9. The reduced viscosity (aP/C, where C is the weight concentration of micelles) under these conditions is independent of concentration. The intrinsic viscosity, 7, is the value of the reduced viscosity at zero concentration. The relationship between the reduced viscosity and concentrationcan be used to give informationabout the shape and degree of solvation of the particles by applying the viscosity data to the equation
where kH is the Huggins coefficient. The equation was developed by Guth and Gold33and explored further by Simha." Application of the equation to polymer suspensions33and nonionic micelles9 has demonstrated its utility. The Huggins coefficient has been described by Batchelor as a key parameter to characterize particle interactions.3'J Tanford has reported a value of k" for hard spheres to be 2.0, while Batchelor determined it to be 1.03. For both repulsive and attractive hard-sphere interactions the value is increased. Labile molecules have values of k~ less than 1.0. The intrinsic viscosity is dependent on the shape of the particles as well as the degree of solvation.32 The relationshipincludes the specific volume of the micelles, V,, as well as that of the solvent,
vs,
(12) 6 is the weight of solvent per gram of particles and Y is a shape factor. It has been reported that neither Y nor b are experimentally determinable. Typically, the approach to analyze the data is to assume first that the particles are hard spheres giving Y a value of 2.5. With this assumption,Aveyard et al. determined that less than 10 solvent molecules per surfactant molecule were solvating spherical micelles of C12E5. One can assume that a large surfactant such as Tween 85 would have more associated solvent II=u(Vm+SVs)
Komives et al.
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o.90
t
95
i
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1
a
im f” lo
5
0
0
2
S wt%Tween86 4
S
lo
Figure 2. Determination of critical microemulsion concentrationat the phase bounary. The maximum water solubilized 0, axis) in solutions of Tween 85 of different concentration (x axis) with isopropyl alcohol (8 v 96) in hexane is extrapolated to show the surfactant concentration at which no water is solubilized. (R= 0.9986.) Temperature was 24.5 f 0.2 o c .
molecules. The values of Aveyard and colleagues for q were around 4-5 for both normal and reversed spherical micelles. Viscosity measurements were made with a Cannon Ubhelohde viscometer D71. Temperature was maintained to f0.1 O C with a circulating water bath. The viscosity was measured by comparing the flow time of the microemulsion through the capillary with that of thecontinuous phase. The continuous phase was taken as hexane with 8% (v) isopropyl alcohol and Tween 85 at the cpc. For the 35 O C measurements, the microemulsion viscosity was compared with hexane containing 8 v % isopropyl alcohol. For flow times less than 500 s, the error in the time measurements was less than f l s and for higher flow times the error was less than f 10 s. 4.1. Determination of Critical Microemulsion Concentration (cpc). The critical microemulsion concentration of Tween 85 at 24.5 OC was measured a t the phase boundary as described by Aveyard et a1.9 Water was added dropwise to mixtures of different surfactant concentrations with isopropyl alcohol (8 v %) and hexane until the solutions first appeared cloudy. The maximum water volume solubilized in solutions of Tween 85 and isopropyl alcohol in hexane was plotted against the surfactant concentration (Figure 2). The intersection of the line with the x axis yields the cpc, which is taken as the concentration of Tween 8 5 in the continuous phase at 25 OC. 4.2. Bulk Partition Coefficients of Pesticides and Isopropyl Alcohol. The partition coefficient of paraoxon between hexane and water (2.33)with 8% isopropyl alcohol was also determined by HPLC (Hewlett Packard 1090, Waldbronn, FRG). The procedure was used as described by DeSchryver and de Reu.35 Gas chromatography (GC) was used to measure the partition coefficient of isopropyl alcohol between water and hexane (24.0). A Hewlett Packard Series I1 gas chromatograph (5890)was used with a column (6-ft X l/s-in. stainless steel, 60/80 Carbopack B/l% SP 1000, Supelco, Inc., PA) and FID detector were used. The oven, injector, and detector temperatures were 140,200,and
0
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0.1
0.16
0.2
036
0.9
W) Figure 3. Determinationof intrinsic viscositiesand Huggins coefficients from viscometry. Specific viscosity, vlP was measured at 35.00 i 0.01 OC of micelle solutions composedof Tween 85 (13 wt % in initial solution, diluted with isopropylalcohol (8 v 8 )in hexane), with isopropyl alcohol (8 v I) in hexane. The concentration of micelles plotted on the x axis is determined as the weight of micelles in g/mL microemulsion. rMi-I
200 O C , respectively. The flow rate of the carrier gas, helium, was 30 mL/min. Concentrations of isopropyl alcohol in the hexane phase after vigorous mixing and settling of the hexane/ water mixture containing alcohol were measured (0.2-pL injections), from which the partition coefficient was calculated. 5. Results and Discussion 5.1. Determination of Critical Microemulsion Concentration (cpc). The plot of maximum water solubilized vs surfactant concentration is shown in Figure 2. From the x-intercept, the cpc can be determined directly to be 1.92f 0.20wt % surfactant (10.5mM). This value is assumed to be the surfactant monomer concentration in the microemulsion. Interestingly, it is similar to the surfactant concentration in the oil phase of a two-phase system with microemulsion in equilibrium with water-in-oil (w/o) microemulsion. The cpc is used to describe the water content of the micelles in terms of W1,as described by eq 6. It is important to use W, to describe the water content, as the cpc is often a significant portion of the total surfactant in nonionic systems.9 It is necessary to assume that the cpc does not change with total surfactant Concentration. The straight line in Figure 2 is a good indication that for the range of surfactant concentrations shown on the plot, the assumption holds. 5.2. Viscosity. As described above, viscosity experiments are useful in elucidating important physicochemical information about reversed micelles and how they interact. By following eq 11, plots of q,,/Cversus C a t 35 OC and fivedifferent water contents are shown in Figure 3. The intrinsic viscosity is found as the intercept with the y axis. The values of intrinsic viscosity and the Huggins coefficients found at the different Wl values are listed in Table 1. The intrinsic viscosity is dependent on both the degree of solvation and the sphericity of the micelles, however no appropriate
Reversed Micellar System for Enzyme Catalysis
The Journal of Physical Chemistry, Vol. 98, No. 1, 1994 373
TABLE 1: Intrinsic Viscosities and Huggins Coefficients at Various W,Values' Wl intrinsic viscosity (1) Huggins coefficient ( k ~ ) 95.7 82.1 52.8 35.6 25.3
* *
3.75 0.13 3.50 0.48 4.05 0.57 5.61 t 2.6 6.36 t 0.16
2.40 0.25 3.87 1.36 3.98 A 1.36 3.06 t 2.9 2.64 i 0.15
a Values detrmined using q 13 from the slope and intercept of plots in Figure 3.
0.08
1
f i
models for reversed micelles have been developed to make a quantitative estimation. Some comparisons to polymers and proteins can suggest an interpretation for the Tween 85 system." The intrinsic viscosity of globular proteins ranges from 3.3 to 4.0 mL/gram, while structural proteins like fibrinogen and collagen range from 27 to 1150 mL/gram. Flexible polymers with 0.04 molecular weight of 10 000 kD have values of intrinsic viscosity around 10mL/gram. As the effective molecular weight of Tween 85 reversed micelles is significantly higher than 10 000 kD, the low value of the intrinsic viscosities of the micelle solutions containing less than about 8% water indicate a spheroid structure for the micelles and rule out coiled or rodlike structures. In addition, the values of intrinsic viscosity are comparable with those of Aveyard et al. for spherical reversed micelles of C I ~ E ~ . ~ 0.02 0.00 o m m a m ~ e o m Also included in Table 1 are the values for the Huggins coefficients of the micelles. By taking the value of kH for hard w ([water] I (ITweensal Imv-aI,) ) spheres to be 2.0 and that of flexible molecules as less than one, Figure 4. Micelle volume fractions were determined using viscometry once again the values obtained for our system are consistent with data. Specific viscosity of micelle solutions composed of 5 wt 96 Tween discrete spherical moieties subject to interparticle interactions. 85 with different water contents in isopropylalcohol (8 v W)and hexane The extent of interactions is also apparent in the dynamic light (upper plot) (R= 0.9833 and 0.9975 for lower and higher portions of scattering dilution curves. line), solutions with Tween 85 (13 wt 56) (middle plot) (R= 0.9886 and 0.9961 for 1 ower and higher portions of line), and with Tween 85 (3.5 A plot of micelle volume fractions calculated from specific wt %) (lower plot, R = 0.9925). Temperature was maintained at 24.50 viscosity data according to eq 7 as a function of W1 is shown in k 0.01 O C with a water bath. Water contentswere determinedwith Karl Figure 4. The surfactant concentration of 5 wt 76 was chosen for Fisher titration. this experiment in order to keep the 4 values below 0.10, as the equation is not valid for more concentrated solutions. Also, an 0.042. This value is consistent with micelle swelling due to important consideration in choosing an appropriate surfactant associated solvent molecules. composition is to be able to examine viscosity changes upon full 5.3. Dynamic Light Scattering and Conductivity. In order to hydration of the micelle head groups. A swelled micelle has the determinemicelle diameters, plots of apparent diffusion coefficient head groups of the surfactant fully hydrated and contains vs micelle volume fraction (Figure 5 ) were extrapolated to give additional bulk water. Solutions with surfactant concentrations the diffusion coefficients in the limit of infinite dilution. These below 5 wt % were unable to form swollen micelles, as shown in values are used to calculate the absolute diameters of the micelles, the curve for 3.5 wt % Tween 85. The two regions of the 5 wt assuming that a t infinite dilution the micellar interactions do not % plot with different slopes undoubtedly represent the region of affect micelle diffusion. Micelle volume fraction is calculated on hydration of the head groups a t the lower water contents, and the the basis of the volumes of surfactant and water, containing steeper slope indicates hydration of the micelles at higher water isopropyl alcohol. W1 is constant in these plots, since the solutions contents. The change in slope is consistent with the results of were diluted with a solution containing the same composition as Day et al. for AOT micelles.22 The micelles formed with AOT, the continuous phase. Micelle solutions at higher water contents however, are fully hydrated at a W,of 3, while the Tween 85 could not be diluted sufficiently without phase separation, and micelles begin to swell a t a W1 of -30. This is reasonable thus data is not reported a t these conditions. At low values of considering the larger size of Tween 85 head groups. The 4, the diffusion coefficient increases with decreasing 4. This molecular weight of the Tween 85 head group is 1.12 kD, whereas would be attributed either to a decrease in micelle diameter or that of AOT is 0.212. Since AOT is an ionic surfactant, the increased mobility of the micelles. Equation 4 does not account molecular weight alone cannot provide a basis for a direct for changes in micelle size, thus, the diffusion coefficient increases comparison of the point of hydration. The W,at which micelles because interactions are less significant as the concentration is are fully hydrated has significance, in that above that value, the decreased. As the volume fraction, and hence, the surfactant water in the micelles can be considered to have the properties of concentration, continues to increase, the diffusion coefficient a bulk aqueous phase. increases with increasing concentration. This is consistent with previous results that higher W,values are obtained at lower Tween Extrapolation of the plot in Figure 4 leads to important 85 concentrations for micelles in equilibrium with an excess water information about the composition of the micelle droplets. phase.'* Extrapolation to zero water content gives an estimation of the The parabolic relationship between apparent diffusion coefvolume fraction of micelles, +,, containing no water. It was ficient and micelle volume fraction, predicted by eq 4, was seen impossible to remove all the water from the system to make an at all seven water contents studied. From the fit of eq 4 to the actual measurement of a dry micelle. The volume of the micelles data, thevalueof X is negative. Following themodelof Batchelor, calculated from the surfactant volume accounting for the monomer this indicates that the micelles are subject toattractiveinteractions. concentration as the cpc, along with the dissolved isopropyl alcohol A plot of the X values obtained at different water contents is gives a volume fraction of 0.033, while the extrapolated value is
i
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1 ~
~
m
374 The Journal of Physical Chemistry, Vol. 98, No. 1. 1994 12
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1
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rd61
Komives et al.
3
t
\
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1
t
0 1
-le
t
1
t
0 0
0.02
0.04
0.08
0.08
0.1
0.12
0.14
C&Ul&dVohrmeEdMhdb
Figure 5. Effect of micelle concentration on diffusion coefficients measured with DLS. The scattering angle was 35" and temperature was 24 1 OC. Equation 4 was used to fit the data by nonlinear regression (Kaleidagraph). Micelle solutions composed of Tween 85 (10 wt %) with added water, isopropyl alcohol (8 v W )and hexane were diluted with a solutioncomposed of Tween 85 (2 wt a),isopropyl alcohol (8 v %) and hexane.
*
shown in Figure 6. The value of X becomes more negative with increasing water content, suggesting that as the micelles become larger, the attractions become more intense. It has been shown that micelles in transition from dispersed droplets to bicontinuous structures are subject to clustering, consequent to the induction of strong attractiveinteractions.37 Above 8% water in the micelles, the formation of bicontinuous38 or intercontiguous39structures is likely, as discussed in a separate paper,5 from phase behavior and conductivity measurements at these conditions. A plot of the measured infinite dilution diffusion coefficients at the different water contents measured is shown in Figure 7. An extrapolation of this plot gives the diffusion coefficient of a dry micelle,whichcan beused with theviscosity data todetermine structural information. From the extrapolation, the diffusion coefficient of a dry micelle was determined to be 1.58 X 10-6 f 1.9 X le7cm2/s. Using the Stokes-Einstein expression in eq 3, this corresponds to a radius of 5.12 nm. An AOT micelle, for comparison,at W,of 3.36had a diameter measured at 1.69nm.22 Considering the molecular weight of AOT is 0.444 kD,while that of Tween 85 is 1.836 kD,the larger size of the Tween 85 micelle is appropriate. The conductivity of the micelle dilutions at different water contents was measured to detect any major structural changes. For instance, a reversion from water/oil to oil/water microemulsions could be viewed by a sharp rise in conductivity with increasing micelle volume fractjon. Pkofdata fmmthree water contents are shown in Figure 1. A a "sincram is seen in the conductivity, even at low-volwrre f r a c t h , kginning at the conductivity of hexane, which Is 0.06 pMHO's. The low value of the conductivityconfirms the estimation of approaching infinite dilution of micelles. The steady rise agrees with previous studies of the conductivity of micelles employing cosurfactants with different chain lengths. The percolatiue behavior of the systems, that is, the significant increase in conductivity over a
t
l6
t
1
t 01
0
4
I
P
w,
I
6
I
40 W 8) 100 ( T ~ S E ~ ~ I I ~ ~ S I - ~ V - ~ ~ ~ )
Figure 7. Effect of water content on the diffusion cocfficieniof micelles meawed as dotermined from DLS data (R= 0.66). Absolute diffusion wfftoisatsshswa were found from the fit of eq 4 to DLS results. The temperature was 24 1 OC.
*
short increase in volume fraction, is expected to be more apparent when the cosurfactant is a short-chain alcohol, as is isopropyl alcohol.14 The previously described explanation for this behavior is that the transition from water/oil to oil/water microemulsion is gradual and cannot be seen in macroscopic changes in the
The Journal of Physical Chemistry, Vol. 98, No. 1, 1994 315
Reversed Micellar System for Enzyme Catalysis 1
1 0
w,
I
and the concentration of micelles is found as follows:
11.m
[Tween 851 - [Tween 851,
W,= 21.61
12
-
[MI=
h
1
0.8
:
0
0
0.6
(13)
In order to calculate the volume of the water pools without knowing the length of a Tween 85 molecule, we have to make several approximations. The volume of the water pool can be found by subtracting the volume of the surfactant layer from the total volume. The volume of the surfactant coat can be estimated from the volume of a micelle with no added water. The radius of such a micelle, from dynamic light scattering, is 5.34 nm, corresponding to a volume of 6.38 X 10-19 cm3 and has an agqregation number of 130. In order to estimate the volume of the surfactant coat for larger micelles, the followingapproximation is made:
1-
8
2r
CI
V, = V,- [(6.38 X 10-'9(2r/130)]
0.4
-
0.2
-
0
0
0,
I
,
,
I
I
(14) From the estimated volumes of the micelle water cores, the radii are calculated. The approximation assumes that the volume of a surfactant molecule in a micelle, including the volumes of associated hexane, isopropyl alcohol and water in the surfactant coat, is constant. While it is possible that the surfactant coat width may change with W1,these numbers represent a reasonable approximation for volume and aggregation number. Clearly, the highest error in the calculations is in the r, values as a result of this approximation. The values of the water pool radii determined for our system are similar to those for other nonionic surfactants.43 6. Conclusion
20.7 25.3 33.7 40.7 47.1 53.0 61.9
5.78 5.95 6.30 6.61 6.93 7.26 7.80
0.060 0.064 0.073 0.083 0.093 0.101 0.115
8.09 8.82 10.47 12.10 13.94 16.03 19.88
132 135 142 143 148 155 170
1.24 1.21 1.15 1.14 1.10
1.05 0.96
3.36 3.73 4.36 4.95 5.42 5.86 6.51
Micelle radii ( r ~ and ) volumes (V,) were calculated from dynamic light scatteringdata;volumefractions (#) were calculatedfrom viscometry data; aggregation numbers (R), micelle concentrations ([MI) and water core radii (rw) were calculated using eqs 12, 15, and 16, respectively. behavior of the solution. Thus, the solutions remain transparent and stable throughout the phase inversion. It has been suggested that percolation in a micelle system results when attractive forces between the particles are ~ignificant.~' One possibility that agrees with significantmicelle interactions is that large micelles exchange between clusters and isolated moieties on a very short time scale, creating an effective continuous interfacial layer, as well as conductive channels, as described previously by O l s ~ o n . ~ l We have also determined the size of the micelle water pools and aggregation numbers from the dynamic light scattering and viscosity data. The values calculated for Tween 8 5 micelles are listed in Table 2. The micelle radii are determined from the data in Figure 7. These values are for spherical micelles, which is an inherent assumption in theStokes-Einstein relation. The 4values are calculated from the lines fit to the data in Figure 4. For these calculations it was assumed that the volume of micelles a t a given water content is not dependent on the concentration of the micelles. The micelle volumes are calculated from the micelle radii in column two, the aggregation numbers are calculated using eq 10,
Tween 85 is a nonionic surfactant that has a number of interesting properties for forming water/oil microemulsions for use in biocatalysis with enzymes. Characterizationof the micelles in terms of size and extent of interactions has been carried out in order to better understand the action of enzymes in the system. Dynamic light scattering along with viscometry was successful in determining micelle size, as well as the interactions between the micelles. As the micelle water content varies from Wl = 20.7 to 61.9,the micelle radius increases from 5.8 to 7.8 nm. At the same time, the micelle volume fraction changes from 0.06 to 0.12,the internal radius changes from 3.4 to 6.5 nm. The data represent theonly physicaldataavailableon theTween 85 reversed micelle system and as such can significantly increase our ability to study enzyme catalysis in this system. Conductivity was also a useful tool to demonstrate the presence of reversed micelles in the system.
Acknowledgment. This work has been funded by PPG, Union Carbide Corporation, and a National Science Foundation PresidentialYoung Investigator Award t0A.J.R. (BCS 9057312). We thank Dr. G. Ayala, Dr. D. Farcasiu, and Dr. E. Beckman for their considerable help during the preparation of this manuscript. Finally, we thank Mr. B. Linton for helping to produce the Tween 85 structure (Figure 1). References and Notes (1) Luisi, P. L.; Magid, L. J. CRCCrir. Rev. Biochem. 1986.20 (4), 409. (2) Martinek, K.; Levashov,A. V.; Klyachko, N. L.; Pantin,V.I.; Berezin, I. V. Biochim. Biophys. Acra 1981,657, 211. (3) Luisi, P.L.; Steinmann-Hofmann,B. Merhods Enzymol. 1987,136, 188. (4)
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376 The Journal of Physical Chemistry, Vol. 98, No. 1, 1994
Additions and Corrections
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ADDITIONS AND CORRECTIONS
1993, Volume 97 Hironobu Kunieda,' Kazuyoshi Nakamura, Ulf Olsson, and BjSrn Lindman: Spontaneous Formation of Reverse Vesicles. Page 9528. The vertical axis of Figure 8 should read as follows: D X 10l0/mZs-l.
