J. Phys. Chem. 1993,97,5752-5761
5752
Temperature and Gas Pressure Induced Microstructural Changes in AOT Water-in-Oil Microemulsions: Characterization through Electron Paramagnetic Resonance Spectroscopy N. S. Kommareddi,? V. T. John,'*t Y. Y. Waguespack,*vs and G. L. McPherson'*t Department of Chemical Engineering and Department of Chemistry, Tulane University, New Orleans, Louisiana 70118 Received: October 27,1992;In Final Form: February 11, I993
EPR spectra of a variety of nitroxide spin labels have been used to characterize microstructural changes in AOT water-in-oil microemulsions at conditions approaching critical phase transitions. Of particular interest is a pressure-induced cloud point, where the dissolution of a light hydrocarbon gas into the liquid solvent (isooctane) reduces the solvent density and leads to micellar instability at a critical density. The pressure approach to a cloud point is compared to the temperature approach. A variety of spin label probes are used to report changes in the aqueous core, the interfacial layer, the micellar tail region, and the intermicellar bulk isooctane region. Both temperature and gas pressure increases result in a decrease of the rigidity (ordering) in the immediate vicinity of the surfactant layer in the interfacial region. The surfactant tails region exhibits maximum sensitivity toward both temperature and gas pressure variations.
Introduction Surfactants dissolved in apolar solvents have the capability of solubilizing large amounts of water. The anionic surfactant sodium bis(2-ethylhexyl) sulfosuccinate (AOT) has two alkyl tails with small side chains and a relatively small hydrophilic head group, giving it the shape of a truncated cone. Thus AOT can pack well at the micellar interface and is well suited for the formation of a water-in-oil microemulsion without the need for added cosurfactant. The size of these dispersed aggregates has been shown to be dependent upon the molar ratio of water to surfactant, w,.I These opticallyclear water-in-oil microemulsions are traditionally, although somewhat incorrectly, referred to as reversed micelles. Reversed micellar systemshave been the subject of intense investigation due to their potential applications in a variety of fields including nanobrticle synthesis,2Jbiocatalysis,4-6 bio~eparations,~-9 and biomembrane mimetics. l o Electron paramagnetic resonance (EPR) spectroscopyemploys stablenitroxidefree radicals as probemolecules. TheEPRspectra of these compounds compriseof three well-resolvedlines' arising from the hyperfine coupling of the unpaired electron with the nitrogen nucleus. Specialized spin labels have been synthesized so that their paramagnetic groups can report from different environments of the microemulsion droplet, the water core, the interface, and the tail regions.12J3 Thus EPR is an excellent tool to study the physiochemical and microstructural properties of these systems, and the technique has been used in earlier research to understand the nature of intramicellar wafer1*J420 and the fluidity of the interfacial and tail regions.I2 In this paper, we describe EPR studies of isooctane-based reversed micellar systems when pressurized with a light hydrocarbon gas, specificallyethylene. Our reason for conductingthese studies is perhaps best described by examining Figure 1, which schematizes the phase behavior of pressurized water-in-oil microemulsions. If we start with the AOT-water-isooctane system, we have a macroscopically single-phase microemulsion as designated by point a in the figure. When this system is pressurized with ethylene,the dissolutionof the gas into the solvent leads to a solvent swelling and a decrease in solvent density. Thus the trajectory a- -b is followed upon pressurization. At a critical pressure/density of 0.5 X IO3 kgm-3, the micelles tend to lose
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' Department of Chemical Engineering. * Department of Chemistry.
5 Present address: Department of Chemistry, Fairfield University, CT.
0022-3654/93/2097-5752$04.00/0
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Figure 1. Schematic representation of the phase behavior of reversed micelles under pressure. The upper phase transition occurs at very high density and represents water squeeze-out; it is inconsequential to this
work. stability and the system deteriorates into a two-phase system, with the micellar constituents being precipitated out of solution (point c). Thus, this is a pressure-induced cloud point and is a phenomenon that can be exploited to recover intramicellar solutes such as proteins, nanoparticles, etc21 An accompanying phenomenon that occurs during pressurization at lower temperatures (273-278 K, for example) is the phenomenon of clathrate formation.22 Here, ethylene clathrates formed from the gas and the intramicellar water drop out of solution, thus removing intramicellar water and decreasing the micelle size.23 It is of fundamental and applied interest to attempt an understanding of micelle behavior approaching the pressureinduced cloud point. Recent studies have shown that the reverse trajectory c- - -b- -a can also be followed.2426 That is, when a light hydrocarbon gas such as ethane or propane is contacted with AOT and water and pressurized, the AOT and water are incorporated into reversed micelles when the gas density exceeds a critical density, which again turns out to be about 0.5 X lo-' kg m-3.27 In such systems it has been shown, through smallangle neutron scattering, that micellar clusters are prevalentjust above the critical density2$ as a result of enhanced droplet interactions in the reduced density fluid. Since it is reasonable to assume that the phenomenon of pressurizingisooctanereversed micelles with ethylene (trajectory a- -b- - -c) leads toessentially the same physiochemical mechanisms near the phase-split condition, it is worthwhile to determine if microstructural changes with pressure can be followed through EPR. Thus, we report our EPR studies using a variety of nitroxide spin labels in reversed micelles. Earlier work by Haering et al.12 established a basis for our research in terms of the choice of the
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0 1993 American Chemical Society
Changes in AOT Water-in-Oil Microemulsions spin labels and spectra conducted at ambient conditions. However, in carrying out these background experiments, a considerable improvement in spectral resolution was noticed when the system was free of dissolved oxygen (the concept of oxygen broadening), a factor not well addressed in the earlier publication.12 Hence we also report the results of EPR studies of reversed micelles at ambient conditions to point out microstructural changes as the water content is varied. Finally, we have also examined the effect of temperature on the micellar microstructure. Previous EPR investigations of temperature effects in reversed micelles have been aimed at probing the polarityI6 and rotational dynamic@ of spin labels solubilized in the aqueous core and the partitioningI7J8 of the spin label between the aqueous core and the surfactant shell. Rigidity of the micellar interface and the dynamics of the surfactant tail region need to be examined from the point of view of temperature-dependent microemulsion behavior. With increasing temperature, a cloud point is reached where the system splits into two phases.Z8 Again, the approach to the phase boundary is characterized by an increase in intermicellar interactiow2*and it is of interest to examine the dynamics of the spin labels in the interfacial and tail region as the droplet interactions are modified through temperature. Our objective is to compare the approach to the pressureinduced cloud point to the temperature-induced cloud point through EPR characterizations of micelle microstructure. Using EPR spin-labeling techniques, the fluidity of the interface is quantified and its response to changing density, temperature, etc. is investigated. Of particular interest to us is the effect of these environmental variations on the fluidity of the interfacial layer, the rotational dynamics of the reporter group, and the polarity of different regions of the reversed micelle.
The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 5753
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F e r e 2. Chemical structures of nitroxide spin labels used to dope AOT reversed micelles shown alongside the surfactant molecule. (1) AOT (2) 5-doxylstearic acid, (3) CATl6, and (4) DSTA. The angle between the diffusion axis (2’)and the magnetic axis (2”’) is denoted by the diffusion tilt angle, q.
at 2-mW power, 3300.0-G center-field, 100-kHz modulation, 1.25-Gpeak-to-peakmodulation amplitude, 100-G sweepwidth, and 2004 sweep time. Digitized spectra were stored on an IBM PC interfaced to the spectrometer. By flowing heated nitrogen gas through the cavity the temperature was controlled to an accuracy of h0.5K. 2.16-mm4.d X 5-mm-0.d. clear fused quartz Experimental Section EPR tubes were used to contain the samples. Materids Surfactant bis(2-ethylhexyl) sulfosuccinatesodium For spectral measurements under pressurized conditions, the salt (AOT), 2,2,4-trimethylpentane (isooctane), 5-, 7-, 12-, and quartz sample tube was first etched inside and outside by a 5% 16-doxylstearicacids, and buffer componentswere obtained from hydrogen fluoride solution to remove any small nicks and cuts, Aldrich Chemical Co., Milwaukee, WI. Spin probes 4-[(N,Nwhich might decrease the pressure resistance of the tube. The dimethyl-N-hexadecyl)ammonio]-2,2,6,6-tetramethylpiperidin- tube and control valve assembly was pressure tested for leaks and 1-oxy1 iodide (CAT16) and 4-(N,N-dimethyl-N-(3-~ulfopropyl)- rupture at pressures up to 7 MPa. In preparing high-pressure ammonio)-2,2,6,6-tetramethylpiperidin-l-oxyl(DSTA) were samples, the EPR tube was connected to the ethylene gas cylinder purchased from Molecular Probes, Eugene, OR. EPR sample through the control valve, with a branch to a digital pressure tubes were obtained from Wilmad Glass Inc., Buena, NJ. gauge to measure the pressure in the tube. After pressurization, Ethylene gas of commercial purity grade was obtained from the cylinder connection was removed. The pressure decrease as Matheson Gas Products, Gonzales, LA. Doubly distilled deionthe gas dissolved into the organic phase was monitored till an ized water was used in all buffer preparations. Buffer components equilibrium pressure was achieved, indicating that the tube included sodium citrate (pH 4.6), sodium phosphate (pH 7.9, contents had reached equilibrium. The tube and control valve and sodium borate (pH 8.5) and were all 0.02 M. All compounds assembly was then lowered into the EPR sample chamber. All except isooctanewere used without further purification/treatment. EPR spectra at high pressure were recorded at a sample M e M s . Isooctane was degassed by four freeze-pumpthaw temperature of 298.15 K. cycles to remove all the dissolved oxygen. The surfactant Ca/c&tiom. We have employed a variety of spin labels to probe different regions of the reversed micellar system. The concentration in all reversed micellar solutions was kept at 0.15 chemical structures of the spin labels used are shown in Figure M. The overall spin label concentration was kept at 0.2 mM so that the molar ratio of surfactant to spin label was 750/1. All 2. The surfactant molecule is shown in structure 1 along with the spin labels. This should provide an approximateidea regarding stock solutions and reversed micellar solutions were prepared under a nitrogen atmosphere by using degassed isooctane. Stock the regions being probed by the nitroxide group on the spin labels, relative to the AOT molecule. Structure 2 represents S-doxylsolutions of different spin labels (except DSTA) were prepared by adding predetermined amounts of spin label to a solution of stearic acid, which belongs to the class of n-doxylstearic acids. 0.15 M AOT in isooctane. Reversed micellar solutions were The presence of the carbonyl group facilitates the anchoring of prepared by adding the required amount of buffer to the spinthese probes at the oil/water interface. 5-Doxylstearic acid and labeled dry micelles in isooctane and vortex mixing the solution. 7-doxylstearicacid probe the AOT tail regions, whereas 12-doxy1 and 16-doxy1 probe the bulk isooctane region just outside the With DSTA, the probe was dissolved in the requisite buffer and AOT tails. Structure 3 represents CAT16, which possesses a reversed micellar solutions were prepared by adding the buffer to the solution 0.15 M AOT in isooctane. The overall DSTA charged head group and its nitroxide moiety probes the interfacial concentration in all cases was 0.2 mM. region. DSTA, shown in structure 4, is zwitterionic and water soluble and is used to probe the microaqueous phase. ReconuOg EPR Spectra. EPR spectral measurements were The EPR spectra for 5-doxyl- and 7-doxylstearic acids in made at 9.25 GHz by using an IBM-Bruker X-band EPR reversed micelles were highly anisotropic and displayed wellspectrometer (Model ER200D-SRC). All spectra were recorded
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5154 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993
resolved maximum and minimum hyperfine splittings. These anisotropic spectra were characterizedby using the apparent order parameter S.29
The following were used for the principal values of the hyperfine splitting tensor:30A, = 6.3 G, A, = 5.8 G, and A,, = 33.6 G. The polarity correction factor involves a, = 1/3(Axx+ A,, +A,) = 15.23 and aN = 1/3(All+ 24,). aN is the hyperfine splitting constant and is a measure of the polarity of the spin label environment. For fairly isotropic EPR spectra, aN was measured as half the separation in gauss between the low-field line (MI = +1) and the high-field line ( M I = -1). Typically, aN increases by about 2.0 G in transferring a spin label from a hydrocarbon environment to an aqueous environment. The spectra from other spin labels, which were fairly isotropic, were characterized through the rotational correlation times3’given by where T R is the rotational correlation time in seconds, ho and h-1 are the peak-to-peakheights of the center line and high-field line, respectively, and A H 0 is the center line peak-to-peak width in gauss. In all EPR spectra the applied magnetic field increases from left to right.
Factors Affecting EPR Spectra Before proceeding with the results it is appropriate to consider some relevant factors that influence the EPR spectra. EtreCt of -gem. The nitroxide radicals used in this study display a simple three-line derivative spectrum representativeof spin labels tumbling freely in solution. However, if the solvent is saturated with air, the three resonance lines are broadened significantly (especiallyin hydrocarbon solventssuch as isooctane). The oxygen-induced line broadening has been described by Jost and Griffith32 as a consequence of interactions between oxygen and the spin label through exchange and dipolar mechanisms. The relevance of oxygen line broadening to our studies can be seen rather clearly from Figure 3A. Spectrum (a) illustrates the EPR signal of 5-doxy1 in degassed isooctane, indicating a rapidly tumbling molecule, with three narrow lines of almost equal amplitudes. Upon addition of surfactant (AOT), 5-doxy1 is incorporatedinto the dry reversed micelles and the motion of the spin probe is significantly restricted as shown in spectrum (b). The degree of restriction of spin label motion is reflected by the high-field line, which has broadened appreciably. The broadening leads to a noticeable reduction in the peak-to-peak amplitude. The high-field line has the shortest transverse relaxation time and hence is most sensitive to changes in the mobility of the spin label. We also notice that there is a slope to the baseline, and we decided to determine if this was due to nitroxide-nitroxide interactions, the Heisenberg spin-exchange phenomenon. As shown by Jost and Griffith,)*the clue to such interactions is a changing baseline as the probe concentration is varied down to levels where the baseline integrity is observed. Accordingly, we carried out experiments where the probe concentrationwas varied from 50 to 500 p M , noting our datum concentration of 200 pM. Spectral integrity was retained over this range, and there is no flattening of the baseline even at the very low concentration of 50 pM. Also, spectral measurements such as the separation between the hyperfine lines and the peakwidthsremained identical with this change in concentration. In their EPR workon reversed micelles, Menger and co-workers4 reported that a 3-4-fold reduction of the concentration of the spin probe (a PROXYL radical) had an insignificant effect on the EPR parameters. These authors concluded that nitroxide-nitroxide interactions are not
wo
0
wo 26
wo 50
Figure 3. (A) EPR spectra of 5-doxy1 in (a) degassed isooctane, (b) 0.15 M AOT dry micelles, (c) 0.15 M AOT dry micelles saturated with air, and (d) 0.15 M AOT dry micelles saturated with pure oxygen. (B) Oxygen-induced line-broadening at w, values 0, 26, and 50.
a factor. Although we cannot completelyrule out complications due to spin exchange in our systems, we are confident that the spectral characteristics of peak separation and peak width do lead to correct interpretations of the nature of the micellar environment. Spectra (c) and (d) illustrate the oxygen line broadening effect that occurs when the solution is saturated with air (spectrum c) and with pure oxygen (spectrum d). The bar graph in Figure 3B shows that there is a dramatic line broadening in the presence of increased concentrations of oxygen with relatively low water content micelles and a minor broadening with relatively high water content (w, > 20) micelles. Since the spin label used, 5-doxyl, is located within the environment of the AOT tails (See Probe Location and Behavior) of the reversed micelles, this means that the dissolved oxygen is not present in the immediatevicinityof thenitroxidemoiety in high water content (w, > 20) micelles. This is easily explained since oxygen has a low solubility in water. With high water content micelles the water molecules seem to penetrate into the AOT tail region, so that the nitroxide moiety in the 5-doxy1 spin label is located in an environment with a substantialaqueouscharacter. In contrast, the water layer does not penetrate into the AOT tails in low water content micelles. The environment about the nitroxide will be essentially pure hydrocarbon, where the solubility of oxygen is quite high. We have carried out all subsequent experimentsin the absence of oxygen to accurately reflect the spectra of the various spin labels. Figure 4 illustrates the effect of increasing water content on the EPR spectra of 5-doxy1 in reversed micelles. At w, = 0, the spectrum is fairly isotropic; however, the degree of anisotropy increases as w, increases. The reason for an increase in the spectral anisotropy has been well described by Haering et a1.,I2 but we repeat the main argumentsfor clarity in interpretation. Brownian
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The Journal of Physical Chemistry, Vol. 97, No. 21, I993 5755
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Figure 6. EPR spectra of 5-doxyl-labeled reversed micelles as affected by pH of the stock buffer. [AOT] = 0.15 M; wo = 26; 0.02 M sodium citrate pH 4.6, 0.02 M sodium phosphate pH 7.5, and 0.02 M sodium borate pH 8.5. The arrows indicate the two spectral components. 2All is a measure of the maximum hyperfine splitting and 2AL is a measure of the minimum hyperfine splitting. These are used to calculate the order parameter, S, and the hyperfine splitting constant, UN, for highly anisotropic spectra. Figure 4. Effect of w0([H20/AOT]) on the EPR spectra of 5-doxyllabeled reversed micelles. pH 8.5 buffer was used in making the reversed micelles. Spectra recorded at 298.15 K.
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, 30
,
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40
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w.a
Figure 5. Isotropic hyperfine splitting constant,
UN (gauss), of 5-doxy1 and order parameter, S, of AOT reversed micelles as a function of w,([H20/AOT]). Order parameters werecalculatedonly when thespectra were sufficiently anisotropic to allow measurement of the maximum and minimum hyperfine splittings. See Figure 6 for determining maximum and minimum hyperfine splittings.
tumbling of the reversed micelles and the lateral diffusion of surfactant molecules along the interface are the two relevant motional processes present. These two processes modulate the hyperfine coupling tensor, A, and the g tensor. When the frequency of the above two averaging processes is >lo8 s-I, the resulting spectra are isotropic as seen for the spectra at w, = 0, and 5. As the size of the reversed micelles increases with increasing wO,l Brownian tumbling decreases and the frequency of the averaging processes falls below lo8s-I, which is not rapid enough to average out the anisotropies. This results in highly anisotropic spectra at high w ovalues. Such spectra are normally discussed in terms of the order parameters given by eq 1. Order parameters provide information regarding the degree of organization in the surfactant tail region, which directly affects the environment of the nitroxide moieties in 5-doxy1 and 7-doxyl. Figure 5 illustrates the a~ and order parameter profiles for 5-doxy1 in reversed micelles, as affected by the water content in the system. The hyperfine splitting constant, a N , is used as a measure of the polarity of the spin label environment. As
increasing amounts of water are added, there is an initial rapid increase in the polarity of the spin label environment until approximatelyw, = 20. The order parameter also initiallysharply increases with w,. We note here that our main observations on the oxygen-free system qualitatively follow the same trends described by Haering et al.lz The main difference is the fact that the spectra of the oxygen-free system are more sensitive to microenvironment changes, resulting in quantitatively different values of the order parameter and the polarity profiles. We have therefore proceeded to examine temperature and pressure effects on the microstructure, aspects not fully discussed (particularly the pressure effect) in the earlier study.I2 Effect ofpH. EPR spectra of n-doxylstearic acids are known to be pH d e p e ~ ~ d e n t .Figure ' ~ , ~ ~ 6 illustrates the EPR spectra of 5-doxy1 in w, = 26 reversed micelles as a function of pH. At low pH values the spin label exists in the nonionized form and at high pH in the fully ionized form. At intermediate pH values two components are observed as indicated by the arrows. To ensure that there was only one type of spin label present, we performed all studies using aqueous buffer at pH 8.5 in making up the micellar solutions. Results and Discussion
ProbeL.wationandBehavior. The spin label DSTA is soluble only in water and is hence expected to reside in the aqueous part of the reversed micelle. The dissociation of the iodide in CAT16 imparts surface-active properties to the spin label. Since the nitroxide moiety is present on the polar piperidine group, CAT16 must be probing theaqueous part of theinterfacial layer. Surfaceactive characteristics are exhibited by the doxyl stearates, since they possess a terminal carboxyl group. The doxyl groups, which host the nitroxide moiety, are located on different carbon atoms along the stearic acid chain. The doxyl group is nearest to the terminal carboxyl in the case of 5-doxylstearic acid, whereas it is the farthest from the terminal carboxyl for 16-doxylstearic acid. With the n-doxylstearates anchored at the interface, their nitroxide moieties are expected to probe regions from within the AOT tails to the bulk isooctane phase. A recent electron spin echo modulation study using n-doxylstearic acids in reversed micelles confirms the location of the paramagnetic moieties and the solubilization of the spin labels at the micellar interface.13 All the spin labels used in this study when free in solution, either isooctane or water, exhibit a typical three-line spectrum
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5756 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993
CAT16
5-OOXYL
7-OOXYL
318.64
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321.53
328.35
Figure 7. EPR spectra of the different spin labels used in this work, when doped into 0.15 M AOT reversed micelles of wo = 26. pH 8.5 buffer used. All spin labels used here, when free in solution,display EPR spectra similar to that seen in Figure 3A(a). DSTA resides in the aqueous core of the reversed micelle. The nitroxide moiety of CAT16 is in the aqueous part of the interface, the nitroxide moieties of 5-doxy1 and 7-doxy1 are in the AOT tail region, and the nitroxide moieties of 12-doxy1 and 16doxy1 are in the bulk isooctane.
with lines of almost equal amplitudes, indicative of a rapidly tumbling molecule (see Figure 3A(a)). These spin labels when incorporated into reversed micelles of w, = 26 produce spectra ranging from representations of totally isotropic motion to those of extremely anisotropic motion. The water-solubleprobe DSTA and the interfacial probe CAT16 give rise to fairly isotropic spectra, as seen in Figure 7. As the location of the nitroxide moiety is moved from the interfacial layer to the tail region of the surfactant, the 5-doxy1 and 7-doxy1 probes exhibit highly anisotropic spectra. As one progresses out of the tail region into the intermicellar bulk isooctane region, it is seen that the spectra again become isotropic as noted from the behavior of 12-doxy1 and 16-doxyl. The reversed micellar environment seems to impose different ordering characteristics to probes located in different regions. It is clear from Figure 7 that the probes located in the AOT tail region of the reversed micelles experiencethe maximum restriction to their mobility. Locations away from the tail region, either toward the aqueous core or toward the intermicellar isooctane phase, result in enhanced mobility. E f f ~ cof t Temperature. Reversed micelles of w, = 26 were chosen as the representative model system for the variabletemperature studies for two reasons. Significant anisotropy was exhibited by 5-doxy1 and 7-doxy1 probes at this wor allowing for order parameter calculation over a wider temperature range. Moreover, our results could be related to previous temperaturedependent phase behavior experimentsdone on the same system.28 Figure 8 shows the effect of increasing temperature on the EPR spectra of 5-doxy1 in reversed micelles. The room temperature spectrum is highly anisotropic and is characteristic of a random distributionof fairly immobilizedspinprobes. As the temperature is increased, the inner and outer hyperfine extrema move closer to each other, and the degree of anisotropy decreases. The effects of increasing temperature on the reversed micellar system include (a) faster tumbling of the reversed micelles and (b) increased lateral diffusion of the surfactant molecules due to increased thermal energy. As discussed earlier, factors a and b would result in a decrease in the spectral anisotropy. Therefore, these factors should help to average out the anisotropies in the spectra and decrease the order parameter. However, an increase in the
Figure 8. Effect of increasing the temperature on the EPR spectra of 5-doxyl-labeled reversed micelles. The nitroxide moiety resides in the AOT tail region. [AOT] = 0.15 M, wo = 26, and pH 8.5. The outer hyperfine extrema move closer to each other as the temperature is increased.
o.2"'""'"'""..'....I 290
300
310
320
330
340
Temperature, K
Figure 9. Order parameter S of AOT reversed micelles, labeled with 5-doxy1 and 7-doxy1, as a function of temperature. [AOT] = 0.15 M, wo = 26, and pH 8.5.
temperature does not render the line shape isotropic. Thus, an increase in overall tumbling of the reversed micelles as well as an increase in the lateral diffusion of the surfactant molecules along the interface is ineffective in completely averaging out the anisotropic magnetic interactions. The changes in the anisotropic nature of the EPR spectra have been quantified through order parameters. The calculated order parameter profiles for 5-doxy1and 7-doxy1spin probes as a function of temperature are shown in Figure 9. It is seen that an increase in temperature results in a decrease in the order parameter in an approximatelylinear fashion. It is thusevident that the tail region near the interface becomes increasingly fluid with temperature, a consequenceof increasing surfactant disorder near the interface. It is interesting that both 5-doxy1and 7-doxy1show similar trends, maintaining the flexibility differences between the two probes and indicating that the flexibility gradient along the tail may be retained with temperature. We next considered temperature effects on the other probes, which exhibited fairly isotropic spectra. Figure 10illustrates the variation in the EPR spectra of CAT16 labeled reversed micelles with temperature. The two predominant effects of increasing temperature are a decrease in the line width and a decrease in the ratioof center to high-field lineamplitudes. Both theseeffects
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A 16.0
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340
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Temperature, K
340.2
344.8
Ft,-re 10. Effect of temperature on the EPR spectra of the interfacial probe CAT16 in 0.15 M AOT reversed micelles of w, = 26. High-field line exhibits significant narrowing and increase in amplitude. 1.5
,
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,
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0 1'2-BOWL
v O-DOXYL A
CATl I
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*c b
0.5
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320
340
3
Temperature. K
Figure 11. Rotational correlationtimes for the spin probes DSTA, CAT16, 12-doxy1,and 1Cdoxyl in 0.15 M AOT reversed micelles of w, = 26, as
affected by the temperature.
indicate a decrease in the rotational correlation time ( T R ) as calculated from eq 2. The rotational correlation time profiles for DSTA, CAT16,12-doxyl, and 16-doxy1 are shown in Figure 11. The water-soluble spin label DSTA experiences an increased mobility as the temperature is increased, with TR varying linearly with temperature. However, for CATl6, TR decreases dramatically with an increase in temperature from 296K to about 320K, beyond which it decreases relatively slowly. Also CATl 6 exhibits the maximum variation in the correlation time. This is to be expected, considering that the nitroxide group is located right at the interface, which is a highly organized region in the reversed micellar system. Changes in the interfacial fluidity and organization should therefore be reflected in the mobility of the interfacial spin probe. Turning to 12-doxyl, which is located outside the AOT tail region, we see that the probe experiences a linear decrease in T1( with an increase in temperature. 16Doxyl, on theother hand, is relatively unaffected by temperature. Since 16-doxy1is situated well outside the AOT tail region in the bulk isooctane phase, it is already near its high-mobility limit and is not very sensitive to changes in temperature.
Figure 12. Effect of temperature on the isotropic hyperfine splitting constant, UN, for the various spin probes in 0.15 M AOT reversed micelles of w, = 26. The AOT reversed micelles were labeled with DSTA, CAT16, 5-doxy1, 7-doxy1, 12-doxy1, and 16-doxyl.
The temperature variation of the (IN profiles for the various probes is described in Figure 12. It is immediately recognized that a polarity gradient exists as one progressesfrom the aqueous core of the reversed micelles to the intermicellar oil phase. The water-soluble probe DSTA and the interfacial probe CATl 6 display the highest polarity, followed by 5-doxy1 and 7-doxy1 located in the AOT tail region. 12-Doxy1and 16-doxy1 located outside the AOT tail region exhibit the lowest polarities. From Figure 12, DSTA appears to be located in observably less polar environments as the temperature is increased. This is counterintuitive but can be explained considering that we did notice a "boiling effect" similar to that noted by Zulaufand Eickel where water from the reversed micelles vaporizes as the temperature is increased. Haering et a1.I2 have shown that the aN values for DSTA decrease sharply with decreasing w,, and, therefore, the decrease in the polarity of the environment around DSTA can be attributed to the boiling effect, which results in loss of water from the reversed micelles. However, CAT16 does not experience a major change in polarity as the temperature is increased. Thisis probably because the polarity profile for CAT16 reaches a plateau at high w,. 12-Doxy1 and 16-doxy1 exhibit a minor variation in their UN value in response to a temperature increase. This is understandable, since the probes are far removed from the water core. Also the boiling effect, inducing water loss, should not affect the environment of these two probes. Due to the anisotropic nature of the EPR spectra, uN values for 5-doxy1 and 7-doxy1 were calculated on the basis of the maximum and minimum hyperfine splittings as described earlier in reference to eq 1. This is perhaps the reason the UN profiles for 5-doxy1 and 7-doxy1 are not linear, in contrast to the trends shown by the other spin labels. The slight dip in the curves is mainly due to uncertainty in the peak positions used to determine the aNvalues rather than a result of the boiling effect. We can say this because the results from the variable w, experiments (Figure 5) show that both the S and ( I N values plateau out after w, = 20. So a slight loss of water from the reversed micelles is not going to change the values for either S or U N . Thus, we can directly link any changes in S and U N to the temperature response of the system, rather than to loss of water. A continued increase in temperature eventually leads to phase separation. We notice that there exist two phases, with the bottom aqueous phase being about 2&25% of the total volume. Since the total water content is only about 6% of the total volume, this implies significant solubilization of oil and surfactant. Analysis of the oil phase through FTIR shows negligiblesurfactant present, while the bottom phase does show the presence of AOT and isooctane. There is no observablemiddle phase of finite thickness although some surfactant can be seen at the oil/water interface. We therefore feel that this transition leads to a Winsor type I where an oil-in-water microemulsion coexists with an oil phase.
5758 The Journal of Physical Chemistry, Vol. 97, NO. 21, 1993
As shown by K a h l ~ e i tand ~ ~ pointed out by McFann and Johnston,*6temperature and salt have opposing effects on the phase diagram and can be related to the AOT content through the so-called "fish"-type phase diagram. If the temperature is increased in a two-phase water-in-oil microemulsion in equilibrium with a water phase (Z), the system transforms to three phases, with a middle phase microemulsion (3). and finally back to a two-phase system of an oil-in-water microemulsion in equilibrium with an oil phase (2). In our case, we start with a single-phase water-in-oil microemulsion (l), but the final state is the 2 state. It is also intuitive that temperature should help AOT solubifization in water, which is the observation noted. Reproducible EPR spectra are hard to obtain at the higher temperatures corresponding to the phase split. Boiling causes the formationof vapor slugs in the EPR tube and affects the instrument tuning. We therefore do not make any conclusions about the microenvironment after phase split and merely examine the microstructural changes during the approach to the temperature-based phase split, conditions at which the data are fully reproducible. Effbct of Pressurized Ethylene. We now turn to the characterization of microstructural changes in reversed micelles subsequent to pressurization of the system using a gas such as ethylene. As discussed in the introduction, the objective is to follow the trajectory a- -bin Figure 1, which is the approachto the pressureinduced phase-split condition. We have again used reversed micelles of w, = 26 as the model system. In independent experiments using a high-pressure glass-windowed apparatus described earlier25 we have found that the pressure for phase split is approximately 4.2-4.5 MPa at ambient temperature; up to this pressure, the water and the surfactant content of the microemulsion phase are totally retained. In order to characterize the microstructure, we therefore follow the EPR spectra of the various probes as the system is pressurized to about 4 MPa. Beyond the phase-split condition (trajectory b- - -c), we have found that the EPR spectra obtained are not meaningful. This is more of a practical problem dealingwith the EPR tube dimensions. When phase-split occurs, a portion of the spin probes and surfactant that come out of solution adsorb to the walls of the EPR tube rather than precipitate to the bottom. The resultant spectra are, therefore, a combination of somewhat immobilized spin probes, of spin probes residing in the precipitated aggregates of AOT, and of spin probes rapidly tumbling in the supernatant oil phase, which is devoid of AOT. We therefore simply concentrate on the spectra during the approach to the phase-split, i.e., trajectory a- - -b. Figure 13 illustrates the effect of ethylene pressure on the EPR spectrum of 5-doxy1in reversed micelles. The spectrum is highly anisotropicat atmospheric pressure, but under progressively pressurized conditions, the degree of anisotropy decreases. The 7-doxy1 probe also exhibits a similar decrease in anisotropy with pressure. We attribute the increased mobility of these spin probes as simply caused by motion in a progressively reduced-density fluid. In isooctane-based reversed micelles, SANS studies have indicated that the isooctane solvent penetrates about 2.5 A of the 9-8,AOT tails,36the spectra for 5-doxy1 and 7-doxy1 at ambient pressures are therefore indicative only of motion in a solvent-free tail environment. But with pressurization, ethylenedissolvesboth in the bulk solvent, reducing its density, and should penetrate well into the tail environment due to the molecular similarity between the ethylhexyl tails and isooctane. Thus, we do expect the density of the tail environment to be also reduced, leading to an increase in the fluidity of the tails almost up to the interface. Calculations of the order parameter for 5-doxy1 and 7-doxy1are shown in Figure 14, and it is seen that the order parameter does decreasewith increasing ethylenepressureand decreasing solvent density. We also note the similarity in trends with the data for the temperature effect (Figure 9). EPR spectra for the interface-resident probe CAT16 as a
-
Kommareddi et al.
4 P
+?I
-
0.1 MPi
1.01
1.77
2.44
3.00
V Figure 13. Ethylene gas pressure induced changes in the EPR spectra of 5-doxyl-labeledreversed micelles. [AOT] = 0.15 M, w, = 26, and pH 8.5. Spectra were recorded at 298.15 K. The hyperfine extrema move closer to each other. Density, g/ml 0.69
0.66
0.63
0.60
0.57
0.54
0.4
S
0.3
0.2
0
1
2
3
4
Ethylene Pressure, MPa
Figure 14. Order parameter, S,for 5-doxyl- and 7-doxyl-labeledreversed micelles as a function of the equilibrium ethylene gas pressure. [AOT] = 0.15 M, w o = 26, and pH 8.5.
function of ethylene pressure are shown in Figure 15. Increasing ethylene pressure results in a slight but noticeable narrowing of the high-field line and a decrease in the ratio of the center to high-field line amplitudes. These observations point toward an increased mobility of the CAT16 probe. Further information can be gained from the data of Figure 16, which summarize the rotational correlationtime profiles for 12-doxyl, 16-doxyl,DSTA, and CAT16 probes under pressurized conditions. Let us examine the data starting from the spin label that resides the furthest away form the water core, Le., 16-doxyl, which should normally reside outsidethe micelle and in the bulksolvent. At atmospheric pressure, 16-doxy1does not experienceany restriction to its motion and has a relatively low rotational correlation time. Increasing the pressure does not affect its mobility significantly, indicating that the immediately surrounding medium is totally isotropic (i.e., does not impose order). For 12-doxyl, which is closer to the micellar tail region, theEPRspectra at ambient conditions indicate moderate restriction, reflected in a higher T R in comparison to 16-doxyl. With pressure, the rotational correlation timedecreases, indicating an enhancementin mobility. We wish to note, however, that while we have used 12-doxy1 and 16-doxy1 to attempt to monitor the intermicellar tail penetration duringcluster formation approaching the phase-split conditions, the spectra do not reveal
Changes in AOT Water-in-Oil Microemulsions
The Journul of Physical Chemistry, Vol. 97, No. 21, 1993 5159 Density, g/ml
P
-
0.69
0.66
0.63
0.60
0.57
0.54
0.1 UP,
15.5
1
14.0 0
1
2
3
4
Ethylene P r e s s u r e , MPo
F i p e 17. Effect of the equilibrium ethylene gas pressure on the isotropic hyperfine splitting constant, ON, for the various spin probes in O.15M AOT reversed micelles of w, = 26. The AOT reversed micelles were labeled with DSTA, CAT16,5-doxyl, 7-doxy1, 12-doxy1,and 16-doxyl.
V
Figure IS. EPR spectra of the interfacial probe CAT16 as affectc- -y the equilibrium ethylene gas pressure, in 0.15 M AOT reversed micelles of w, = 26. Note narrowing of the high-field line. Denrity. g/ml 0.69
0.86
1 .o 0
D
c 01
,
0.57
0.60
0.63 I
.
,
.
(
0.54 .
~.
t
cK
0.0
1
2
3
4
Ethylene Pressure. MPo
Figure 16. Rotational correlationtime profiles for the spin probes DSTA, CAT16, 12-doxy1,and 16-doxy1in 0.15 M AOT reversed micelles of w, = 26, asaffected bytheequilibriumethylenegaspressure. Theinterfacial probe, CAT16, exhibits the maximum variation.
any direct evidence of such clusters. That is, we do not see a restriction in the mobility of the probes at the higher pressures as individual micelles interact. This does not imply that micellar clusters are not present; rather, the implication is that the consequences of such clustering are not revealed through the EPR spectra of 12-doxy1 and 16-doxyl. Again, since that AOT tails are structurally similar to isooctane, it is entirely possible that the cluster environment of 12-doxy1 and 16-doxy1 is very similar to the environment exposed to isooctane. Hence, we would simply see a continuation of the effects at lower pressures, Le., an increase in mobility as the solvent density is reduced. Moving closer to the micellar core in Figure 16, we see that CAT16, theinterfacialprobe,exhibitsa strong increasein mobility upon pressurization. This is interesting as it could reflect the effect of ethylene penetration up to the interface. It is noteworthy that themobility characteristicsof DSTA, thecore-resident probe, are not perturbed by ethylene pressure. Considering the relative insolubility of the nonpolar gas in water, one would not expect a major change in DSTA's environment. Figure 17 illustrates the variation of the hyperfine splitting
constant, ON, for the various probes, with ethylene pressure. Spin probes DSTA, CAT16,12-doxyl, and 16-doxy1do not experience any major change in the polarity of their respective environments with pressure. However, 5-doxy1 and 7-doxy1 show a minor variation in polarity at pressures above 2.5 MPa. It should be noted that (INvalues for 5-doxy1 and 7-doxy1 are calculated on the basis of peak positions as discussed before in reference to eq 1, and there exists some uncertainty in locating exact peak positions. This is especially true for the low-field signal, where the two well-resolved peaks merge as pressure is increased, as shown in Figure 13 for 5-doxyl. The order parameter and UN profiles for 5-doxy1and 7-doxy1 can be better understood through a comparison of Figures 14 and 17. At pressures below 2.5 MPa, the order parameter, S,decreases, while the polarity factor aN remains fairly constant. From eq 2, it is seen that S is inversely proportional to ON and an increase in UN would result in a reduced SIprovided ,411 is fairly constant. We see in Figure 14 that the calculated values of S decrease consistently with pressures approaching the phase split. Therefore, it seems reasonable that the increase in UN as phase-split conditions are approached is also realistic. To help analyze the EPR spectra as a function of pressure, we invoke comparisonswith experimental data on micelle formation in compressed gases; systems that have been extensivley studied by other resear~hers.3~93~ Yazdi and c o - ~ o r k e r shave ~ ~ used solvatochromicprobes to demonstrate that, in clearly single phase systemsremoved from the vicinity of the phase boundary, pressure has no significant effect on the polarity of water environment, the degree of water motion, and the micellar size. Our results with the water-soluble probe DSTAalso indicate that there is no change in the water environments with pressure. Figure 16 illustrates that the correlation time (reflecting the degree of water motion) is essentially unchanged, and Figure 17 illustrates that there is no major change in the hyperfine splitting constant (polarity in the micellar core). But it is interesting to note the spectral changes of 5-doxy1and 7-doxy1 with pressure, especially at pressures approaching the phase split where theaNvaluesincrease. If we were to rationalize this observation from a mechanistic viewpoint, we could say that the clusters present near the phase transition provide a route to a rapid exchange of water between individual micelles. As a result, at the higher pressures near the phase split, the 5-doxy1 and 7-doxy1 experience a small increase in polarity due to interaction with water that is rapidly exchanging between interconnected pools of water in a cluster. Some evidence of a similar phenomenon in compressed gas micelles may also be
5760 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 obtained from interpretations of UV probe spectral data in such systems.37 Fulton and have shown that the absorbance maximum of a UV probe, thymol blue, undergoes a red shift when the w, of micelles in compressed ethane is increased from 1 to 3 to 5 . For the cases with w, = 3 and 5,there is a small but perceptible red shift as the pressure is decreased toward a phase boundary, indicative of an increase in the polarity as the phase boundary is approached. Perhaps the clearest indication in the literature of an increase in the water exchange rate as a phase boundary is reached is data on the temperature-basedphase split. For example, Lang and coworkers28 used fluorescence quenching experimentsto verify an increase in the exchange rate of intramicellar constituents and in the aggregation number as the temperature-based phase split is reached. Measurements of the electrical conductivity and percolation characteri~tics3~~~ also verify the existence of clusters with rapidly exchanging water near the temperature-based phase split. We continue on the comparison between the approach to the temperature- and the pressure-induced phase splits. For 5-doxy1 or 7-doxyl, we see that an increase in temperature from 298 to 320 K produces approximately the same change in the order parameter as increasing the ethylene pressure from 0.1 to 2.5 MPa. However, the mechanisms by which the fluidity of the surfactant tail region is changed, as reflected by the order parameter, do not appear to be the same. The EPR spectra for 5-doxy1 show differences as the order parameter is decreased, due to an increase in either temperature or pressure. This can be seen by comparing the EPR spectra of Figures 8 and 13. The striking differences in the spectra are seen in the low-field line components. In the variable-temperature case, the two low-field line components are of almost equal amplitude, and they merge to give a broad line at high temperatures. In the variable-pressure spectra on the other hand, the lower of the two low-field line components diminishes preferentially and merges into a single and comparatively sharp line. To understand these differencesin spectra and provide insight into structural changes,we use arguments derived from extensive studies on the mobility of spin labels attached to macromolecules in solution. In particular, we borrow the results of spectral simulations using thevery anisotropicreorientation (VAR) model of Meirovitch et ala4’and relate them to the experimentalspectra represented in Figures 8 and 13. The VAR model has been used previously to analyze EPR spectra of nitroxide spin labels chemically attached to macromolecules such as proteins slowly reorienting in solution with an axially symmetric rotational diffusion tensor characterized by the rotational diffusion rates R I and Rll. The same model should apply to our system also if we consider the spin-labeled reversed micelle to be a macromolecule reorienting slowly in solution. The model also assumes that the rotation of the nitroxide about a mean diffusion axis is very rapid and is characterized by an internal rotational diffusion coefficient R I . RL provides a reasonable estimate of the overall reorientation of the macromolecule and it is assumed that RI >> R I Under these conditions the effect of RIon the EPR spectrum is similar to that of RIIand RI is regarded as an effective RII. Apart from the above-mentioned parameters, of further interest to us is the “diffusion tilt angle” P,which is the angle made between thediffusionaxis z’and themagneticz”’axis (see Figure 2). For w, = 26 reversed micelles, Brownian reorientational rates oftheorderof 106s-’areexpected, basedon therotationalStokesEinstein relationship, and these rates are relatively slow on the EPR time scale. Therefore, any type of motional averaging occurring would be mainly due to internal modes of reorientation. The motional rate RIIand the diffusion tilt angle P reflect the effect of internal motions on the EPR spectra. Meirovitch et ala4’have done spectral simulations using the VAR model for different diffusion tilt angles P and varying motional rates RII,keeping R I constant at 5 X lo6 s-1. On
.
Kommareddi et al. comparing the variable-temperature spectra to their simulated spectra, we infer that the trend exhibited in Figure 8 can be accounted for by assuming that R I , RII,and P increase with temperature, with R I and RIIbeing the major contributors. We can easily see that an increase in temperature would cause the above changes. An increase in RL is a direct result of increased overall tumbling of the reversed micelle, an increase in RII corresponds to more rapid rotation of the doxyl side chain about the long molecular axis (alkyl chain), and an increase in P suggests greater distortion along the chain segment attached to the doxyl group. On the other hand, the variable-pressure spectra represented by Figure 13 can be interpreted by assuming that P increases with gas pressure and R I and Rll remain constant. This suggests that the effect of pressurization with ethylene is primarily to enhance the motion of the doxyl group relative to the long molecular axis of the spin label, which would result in an increased tilt angle P. This could reaffirm our earlier suggestion that ethylene penetrates into the intramicellar tail regions and causes greater flexibility in that region. Thus, temperature and pressure seem to induce disorder in the surfactant tail regions through slightly different mechanisms. Finally, we also state that the system after the pressure-induced phase split is macroscopically dissimilar to the system after the temperature-inducedphase split. Visible observation of the EPR tube contents after phase split shows an aqueous phase and an oil phase separated by a thin highly concentratedsurfactant layer. This leads us to believe that the system evolves to a Winsor type I11 like system after phase split, with a middle phase microemulsion, where the surfactant tails take on the role of the continuous medium. Similar three-phase systems have been observed in the literature on compressed gas micelles, especially in the presence of appreciablesalt.26 In contrast, the temperature based phase split leads to a type I system, as stated earlier. It would be interesting to correlate the microstructural changes during the approach to phase split to the final state after phase split. But such extrapolation of the Meirovitch model to explain these final differences in phase behavior would be speculative. Again, experimentalproblems prohibit study of the system nature after the phase split. The cavity-resident portion of the EPR tube consists of the spin probe in different environments, in the different phases, and adsorbed to the tube walls. Spectral reproducibility is not good, and we therefore have limited our observations to the approach to phase split.
Conclusions
Our EPR experimentswith the various spin probes in reversed micelles subjected to different thermodynamic conditions reveal interesting characteristics of these microemulsion systems. From a spectroscopic viewpoint, we see that oxygen-induced line broadening is prevalent for spin probes residing in nonpolar regions of the reversed micelles and that highly resolved spectra can be obtained by degassing the samples. We have also verified the previously observed maximum in the spectrum anisotropy as one proceeds from the core of the reversed micelles out to the tail regions that are in intimate contact with the bulk organic phase. The response of the microemulsion droplet to changes in thermodynamic conditions is reflected by spin-probe responses to temperature and pressure. Increasingthe temperature results in higher mobility of the spin labels. Spectral analogies with the characteristics for spin-labeled macromolecules reveal that the high mobilities can be traced to an enhanced tumbling rate of the micelles, an increased lateral diffusion of surfactant molecules along the oil-water interface, and a reduction in rigidity of the interfacial and surfactant tail regions. The pressureeffect, while similar in overall characteristics,does have some subtle but critical differences. On pressurizing the microemulsion system with ethylene, we see that the gas perturbs the highly structured interfacial region and decreases the rigidity of the surfactant
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The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 5761
tails. We attribute this effect to penetration of ethylene into the surfactant tails and the resultant effect of tail mobility in a reduced density fluid. An interesting albeit intuitive observation is that the dynamics of spin probes in the microaqueous water pools are unaffected by pressure, probably due to the negligible solubility of ethylene in water. While both temperature and pressure elevation induce increased fluidity in the surfactant tail region, the mechanisms appear to be different. Temperature elevation mainly causes an enhanced tumblingof the micelles and increased lateral diffusion of the surfactant molecules. On the other hand, pressure elevation does not significantly affect the tumbling rate or the lateral diffusion but creates disorder in the surfactant tail region by increasing the flexibility for movement about the long molecular axis of the surfactant molecules. Thus, although direct evidence of micellar clustering has not been obtained through these studies, we do see that EPR spectroscopy does reveal importantcharacteristics of the approach to the temperature- or pressure-induced phase transitions. In conjunction with other characterization techniques such SANS and NMR, it is possible to obtain a fairly detailed picture, not just of micelle microstructure,but also of microstructuralchanges, when these systems are subjected to thermodynamic conditions that lead to phase transitions.
Acknowledgment. Partial support from the National Science Foundation (Grant BCS-9202123)is gratefully acknowledged. References and Notes (1) Zulauf, M.; Eicke, H.-F. J . Phys. Chem. 1979, 83, 480. (2) Steigenvald,M.L.;Brus,L.E.InStructureandReactiuityinReuersed Micelles; Pileni, M. P., Ed.; Elsevier Press: New York, 1989. (3) Petit, C.; Lixon, P.; Pileni, M.-P. J. Phys. Chem. 1989, 93, 5854. (4) Leser, M. E.; Wei, G.; Liithi, P.; Haering, G.; Hochkoppler, A.; Bkhliger, E.; Luisi, P. L. J . Chim. Phys. 1987, 84, 1113. (5) Martinek, K.; Berezin, I. V.; Khmelnitski, Yu. L.; Klyachko, N. L.; Levashov. A. V. Biocaralvsis 1987. 1 . 9. (6) Luisi, P. L.; Giomini, P.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acra 1988, 947, 209. (7) Gdklen, K. E.; Hatton, T. A. Biotechnol. Prog. 1985, 1, 69. (81 Leser. M. E.; Wei, G.; Luisi, P. L.; Maestro, M. Biochem. Biophys. . . Res.'Commun. 1986, 135, 629.
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