J. Phys. Chem. 1994,98, 1185-1191
1185
Fluorescence Probe Studies of Mixed Micellar and Lyotropic Phases Formed between an Anionic Bile Salt and a Cationic Detergent Karen Wut and Linda B. McGown’ P. M . Gross Chemical Laboratory, Department of Chemistry, Duke University, Box 90346, Durham, North Carolina 27708-0346
Received: May 6, 1993; In Final Form: November 12, 1993”
Fluorescent probes, including pyrene, benzo[ghi]perylene (BgP), and perylene, were used to study organized media formed between the anionic trihydroxy bile salt sodium taurocholate (NaTC) and the cationic detergent cetyltrimethylammonium bromide (CTAB), over a wide concentration range that includes micellar and lyotropic phases. Solutions of the individual amphiphiles were studied as well. The location of a probe in the mixed micelles was found to depend on the solubility and size of the probe. The microenvironment of pyrene is dominated by N a T C in the mixed micelles, whereas the larger and less soluble perylene and BgP probes interact more favorably with the hydrophobic tails of the CTAB molecules. The photophysical responses of the probes reflect their different locations, providing different perspectives on the transitions in micellar structure. Bromide counterion a t the micellar surfaces was found to be an important factor in the photophysical responses, along with accessibility to bulk solution. A scheme for mixed micellization is proposed that extends from large excesses of one amphiphile to large excesses of the other, over a wide range of total amphiphile concentration. Interestingly, the lyotropic phases formed between N a T C and CTAB have high bulk viscosity, but the probe microenvironment is less viscous than in the mixed micellar phases.
Introduction Bile salts are biological amphiphiles that have unique aggregation behavior. An interesting property of bile salts is their ability to form mixed micellar phases with other amphiphilic compounds. Due to their physiological importance,mixed micelles formed between bile salts and lipids such as lecithin, cholesterol, and potassium oleate have been well studied.’ The most widely investigated system is the bile salt-lecithin mixture, for which several models have been proposed. In one model, simple micelles coexist with disklike mixed micelles that contain a core of lecithin bilayer surrounded by bile salt monomers.2 A modification of this model, referred to as the ‘mixed-disk” model, includes hydrogen-bonded bile salt dimers along the hydrophobic lecithin tails in the lecithin bilayer c ~ r e . ~Investigations -~ of mixed micelles formed between bile salts and other lipids such as potassium oleate and monolein suggest that the micelle “swells” or increases in size with increasing lipid concentration.’ Bile salts can also combine with conventional detergents to form mixed micelles and, at higher concentrations, lyotropic phases.”I0 Mixed micelle formation between anionic bile salts and cationic detergents has been difficult to study, however, due to the tendency for precipitation or phase separation in such systems.lIJ2 Formation of very large aggregates and coacervation have both been observed in equimolar (or near-equimolar) mixtures of alkyltrimethylammonium bromides with dihydroxy bile salts.” These phenomena in mixtures of cationic amphiphiles with anionic amphiphiles are attributed to the electroneutrality of the aggregates at equimolar concentrations, which decreases the repulsion between them and promotes further aggregation. The absence of these phenomena for the trihydroxy bile salts, which form relatively small, spherical aggregates at all molar ratios,llJ3 is due to the increased solubility afforded by the additional hydroxyl group on the steroid backbone. This is consistent with the higher critical micelle concentration (cmc) and smaller size of the aggregates of trihydroxy bile salts relative to the dihydroxy salts. Corresponding author. Present address: The Prccter and Gamble Co.,Sharon Woods Technical Center, 11450 Grooms Road, Cincinnati, OH 45242. e Abstract published in Advance ACS Abstracts, January 1, 1994.
For trihydroxy bile salts, a model has been proposed for an alkyltrimethylammonium*holate mixed micelle’ that is similar to the disklike model proposed for the bile salt-lecithin system.2 The model is approximately spherical with an approximate diameter of 40 A and a stoichiometry of 2 detergent monomers to 1 bile salt monomer. It was not determined whether or not individual bile salt micelles and detergent micelles coexist along with the mixed micelles as was proposed for the bile salt-lecithin system. At high concentrations, conventional detergents go beyond discretemicellization to form more highly ordered, lyotropic liquid crystalline phases.14Js Bile salts alone do not form lyotropic phases, but they can be incorporated into lyotropic phases of other amphiphiles.I6J7For example, the followingternary systems exhibit lyotropicphases as well as mixed micellar phases: sodium cholatelecithin-~ater,~~-~~ bile salt-monoolein-water,21 and sodium cholate-sodium oleatewater.22 The hexagonal phases of these systems are different from those of conventional detergents.17J8 A proposedstructure for such a phase is comprised of lecithin cylinders surrounded by bile salt monomers which are then packed in a hexagonal array.17J8,23,24 In contrast, the hexagonal phase of the cationic detergent hexadecyl (or cetyl) trimethylammonium bromide (CTAB), which forms at concentrations above approximately 700 mM, is comprised of long rods of surfactant.14 Studies of mixed micellization of bile salts have generally employed measurements of physical properties such as viscosity, conductivity, surface tension, and light scattering. Fluorescence probe techniques can be used to provide a different perspective on these aggregation phenomena that reports on changes in aggregate structure through modifications of the local environment of the probe. This paper describes fluorescence probe studies of mixtures of CTAB with sodium taurocholate (NaTC), which is an anionic trihydroxy bile salt. A previous probe study demonstrated mixed micellization between NaTC and CTAC, which is similar to CTAB but with chloride counterion, at concentration ranges around the cmc of NaTC and showed that the mixed micelles provide binding environments for fluorescent probes that are different from those in the simple micelle^.^ In the present work with CTAB, a much
0022-365419412098-1185%04.50/0 0 1994 American Chemical Society
1186 The Journal of Physical Chemistry, Vol. 98, No. 4, I994
wider concentration range was studied, extending from micellar to lyotropic phases. Polycyclic aromatic hydrocarbons (PAHs), which have been used extensively in fluorescence probe studies of micellar media,25-29served as the probes. Measurements of fluorescence intensity, vibronic band intensity ratio, lifetime, and anisotropy were used to study the various phases and the binding microenvironments of the solubilized probes in these phases. Measurements of scattered light intensity provided information about the relative size of the aggregates in the absence and presence of probe. An aggregation scheme is presented that covers the entire range of molar proportions and concentrations used. These studies are important to further not only the understanding of bile salt aggregation and interactions with other molecules of biological or pharmaceutical interest but also the ongoing studies of applications of organized bile salt media for chemical separation^^^ and analy~is.~’”~ Previous work has shown that bile salt micelles can improve the accuracy of luminescence analysis of complex samples by performing an “in situ” extraction of analytes, thereby providing a relatively uniform microenvironment for a given solute in which the solute is well isolated from the bulk solution and protected from intermolecular interactions that are commonly experienced in conventional Enhanced sensitivity for some fluodetergent rescent analytes has also been dem~nstrated.~) It is of interest to explore mixed micellar and lyotropic phases of bile salts for these and related purposes. Experimental Section Pyrene and benzo[ghi]perylene (BgP), both >98% purity, were purchased from AccuStandard. Perylene (Gold Label, >99%) was purchased from Aldrich Chemical Co. The compounds were used as received. Stock solutions of the probes were prepared in absolute ethanol (Aaper Alcohol and Chemical Co.). Sodium taurocholate (NaTC, >98%) was purchased from Calbiochem (La Jolla, CA). Cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC, both >99% purity) were obtained from Sigma Chemical Co. (St. Louis, MO). All of the surfactants were used without further purification. All aqueous solutions were prepared in HPLC-grade deionized water. The micellar solutions of the probe compounds were prepared by gentle evaporation of the ethanol from the appropriate volume of probe stock solution, followed by dilution with the aqueous micellar stock solution in a volumetric flask and sonication for approximately 1.5 h. The lyotropic liquid crystalline phases were prepared by adding water to a volumetric flask with the appropriate amounts of solid surfactant and pre-evaporated fluorescent probe and sonicating for 2 h. Following sonication, the samples were transferred into test tubes and centrifuged for 2 h. The liquid crystal samples were then extracted from the test tubes with a syringe and dispensed into quartz cuvettes. Fluorescence measurements were made with a multifrequency, phase-modulation spectrofluorometer (Model 48000S, SLM Instruments, Inc., Urbana, IL). The instrument uses a 450-W xenon arc lamp as the excitation source, excitation and emission monochromators for wavelength selection, a Pockels cell electrooptic modulator for modulation of the excitation beam for fluorescence lifetime measurements, and steady-state or phasesensitive PMT detection (Hamamatsu R928P). A reference channel with a glycogen scattering solution was used for ratiometric correction of both steady-state and dynamic-state, frequency-modulated (lifetime) measurements. An IBM-PC AT with SLM instrument software (Version 1.41) was used for online data acquisition and analysis. The temperature of the sample chamber was maintained at 25.0 f 0.1 O C for all measurements with a Haake A81 temperature controller. All solutions were contained in strain-free quartz curvettes. The solutions were not deoxygenated prior to measurement.
Wu and McGown For both excitation and emission spectra, the fluorescence intensity was measured a t 1-nm intervals. Each intensity is the average of five samplings, performed internally by the instrument over a period of 1.5 s. The entrance and exit slits on both monochromators were set to a 4-nm bandpass. The fluorescence intensities at single wavelengths for vibronic band ratios were the averages of four 50-average measurements, using 2-nm bandpass settings for the monochromator slits. Fluorescence anisotropy of perylene was measured in the L-format instrumental c ~ n f i g u r a t i o n ,using ~ ~ excitation and emission wavelengths of 440 and 473 nm, respectively. The monochromator slits were set to a 4-nm bandpass. The scattered light intensity measurements were performed by setting both the excitation and emission monochromators to 540 nm, where the scattered light was detected at 90’ to the excitation beam. Each intensity is the average of four 75-average measurements. Fluorescence lifetimes were determined using five 75-average measurements of phase shift and demodulation of both the sample and reference a t several different modulation frequencies (1,2, 3,5,6,8 MHz for pyrene and BgP; 40,25,10 MHz for perylene). A scattering solution of kaolin in water was used as the fluorescence lifetime reference and was intensity matched with the sample. The excitation monochromator entrance and exit slits were set at 16 and 1 nm, respectively, and the emission monochromator slits were both set at 16 nm. A linear Glan-Thompson polarizer was set at the magic angle in the emission beam to correct for photoselection effects. Nonlinear least-squares analysis of the multifrequency phase-modulation data was performed using either the SLM instrument software or global analysis software (Globals Unlimited). Results
Fluorescence probe studies were conducted in various mixtures of NaTC with CTAB, including the following: (1) pyrene and benzo[ghi]perylene (BgP) in 10, 30, and 120 mM NaTC with 80 mM CTAB (chosen on the basis of preliminary studies, which indicated that 80 mM CTAB solutions showed unusually high bulk viscosity and scattered light intensity); (2) pyrene and perylene in 10 and 30 mM NaTC with CTAB ranging from 40 to 200 mM; (3) pyrene and perylene in solutions of NaTC with CTAB in which the total surfactant concentration (expressed as total moles of surfactant monomer per liter of solution) was kept at 30 or 200 mM and the mole fractions of the two compounds were varied; (4) perylene in lyotropic phases formed between NaTC and CTAB. The concentrations of NaTC and CTAB are expressed in all cases as the total concentration in terms of the monomer. For the most part, the concentrations used in the present work meet or exceed the cmcs of the components. The generally reported cmc of CTAB is 0.9-1 mM. A cmc in the region of 8-12 mM has been identified for NaTC, with an earlier critical region around 3 mM that may correspond to dimer f o r m a t i ~ n . ~ ~ . ~ ~ Studies of Mixed Micellization between NaTC and 80 m M CTAB. Scattered Light Intensity. Figure 1 shows the scattered light intensities of individual solutions of NaTC and CTAB and mixtures of the two, in the presence and absence of 2 pM BgP. The scattered light intensities of the mixed micellar solutions are much larger than the sums of the scattered light intensities of the individual micellar solutions, which are also shown in Figure 1, indicating the formation of mixed micelles that are larger than the individual NaTC and CTAB micelles. The decrease in intensity of the mixed micellar solutions between 30 and 120 mM NaTC suggests a structural change in the mixed micelles to a smaller or more compact form. The addition of 2 p M BgP had little effect on the scattered light intensities of the mixed micellar solutions, demonstrating that the formation of mixed micelles is not a function of the probe
Mixed Micellar and Lyotropic Phases
The Journal of Physical Chemistry, Vol. 98, No. 4, 1994 1187
‘‘1. m
a
am
c
a,
a
m
m m m i a
NaTC CONCEKIIIAllON (mMI
o
a,
a m
m
i m m i a
Narc CONCENlRATlON (mM)
Figure 1. Scattered light intensity, in the absence (left) and presence (right) of BgP, vs NaTC concentrationmeasured at La= Lm= 540 nm. Solid square, NaTC only; solid triangle, NaTC/CTAB mixture; open circle, sum for individual solutions of NaTC and CTAB at the same concentrations as in the mixture.
and that the mixed micelles are sufficiently large to incorporate BgP without significantly changing the size of the micellar unit. In contrast, the scattered light intensity in NaTC alone is much larger in the presence of BgP. The intensity is highest at 10 mM NaTC, which is its cmc, due either to microcrystalline suspensions of BgP in the bulk aqueous solution or to the formation of large aggregates of NaTC with BOP. At higher NaTC concentrations the intensity decreases as the BgP is more completely solubilized within the NaTC micelles, although the intensities are still higher than in the absence of BgP due to distortion of the NaTC micelle by the BgP molecule. VibronicBandlntensity Ratios of Pyrene and BgP. The relative polarity of probe microenvironmentscan be studied by examining the vibronic band intensity ratios (VBIRs) of certain fluorescent probes,37including pyrene and BOP. For both of these probes, band I11 corresponds to an allowed transition which is not sensitive to solvent polarity and band I corresponds to a forbidden transition which is enhanced as polarity increases.3840 In simple solvents, an increase in the intensity ratio of band I to band I11 of these probes indicates an increase in solvent polarity. In micellar media and other structures, VBIRs do not necessarily reflect simple, bulk polarities such as those determined in simple solvents. In the following discussions, the term ’polarity” will be used to refer to the microenvironment of the probe as indicated by the VBIR, with the understanding that these values also reflect the nature of the specific binding interactions and electronic distortions of the probe in the host structure. The VBIRs of pyrene (1.5 pM) and BgP (2.0 pM) in NaTC, CTAB, and mixtures of the two are shown as a function of NaTC concentration in Figure 2. In NaTC alone, the VBIRs decrease steadily as NaTC concentration is increased. For both probes, the VBIRs decrease slightly upon addition of 10 mM NaTC to the 80 mM CTAB solution, followed by an increase when the NaTC concentration is increased to 30 mM. When the NaTC concentrationis further increased to 120mM, the VBIRdecreases dramatically for pyrene but remains the same for BgP. The VBIRs for both probes are higher, indicating a more polar microenvironment, in the mixed micelles than in the simple NaTC micelles; this is particularly dramatic for pyrene in 30 mM NaTC. The VBIRs of the two probes cannot be directly compared to each other because each probe has a different numerical scale. They can be qualitatively compared, however, by using a ‘polarity ruler” constructed for each probe by measuring its VBIR in a
i
0.850
a0
4)
60
83ImIa014,
range of simple solvents. This is shown in Table 1. The two probes experience very apolar environments in 120 mM NaTC, with the environments of pyrene just slightly more apolar than those of BgP. The probes experiencesimilar, polar environments in 10mM NaTC, but in 30 mM NaTC the environment of pyrene is significantly less polar than that of BgP. Pyrene experiences a significantlymore polar environment than BgP in CTAB, which suggests that pyrene is closer to the outer, interfacial region of the micelle.
Wu and McGown
1188 The Journal of Physical Chemistry, Vol. 98, No. 4, 1994
330 A
A
Lm
1.000
e m
f
4
40
A
80
120
160
I
200
CTAB Concentration (mu)
P8
Figure 4. I/III vibronic band intensity ratio of pyrene vs CTAB concentration, measured at bx= 338 nm, L m , I = 372 nm, and Lm,III =
A
B
383 nm: NaTC at 0 mM (open circles), 10 mM (solid triangles), and 30 mM (solid circles).
0
im m 0
0
C
o
20
Po
N
a
m i m i m i a ,
~ concmntmnon C
Figure 3. Fluorescence lifetime vs NaTC concentration determined for = 338 nm, b m = 385 nm) and BgP (bx= 367 nm, L m = pyrene (bx 422 nm). Solid triangles, pyrene in NaTC alone; open triangles, pyrene in NaTC/CTAB mixtures; solid squares, BgP in NaTC alone; open squares, BgP in NaTC/CTAB mixtures. Total CTAB concentration in mixtures is 80 mM.
In the mixed micellar solutions,BgP experiences a lower polarity than pyrene at low NaTC concentrations (10 and 30 mM), indicating that the BgP is located deeper in the hydrophobic, micellar core. The opposite is true at 120 mM NaTC, at which BgP experiences a much more polar environment than pyrene in the NaTC-dominated structures. Fluorescence Lifetimes of Pyrene and BgP. Fluorescence lifetimes are shown in Figure 3 for pyrene (1.5 pM) and BgP (2.0 pM) in the micellar solutions. The lifetimes are much longer in the micellar phases than in ethanol, in which lifetimes of 21.7and 18.8 ns were obtained for pyrene and BgP, respectively, due primarily to protection of the probe in the micelles from dissolved oxygen and other nonradiative deexcitation processes in the bulk solution. Greater rigidity or microviscosity in the micellar microenvironment compared with the bulk solution may also contribute to the increase in lifetime. For both probes, the fluorescence lifetime is generally highest in NaTC alone and lowest in CTAB alone, which suggests that the probes are most prone to quenching and/or nonradiative deexcitation in the CTAB micelles. As NaTC is added to the CTAB solutions, the probe lifetimes approach those in NaTC alone, converging around 120 mM NaTC. Studies of Mixed Micelles between NaTC and CTAB at Various Concentrations. Vibronic Band Intensity Ratio of Pyrene. The VBIRs for 1.5 pM pyrene in mixtures of NaTC with CTAB and in CTAB alone are shown in Figure 4. The standard deviations of the intensity measurements used to calculate the ratios were approximately 1%. Formation of pyrene excimer was not observed. The VBIRs of pyrene in the absence of CTAB are 1.1 8 for 10 mM NaTC and 0.95 for 30 mM NaTC. The microenvironmentalpolarity for pyrene is highest in CTAB alone. The VBIR is relatively constant up to 160 mM CTAB and then increases at 200 mM, indicating a possible phase change at 160 mM CTAB. For 10 mM NaTC in CTAB, the VBIR is essentially constant and independent of CTAB concentration. For 30 mM NaTC in CTAB, the VBIR is low in NaTC alone
0.5004 40
80
120
160
I
200
CTAB Concentration (mM)
Figure 5. Relative fluorescence intensity of perylene vs CTAB " e n tration, measured at Lx= 441 nm and L m = 473 nm: NaTC at 0 mM (open circles), 10 mM (solid triangles), and 30 mM (solid circles).
and increases to approach the values observed in CTAB alone at higher CTAB concentrations. Fluorescence Intensity, Lifetime, and Anistropy of Perylene. The fluorescence intensity of 2.5 pM perylene in mixtures of NaTC with CTAB and in CTAB alone is shown in Figure 5.The standard deviations in the intensity measurements were approximately 0.5%. The curve for 10 mM NaTC in CTAB parallels the curve for CTAB alone but with slightly higher intensities; both curves exhibit maxima at 80 mM CTAB, suggesting a structural transition or unique phase in the region of 80 mM CTAB that is relatively unaffected by the presence of small amounts of NaTC. The curve for 30 mM NaTC in CTAB is different, decreasing from high intensities at both ends to a minimum at 80 mM CTAB that is below the other curves. The molar monomer ratio is close to 1:2NaTC:CTAB in the 30 mM NaTCf80 mM CTAB solution, which is the ratio of the disklike micelles that was proposed for the alkyltrimethylammoniumcholate mixed micelle.11J3 Fluorescence lifetimes were determined for the same solutions and showed very little variation with CTAB or NaTC concentration. All of the values were in the range of 5.7-6 ns. The anisotropy of perylene vs CTAB concentration is shown in Figure 6. Standard deviations in the anisotropy measurements are in the range of 3%-7%. In the absence of NaTC, the anisotropy shows a slight but steady increase with increasing CTAB concentration, with the exception of an anomalously high value a t 160 mM. The anisotropy is higher in the presence of 30 mM NaTC than in CTAB alone at all CTAB concentrations. In the presence of 10 mM NaTC, the anisotropy peaks at 80 mM CTAB and then decreases, intersecting the curve for CTAB alone at 160 mM. Mole Fraction Studies. Vibronic Band Intensity Ratio of Pyrene. The VBIRs of 1.5 pM pyrene at total surfactant concentrations of 30 and 200 mM are shown in Figure 7 for mixtures of CTAB with NaTC. The standard deviations for the VBIRs were in the range of 0.5%-2.0%. The curves for the two total concentrations parallel each other, with the VBIR decreasing linearly up to 0.6 mole fraction NaTC and then leveling off as
Mixed Micellar and Lyotropic Phases
The Journal of Physical Chemistry, Vol. 98, No. 4, 1994 1189 YY,"
r
0.040
g 2
0.020
0.000 120
80
40
160
200
CTAB Concsntrotion (mM)
Figure 6. Fluorescence anisotropy of perylene vs CTAB concentration, measured at Lx= 441 nm and Lm= 473 nm: NaTC at 0 mM (open circles), 10 mM (solid triangles), and 30 mM (solid circles).
0.0
02
04
0.4
0.8
10
NalC M e Fractbn
Figure 9. Fluorescence anisotropy of perylene vs NaTC mole fraction, measured at LX= 442 nm and Lm= 473 nm. The total concentration (NaTC + CTAB, expressed as monomer) is 30 mM (open symbol) or 200 mM (closed symbol).
NaTC Mole Fraction
Figure 7. I/III vibronic band intensity ratio of pyrene vs NaTC mole fraction, measured at Lx= 338 nm, Lm,l= 372 nm, and Lm,III = 383 nm. The total concentration (NaTC + CTAB, expressed as monomer) is 30 mM (open symbol) or 200 mM (closed symbol).
"
-
ao
.
,
,
.
02
I
.
.
M
. . 0..8 - 1.0.
0.4
NoTC Mole Froctlan
Figure 8. Relative fluorescence intensity of perylene vs NaTC mole fraction, measured at Lx= 442 nm and Lm= 473 nm. The total concentration (NaTC + CTAB, expressed as monomer) is 30 mM (open symbol) or 200 mM (closed symbol).
100%NaTC is approached. Although the curves show the same trends, the VBIR is uniformly higher for the 30 mM profile, indicating a more polar probe environment at the lower total concentration. The change in the slopes of the curves near 0.6 mole fraction NaTC at both total concentrations may correspond to a change in the micellar structure toward a structure more similar to the bile salt micelle at high mole fractions of bile salt. Fluorescence Intensity, Lifetime, and Anistropy of Perylene. The fluorescence intensity of 2.5 pM perylene is plotted as a function of mole fraction NaTC in Figure 8 for mixtures of CTAB with NaTC. The intensities converge at 0.6 mole fraction NaTC, which was also a breaking point in the slopes of the VBIR curves (Figure 7). At 30 mM total concentration, intensity is lowest in 100%NaTC at 30 mM and highest in 100%CTAB at 200 mM total concentration. NaTC appears to dominate the perylene microenvironment above 40 mM NaTC, which corresponds to 0.2 mole fraction NaTC in the 200 mM total concentration curve,
but does not significantly affect the microenvironment below 24 mM NaTC, which corresponds to 0.8 mole fraction NaTC in the 30 mM total concentration curve. Fluorescence lifetimes were determined for the same solutions and showed very little variation with mole fraction. Although the lifetimes did tend to mimic the intensity curves, all of the values were in the range of 5.5-6 ns. The anisotropy of perylene vs mole fraction NaTC is shown in Figure 9. The curves are similar for both total concentrations, with slightly higher values at 200 mM. In both curves, the anisotropy increases as the NaTC mole fraction increases from 0 to 0.4 and then levels off. The curves finally diverge as they approach 100%NaTC, with the 30 mM curve decreasing a little and the 200 mM curve increasing by a large amount. Counterion Effects. Solutions of 10 mM NaTC and 30 mM NaTC in cetyltrimethylammonium chloride (CTAC) in the concentration range of 40-200 mM were examined in separate experiments to determine the effects of counterion on the binding microenvironments. In general, trends exhibited by the probes in CTAC were similar to those in CTAB but the magnitudes were different. The VBIRs of pyrene were generally higher for CTAC, indicating a more polar environment. The anisotropy of perylene was lower for CTAC, indicating a less rigid environment or greater freedom of rotation. Light scattering measurements showed that the size of the mixed micelles increaseswith increasing CTAC concentration but then decreases for both 10 and 30 mM NaTC at 200 mM CTAC, suggesting a phase transition not seen in the CTAB solutions. The counterion effect may be described in terms of ion polarizability, in which increased polarizability leads to enhanced binding interactions. This would explain the anisotropy and polarity data in which the stronger binding of the bromide ion results in a larger aggregate that more effectively excludes the bulk solution from the micellar interior, creating a more rigid and apolar microenvironment in the mixed micelles. Lyotropic Phases. The results of fluorescence measurements of 2.5 pM perylene are shown in Table 2 for several lyotropic phases and mixed micellar solutions. The liquid crystallinity of the lyotropic solutions was confirmed by optical microscopy. The intensity of perylene is much higher in the CTAB lyotrope than in any of the other phases, and lowest in the mixed lyotropic phases. The anisotropy, on the other hand, is lowest in the pure CTAB phases and highest in the mixed micellar phases. The lifetime, which shows only small differences among the various phases, is consistently lower in the lyotropes than in the micellar phases. Discussion The results of these experiments provide strong evidence for mixed micellization between NaTC and CTAB over a range of
1190 The Journal of Physical Chemistry, Vol. 98, No. 4, I99 4
TABLE 2 Fluorescence Characteristics of 2.5 pM Perylene in NaTC/CTAB Lyotropic Phases and Mixed Micellar Phases intensity anisotropy lifetime Ins) Lyotropic Phases 1.44 0.028 5.6 710 mM CTAB 710 mM CTAB/30 mM NaTC 0.78 0.31 5.6 710 mM CTAB/100 mM NaTC 0.80 0.030 5.6 Micellar Phases 1.05 0.023 5.8 80 mM CTAB 80 mM CTAB/lO mM NaTC 1.25 0.041 5.7 0.97 0.047 5.8 80 mM CTAB/30 mM NaTC concentrations that extends well above the cmcs of the individual components. The mixed micelles are substantially larger than the simple CTAB or NaTC micelles, increasing in size as equimolar proportions of the two amphiphiles are approached, but in all cases still much larger than the individual micelles. It is interesting to note the effect of increasing NaTC concentration from 10 mM, which is in the midst of its cmc region, to 30 mM. At the lower concentration, NaTC behaves primarily like a monomer that is solubilized along with the probes in CTAB-like micelles. At the higher concentration, NaTC has a much stronger influence on the structure of the mixed micelles even a t stoichiometric far from 1:2 NaTC:CTAB. A transition at 80 mM CTAB is indicated by the VBIR of pyrene and the fluorescence intensity of perylene, both in the absence and presence of NaTC. A second transition is indicated at 160 mM CTAB by the VBIR of pyrene and the anisotropy of perylene. This concentration has been previously identified as a transition point in the aggregation of CTAB,41from a region of changes that are tentatively attributed to variations in counterion binding to a region of relatively constant properties that endure up to approximately 250-300 mM. In the development of a model for the mixed micellization, it is important to first consider the possibility of coexistence of bile salt and CTAB micelles. Such coexistence has been reported for bile salt-lecithin system^^^,^^ but has not been established for the bile salt-alkyltrimethylammonium systems. In the present work, coexistence is contraindicated by the recovery of only one fluorescence lifetime component for the probes in any of the solutions, although this could also indicate that the particular probe has a strong perference for one particular aggregate. Coexistence could explain the increasingly NaTC-like character of the probe binding sites as NaTC concentration is increased, but not all of probe characteristics showed this trend. Moreover, the scattered light intensity of all of the mixtures in the absence of probe greatly exceeds the sum of the intensities of solutions of the individual components at the same concentrations, indicating the formation of larger aggregates. Therefore, coexistence of simple micelles is not considered to be an important factor in the NaTC/CTAB system. Based on theseconsiderations, we propose the following scheme for aggregation in mixtures of NaTC with CTAB. In the absence of NaTC, CTAB forms conventional, spherical micelles. When small amounts of NaTC are added, the NaTC monomers are incorporated into the interfacial regions of the CTAB micelle, expanding the micelle and perhaps even forming hydrogen-bonded dimers in the interfacial region as NaTC concentration increases. As the 1:2 NaTC:CTAB ratio is approached, “disklike” mixed micelles form in which a CTAB lamellar core is surrounded by NaTC monomers with their hydrophobic surfaces facing inward. As still more NaTC is incorporated into the mixed micelles, the “mixed-disk” mixed micelles form, in which hydrogen-bonded NaTC dimers are mixed with the hydrophobic tails of CTAB in the lamellar core of the disklike micelle. At even higher excesses of NaTC, smaller NaTC-like micelles form that solubilize both CTAB monomers and the fluorescent probes. The size and solubility of a probe influence its location in a
Wu and McGown micelle, and therefore determine its response tochanges in micellar structure and composition. For example, when the NaTC concentration is held constant at its cmc of 10 mM and CTAB concentration is increased from 0 to 200 mM, the VBIRof pyrene is unaffected by the increasing CTAB concentration while the intensity of perylene is largely unaffected by the addition of NaTC. In the mole fraction studies, the VBIR of pyrene steadily decreases as the mole fraction of NaTC is increased from 0 to 1, while perylene intensity and anisotropy remain relatively constant over fairly broad ranges of mole fraction. We conclude from these results that the small, relatively soluble pyrene shows the greatest affinity for NaTC and is therefore most sensitive to structural perturbations in the outer regions of the micelle, while the larger, highly insoluble BgP and perylene probes prefer to be associated with the hydrophobic tails of CTAB and remain in more constant, interior regions. These differences among the probes provide an explanation for their different photophysical responses to the changes in mixed micellar structure. In the simple CTAB micelles, BgP and perylene are buried deep within the hydrophobic core while pyrene resides closer to the interfacial regions. The addition of small amounts of NaTC creates a more protected environment for all three probes by tighteninguptheinterfacialregionsin thespherical micelle, thereby reducing penetration of the bulk solution and diluting the surface coverage of bromide ions. This results in increases in the fluorescence lifetimes and decreases in the VBIRs of BgP and pyrene. As the NaTC:CTAB ratio approaches 1:2, there is a structural transition to the disklike mixed micelles. BgP and perylene remain associated with the CTAB tails in the lamellar core, in a more rigid, protected environment. Pyrene resides near the outer NaTC layer. The lifetimes of pyrene and BgP increase, indicating even better protection from bulk solution than in the spherical, CTABlike structures. However, the increase in the VBIRs of these probes, as well as the decrease in perylene intensity, reflects the disk structure in which the bromide ions are concentrated at either end of the disk and are no longer diluted by interspection of NaTC molecules. As NaTC concentration is further increased to form the mixed disk micelles and, finally, the NaTC-like micelles, pyrene experiences increasingly NaTC-like microenvironments while BgP and perylene remain in relatively constant environments that are dominated by the solubilized CTAB tails. The influence of bromide counterions is an important consideration. The effects of bromide ions in micellar systems do not follow typical collisional kinetics due to the restricted motion of the probes within the micelle as well as the strong association of the bromide ions with the external surface of the mi~elle.2~ The bromide ions can therefore cause increases in VBIR and decreases in intensity of the probes without significantly affecting the probe lifetimes, because the bromide-probe interactions are essentially static on the time scale of the fluorescence event. The large increase in the lifetimes of BgP and pyrene as NaTC is added to CTAB is primarily a result of the increased protection from dissolved oxygen and other quenching interactions as the micelles become larger and better protected from bulk solution. Perylene does not show the same increase because its lifetime is too short ( < l o ns) to be much affected by the collisional processes. The disklike models may also be extended to the hexagonal lyotropic phases, in which the CTAB monomers are organized into long rods and the bile salts are wrapped around the rods up to a certain concentration, above which a more favorable structure is formed with bile salt dimers surrounded by the rodlike detergent monomers. Although this work does not provide sufficient information to formulate a model for the lyotropes, it is clear that perylene senses the presence of NaTC in the CTAB l y o t r o p , even a t NaTC concentrations as low as 30 mM. Upon addition of NaTC, the perylene fluorescence becomes much less intense,
Mixed Micellar and Lyotropic Phases slightly more anisotropic and perhaps slightly shorter-lived. This may be due to increased proximity of the probe to the NaTC monomers that coat the surface of the rods, holding the probes more rigidly but also in greater contact with the bulk solution and bromide counterions that may be near the surface. Originally, the rationale for studying lyotropic phases was to identify phases that offer a greater degree of rigidity and protection in the binding microenvironment, in order to enhance the luminescence of solubilizates and minimize matrix effects from the bulk solution. However, despite the high bulkviscosity of the lyotropic phases, on the molecular level the perylene microenvironments have significantly lower microviscosity in the lyotropic phases than in the mixed micellar phases. Only in the individual CTAB micelles is the microviscosity lower than in the lyotropes, which highlights the influence of the bile salts on the probe microenvironments in the mixed media. In fact, perylene is in a more rigid and protective environment in the mixed micellar phases than in the mixed lyotropes, as evidenced by the greater anisotropies,higher intensities, and longer lifetimes. This suggests that the micellar media offer more possibilitiesfor rigid, protective microenvironments for fluorescent solubilizatesthan the lyotropic phases. Acknowledgment. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, U.S. Department of Energy (Grant DEFG0588ER1393 1) and by the Environmental Protection Agency (Grant R8 17127-01). References and Notes (1) Nair, P. P., Kritchevsky, D., Eds. Bile Acids; Plenum Press; New York, 1971. (2) Carey, M. C.; Small, D. M. J. Clin. Invest. 1978, 61, 998. (3) Mazer, N. A.; Benedek, G. B.; Carey, M. C. Biochemistry 1980,19, 601. (4) Mazer, N. A.; Kwasnick, R. F.; Carey, M. C.; Benedek, G. B. Micellization, Solubilization, and Microemulsions, Vol. 1; Mittal, K. L., Ed.; Plenum Press: new York, 1977. ( 5 ) Zimmerer, R. O., Jr.; Lindenbaum, S. J . Pharm. Sci. 1979,68,581. (6) Velazquez, M. M.; Garica-Mateos, I.; Lorente, F.; Valero, M.; Rodriquez, L. J.; J . Mol. Liq. 1990, 45, 95. (7) Nithipatikom, K.; Mc Gown, L. B. Photochem. Photobiol. 1988,47, 797.
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