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Novel Fluorescence Spectral Deconvolution Method for Determination of Critical Micelle Concentrations Using the Fluorescence Probe PRODAN J. E. Wong, T. M. Duchscherer, G. Pietraru, and D. T. Cramb* Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4 Received December 14, 1998. In Final Form: April 29, 1999 A simple yet novel method for the determination of critical micelle concentrations is introduced. In this method, the fluorescence spectrum of a probe molecule is deconvoluted into constituent spectra consisting of that from the surfactant free probe and that from the micelle-bound probe molecule. There is no assignment of arbitrary shape functions to the spectra in this process. Using the probe molecule PRODAN, the deconvolution method is compared with the more traditional fluorescence spectral maximum shift method for the cmc determination of aqueous surfactants including sodium dodecyl sulfate (SDS), Brij 35, and reverse micelles of Triton X-100 (TX-100) in cyclohexane/hexanol solution. Finally, to help determine its position in the micellar environment, fluorescence quenching experiments were performed on PRODAN in aqueous SDS and TX-100 reverse micelle solutions.
Introduction The critical micelle concentration (cmc)1 is the first identifiable physical change in a solution containing surfactants, as their concentration is increased. In the simplest terms, surfactants in solution below their cmc are thought of as monomeric and dispersed. At concentrations above their cmc, surfactants self-associate, in spheroid micelles or micelles of other morphologies, to lower their free energy. Thus, the critical micelle concentration, for a surfactant in a given solvent, is a primary means of surfactant characterization. There have been many methods developed to measure cmc’s. Some of the most common methods are surface tension, conductivity, dynamic light scattering, and spectrophotometry.2 Historically, surface tension measurements, using the Wilhelmy plate method,3 have been the most abundant. More recently, however, much attention has been paid to spectroscopic methods of cmc determination.2 This may have resulted from the relative ease with which these experiments can be performed using even modest spectrometers. Spectroscopic cmc determination relies on the use of a probe molecule which readily partitions into micelles and whose spectral characteristics change in some way within the micelle. The changes in the probe molecule could be a shifting of the wavelength for maximum absorption or emission, changes in the fluorescence quantum yield, or all of these. The most successful probe molecules are somewhat amphiphilic and possess excited states with large electric dipole moments. Their amphiphilicity will enhance their universal solubility, and their excited-state dipoles will result in sensitivity to the polarity of their local environment. The latter will be manifest in large spectral shifts as the molecules partition into the micelles. Recently novel spectrophotometric methods for cmc determination have been introduced. These include the * Corresponding author. E-mail:
[email protected]. (1) Hunter, R. J. Foundations of Colloid Science; Oxford University Press: New York, 1991; Chapter 10. (2) McIntire, G. L. Crit. Rev. Anal. Chem. 1990, 21, 257. (3) Volkov, A. G.; Deamer, D. W.; Tanelian, D. L.; Markin, V. S. Liquid Interfaces in Chemistry and Biology; Wiley-Interscience: New York, 1998; Chapter 2.
absorbance deviation method,4 fluorescence anisotropy,5 and low cmc determination by molar fluorescence.6 The cmc’s determined using internal and external fluorescence probes have been compared.7 Additionally, critical micelle concentration and microviscosity have been examined using fluorescence quantum yields.8 Pyrene has been extensively used9 for spectrophotometric cmc determination of surfactants ranging from the common to more exotic polymeric systems. It has welldocumented1 fluorescence spectral characteristics, which change between polar and nonpolar environments. The disadvantage of using pyrene is its poor solubility in water and thus its propensity to form spectrum-obfuscating microcrystals. Because of their severely hydrophobic nature, molecules such as pyrene cannot be used to examine the interiors of reverse micelles. Fluorescence spectral shifting is perhaps more sensitive to the onset of micelle formation than changes in fluorescence quantum yield. Unfortunately, fluorescence quantum yields can be very sensitive to probe-surfactant dimerization.10 In contrast, shifts in fluorescence maxima are most pronounced when the probe molecule partitions into a completely distinct molecular environment. The limitation with spectral shifting is that, very near the cmc and for low cmc surfactants, there will always exist a bimodal (or higher) distribution of the amphiphilic probe molecules between the micelles and the solvent. This may make the fluorescence shift at surfactant concentrations near the cmc difficult to discern. Given the ubiquity of fluorescence spectrometers in institutional and industrial laboratories, an uncomplicated (4) Ysambertt, F.; Vejar, F.; Paredes, J.; Salager, J.-L. Colloids Surf., A 1998, 196, 189. (5) Zhang, X.; Jackson, J. K.; Burt, H. M. J. Biochem. Biophys. Methods 1996, 31, 145. (6) Mehreteab, A.; Chen, B., J. Am. Oil Chem. Soc. 1995, 72, 49. (7) Goon, P.; Manohar, C.; Kumar, V. V. J. Colloid Interface Sci. 1997, 189, 177. (8) Myagishi, S.; Kurimoto, H.; Ishihara, Y.; Asakawa, T. Bull. Chem. Soc. Jpn. 1994, 67, 2398. (9) Rager, T.; Meyer, W. H.; Wegner, G.; Winnik, M. A. Macromolecules 1997, 30, 4911. (10) Casero, I.; Sicilia, D.; Rubio, S.; Pe´rez-Bendito, D. Talanta 1997, 45, 167.
10.1021/la981716z CCC: $18.00 © 1999 American Chemical Society Published on Web 07/01/1999
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method to discern more accurately critical micelle concentrations using fluorescence maxima shifting is desirable. This method exploits the microscopic chemical environments experienced by the probe molecule in a surfactant solution near the cmc. Furthermore, a consistent comparison of cmc’s for different surfactants is best achieved using a single, “universal” fluorescence probe molecule. In this article, we use 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) as a “universal” cmc probe molecule. Moreover, we present a novel method of cmc determination using the environmentally sensitive fluorescence spectrum of this probe. We will show that, using a spectral deconvolution, PRODAN can be applied to the cmc determination for many types of surfactants, including reverse micelle systems. Experimental Methods Reagents. The fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (PRODAN, Molecular Probes, Eugene, Oregon) was used as received. Solutions were prepared using methanol (BDH), water (Barnsted Nanopure), n-pentane (BDH), n-heptane (BDH), toluene (BDH), and cyclohexane (BDH). The surfactants sodium dodecyl sulfate (SDS, Sigma), polyethoxy 23 lauryl ether (Brij 35, Sigma), and t-octylphenoxypolyethoxyethanol (Triton X-100, Sigma) were used without further purification. Hexanol (BDH) was used as a cosurfactant to TX100. Recrystallized sodium iodide (NaI, Fisher Scientific) and liquid diiodomethane (CH2I2, Aldrich) were used as quenchers. Procedure To Prepare Solutions. PRODAN Solutions Using Different Solvents. A stock solution of PRODAN with a concentration of 1.0 × 10-4 M in methanol was prepared. To prepare each solution, an aliquot of the stock solution was measured into a glass vial. The methanol was then evaporated off using dry nitrogen. A specific volume of distilled, deionized water, n-pentane, n-heptane, methanol, or toluene was added to prepare the solutions. This will be called the dry method of preparation. The solutions were kept in glass vials, covered with aluminum foil, and stored in the dark until used. Preparation of Aqueous Micelle Solutions. Stock solutions of SDS (2.5 × 10-2 M) and Brij 35 (1 × 10-3 M) were prepared. For cmc determination a series of dilutions of aqueous SDS were added to PRODAN using the dry preparation method. For the fluorescence quenching experiments, solutions were prepared with a SDS concentration of 10 mM. An iodide concentration in the range 0-0.15 M was made from a stock solution of sodium iodide in water (1.000 M). All of the solutions were sonicated for 10 min. Preparation of Triton X-100 Reverse Micelle Solutions. A stock solution of 4:1 Triton X-100/hexanol was prepared. This solution was diluted with cyclohexane to make a solution that had a TX100 concentration of 10 mM. A stock solution of PRODAN in cyclohexane was also prepared with a concentration of 1.25 × 10-4 M. A series of solutions were prepared containing TX-100/hexanol, PRODAN, and cyclohexane. The concentrations of TX-100 ranged from 0.4 mM up to 4.0 mM. The concentration of PRODAN was 10 µM. Each solution was sonicated for 10 min. A 0.01 M solution of CH2I2 in cyclohexane was prepared and used in the quenching experiment of 4.0 mM TX-100/hexanol/ cyclohexane. Spectroscopic Measurements. Fluorescence spectra were recorded using a Photon Technologies Inc. (Canada) spectrofluorometer, with a band width set to 2 nm and dwell times ranging from 0.05 s/point to 1 s/point, depending on the intensity of the fluorescence signal. A constant step size of 0.25 nm was used in all fluorescence and excitation spectra. Absorption spectra over the range 180-810 nm were measured using a Hewlett-Packard 8453 UV-vis diode array spectrophotometer.
Results and Discussion PRODAN Spectral Characteristics and Advantages. Lately, PRODAN has been employed as an
Figure 1. Fluorescence spectra of PRODAN (1.6 µM) in various solvents. The excitation wavelength was 360 nm in all cases.
environmentally sensitive probe of membrane dynamics,11 actin unfolding and interactions,12 and the hydrophobicity of bovine serum albumin.13 The advantage of PRODAN in these applications is its large solvatochromic fluorescence shift. To illustrate this, we have recorded the fluorescence spectra of PRODAN in solvents of varying polarities and dielectric constants. These are presented in Figure 1. It is notable that the effect of these solvents on PRODAN is two-fold. Primary is the shift in fluorescence maximum according to the solvent polarity in the sense λflmax(water) > λflmax(methanol) > λflmax(toluene) > λflmax(heptane) ≈ λflmax(pentane). This can be explained by the fact that the large electric dipole moment of the emitting electronic state involves charge transfer14 and is stabilized in more polar solvents. The other effect is on the fluorescence quantum yield. In this case, methanol or toluene solvation results in a quantum yield more than 10 times that of the other solvents. PRODAN sometimes exhibits wavelength dependent dual fluorescence. This is particularly apparent in aqueous solution where an enigmatic band centered around 430 nm appears. This band has been attributed either to watersoluble impurities15 or to PRODAN microcrystals.16 The arguments in favor of microcrystals are strong at present, but conclusive proof that the microcrystals exist at concentrations above the apparent aqueous solubility limit (∼1 µM) are elusive. To avoid spurious spectral effects due to undissolved PRODAN, concentrations are kept near or below the solubility limit, where possible. In the following sections, we will exploit the solvatochromic effect in PRODAN to help determine the critical micelle concentration of a number of surfactant systems. It is noteworthy that although PRODAN is soluble in all of the solvents used, it partitions from aqueous solution (11) Hutterer, R.; Schneider, F. W.; Fidler, V.; Grell, E.; Hof, M. J. Fluoresc. 1997, 7, 161S. (12) Zechel, J. Biochem. J. 1993, 290, 411. (13) Haskard, C. A.; Chan, E. C. Y. Agric. Food Chem. 1998, 46, 2671. (14) Parusel, A. B. J.; Schneider, F. W.; Kohler, G. J. Mol. Struct. (THEOCHEM) 1997, 398, 341. (15) Bunker, C. E.; Bowen, T. L.; Sun, Y.-P. Photochem. Photobiol. 1993, 58, 499. (16) Sun, S.; Heitz, M. P.; Perez, S. A.; Colon, L. A.; Bruckenstein, S.; Bright, F. V. Appl. Spectrosc. 1997, 51, 1316.
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Langmuir, Vol. 15, No. 19, 1999 6183 Table 1. Parameters from the Fit of a Single Gaussian Function to the Fluorescence Spectrum of 3.5 µM PRODAN in the Presence of Aqueous SDS at Various Concentrations
Figure 2. Fit of a Gaussian function (red line) to the fluorescence spectrum (black line) of PRODAN (3.5 µM) in 9.0 mM SDS solution. The excitation wavelength was 360 nm.
SDS concn (mM)
band center for fluorescence maximum (cm-1)
fluorescence spectrum full width at half-maximum (cm-1)
fluorescence maximum wavelength (nm)
1.0000 6.1000 7.5000 7.9000 8.0000 9.0000 11.0000 14.0000 16.0000
18 980.0000 18 997.0000 19 067.0000 19 136.0000 19 157.0000 19 556.0000 19 647.0000 19 645.0000 19 650.0000
2470 2480 2582 2640 2599 2847 2855 2850 2857
526.8704 526.3878 524.4664 522.5753 522.0024 511.3520 508.9836 508.9836 508.9836
ditionally, there is an increase in fluorescence quantum yield at high surfactant concentration. This could indicate that PRODAN is in an environment where it has lower exposure to water-soluble fluorescence quenchers, such as dissolved O2. Alternatively, in a micelle PRODAN is less susceptible to nonfluorescent internal conversion promoted in a structured aqueous environment. Together, the shift and quantum yield imply that PRODAN has partitioned inside the micelle but not into a completely nonpolar environment. As has been suggested by Karukstis et al.,17 such an environment could exist near the micelle surface. We will revisit the micellar location of PRODAN later in this document. Let us consider the fluorescence spectrum of PRODAN as a function of increasing surfactant concentration. There is an apparent shift of the fluorescence maximum to the blue as the surfactant mole fraction is increased. To minimize the uncertainty in determining the fluorescence intensity maximum, we have chosen to fit a Gaussian line-shape to the spectra. A Gaussian function has the form18
() (
ν Ifl ∝ a ν0
Figure 3. Plot of the wavelength for maximum PRODAN fluorescence intensity versus SDS concentration. The maxima were determined from the fit of a Gaussian function to the fluorescence spectrum. For data, see Table 1.
into more hydrophobic environments (e.g. micelles) and from cyclohexane into more hydrophilic environments (e.g. reverse micelles). cmc Determination Using PRODAN (Spectral Shift). For the sake of comparison between our spectral deconvolution method and the standard fluorescence shift method, we will first examine cmc determination using the standard fluorescence shift technique. To measure cmc using PRODAN, one assumes that any shifting of the fluorescence maximum wavelength arises from dye molecules experiencing a different environment. This different environment is presumed to be created through micelle formation. Moreover, the magnitude of the spectral shift should indicate the polarity of the modified environment. We have found that the shift of the fluorescence maximum for PRODAN, in aqueous SDS solutions with high surfactant concentration, is approximately 15 nm to the blue of its maximum in water. According to Figure 1, this suggests that PRODAN experiences an environment somewhere between those of water and methanol. Ad-
)
{ν - ν0}2
2
exp -ln 2
∆ν2
(1)
where ν0 is the band center and ∆ν is the full spectral width at half-maximum, both in units of inverse centimeters. The constant a represents the magnitude of the maximum. This function fits the spectral data almost perfectly, as can be seen in Figure 2. The data for these fits are presented in Table 1. One can plot the apparent shift in fluorescence maximum versus surfactant concentration to help determine the critical micelle concentration. This is presented for sodium dodecyl sulfate in Figure 3. It must be noted that the procedure we are presenting in this subsection is a best case scenario for PRODAN in SDS, because we have taken extreme care in the determination of the fluorescence maxima. Attempting to fit line shapes in order to model cmc’s for other surfactants was less successful, even when multiple Gaussians were used. It must be further noted that a Gaussian may not always be the best model line shape. Finally, it may be very difficult to predict when it may be appropriate to use multiple Gaussian functions to model asymmetric spectral features. For the estimation of cmc’s, one must decide where the change in fluorescence maximum is large enough to signify (17) Karukstis, K. K.; Frazier, A. A.; Loftus, C. T.; Tuan, A. S. J. Phys. Chem. B 1998, 102, 8163. (18) Hollas, J. M. High-Resolution Spectroscopy; Butterworths: New York, 1983.
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Table 2. Parameters from the Deconvolution of the Fluorescence Spectrum of 3.5 µM PRODAN in the Presence of Aqueous SDS at Various Concentrationsa SDS concn (mM)
R
β
1.0000 6.1000 7.5000 7.9000 8.0000 9.0000 11.0000 14.0000 16.0000
9.0510 × 10-3 9.0510 × 10-3 0.0321 0.0491 0.0450 0.4200 0.9007 1.0000 1.0000
0.91 0.86 0.82 0.82 0.82 0.34 0.06 0.000 0.000
a R Represents the contribution of micellar PRODAN, and β represents the contribution of PRODAN in a pure aqueous environment.
a change in the local solution structure. From Figure 3, it is evident that this change occurs somewhere between 0.007 and 0.0085 M. This uncertainty arises primarily from the gradual change in slope near the cmc. cmc Determination Using PRODAN (Deconvolution). As shown in the previous section, it can be difficult to discern probe spectral shifts near the cmc. There is, however, a method in which one can determine the contributions of the micelle-solvated and free probe fractions to the overall fluorescence spectrum. This involves assuming that the probe will exist in two possible environments, that is, within the micelles and excluded from the micelle. A well-chosen probe molecule will display a large fluorescence maximum spectral shift between these environments. The measured spectrum in the presence of surfactant can then be deconvoluted into contributions from the micellar and nonmicellar environments. Algebraically this is indicated as
Ifl(λ) ) RImicelle (λ) + βInonmicelle (λ) fl fl
Figure 4. Deconvolution of the fluorescence spectrum of PRODAN (3.5 µM) in 9.0 mM SDS solution. See text for details of the deconvolution procedure. The excitation wavelength was 360 nm.
(2)
where R and β are coefficients multiplying contributions from “pure” micellar probe and “pure” nonmicellar probe spectra, respectively. In the deconvolution procedure, the micellar probe spectrum is recorded in the presence of surfactant well above the cmc and the nonmicellar probe spectrum is recorded in the absence of surfactant. Thus, there is no need for the introduction of an arbitrary function to represent the spectra. This technique will work regardless of the spectral band shape. In the following, we describe PRODAN as a determinant of the sodium dodecyl sulfate cmc using our spectral deconvolution method. The fluorescence spectra of aqueous PRODAN at 3.5 µM were collected over a series of surfactant concentrations. Since PRODAN partitions strongly into micelles,19 one can approximate the spectrum at high surfactant concentration as that resulting from completely micellized probe molecules. For this, we have chosen a concentration ([SDS] ) 16.0 mM) at which PRODAN exhibits no further shifting of its fluorescence maximum. The fluorescence spectra are then fitted by eq 2 using a nonlinear least squares computer program. The deconvolution constants R and β are presented in Table 2. A sample spectrum, its calculated analogue, and the deconvoluted contributions are presented in Figure 4. The recorded spectra are modeled very well by this method. Besides the fact that this method represents a more physically realistic rationalization of the micelle-induced spectral behavior than a solely shifting spectrum, it may provide a more accurate determination of critical micelle (19) Karukstis, K. K.; Suljak, S. W.; Waller, P. J.; Whiles, J. A.; Thompson, E. H. Z. J. Phys. Chem. 1996, 100, 11125.
Figure 5. Plot of the micelle contribution fit coefficient for the PRODAN fluorescence spectrum versus SDS concentration. The coefficients were determined as described in section 3.3. For data, see Table 2.
concentrations than the fluorescence shift method presented in the previous section. This statement can be supported by considering Figure 5, in which the R constant is plotted versus surfactant concentration. In this figure, it is clear that even small contributions to the overall spectrum by the micelle-shifted spectrum indicate the onset of micelle formation. The onset is sharper using this method and thus provides a more accurate measure of the cmc. Here we find a cmc for SDS of 7.6 ((0.2) × 10-3 M. PRODAN was used in the determination of the cmc of other surfactants. These include the neutral poly(oxyethylene)23 lauryl ether (Brij 35) and reverse micelles of Triton X-100 in a mixture of hexanol and cyclohexane. The cmc’s determined for these surfactants and for SDS are given in Table 3. Brij-35 was chosen as an example both of a neutral surfactant and of a low-cmc surfactant in aqueous solution. From the literature, it was curious to discover that
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Table 3. Critical Micelle Concentrations Determined Using the Fluorescence Deconvolution Method Compared with Reliable Literature Values surfactant SDS Brij 35 TX-100 (reverse micelle) a
cmc (this work) (mM) cmc (lit.) (mM) 7.6(0.2) 0.08 (0.003) 1.0 (0.2)
8.0a 0.07a 1.09b
Reference 20. b Reference 23.
although Brij-35 had a quoted cmc of 7 × 10-5 M,20 it was often used at a concentration of 1 × 10-3 M or greater.21,22 Indeed, when we attempted to determine the cmc of Brij35 using the fluorescence probe molecules indole or dibucaine, there was no discernible cmc until a surfactant concentration of 1 × 10-2 M was reached. This may have resulted from an unfavorable Brij-35 micellar partition coefficient for these probe molecules. The attempt to determine the cmc of Brij-35 was more successful using PRODAN and the spectral deconvolution method. Our method identifies the onset of micelle formation occurring at 8.0 ((0.3) × 10-5 M. To be a reasonably universal cmc indicator, PRODAN would have to be applicable also to reverse micelles. To test this, the system of Triton X-100 in a hexanol/ cyclohexane mixture was chosen. This system will have micelles with interiors that are less lipophilic than the solvent. It should be noted that TX-100 is actually a mixture of surfactants with slightly different numbers of ethoxy (CH2CHO) units resulting in an average ethoxy polymer length of 9.5 units (i.e. (CH2CHO)9.5). This may eventuate a less sharp cmc. Also, it is possible that the reverse micelles created under the experimental conditions used in this work contain some hexanol.23 Here, one utilizes the fluorescence spectra of PRODAN in cyclohexane/ hexanol and that within the TX-100 micelle to deconvolute the mixed spectrum at intermediate surfactant concentrations. The difference between this system and aqueous micelles is that a contribution from a red-shifted spectrum indicates the onset of micelle formation, because of the more polar micelle interior. The favorable partitioning of PRODAN into a TX-100 reverse micelle probably arises from dispersive interactions with the aromatic group of TX-100. This argument is reinforced by our quenching studies (section 3.5), which suggest that PRODAN may be positioned near the surface of the reverse micelles. The two spectral contributions to the reverse micelle environment are well-separated, with the fluorescence maximum for surfactant-free PRODAN at 400 nm, whereas above the cmc the fluorescence maximum shifts to 414 nm. The spectral deconvolution method yields a cmc of 1.2 ( 0.2 mM for Triton X-100 in a hexanol/ cyclohexane mixture, as shown in Figure 6. This value is in very good agreement with that previously determined (1.09 mM) using methylene blue and the absorption ratio method.23 Using the cationic probe methylene blue, Pramanuk and Mukherjee23 observed a red-shifted feature in the absorbance spectrum, which gains substantial intensity in the presence of surfactant above the cmc. They attributed this to either self-π-stacking of the probe on the micelle surface or interactions of the probe with the TX-100-substituted benzyl group, leading to a charge(20) McIntire, G. L. Crit. Rev. Anal. Chem. 1990, 21, 257. (21) Ashby, K. D.; Das, K.; Petrich, J. W. Anal. Chem. 1997, 69, 1925. (22) Roberts, E. L.; Chou, P. T.; Alexander, T. A.; Agbaria, R. A.; Warner, L. M. J. Phys. Chem. 1995, 99, 5431. (23) Pramanick, D.; Mukherjee, D. J. Colloid Interface Sci. 1993, 157, 131.
Figure 6. Plot of the micelle contribution fit coefficient for the PRODAN fluorescence spectrum versus the log of TX-100 concentration. The coefficients were determined as described in section 3.3.
transfer excited state. Qi and Ma24 extended this study to include the anionic probe methyl orange and found that the effect, if any, is much less pronounced for this species. From our absorption, excitation, and fluorescence spectra, we observed no evidence of ground or excited-state chargetransfer interactions between PRODAN and TX-100 over the concentration range employed. The solvatochromic shift we observe in fluorescence above the cmc is consistent with a minimal stabilization of the large excited-state electric dipole moment for this system. This is further evidence of PRODAN being localized near the reverse micelle surface. Wilhelmy Plate Method for cmc Determination. To ascertain whether PRODAN was itself affecting the critical micelle concentration values, cmc’s for SDS, Brij35, and TX-100 were evaluated using the Wilhelmy plate method of surface tension measurement. Presumably, if PRODAN is itself surface-active at the concentration used in the fluorescence experiment, cmc’s determined in its presence and absence will differ. For PRODAN at a concentration of 3.5 µM, there was no indication of surface activity for either SDS or Brij 35, whose cmc’s were measured as 8.2 and 40 µM, respectively. Interestingly, the cmc of TX-100 reverse micelles in hexanol/cyclohexane could not be determined using the Wilhelmy plate method. Position of PRODAN in Micelles. In previous studies,25,26 it has been demonstrated that the location of fluorescent probe molecules within micelles could be determined using fluorescence quenching. In the present work, fluorescence quenching experiments were performed to help determine the location of PRODAN in SDS micelles and TX-100 reverse micelles. It was expected that determining the position of PRODAN within a SDS micelle would be relatively simple using fluorescence quenching. The results of our studies to date suggest that the PRODAN/SDS/counterion (NaI) system is far from simple. As has been reported previously,19 adding NaI as a quencher to a PRODAN/SDS solution actually results in an increase in fluorescence yield. This was attributed to a loss of surface fluidity and a subsequent redistribution of PRODAN deeper inside (24) Qi, L.; Ma, J. J. Colloid Interface Sci. 1998, 197, 36. (25) Luoro, S. R. W.; Tabak, M.; Nascimento, O. R. Biochim. Biophys. Acta 1994, 1189, 243. (26) Lakowics, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1983; Chapter 9.
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Figure 7. Stern-Volmer plot of the quenching efficiency of CH2I2 on 10 µM PRODAN in TX-100 reverse micellar solution (4.0 mM) and in surfactant-free cyclohexane/hexanol solution. The lines represent fits to the Stern-Volmer equation. Error bars represent 1 standard deviation unit determined from the fluorescence intensity computation. See text for details.
the micelle. When the concentration of NaI reached 0.1 M, the increase in fluorescence yield was overcome by the sheer quantity of I- present and began to diminish. We have also observed this behavior for PRODAN at 5 µM and for SDS at 10 mM. However when 1 µM PRODAN is used, the fluorescence yield increases until a NaI concentration of 0.3 M is reached. It is possible that, at 5 µM, some PRODAN remains in the microscrystalline phase, as suggested by Bright and co-workers,16 and is affecting the quenching behavior. At 1 µM, the initial aqueous solution contains more free PRODAN, which may have an easier time entering the micelles. The addition of NaI to the solution will undoubtedly affect the cmc of SDS. Thus, these experiments were repeated at constant ionic strength using NaCl to balance the NaI added. In this case, the behavior was more reasonable but still complex. We have performed parallel quenching experiments in the absence of surfactant. In this work, the expected loss of fluorescence with increase in NaI concentration is observed. We will continue to work toward the elucidation of this interesting behavior, but its presentation is beyond the scope of the present paper. For the reverse micelle system, the quencher CH2I2 was used. A series of steady-state fluorescence measurements were performed in which CH2I2 was added (1) to PRODAN solutions free of TX-100 and (2) to solutions with 4 mM TX-100. The resulting reductions in fluorescence intensity were determined using an alternate form of eq 2, where only one spectral component was used. It was found that CH2I2 partitions inside the reverse micelle. The SternVolmer plots for the reverse micelle system versus surfactant-free solutions, found in Figure 7, confirm this. We find that the Stern-Volmer quenching constants are 671 and 356 M-1 for reverse micellar and surfactant-free solutions, respectively. Since we also know the quenching efficiency versus concentration in surfactant-free solution, we can estimate the apparent concentration of CH2I2 in the reverse micelles. This is done by extrapolating the efficiency in the Stern-Volmer plot with surfactant to the apparent concentration needed for this efficiency in the surfactant-free Stern-Volmer plot. In so doing, one finds that the apparent quencher concentration in the presence of TX-100 is 1.9 times that in the absence of
Wong et al.
TX-100. The increase arises from the partitioning of CH2I2 inside the micelles. If every CH2I2 molecule partitions into a micelle, then the reduced volume experienced is just the concentration of micelles times the micellar volume. If we assume an aggregation number of 40 and a diameter of 21 nm,27 then the volume of the TX-100 reverse micelles is 0.1 L per liter of water. This reduced volume would result in a 10-fold increase in quenching efficiency due to increased collisional frequency. In surfactant-free solution, quenching may proceed through simultaneous interactions with multiple quenchers at long distances. Since PRODAN will encounter only one quencher at the micellar concentrations used, the quenching efficiency does not attain the full possible increase. This may also suggest that both CH2I2 and PRODAN are located near the surface of the TX-100 reverse micelle or at least that CH2I2 and PRODAN are separated within the reverse micelle. The position of PRODAN in the TX-100 reverse micelle may be similar to that found by Karukstis et al.17 for the Aerosol OT/heptane/water reverse micelle system. However, for TX-100 there does not appear to be a contribution from PRODAN in the reverse micelle core. If PRODAN did partition into the micelle core, this would have resulted in a stronger quenching propensity than observed. If CH2I2 and PRODAN were on opposite sides of the sphere surface, this would be the equivalent of a free solution concentration around 0.05 M, using spherical space-filling arguments. The assumed diameter of the reverse micelle, 21 nm, would be the maximum separation possible between CH2I2 and PRODAN. At a 1:1 mole ratio of CH2I2 to micelle (i.e. [CH2I2] ) 1.0 × 10-4 M), the observed Stern-Volmer ratio (F0/F ) 1.07) is still less than that predicted from an apparent concentration of 0.05 M in surfactant-free solution (F0/F ) 19). Thus, it would appear that there must be incomplete partitioning of the quencher into the micelle. Conclusions Using the probe molecule PRODAN, a novel spectral deconvolution method is introduced and compared with the more traditional fluorescence spectral maximum shift method for cmc determination of aqueous surfactants including sodium dodecyl sulfate and Brij 35 and reverse micelles of Triton X-100 in cyclohexane/hexanol solution. Fluorescence quenching experiments were performed on PRODAN in aqueous SDS and TX-100 reverse micelle solutions to help determine its position in the micellar environment. It was found that PRODAN may have a propensity to partition into the surface regions of both aqueous and reverse micelles. Acknowledgment. Financial support from the Natural Sciences and Engineering Research Council of Canada, the Imperial Oil Charitable Foundation (University Research Grant), and the Government of Canada Summer Term Employment Program (T.M.D.) is gratefully acknowledged. D.T.C. is indebted to Professor G. Liu for the extensive use of his spectrofluorometer and Professor C. Langford for the use of his diode array absorption spectrometer. We thank Drs. L. Schramm and E. Stasiuk of the Petroleum Recovery Institute (Calgary) for performing the Wilhelmy plate cmc determination. LA981716Z (27) Zhu, D.-M.; Feng, K.-I.; Schelly, Z. A. J. Phys. Chem. 1992, 96, 2382.
