Fluorescence Probe Studies of Hydrophobic Domains in a Model

Mitchell A. Winnik*. Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6. Richard D. Jenkins. Technical Center, Union Car...
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Langmuir 1999, 15, 1644-1650

Fluorescence Probe Studies of Hydrophobic Domains in a Model Hydrophobically Modified Alkali-Swellable Emulsion (HASE) Polymer with C20H41 Groups Kazunaga Horiuchi,† Yahya Rharbi, John G. Spiro, Ahmad Yekta, and Mitchell A. Winnik* Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6

Richard D. Jenkins Technical Center, Union Carbide Asia Pacific, Inc., 16 Science Park Drive, #04-01/02 The Pasteur, Singapore 118227

David R. Bassett Union Carbide Corporation, UCAR Emulsion Systems, Research and Development, 410 Gregson Drive, Cary, North Carolina 27511 Received June 8, 1998. In Final Form: December 4, 1998 The fluorescence and fluorescence decay profiles of pyrene and 1-ethylpyrene in solutions of a hydrophobic alkali-swellable emulsion (HASE) polymer were examined to characterize the association structure formed from the hydrophobic substituents. The HASE polymer was obtained by the copolymerization of ethyl acrylate (EA), methacrylic acid, and a macromonomer containing a C20H41 group separated from the backbone by 32 ethylene oxide units. We examine solutions neutralized with 1 equiv of NaOH, at polymer concentrations where virtually all of the added probe is partitioned into hydrophobic domains of the polymer. Both monomer and excimer emission are observed, and IE/IM increases in proportion to the amount of probe added to the system. Individual monomer fluorescence decay profiles fit well to the traditional micelle Poisson quenching model, but attempts to calculate the hydrophobe aggregation number NR led to values that changed markedly with the ratio of probe to polymer. These results were rationalized in terms of a polymer structure in water containing various hydrophobic domains of different composition. These domains vary from nonionic micelle like structures containing upward of 60-80 C20H41 groups, where the first probes added to the system are located, to mixed structures containing both C20H41 and EA groups from the polymer backbone.

Introduction This is the third in a series of papers from our laboratory that examines the properties in solution of a hydrophobically modified alkali-swellable emulsion (HASE) polymer,1-3 a type of associating polymer employed as an associative thickener in paints and other coatings formulations.1,4 This class of hydrophobically modified polyelectrolytes is prepared by emulsion polymerization at † Permanent address: Fuji-Xerox Co. Ltd., 1600 Takematsu, Minamiashigara-shi, Kanagawa 250-01, Japan.

(1) (a) Water Soluble Polymers; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1986. (b) Polymers in Aqueous Media; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1989. (c) Hydrophobic Polymers: Performance with Environmental Acceptance; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1996. (d) Polymers as Rheology Modifiers; Glass, J. E.; Schulz, D. N., Ed.; American Chemical Society: Washington, DC, 1991. (2) (a) Jenkins, R. D. Ph.D. Thesis, Lehigh University, Bethlehem, PA, 1990. (b) Jenkins, R. D.; Silebi, C. A.; El-Aasser, M. S. Polym. Mater. Sci. Eng. 1989, 61, 629. (c) Jenkins, R. D.; Silebi, C. A.; El-Aasser, M. S. In Advances in Emulsion Polymerization and Latex Technology: 21st Annual Short Course, 1989; El-Aasser, M. S., Ed.; Lehigh University: Bethlehem, P. A., 1990; Vol. 61, Chapter 17, p 629. (3) (a) Nishida, S.; El-Aasser, M. S.; Klein, A.; Vanderhoff, J. W. In Emulsion Polymers and Emulsion Polymerization; American Chemical Society: Washington, DC, 1981; pp 291-314. (b) Jenkins, R. D.; DeLong, L. M.; Bassett, D. R. In Hydrophobic Polymers; J. E. Glass, Ed.; American Chemical Society: Washington, DC, 1996; p 425. (c) Shay, G. D.; Kravitz, F. K.; Brizgys, P. V. In Polymers as Rheology Modifiers; Schulz, D. N., Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1989; pp 121-141. (d) Shay, G. D. In Polymers in Aqueous Media; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1987; pp 457494.

low pH to yield an acid-rich copolymer in the form of a dispersion of latex particles. Each particle contains many polymer molecules. As base is added to the dispersion, the -COOH groups of the polymer are neutralized. The particles swell and then dissolve, yielding a soluble polyelectrolyte. The specific polymer we examine here is a copolymer of ethyl acrylate (EA) and methacrylic acid (MAA), with an EA-MAA molar ratio of approximately 50/50, containing a small fraction of a comonomer containing a C20H41 group as a hydrophobic substituent. The C20H41 group is separated from the polymer backbone by a spacer chain containing an average of 32 ethylene oxide (EO) units. The structure of the polymer is shown in Chart 1. We refer to this material as C20E32. This and other related polymers were prepared by R.D.J. at Union Carbide with the idea that these complex materials might be studied by a number of different techniques and that those employing these different techniques would all be studying common samples. Studies of the rheology of these polymers in aqueous solution are beginning to appear.5,6 In previous experiments,5b we have used a combination (4) Winnik, M. A.; Yekta, A. Curr. Opin. Colloid Interface Sci. 1997, 2, 424-436. (5) (a) Kumacheva, E.; Rharbi, Y.; Winnik, M. A.; Guo, L.; Tam, K. C.; Jenkins, R. D. Langmuir 1996, 13, 182-186. (b) Horiuchi, K.; Rharbi, Y.; Yekta, A.; Winnik, M. A. Can. J. Chem. 1999, in press. (6) (a) Tirtaatmadja, V.; Tam, K. C.; Jenkins, R. D. Macromolecules 1997, 30, 1426-1433. (b) Tirtaatmadja, V.; Tam, K. C.; Jenkins, R. D. Macromolecules 1997, 30, 3271-3282. (c) English, R. J.; Gulati, H. G.; Jenkins, R. D.; Khan, S. A. J. Rheol. 1997, 41, 427-444.

10.1021/la9806653 CCC: $18.00 © 1999 American Chemical Society Published on Web 02/04/1999

Fluorescence Studies of a HASE Polymer Chart 1

of viscosity, dynamic light scattering, and steady-state fluorescence experiments to examine global features of the polymer behavior in water as a function of the degree of neutralization of the polymer. Conductometric and potentiometric titrations indicated that at partial neutralization the pH of the solution was not very sensitive to the degree of neutralization of the polymer. We learned that the best way to describe the state of the polymer in the presence of added base is in terms of the degree of neutralization R, defined as R ) [NaOH]/[COOH], where [NaOH]/[COOH] describes the ratio of moles of NaOH added to the solution per mole of -COOH groups present in the polymer. Although the value of R is independent of polymer concentration, it differs from the true degree of dissociation of the carboxylic acid groups, especially at R ) 0 and for values of R close to or greater than 1.0. When pyrene (Py) is added as a fluorescent probe to solutions of this polymer, it partitions between the polymer phase and the water. At low R, the Py dissolves in the EA-MAA copolymer, where it exhibits very little excimer emission. As more base is added to the system, there is a pronounced increase in excimer emission at a degree of neutralization that corresponds to a sharp increase in the solution viscosity. All of the evidence available points to a rearrangement of the system accompanying deprotonation of the -COOH groups, to form a soluble polyelectrolyte in which the C20H41 groups associate into micelle like aggregates. The Py probe molecules become localized in these micelles, and the solutions, when irradiated, give rise to a strong excimer emission. In our earlier report,5b we pointed out that interpretation of Py fluorescence decay measurements were difficult because under the conditions of those measurements a significant fraction of the Py was located outside the micelles in the aqueous phase. In the present paper, we reexamine the fluorescence decay behavior of Py in these HASE polymer solutions under conditions in which virtually all of the Py is partitioned into the polymer phase. These results are compared with the fluorescence decay behavior of 1-ethylpyrene (EtPy), a probe with spectroscopic properties similar to Py but with a shorter lifetime and reduced water solubility. Both Py and EtPy form excimers through quenching of the monomer fluorescence, and in simple surfactant micelles, the probability of quenching can be interpreted in terms of a Poisson distribution of the Py or EtPy molecules among the micelles. 5,7-9 (7) (1) Alami, E.; Almgren, M.; Brown, W. Macromolecules 1996, 29, 2229-2243. (2) Alami, E.; Almgren, M.; Brown, W. Macromolecules 1996, 29, 5026-5035.

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Application of fluorescence quenching methodology to characterize the micelle-like aggregates formed in associating polymer systems is more difficult and less straightforward than that for normal surfactant micelles.7 In our hands, the Py excimer technique has worked well for a class of hydrophobe-end-capped (HEUR) polymers with a poly(ethylene glycol) backbone,8 indicating a mean aggregation number of 20 C16H33O- groups per micelle, spanning a range of polymer concentrations (all less than 2 wt %) over which the solution viscosity varied by more than 100 times. In the HASE polymer described here, the fluorescence quenching behavior is more complicated. Individual decay curves give excellent fits to the model which describes fluorescence quenching kinetics in micellar systems. When data from different experiments are compared, however, more complex behavior is revealed. In this paper, we examine this behavior with careful statistical analyses of the individual decays. We describe the effects of varying both polymer and probe concentrations and provide a comparison of Py and EtPy as fluorescence probes in this system. Materials and Methods Materials. The polymer examined in the present work, denoted C20EO32 (28RDJY72-6), is a copolymer of MAA, EA, and a C20H41O end-capped macromonomer whose structure is shown above, with molar ratios x/y/z of 49.06: 50.04:0.90. Full details of the synthesis and characterization of this and similar HASE polymers have been reported by R.D.J. 3b In short, a macromonomer was prepared first by ethoxylation to 32 mol of the C20 alcohol, followed by reaction with R,R-dimethyl m-isopropenyl benzyl isocyanate. This macromonomer was introduced into a conventionally seeded semicontinuous emulsion polymerization under monomer-starved conditions with appropriate amounts of MAA and EA. In this work, we designate this polymer C20E32. According to the structure and composition of the polymer, it contains 2.12 × 10-2 g C20H41 groups/g polymer (7.53 × 10-5 mol/g polymer). The latex emulsion was dialyzed against 3.0 L of deionized water for more than 2 weeks with a cellulose membrane, formulated to cutoff from Mw 12 000 to 14 000 (Fisher Scientific). A series of different polymer concentrations was prepared by dilution of the dialyzed HASE solution with deionized water. Salt was added to give an ionic strength of 1.0 mM NaCl. To minimize concentration changes of the polymer solution, 1.0 M NaOH was used to neutralize the polymer. Pyrene (Aldrich) was purified by recrystallization and twice repeated sublimation. 1-Ethylpyrene (Molecular Probes) was used without purification. Solubilization of Fluorophores in C20E32 HASE Solution. For fluorophore solubilization into HASE solutions, a dilute acetone solution containing a known amount of the fluorophore was placed in a flask, and then the acetone was evaporated completely under a gentle flow of nitrogen. To equilibrate the fluorophore in the HASE solution, the mixture was stirred at room temperature for 1 week. Subsequent centrifugation of the solution at 15 000 rpm for 60 min caused the insoluble Py (8) (a) Yekta, A.; Duhamel, J.; Brochard, P.; Adiwidjaja, H.; Winnik, M. A. Macromolecules 1993, 26, 1829-1836. (b) Yekta, A.; Xu, B.; Duhamel, J.; Adiwidjaja, H.; Winnik, M. A. Macromolecules 1995, 28, 956-966. (9) (a) Surfactant Solutions, New Methods of Investigation; Zana, R., Ed.; Marcel Dekker Inc.: New York, 1997; pp 241-294. (b) Infelta, P. P. Chem. Phys. Lett. 1979, 61, 88. (c) Yekta, A.; Aikawa, M.; Turro, N. J. Chem. Phys. Lett. 1979, 63, 543.

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Figure 1. Pyrene (Py) solubilization in C20E32 HASE solutions at R ) 0.4 and R ) 1.0, measured by UV absorbance at 338 nm.

or EtPy crystals to sediment without affecting the dispersion or solution stability. The fluorophore concentration was monitored by UV for solutions in 10 mm quartz cuvettes, using a PerkinElmer Lambda 6 dual-beam spectrometer. The concentration was monitored at the strongest absorption peak at 338 nm for Py or 345 nm for EtPy, with a correction for the background absorption (turbidity) of the polymer. Steady-State Fluorescence Measurements. A SPEX Fluorolog 2, with double-grating monochromators for both the excitation and emission, was used to observe steadystate fluorescence spectra in a right-angle geometry. The excimer-to-monomer ratios (IE/IM) were calculated from the area integrations of the emission spectrum, from 360 to 400 nm for IM and from 450 to 550 nm for IE. Dynamic Fluorescence Measurements and Fluorescence Decay Analyses. Fluorescence decay profiles were measured by the single photon timing technique, using a 50 kHz flash lamp filled with deuterium gas. The wavelengths of the excitation and emission monochromators were fixed, respectively, at 339 and 371.5 nm for Py or 346 and 375.5 nm for EtPy. A long-pass filter was used to prevent scattered excitation light from reaching the detector. In the fit of the experimental data to the micellequenching model (see below), the mimic technique10 was employed using p-bis[2-(5-phenyloxazolyl)]benzene (lifetime: 1.1 ns) as the reference for calculating the exciting lamp profile, and a small correction was made for light scattering from the solutions.10 The data analysis yields the parameters τ, 〈n〉, and k, whose significance is described in a subsequent section of this paper. Following the determination of reliable τ values, we estimated 95% confidence intervals for the parameters 〈n〉 and k. As discussed below, the observed trends appear to be “real”, in the sense that changes in the model parameters greatly exceed the random errors. Results and Discussion Fluorophore Solubilization in C20E32 HASE Solution. The results of Py solubilization experiments are shown in Figure 1 as a function of the polymer concentration for two different values of R, the neutralization degree of the MAA components. The extent of Py solubilization increased proportionally to the polymer concentration in (10) (a) O’Connor, D. V.; Phillips, D. Time-Correlated Single Photon Counting; Academic Press: London, 1984. (b) James, D. R.; Demmer, D. R. M.; Verrall, R. E.; Steer, R. P. Rev. Sci. Instrum. 1983, 54, 1121.

Horiuchi et al.

Figure 2. Excimer-to-Monomer ratio (IE/IM) in the steadystate fluorescence using Py or EtPy as a probe. The polymer concentrations are Cpol ) 0.8 g/L at complete neutralization, R ) 1.0.

both solutions, at R ) 0.4 and 1.0. The higher solubility seen at R ) 0.4 is due to Py solubilization in the hydrophobic domain of the EA-MAA copolymer at low extent of neutralization. The system at R ) 1.0 corresponds to the maximum viscosity of the solution, where micellar structures formed by the C20H41 hydrophobes are responsible for the polymer association. From the data in Figure 1, we calculate that there are approximately 0.05 Py/C20H41 at R ) 1.0. When EtPy is dissolved in this solution at R ) 1.0, it is less soluble than Py. At saturation, there are approximately 0.035 EtPy/C20H41. Steady-State Fluorescence Spectra. The excimerto-monomer ratios (IE/IM) as a function of probe concentration for a C20E32 HASE solution with Cpoly ) 8.0 g/L and R ) 1.0 are plotted in Figure 2. The IE/IM ratios increase linearly with the concentration of Py and EtPy. The results indicate that the fluorophores dissolve in the polymeric micelle aggregates. These results also appear to indicate that the local concentration of Py or EtPy in the micelles increases linearly with the amount of probe added. As we will see from the fluorescence decay measurements described below, the actual situation is in fact more complicated. Analysis of Fluorescence Decay Profiles. The fluorescence decay associated with the monomer emission exhibits self-quenching behavior through excimer formation that follows the Poisson quenching model which is typical of fluorescence quenching of micelle-bound species.5,7-9

{

t I(t) ) I(0) exp - - 〈n〉[1 - exp(- kt)] τ

}

(1)

There are four fitting parameters: the initial intensity I(0), the unquenched lifetime τ, the mean number of fluorophore molecule per micelle 〈n〉, and the first-order rate k constant for excimer formation in a micelle k. The equation assumes that the fluorophore is solubilized uniformly in micellar domains in the solution, that the micellar distribution does not change during the fluorescence event, and that the excimer forms in an intramicellar process without any exchange of fluorophore between micelles. From the quantitative relation between fluorophore and polymer concentrations, one can calculate the aggregation number of hydrophobes forming a micelle.

Fluorescence Studies of a HASE Polymer

〈n〉 )

[Py] [Py] NR ) [micelle] Cpoly qr

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(2)

Here [Py] is the concentration in moles per liter of the fluorophore, Cpoly is the polymer concentration in grams per liter, NR is the aggregation number, and qr is the hydrophobic chain content in the polymer in moles of hydrophobic groups per gram of polymer. Experimental Difficulties in the C20E32 HASE System. Before measurements to determine the aggregation number of the associated micellar structures are discussed, a few remarks should be made about some experimental difficulties for this HASE polymer system. First, at low polymer concentration, there is considerable fluorescence emission from Py in the water phase where its limiting solubility is 7 × 10-7 mol/L at 22 °C.11 Figure 3 shows values of unquenched Py lifetimes at various polymer concentrations. The values are derived by fitting experimental fluorescence decay profiles with eq 1, and the x axis represents the Py concentration divided by the saturation concentration for each solution, where the polymer concentrations are kept constant. All samples are at R ) 1.0. If the Py molecules were confined to the micelle phase, the unquenched lifetime τ should be independent of the Py concentration. In fact, we observe a constant lifetime of about 200 ns above Cpoly ) 3.2 g/L. However the lifetimes at Cpoly ) 1.6 g/L and Cpoly ) 0.8 g/L clearly deviate from 200 ns and vary with Py concentration. These deviations can be explained by a decrease of the Py concentration in hydrophobic domains with a corresponding increase of the Py concentration in water. This conclusion is confirmed by steady-state fluorescence measurements,5b which show a blueshift in the Py excitation spectrum corresponding to increasing amounts of Py in the water phase at lower polymer concentrations. The fluorophore solubility in the water phase is lower for EtPy than for Py, but EtPy also has a lower saturation solubility in the presence of the HASE polymer. Thus the use of EtPy as a probe here is not as advantageous as one might hope. A second technical problem we encountered is that we find a weak short-lived emission from the HASE polymer itself. In Figure 4 we show a fluorescence decay profile of C20E32 at Cpoly ) 8.0 g/L in the absence of Py or EtPy. The inset in Figure 4 indicates that the rapid portion of the decay is different from the lamp profile. This impurity fluorescence can be a problem for experiments at very low Py or EtPy concentrations but appears not to be important for probe concentrations of 2.0 × 10-6 mol/L or higher, which we use for all the experiments described here. Seeking the Aggregation Number in the C20E32 HASE System. Fluorescence decay profiles for C20E32 at Cpoly ) 8.0 g/L (R ) 1.0) are shown in Figure 5 for various Py concentrations. The sharp decay at early times in the fluorescence decay is consistent with intramicellar selfquenching and, as expected from eq 1, becomes more pronounced with increasing Py concentration. The tail in the decay at long times is consistent with emission from unquenched Py in micelles that contain only a single Py molecule. One observes the same behavior in Figure 5b, where we use EtPy as the probe. When the fluorescence decay curves in parts a and b of Figure 5 are fitted to eq 1, we obtain the values of the unquenched lifetimes for Py and EtPy shown in Figure 6. The limiting solubility in the polymer solution is higher (11) (a) Wilhelm, M.; Zhao, C. L.; Wang, Y.; Xu, R.; Winnik, M. A. Macromolecule 1991, 24, 1033. (b) Schwartz, F. P. J. Chem. Eng. Data 1977, 22, 273.

Figure 3. Lifetimes of Py fluorescence at different polymer concentrations, examined with various Py concentrations at complete neutralization, R ) 1.0.

Figure 4. Fluorescence decay measurement of the HASE C20E32 polymer in the absence of fluorescent probe (Py and EtPy). The reference spectrum for the reconstruction of the lamp profile at the emission wavelength was taken with a standard sample of lifetime 1.1 × 10-9 s. The polymer concentration is Cpoly ) 8 g/L at R ) 1.0.

for Py than for EtPy, and thus, for this concentration of polymer, no data are available for bulk concentrations of EtPy greater than 2.2 × 10-5 M. Over the whole range of the probe concentrations in these aerated solutions, we find constant values of the lifetime τ equal to 200 ns for Py and 158 ns for EtPy. One can compare these values to those of 128 ns for Py in water and 90 ns for EtPy in water, determined in separate experiments. It is possible now to state that the fluorophores associate with hydrophobic domains in the system and that the unquenched monomer emission exhibits no obvious changes in its spectroscopic properties with a change in probe concentration. Generally speaking, this type of behavior is an appropriate prerequisite for the determination of the other parameters, k and 〈n〉, by fitting fluorescence decay profiles to eq 1. We emphasize this point because of the unusual concentration dependence we find for the fitting parameters k and 〈n〉. For this reason, we also carried out detailed error analyses of the fitting procedure, so that we could distinguish statistical problems with the fitting from mechanistic problems with the Poisson quenching model. Figure 7 shows 95% joint confidence intervals for the parameters 〈n〉 and k, as obtained in typical fluorescence decay measurements with Py or EtPy. For both of the Py and EtPy experiments, the heights of the ellipses are ca. 0.03 for 〈n〉 and the widths are ca. 6 × 10-4 ns-1 for k; these

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Figure 5. Fluorescence decay profiles of (a) Py and (b) EtPy in HASE C20E32 solution, for Cpoly ) 8 g/L at R ) 1.0.

Figure 6. Fluorescence lifetimes obtained by fitting the fluorescence decays in parts a and b of Figure 5 to eq 1.

Figure 7. Confidence ellipses (95%) obtained by error analysis for the parameters 〈n〉 (the number of Py or EtPy per micelle) and k (the first-order quenching rate constant), calculated using the constant lifetimes τ shown in Figure 6.

uncertainties exceed by only 25% the magnitude of the error bars computed by the more usual techniques,12a employing the diagonal elements of the asymptotic co-

Horiuchi et al.

Figure 8. (a) First-order quenching rate constant k as a function of Py or EtPy concentration, for Cpoly ) 8 g/L at R ) 1.0. (b) The number of the fluorophore molecules per micelle 〈n〉 as a function of Py or EtPy concentration. The error bars represent those calculated by the error analysis shown in Figure 7.

variance matrixes. In other words, correlations between 〈n〉 and k do not appear to be troublesome. Indeed, analysis of the χ2 surfaces obtainable12b by taking into account the correlations between all three regressors [the third one corresponds to the initial intensity I(0) of eq 1] still does not indicate a larger imprecision in the fitted data. The preceding error estimates were based on the most common linear approximation technique employed in nonlinear regression computations: using the inverse of the symmetrized Jacobian matrix to estimate the variances and covariances of the model parameters.12c It is well-known (e.g., ref 12c) that this approximation may underestimate the uncertainties. Therefore, we also conducted a few Monte Carlo simulations to develop more reliable error bounds. Namely, we simulated the experiments represented in Figure 7 by adding Poisson noise to the theoretical decay curves and then deconvolving the resulting “data”. Perhaps not surprisingly, the latter computations suggested that the values obtained for 〈n〉 and k were more precise, rather than less precise, than one would surmise from the error estimates given in the previous paragraph. This result implies only that the conventional analyses detected imprecision beyond the Poisson error of counting. Thus, we may now proceed to discuss the experimental results further, knowing that they are statistically meaningful. Although individual fluorescence decay profiles give statistically meaningful values of 〈n〉 and k, these parameters show an unusual dependence on probe concentration that we have never before observed with micellar surfactant or linear AT polymer systems. In Figure 8, we see that both k and 〈n〉 decrease with decreasing fluorophore concentration. Moreover, there are unexpectedly (12) (a) O’Connor, D. V.; Phillips, D. Time-Correlated Single Photon Counting; Academic Press: 1984; pp 173-174. (b) Press, W. H.; Teukolsky, S. A.; Vetterling, W. T.; Flannery, B. P. Numerical Recipes in FORTRAN - The Art of Scientific Computing; Cambridge University Press: Cambridge (England), 1992; pp 690-694. (c) Donaldson, J. R.; Schnabel, R. B. Technometrics 1987, 29, 67-82.

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Figure 10. Apparent aggregation numbers of the micelle structures calculated from the data in Figure 8b, as described in the text.

Figure 9. (a) First-order quenching rate constant k as a function of Py concentration divided by the saturated Py concentration for each polymer solution, Cpoly ) 8, 6.4, 3.2, 1.6, and 0.8 g/L at R ) 1.0. (b) The number of Py molecules per micelle 〈n〉 as a function of Py concentration divided by the saturated Py concentration for each polymer solution, Cpoly ) 8, 6.4, 3.2, 1.6, and 0.8 g/L at R ) 1.0.

large values of 〈n〉 found for low fluorophore concentrations. The error bars shown in Figure 8 are obtained from the 95% joint confidence ellipses and are very small compared to the changes seen for the k and 〈n〉 parameters. We need to consider a number of different reasons for this behavior. First we examine three potential artifacts that could lead to large apparent 〈n〉 values at low fluorophore concentrations. One, we know that there is an impurity fluorescence associated with the HASE polymer. This relatively weak emission would contribute a rapid component to the decay profile, but for probe concentrations in excess of 2 × 10-6 M, its contribution to the measured intensity would be very small. Two, another source of a short-lived fluorescence signal, probe emission from the water phase, is also insignificant at the polymer concentrations we examine. Three, an impurity associated with the polymer itself might give rise to an artifact. In this way, probes located in some environments within the polymer would experience quenching in competition with their fluorescence, providing a fast component to their decay profile. This possibility is particularly difficult to rule out. Note that these three potential artifacts lead to a rapid component to the fluorescence decay of the probe which is not associated with excimer formation. Our view is that these possible artifacts make at most a very small contribution to the results we report. The problem is more intimately connected to the distribution of Py or EtPy in the system. It is useful at this point to recall that plots of IE/IM for the two probes appear normal, except for an unusual positive intercept in the limit of low probe concentration. To emphasize that the unusual behavior we observe for 〈n〉 and k depends on the Py concentration and not the polymer concentration, we present plots of these data vs [Py]/[Py]sat in Figure 9. We observe that at polymer concentrations sufficiently high that a negligible fraction of the Py is in the water phase, the magnitude of these

parameters depends on the fractional saturation of the solution with Py. This type of behavior suggests that there are multiple binding regions in the polymer with different affinities for Py. As more Py is added to the system, the fluorescence signal becomes increasingly weighted in favor of those sites with smaller affinity for Py. Another way to examine these data is to calculate apparent hydrophobe aggregation numbers NRapp from the data in Figure 8b. According to eq 2, a plot of 〈n〉 vs [Py] or [EtPy] should be linear with a slope proportional to NR. We estimate apparent values of this aggregation number from the slope of the curve at each probe concentration and plot these values in Figure 10. The apparent aggregation numbers, describing the apparent number of hydrophobes that form a micelle, decrease drastically with increasing fluorophore concentration. We emphasize that these results are reproducible, and not only for experiments carried out at a single polymer concentration. With Py as a probe, we have carried out experiments at different polymer concentrations (Cpoly ) 3.2, 4.8, and 6.4 g/L at R ) 1.0) and find that the parameters k and 〈n〉, for a given Py concentration, are independent of polymer concentration. Nevertheless, the actual numbers in Figure 10 are probably not very meaningful, except as they point to different aspects of the system sampled by the probes at different probe concentrations. Possible Explanations for Our Results. Not all fluorescence quenching experiments in micellar systems fit well to eqs 1 and 2. Two problems which have been identified in the past include the case of the exchange of the probe between micelles during its excited state13 and the case of polydispersity in micelle size.14 Under these circumstances, one can introduce additional parameters into the expression for fitting the fluorescence decay profile and, in this way, describe the exchange rate of probes between micelles. In the case of a distribution of micelle sizes, an appropriate model has been developed for the case where this distribution is monomodal and not too broad.14 Recently Almgren and co-workers 15 have been interested in using fluorescence quenching to study nonideal (13) (a) Tachiya, M. Chem. Phys. Lett. 1975, 33 (2), 289-292. (b) Dederen, J. C.; Auweraer, M. V. D.; De Schryver, F. C. Chem. Phys. Lett. 1979, 68 (2,3), 451-454. (c) Dederen, J. C., Auweraer, M. V.; De Schryver, F. C. J. Phys. Chem. 1981, 85, 1198-1202. (14) (a) Almgren, M.; Lofroth, J. E. J. Colloid Interface Sci. 1981, 81, 486. (b) Almgren, M.; Lofroth, J. E. J. Chem. Phys. 1982, 76, 2734. (15) (a) Almgren, M.; Hansson, P.; Wang, K. Langmuir 1996, 12, 3855. (b) Almgren, M.; Wang, K.; Asakawa, T. Langmuir 1997, 13, 4535.

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mixed micellar systems. These experiments employed Py as a fluorophore and a quencher which is also a surfactant. For example, dodecylpyridinium chloride (C12PC) was used with Py to characterize micelles formed from the nonionic surfactant octaethylene glycol dodecyl ether (C12E8). They found, for small amounts of C12PC, that the apparent aggregation number (Nagapp) increased linearly with increasing quencher concentration. This result was interpreted in terms of a net thermodynamic attraction between the ionic surfactant and the nonionic molecules. The authors point out that addition of more than one charged surfactant to the nonionic surfactant micelle will be hindered by an electrostatic repulsion, leading to a perturbation in the quencher distribution away from purely Poisson statistics.15a More recently, they examined fluorocarbon-hydrocarbon surfactant mixtures, comparing a fluorocarbon surfactant-quencher with a hydrocarbon surfactant-quencher of similar structure. In many of the examples reported here Nagapp values decreased with increasing surfactantquencher concentration. In the extreme case of surfactant demixing to form separate fluorocarbon and hydrocarbon micelles, the fluorocarbon quencher became localized in the fluorocarbon micelles.15b The results presented in Figures 8-10 represent much more serious deviations from simple Poisson quenching statistics than those reported by Almgren et al.15 Although our system is in many ways more complex than binary surfactant mixtures, the analysis of the binary systems presented earlier15 provides insights which are helpful to us in rationalizing our results. We need to consider the factors that affect the distribution of Py or EtPy among the hydrophobic domains in our polymer in aqueous solution. A linear increase in IE/IM with increasing [Py] or [EtPy], coupled with a positive intercept at low probe concentration, suggests that the difficulties with the model are due to an enhanced probability of excimer formation at low probe concentration. This implies the existence of very hydrophobic domains that lead to a significant probability of finding two probes in the same domain. Alternatively, if the interaction between the probe and (some of) the micelles were repulsive,15 there could be an enhanced probability that a second probe molecule would prefer to enter a micelle already containing a probe than to enter an unoccupied micelle. This situation would also lead to an enhanced probability of excimer formation. For further insights into the nature of these domains, we consider the saturation concentrations of the two probes in the presence of the HASE polymer at R ) 1.0. The limiting solubility in the system is 1 Py/20 C20H41 groups and 1 EtPy/30 C20H41 groups. The data in Figure 8 span the range of about 30-300 C20H41 groups/EtPy and 20300 C20H41 groups/Py. If the system formed micelle-like structures with small or even modest aggregation numbers, such as 20-30 hydrophobes/micelle, then the probability of finding two probes in the same micelle would be

Horiuchi et al.

very small at low probe-to-hydrophobe concentration ratios. Thus we think it likely that the C20H41 groups associate to form some micelle-like structures with 6080 groups per aggregate. These numbers are in the range of typical aggregation numbers for nonionic surfactants and are not unexpected for strong hydrophobes separated from the polymer backbone by long EO tethers. From this point of view, the initial Py probes added to the HASE polymer solution partition into large micelles formed by the C20H41 groups. There are other hydrophobic domains in the system, particularly regions of the polymer backbone rich in EA groups. Even at R ) 1, a small fraction of the MAA groups will be protonated. We know from studies at low values of R that the EA-MAA copolymer is capable of solubilizing Py from aqueous solution. In our current, still-qualitative model, we imagine a significant distribution of hydrophobic domains, ranging from large micelles comprised of C20H41 groups, and a variety of mixed micelles consisting of varying mixtures of C20H41, EA, and protonated MAA groups. EtPy, the more hydrophobic probe, preferentially partitions into the more hydrophobic of these domains. Thus, for comparable concentrations of the two probes, we find larger values of 〈n〉, k, and IE/IM for EtPy than for Py. Conclusions Fluorescence probe experiments on solutions of a HASE polymer in water indicate the presence of hydrophobic micelle like domains in the solution. The hydrophobes in this polymer are C20H41 groups, separated from the polymer backbone by a long oligomeric chain consisting of 32 EO units. With Py and EtPy as probes, both monomer and excimer emission are observed, and IE/IM increases in proportion to the amount of probe added to the system. Furthermore, individual monomer fluorescence decay profiles fit well to the traditional model based upon a Poisson distribution of fluorophores and quenchers among micelles. Attempts to calculate the hydrophobe aggregation number NR led to values that changed markedly with the ratio of probe to polymer. These results were rationalized in terms of a structure containing various hydrophobic domains of different composition, coupled with partitioning of the probes among them. These domains vary from nonionic micelle like structures containing upward of 60 C20H41 groups, where the first probes added to the system are located, to mixed structures containing C20H41, EA groups, and perhaps some protonated MAA groups as well. Acknowledgment. This research owes much to helpful support by Mr. K. Nishikawa. The authors thank Drs. K. C. Tam and V. Tirtaatmadja at Nanyang University, Singapore, for useful discussions. The authors thank FujiXerox Co. Ltd. Japan, NSERC Canada, and the Singapore/ Ontario Collaborative Research for their support of this research. LA9806653