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Time-Resolved Fluorescence Anisotropies in Mixed Surfactant Solutions
Journal of Colloid and Interface Science 218, 260 –264 (1999) Article ID jcis.1999.6414, available online at http://www.idealibrary.com on
Time-Resolved Fluorescence Anisotropies in Mixed Surfactant Solutions Matthew E. McCarroll,† ,1 Alan G. Joly,* Zheming Wang,* Donald M. Friedrich,* and Ray von Wandruszka† ,2 *Environmental and Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352; and †Department of Chemistry, University of Idaho, Moscow, Idaho 83844 –2343 Received February 18, 1999; accepted July 9, 1999
micellar solutions of the nonionic surfactant Triton X-114 (TX-114) produces micelles with a relatively fluid core. The behavior of micellized perylene suggested that it was displaced from the structured palisade layer, where it has limited rotational freedom, to the more randomly organized interior. The anisotropies measured in this manner, however, necessarily represent an average of the rotational diffusion of the fluorophore. This may be affected by the viscosity of its microenvironment and by its inability to rotate freely (i.e., in the case of hindered rotation). Steady-state measurements cannot differentiate between these two effects. If the fluorophore in a system is present in more than one environment, the average steadystate anisotropy is given by
Time-resolved fluorescence anisotropy decays of solutions of Triton X-114 (TX-114) with various amounts of sodium dodecyl sulfate (SDS) were measured using the emission both from the surfactant itself and from added perylene. In the former case, the monomer and aggregate species of the surfactant were spectroscopically isolated and were shown to have significantly different rotational correlation times. The rotational diffusion of perylene in micellar TX-114 with small amounts of added SDS appeared to have a component with a very short correlation time. The anisotropy decay curves showed the existence of limiting anisotropies (r `), indicating hindered probe rotation in the micellar environment. At higher SDS concentrations, the fast-decaying component slowed down and the limiting anisotropy decreased substantially, suggesting some migration of the probe to the interior of the micelle. © 1999 Academic Press Key Words: mixed micelles; perylene fluorescence; time-resolved fluorescence anisotropy; subslip behavior.
r avg 5
1 Current address: Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803. 2 To whom correspondence should be addressed.
0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
i i
[1]
i
where f i and r i are the ith fractional emission intensity and anisotropy, respectively. In most cases it is not possible to resolve the individual components that contribute to r avg. In contrast, this resolution can often be achieved by the measurement of the time-resolved anisotropy, r(t), of the fluorophore. Specifically, the time decay of r(t) can reveal whether the fluorophore is free to rotate in solution or whether this rotation is hindered. This attribute makes r(t) especially relevant to micellar systems, where the nature of binding interactions between a solubilized probe and the micelle is of interest. To determine r(t), the decays of the fluorescence intensity are measured for the parallel [I \ (t)] and perpendicular [I ' (t)] polarized emission components, following a polarized excitation pulse. The value of r(t) is then found from
INTRODUCTION
The addition of ionic surfactants to solutions of their nonionic counterparts generally results in changes in both aggregation and solubilization characteristics of these solutions. As regards the former, the presence of a minor proportion of an ionic surfactant leads to an increase in the cloud point (1–11), which is ascribed to electrostatic repulsion experienced by micelles with an ionic component. The micellization of nonpolar probe molecules such as pyrene and perylene (3) in mixed micelles exhibits mechanistic differences compared with their solubilization in solutions of pure nonionic surfactants. This is the focus of the present investigation. In previous studies in this laboratory (3, 4, 12), the steadystate fluorescence anisotropy, r, has been employed to monitor microenvironmental changes in nonionic surfactant solutions. Both native surfactant fluorescence and added fluorescent probes were used in this work. The results indicated that the addition of small amounts of sodium dodecyl sulfate (SDS) to
O fr,
r~t! 5
I \ ~t! 2 I ' ~t! . I \ ~t! 1 2I ' ~t!
[2]
In the case of a spherical fluorophore in a homogeneous solution, r(t) is also given by
260
r~t! 5 r 0 e 2t/F ,
[3]
TIME-RESOLVED FLUORESCENCE ANISOTROPIES
where r 0 is the intrinsic anisotropy, t is time, and F is the rotational correlation time of the emitter. In simple cases of electric dipole-allowed excitation and fluorescence transitions in spherical or symmetric ellipsoidal solutes, the decay of the anisotropy can elucidate both the angle between the excitation and emission oscillator of the fluorophore and the rate at which it rotates in solution. In the case of an anisotropic rotation, r(t) decays as a multiple exponential, r~t! 5 r 0
O fe i
2t/F i
,
[4]
i
where f i is the fractional intensity of the ith component of the decay. In cases where the fluorophore is bound to a larger body, or experiences hindered rotation, the anisotropy may not decay to zero. In this case, the fluorophore is said to have a limiting anisotropy, r ` , and the decay can be represented by r~t! 5 ~r 0 2 r ` !e 2t/F 1 r ` .
[5]
Fluorophores are especially noted to have limiting anisotropies in membrane systems and micellar solutions. Previous work in this laboratory has dealt with steady-state anisotropies in micellar solutions of nonionic surfactants, using both native and probe fluorescence (3, 4, 9, 12). In both cases, r was found to be strongly dependent on the presence and concentration of added ionic surfactants. Specifically, r of perylene in micellar TX-114 solutions was shown to decrease sharply on addition of SDS. It was suggested that the probe migrated from the ordered palisade region of the micelle to the interior portions of the core, where it can rotate in a less hindered fashion. In the present study, this premise is further examined by monitoring the time-resolved anisotropy of the system in question. MATERIALS AND METHODS
Reagents and solutions. TX-114 was obtained from Sigma and used without further purification. This surfactant has a critical micelle concentration (CMC) of 2.8 3 10 24 M and a cloud point of 23°C. SDS (99%; CMC, 8 3 10 23 M) was purchased from J. T. Baker and used without further purification. Perylene (Aldrich, 99.5%) was purified by cold finger sublimation. Chloroform (ACS grade) was obtained from Fisher and used as received. Doubly deionized water, treated with a 0.22-mm Millipore filter system to a resistivity of at least 18 MV cm, was used to prepare all solutions. Procedures. Aqueous stock solutions of TX-114 (0.010 M) and SDS (0.10 M) were prepared. For surfactant solutions containing perylene, the appropriate amount of the probe in chloroform was placed in a dry volumetric flask, evaporated with a stream of nitrogen, and diluted to volume with the pertinent surfactant solution. In the solutions containing the
261
highest SDS concentrations, a weighed quantity of solid SDS was diluted with the appropriate nonionic surfactant solution. All solutions were sonicated for 15 min and allowed to equilibrate at least 1 h prior to measurement. Fluorescence decay measurements. The fluorescence decays were measured using a system comprising ultrafast lasers and a Hamamatsu streak camera (Model C5680-21, 3-ps response), operated in the synchronous scanning mode at 76MHz pulse rate. The sample solution was contained in a 10-cm-long, 2.5-cm-diameter cylindrical quartz cell. A long cell was necessary to avoid detection of delayed excitation caused by reflection of the laser pulses from the back window of the cell. The excitation beam was positioned near the cylindrical side wall and the emission viewed at 90°, very close to the front window. By this method, signal loss due to inner filtering was minimized. The fluorescence was passed through a cutoff filter (to block scattered excitation light) and a UV sheet polarizer before being focused on the entrance slit of the streak camera. A consequence of the use of filters was that the experimental g factor in these measurements was essentially unity, and no correction for vertical or horizontal polarization bias was required. In the case of native surfactant fluorescence (TX-114), the excitation source was a Coherent 702 dye laser (12-ps FWHM pulses) pumped by a 76-MHz Coherent Antares Nd:YAG laser. The polarized output (l) of the dye laser passed through a BBO frequency doubling crystal to generate pulses at the second harmonic (l/2) which was separated from the fundamental by a Pellin–Broca dispersing prism and UV transmitting color filters. The monomer or aggregate form of the surfactant could be excited selectively by tuning the dye laser output to 580 or 620 nm (second harmonic at 290 or 310 nm), respectively. The spectral properties of TX-114 aggregates have been reported elsewhere (4, 18). In the case of perylene fluorescence, a Clark-MXR Ti:sapphire laser (NJA-5, 76 MHz) producing 0.1-ps pulses at 800 nm was used to excite perylene with the polarized second harmonic at 400 nm. In either case, the instrument response, determined by using a scattering solution of alumina particles in water, was measured to be under 12 ps FWHM. RESULTS AND DISCUSSION
Native Surfactant Fluorescence Figure 1 shows the time-dependent anisotropy decays of the monomer and aggregate species in 0.01 M TX-114. Spectroscopic separation of the two forms of the surfactant was achieved by selective excitation (monomer, 290 nm; aggregate, 310 nm) and the use of cutoff filters for the emission (monomer peak, 302 nm; aggregate peak, 345 nm) (4). The noisy signal observed for the aggregate is attributable to the relatively low light intensity obtained from the laser system at 302 nm. The aggregate emission anisotropy was represented by a two-com-
262
MCCARROLL ET AL.
FIG. 1. Fluorescence anisotropy decay of monomer (E) and aggregate (3) species in 10.0 mM TX-114.
ponent decay, where the major component ( f 1 5 0.7) had a rotational correlation time, F, of 3500 ps, and the minor component ( f 2 5 0.3) had F 5 200 ps. The longer component did not appear to have a limiting anisotropy, suggesting free, but slow, rotation. The surfactant monomer had a much more rapid decay ( f 1 5 0.4, F 1 5 20 ps; f 2 5 0.6, F 2 5 200 ps), reflecting its relatively free motion in solution. Importantly, the substantial difference between the two decay curves and the corresponding correlation times furnished direct confirmation for the assignment of the respective emissions to the aggregate and monomer forms of the surfactant. In addition, the intrinsic anisotropy (r 0 5 0.22) obtained from r(t) at t 5 0 is in agreement with a previously reported value measured in this laboratory using steady-state methods (3).
Perylene Fluorescence in Mixed Micelles Previous work in this laboratory (3) has shown that the steady-state anisotropy of perylene decreases substantially at higher SDS concentrations. This was ascribed to migration of the probe from the palisade layer toward the interior of the micelle. Figure 2 shows the anisotropy decays for three solutions of TX-114 with varying amounts of SDS. The solutions are comparable to those reported earlier (3) and correspond to steady-state anisotropies of 0.078, 0.075, and 0.050. It can be seen that the r(t) at t 5 0 was different in the three solutions, having values of 0.22, 0.31, and 0.28 for 0.010, 0.10, and 1.0 mM SDS, respectively. This anisotropy is normally interpreted as r 0 , but the value of this parameter for perylene is known to be 0.36 (13). In general, one does not expect r 0 of a fluoro-
FIG. 2. Fluorescence anisotropy decay of perylene in 7.8 mM TX-114 with 0.010 (1), 0.10 (F), and 1.0 (‚) mM SDS.
263
TIME-RESOLVED FLUORESCENCE ANISOTROPIES
Figure 3 shows the experimental decays and mathematical fits obtained with Eq. [6]. All three solutions had a limiting anisotropy and a two-component decay. The parameters that produced adequate fits are summarized in Table 1. The observed variations in perylene anisotropy resulted primarily from changes in r ` and the faster rotational component, assigned to the in-plane rotation of the probe (correlation time F ip). At lower SDS concentrations, an increase in this concentration resulted in a rise in F ip, while the limiting anisotropy changed little. This suggests that the increased presence of SDS in the palisade layer initially caused its microviscosity to rise, at least to the extent that the fast in-plane motion of the probe was slowed. The mechanism for this process cannot be fully explained, but it may the rationalized by considering that incorporation of SDS brought the charged, strongly hydrated, headgroup of the anionic surfactant into close proximity to the TX-114 palisade layer. This both increased the polarity of this region and disrupted its orderly arrangement, especially with regard to the stacking of the aromatic moieties. A flat probe molecule such as perylene lodged there may therefore have become more encumbered in its in-plane motion, while outof-plane (end-over-end) rotations remained equally inhibited in both scenarios. At the highest concentration of SDS, however, both the limiting anisotropy and the slower rotational component decreased notably, indicating a greater fluidity in the probe environment. As has been suggested before (3), in the presence of a larger proportion of SDS (;13% in this case), the C-12 chains of this surfactant may cause the probe to slip out of the palisade layer and migrate into the more disordered core of the micelle. It is informative to compare steady-state and time-resolved anisotropy data for these systems. The steady-state anisotropies can be calculated from the rotational correlation times and the fluorescence lifetime using
FIG. 3. Fluorescence anisotropy decay ({) and theoretical fit (—) for perylene in 7.8 mM TX-114 containing 0.010 (A), 0.10 (B), and 1.0 (C) mM SDS.
phore to change in response to environmental parameters. Factors that can lead to its decrease include energy transfer and environmental effects that alter the geometry of the fluorophore. The former is precluded by the low concentration (1.0 3 10 26 M) of perylene, while its rigidity makes geometric alterations unlikely. Another possible explanation for the low value of the zero-time anisotropy is the existence of a decay component faster than could be resolved in the experiment (;14 ps). This is also supported by the observation that the decays could not be fit with Eq. [4] or [5], when using r(t) at t 5 0 as r 0 . When the known value of 0.36 was used, good fits were obtained. This did, however, necessitate the use of an equation combining the effects of a multicomponent decay with those of a limiting anisotropy. To achieve this, the anisotropy decays were fit with the equation r~t! 5 ~r 0 2 r ` !
O ae i
2t/F
1 r 0.
r avg 5
O f 1 1rt /F 1 r , 0
i
i
i
[7]
`
where the symbols have the same meaning as in previous expressions. Using Eq. [7] and the parameters obtained from the anisotropy decays, steady-state anisotropies were calculated and are listed in Table 1. The calculated values corre-
[6]
i
TABLE 1 Rotational Diffusion Parameters of Perylene in 7.8 mM TX-114 with Added SDS SDS (mM)
r`
F1 (ps)
F2 (ps)
a1
a2
r (steady state) av
r(t) av
Correlation coefficient
0.010 0.10 1.0
0.07 0.06 0.036
0.5 8 6
240 240 180
0.50 0.52 0.50
0.50 0.48 0.50
0.078 0.075 0.050
0.078 0.068 0.042
0.98 0.98 0.98
264
MCCARROLL ET AL.
spond well with the measured ones, bearing out the procedures used to fit the anisotropy decays. The origin of the proposed fast component of the r(t) decay warrants careful evaluation. First it needs to be established whether it was real or possibly arose from laser jitter. Numerical simulation of this instrumental artifact showed that during the jittering period (,100 ps), the anisotropy stayed high because fresh oriented samples were being photoselected continuously during the interval. The result would be a delay in anisotropy decay, a very slight indication of which may be seen at the beginning of the plot in Fig. 1. In any event, the effect cannot be represented by a separate exponential component, and it is therefore indicated that the short anisotropy decay time probably had a molecular origin. Thus assuming the physical reality of the fast r(t) decay component, it remains to be determined whether it arose from the combined motions of a single perylene population or from probe molecules in two different environments. To simulate the rotational diffusion of perylene and its anisotropy relaxation, the diffusion constants must be estimated on the basis of the molecular geometry (i.e., spherical top, symmetric top, or asymmetric rotor) and a selected (stick or slip) friction model (19, 20). The microscopic viscosity around the solute molecule also must be known or estimated as a fitting parameter. Kim and Hochstrasser (21) modeled fluorene relaxation as an asymmetric rotor in both the slip and stick friction limits. The slip model (20) predicted a biexponential relaxation of r(t) with two time constants, 11.1h and 5.0h, where h is the viscosity of a series of alcohols (C 1–C 11). However, the r(t) relaxation could be adequately fit to a single exponential. The observed relaxation time scaled with the bulk alcohol viscosity as t obs 5 5h, although the hydrodynamic model predicted slightly greater contribution from the slower relaxation term (0.212) relative to the faster term (0.188). It was suggested (21) that the subslip behavior was due to the aromatic molecule being preferentially solvated in the hydrophobic regions of the alcohol solvents, having a lower viscosity than the bulk which is determined by H bonding in the hydrophilic regions. This subslip hydrodynamic behavior was later confirmed by Hartman and Waldeck (22) and treated by a model that included both hydrodynamic and dielectric friction. These considerations are relevant to the present perylene relaxation since (1) although fluorene is considerably more asymmetric than perylene, only one exponential relaxation term was needed to fit the data; and (2) the observed relaxation rate adhered to the faster (subslip) exponential term. Further work by Brocklehurst and Young (23) and Jiang and Blanchard (24) demonstrated that the rotational diffusion of perylene in alkanes can be very complex, apparently switching from a stick mechanism for small molecule solvents to a slip mechanism in larger alkanes. At low temperatures, biexponential relaxation was observed (23), but near room temperature only single exponential decays occurred for perylene in alkanes (24).
In view of these considerations, one would expect perylene to exhibit a single exponential relaxation at room temperature in the hydrophobic regions of the micelles. Therefore, experimental observation of biexponential r(t) relaxation in the mixed SDS/TX-114 micelles should be interpreted as evidence for the coexistence of at least two distinct hydrophobic regions characterized by a high microviscosity and a low microviscosity, both different from the bulk. This may again be ascribed to a mechanism in which the inclusion of SDS in TX-114 micelles led to a partial dislodging of perylene from the micellar palisade layer, establishing a small population of probe molecules located in the core of the micelle. ACKNOWLEDGMENTS The research described in this paper was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle under Contract DE-AC06-76RLO 1830. The authors acknowledge additional financial support from the Idaho NSF EPSCoR program.
REFERENCES 1. Maclay, W. N., J. Colloid Sci. 11, 272 (1956). 2. Nishikido, N., Akisada, H., and Matura, R., Mem. Fac. Sci. Kyushu Univ. Ser. C 10, 92 (1977). 3. McCarroll, M., Toerne, K., and von Wandruszka, R., Langmuir 14, 2965 (1998). 4. McCarroll, M. E., and von Wandruszka, R., J. Fluorescence 7, 185 (1997). 5. Hinze, W. L., and Pramauro, E., Crit. Rev. Anal. Chem. 24, 133 (1993). 6. Corti, M., Minero, C., and Degiorgio, V., J. Phys. Chem. 88, 309 (1984). 7. Schott, H., Royce, A. E., and Han, S. K., J. Colloid Interface Sci. 98, 196 (1984). 8. Marszall, L., Langmuir 4, 90 (1988). 9. Komaromy-Hiller, G., and von Wandruszka, R., J. Colloid Interface Sci. 177, 156 (1996). 10. Komaromy-Hiller, G., Calkins, N., and von Wandruszka, R., Langmuir 12, 916 (1996). 11. Valaulikar, B. S., and Manohar, C., J. Colloid Interface Sci. 108, 403 (1985). 12. McCarroll, M. E., Toerne, K., and von Wandruszka, R., Langmuir 21, 6096 (1998). 13. Lakowicz, J. R., “Principles of Fluorescence Spectroscopy,” Chap. 5. Plenum, New York, 1983. 14. Chou, S., and Wirth, M. J., J. Phys. Chem. 93, 7694 (1989). 15. Maiti, N. C., Mazumdar, S., and Periasamy, N., J. Phys. Chem. 99, 10708 (1995). 16. DeLong, L. J., and Nichols, J. W., Biophysical J. 70, 1466 (1996). 17. Chen, L. A., Dale, R. E., Roth, S., and Brand, L., J. Biol. Chem. 252, 2163 (1977). 18. Ikeda, S., and Fasman, G. D., J. Polym. Sci. Part A-1 8, 991 (1970). 19. Hu, C-M, and Zwanzig, R., J. Chem. Phys. 60, 4354 (1974). 20. Youngren, G. K., and Acrivos, A., J. Chem. Phys. 63, 3846 (1975). 21. Kim, Y.-R., and Hochstrasser, R. M., J. Phys. Chem. 96, 9595 (1992). 22. Hartman, R. S., and Waldeck, D. H., J. Phys. Chem. 98, 1386 (1994). 23. Brocklehurst, B., and Young, R. N., J. Phys. Chem. 99, 40 (1995). 24. Jiang, Y., and Blanchard, G. J., J. Phys. Chem. 98, 9411 (1994).
Time-Resolved Fluorescence Anisotropies in Mixed Surfactant Solutions Matthew E. McCarroll,† ,1 Alan G. Joly,* Zheming Wang,* Donald M. Friedrich,* and Ray von Wandruszka† ,2 *Environmental and Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352; and †Department of Chemistry, University of Idaho, Moscow, Idaho 83844 –2343 Received February 18, 1999; accepted July 9, 1999
micellar solutions of the nonionic surfactant Triton X-114 (TX-114) produces micelles with a relatively fluid core. The behavior of micellized perylene suggested that it was displaced from the structured palisade layer, where it has limited rotational freedom, to the more randomly organized interior. The anisotropies measured in this manner, however, necessarily represent an average of the rotational diffusion of the fluorophore. This may be affected by the viscosity of its microenvironment and by its inability to rotate freely (i.e., in the case of hindered rotation). Steady-state measurements cannot differentiate between these two effects. If the fluorophore in a system is present in more than one environment, the average steadystate anisotropy is given by
Time-resolved fluorescence anisotropy decays of solutions of Triton X-114 (TX-114) with various amounts of sodium dodecyl sulfate (SDS) were measured using the emission both from the surfactant itself and from added perylene. In the former case, the monomer and aggregate species of the surfactant were spectroscopically isolated and were shown to have significantly different rotational correlation times. The rotational diffusion of perylene in micellar TX-114 with small amounts of added SDS appeared to have a component with a very short correlation time. The anisotropy decay curves showed the existence of limiting anisotropies (r `), indicating hindered probe rotation in the micellar environment. At higher SDS concentrations, the fast-decaying component slowed down and the limiting anisotropy decreased substantially, suggesting some migration of the probe to the interior of the micelle. © 1999 Academic Press Key Words: mixed micelles; perylene fluorescence; time-resolved fluorescence anisotropy; subslip behavior.
r avg 5
1 Current address: Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803. 2 To whom correspondence should be addressed.
0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
i i
[1]
i
where f i and r i are the ith fractional emission intensity and anisotropy, respectively. In most cases it is not possible to resolve the individual components that contribute to r avg. In contrast, this resolution can often be achieved by the measurement of the time-resolved anisotropy, r(t), of the fluorophore. Specifically, the time decay of r(t) can reveal whether the fluorophore is free to rotate in solution or whether this rotation is hindered. This attribute makes r(t) especially relevant to micellar systems, where the nature of binding interactions between a solubilized probe and the micelle is of interest. To determine r(t), the decays of the fluorescence intensity are measured for the parallel [I \ (t)] and perpendicular [I ' (t)] polarized emission components, following a polarized excitation pulse. The value of r(t) is then found from
INTRODUCTION
The addition of ionic surfactants to solutions of their nonionic counterparts generally results in changes in both aggregation and solubilization characteristics of these solutions. As regards the former, the presence of a minor proportion of an ionic surfactant leads to an increase in the cloud point (1–11), which is ascribed to electrostatic repulsion experienced by micelles with an ionic component. The micellization of nonpolar probe molecules such as pyrene and perylene (3) in mixed micelles exhibits mechanistic differences compared with their solubilization in solutions of pure nonionic surfactants. This is the focus of the present investigation. In previous studies in this laboratory (3, 4, 12), the steadystate fluorescence anisotropy, r, has been employed to monitor microenvironmental changes in nonionic surfactant solutions. Both native surfactant fluorescence and added fluorescent probes were used in this work. The results indicated that the addition of small amounts of sodium dodecyl sulfate (SDS) to
O fr,
r~t! 5
I \ ~t! 2 I ' ~t! . I \ ~t! 1 2I ' ~t!
[2]
In the case of a spherical fluorophore in a homogeneous solution, r(t) is also given by
260
r~t! 5 r 0 e 2t/F ,
[3]
TIME-RESOLVED FLUORESCENCE ANISOTROPIES
where r 0 is the intrinsic anisotropy, t is time, and F is the rotational correlation time of the emitter. In simple cases of electric dipole-allowed excitation and fluorescence transitions in spherical or symmetric ellipsoidal solutes, the decay of the anisotropy can elucidate both the angle between the excitation and emission oscillator of the fluorophore and the rate at which it rotates in solution. In the case of an anisotropic rotation, r(t) decays as a multiple exponential, r~t! 5 r 0
O fe i
2t/F i
,
[4]
i
where f i is the fractional intensity of the ith component of the decay. In cases where the fluorophore is bound to a larger body, or experiences hindered rotation, the anisotropy may not decay to zero. In this case, the fluorophore is said to have a limiting anisotropy, r ` , and the decay can be represented by r~t! 5 ~r 0 2 r ` !e 2t/F 1 r ` .
[5]
Fluorophores are especially noted to have limiting anisotropies in membrane systems and micellar solutions. Previous work in this laboratory has dealt with steady-state anisotropies in micellar solutions of nonionic surfactants, using both native and probe fluorescence (3, 4, 9, 12). In both cases, r was found to be strongly dependent on the presence and concentration of added ionic surfactants. Specifically, r of perylene in micellar TX-114 solutions was shown to decrease sharply on addition of SDS. It was suggested that the probe migrated from the ordered palisade region of the micelle to the interior portions of the core, where it can rotate in a less hindered fashion. In the present study, this premise is further examined by monitoring the time-resolved anisotropy of the system in question. MATERIALS AND METHODS
Reagents and solutions. TX-114 was obtained from Sigma and used without further purification. This surfactant has a critical micelle concentration (CMC) of 2.8 3 10 24 M and a cloud point of 23°C. SDS (99%; CMC, 8 3 10 23 M) was purchased from J. T. Baker and used without further purification. Perylene (Aldrich, 99.5%) was purified by cold finger sublimation. Chloroform (ACS grade) was obtained from Fisher and used as received. Doubly deionized water, treated with a 0.22-mm Millipore filter system to a resistivity of at least 18 MV cm, was used to prepare all solutions. Procedures. Aqueous stock solutions of TX-114 (0.010 M) and SDS (0.10 M) were prepared. For surfactant solutions containing perylene, the appropriate amount of the probe in chloroform was placed in a dry volumetric flask, evaporated with a stream of nitrogen, and diluted to volume with the pertinent surfactant solution. In the solutions containing the
261
highest SDS concentrations, a weighed quantity of solid SDS was diluted with the appropriate nonionic surfactant solution. All solutions were sonicated for 15 min and allowed to equilibrate at least 1 h prior to measurement. Fluorescence decay measurements. The fluorescence decays were measured using a system comprising ultrafast lasers and a Hamamatsu streak camera (Model C5680-21, 3-ps response), operated in the synchronous scanning mode at 76MHz pulse rate. The sample solution was contained in a 10-cm-long, 2.5-cm-diameter cylindrical quartz cell. A long cell was necessary to avoid detection of delayed excitation caused by reflection of the laser pulses from the back window of the cell. The excitation beam was positioned near the cylindrical side wall and the emission viewed at 90°, very close to the front window. By this method, signal loss due to inner filtering was minimized. The fluorescence was passed through a cutoff filter (to block scattered excitation light) and a UV sheet polarizer before being focused on the entrance slit of the streak camera. A consequence of the use of filters was that the experimental g factor in these measurements was essentially unity, and no correction for vertical or horizontal polarization bias was required. In the case of native surfactant fluorescence (TX-114), the excitation source was a Coherent 702 dye laser (12-ps FWHM pulses) pumped by a 76-MHz Coherent Antares Nd:YAG laser. The polarized output (l) of the dye laser passed through a BBO frequency doubling crystal to generate pulses at the second harmonic (l/2) which was separated from the fundamental by a Pellin–Broca dispersing prism and UV transmitting color filters. The monomer or aggregate form of the surfactant could be excited selectively by tuning the dye laser output to 580 or 620 nm (second harmonic at 290 or 310 nm), respectively. The spectral properties of TX-114 aggregates have been reported elsewhere (4, 18). In the case of perylene fluorescence, a Clark-MXR Ti:sapphire laser (NJA-5, 76 MHz) producing 0.1-ps pulses at 800 nm was used to excite perylene with the polarized second harmonic at 400 nm. In either case, the instrument response, determined by using a scattering solution of alumina particles in water, was measured to be under 12 ps FWHM. RESULTS AND DISCUSSION
Native Surfactant Fluorescence Figure 1 shows the time-dependent anisotropy decays of the monomer and aggregate species in 0.01 M TX-114. Spectroscopic separation of the two forms of the surfactant was achieved by selective excitation (monomer, 290 nm; aggregate, 310 nm) and the use of cutoff filters for the emission (monomer peak, 302 nm; aggregate peak, 345 nm) (4). The noisy signal observed for the aggregate is attributable to the relatively low light intensity obtained from the laser system at 302 nm. The aggregate emission anisotropy was represented by a two-com-
262
MCCARROLL ET AL.
FIG. 1. Fluorescence anisotropy decay of monomer (E) and aggregate (3) species in 10.0 mM TX-114.
ponent decay, where the major component ( f 1 5 0.7) had a rotational correlation time, F, of 3500 ps, and the minor component ( f 2 5 0.3) had F 5 200 ps. The longer component did not appear to have a limiting anisotropy, suggesting free, but slow, rotation. The surfactant monomer had a much more rapid decay ( f 1 5 0.4, F 1 5 20 ps; f 2 5 0.6, F 2 5 200 ps), reflecting its relatively free motion in solution. Importantly, the substantial difference between the two decay curves and the corresponding correlation times furnished direct confirmation for the assignment of the respective emissions to the aggregate and monomer forms of the surfactant. In addition, the intrinsic anisotropy (r 0 5 0.22) obtained from r(t) at t 5 0 is in agreement with a previously reported value measured in this laboratory using steady-state methods (3).
Perylene Fluorescence in Mixed Micelles Previous work in this laboratory (3) has shown that the steady-state anisotropy of perylene decreases substantially at higher SDS concentrations. This was ascribed to migration of the probe from the palisade layer toward the interior of the micelle. Figure 2 shows the anisotropy decays for three solutions of TX-114 with varying amounts of SDS. The solutions are comparable to those reported earlier (3) and correspond to steady-state anisotropies of 0.078, 0.075, and 0.050. It can be seen that the r(t) at t 5 0 was different in the three solutions, having values of 0.22, 0.31, and 0.28 for 0.010, 0.10, and 1.0 mM SDS, respectively. This anisotropy is normally interpreted as r 0 , but the value of this parameter for perylene is known to be 0.36 (13). In general, one does not expect r 0 of a fluoro-
FIG. 2. Fluorescence anisotropy decay of perylene in 7.8 mM TX-114 with 0.010 (1), 0.10 (F), and 1.0 (‚) mM SDS.
263
TIME-RESOLVED FLUORESCENCE ANISOTROPIES
Figure 3 shows the experimental decays and mathematical fits obtained with Eq. [6]. All three solutions had a limiting anisotropy and a two-component decay. The parameters that produced adequate fits are summarized in Table 1. The observed variations in perylene anisotropy resulted primarily from changes in r ` and the faster rotational component, assigned to the in-plane rotation of the probe (correlation time F ip). At lower SDS concentrations, an increase in this concentration resulted in a rise in F ip, while the limiting anisotropy changed little. This suggests that the increased presence of SDS in the palisade layer initially caused its microviscosity to rise, at least to the extent that the fast in-plane motion of the probe was slowed. The mechanism for this process cannot be fully explained, but it may the rationalized by considering that incorporation of SDS brought the charged, strongly hydrated, headgroup of the anionic surfactant into close proximity to the TX-114 palisade layer. This both increased the polarity of this region and disrupted its orderly arrangement, especially with regard to the stacking of the aromatic moieties. A flat probe molecule such as perylene lodged there may therefore have become more encumbered in its in-plane motion, while outof-plane (end-over-end) rotations remained equally inhibited in both scenarios. At the highest concentration of SDS, however, both the limiting anisotropy and the slower rotational component decreased notably, indicating a greater fluidity in the probe environment. As has been suggested before (3), in the presence of a larger proportion of SDS (;13% in this case), the C-12 chains of this surfactant may cause the probe to slip out of the palisade layer and migrate into the more disordered core of the micelle. It is informative to compare steady-state and time-resolved anisotropy data for these systems. The steady-state anisotropies can be calculated from the rotational correlation times and the fluorescence lifetime using
FIG. 3. Fluorescence anisotropy decay ({) and theoretical fit (—) for perylene in 7.8 mM TX-114 containing 0.010 (A), 0.10 (B), and 1.0 (C) mM SDS.
phore to change in response to environmental parameters. Factors that can lead to its decrease include energy transfer and environmental effects that alter the geometry of the fluorophore. The former is precluded by the low concentration (1.0 3 10 26 M) of perylene, while its rigidity makes geometric alterations unlikely. Another possible explanation for the low value of the zero-time anisotropy is the existence of a decay component faster than could be resolved in the experiment (;14 ps). This is also supported by the observation that the decays could not be fit with Eq. [4] or [5], when using r(t) at t 5 0 as r 0 . When the known value of 0.36 was used, good fits were obtained. This did, however, necessitate the use of an equation combining the effects of a multicomponent decay with those of a limiting anisotropy. To achieve this, the anisotropy decays were fit with the equation r~t! 5 ~r 0 2 r ` !
O ae i
2t/F
1 r 0.
r avg 5
O f 1 1rt /F 1 r , 0
i
i
i
[7]
`
where the symbols have the same meaning as in previous expressions. Using Eq. [7] and the parameters obtained from the anisotropy decays, steady-state anisotropies were calculated and are listed in Table 1. The calculated values corre-
[6]
i
TABLE 1 Rotational Diffusion Parameters of Perylene in 7.8 mM TX-114 with Added SDS SDS (mM)
r`
F1 (ps)
F2 (ps)
a1
a2
r (steady state) av
r(t) av
Correlation coefficient
0.010 0.10 1.0
0.07 0.06 0.036
0.5 8 6
240 240 180
0.50 0.52 0.50
0.50 0.48 0.50
0.078 0.075 0.050
0.078 0.068 0.042
0.98 0.98 0.98
264
MCCARROLL ET AL.
spond well with the measured ones, bearing out the procedures used to fit the anisotropy decays. The origin of the proposed fast component of the r(t) decay warrants careful evaluation. First it needs to be established whether it was real or possibly arose from laser jitter. Numerical simulation of this instrumental artifact showed that during the jittering period (,100 ps), the anisotropy stayed high because fresh oriented samples were being photoselected continuously during the interval. The result would be a delay in anisotropy decay, a very slight indication of which may be seen at the beginning of the plot in Fig. 1. In any event, the effect cannot be represented by a separate exponential component, and it is therefore indicated that the short anisotropy decay time probably had a molecular origin. Thus assuming the physical reality of the fast r(t) decay component, it remains to be determined whether it arose from the combined motions of a single perylene population or from probe molecules in two different environments. To simulate the rotational diffusion of perylene and its anisotropy relaxation, the diffusion constants must be estimated on the basis of the molecular geometry (i.e., spherical top, symmetric top, or asymmetric rotor) and a selected (stick or slip) friction model (19, 20). The microscopic viscosity around the solute molecule also must be known or estimated as a fitting parameter. Kim and Hochstrasser (21) modeled fluorene relaxation as an asymmetric rotor in both the slip and stick friction limits. The slip model (20) predicted a biexponential relaxation of r(t) with two time constants, 11.1h and 5.0h, where h is the viscosity of a series of alcohols (C 1–C 11). However, the r(t) relaxation could be adequately fit to a single exponential. The observed relaxation time scaled with the bulk alcohol viscosity as t obs 5 5h, although the hydrodynamic model predicted slightly greater contribution from the slower relaxation term (0.212) relative to the faster term (0.188). It was suggested (21) that the subslip behavior was due to the aromatic molecule being preferentially solvated in the hydrophobic regions of the alcohol solvents, having a lower viscosity than the bulk which is determined by H bonding in the hydrophilic regions. This subslip hydrodynamic behavior was later confirmed by Hartman and Waldeck (22) and treated by a model that included both hydrodynamic and dielectric friction. These considerations are relevant to the present perylene relaxation since (1) although fluorene is considerably more asymmetric than perylene, only one exponential relaxation term was needed to fit the data; and (2) the observed relaxation rate adhered to the faster (subslip) exponential term. Further work by Brocklehurst and Young (23) and Jiang and Blanchard (24) demonstrated that the rotational diffusion of perylene in alkanes can be very complex, apparently switching from a stick mechanism for small molecule solvents to a slip mechanism in larger alkanes. At low temperatures, biexponential relaxation was observed (23), but near room temperature only single exponential decays occurred for perylene in alkanes (24).
In view of these considerations, one would expect perylene to exhibit a single exponential relaxation at room temperature in the hydrophobic regions of the micelles. Therefore, experimental observation of biexponential r(t) relaxation in the mixed SDS/TX-114 micelles should be interpreted as evidence for the coexistence of at least two distinct hydrophobic regions characterized by a high microviscosity and a low microviscosity, both different from the bulk. This may again be ascribed to a mechanism in which the inclusion of SDS in TX-114 micelles led to a partial dislodging of perylene from the micellar palisade layer, establishing a small population of probe molecules located in the core of the micelle. ACKNOWLEDGMENTS The research described in this paper was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle under Contract DE-AC06-76RLO 1830. The authors acknowledge additional financial support from the Idaho NSF EPSCoR program.
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