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Langmuir 1996, 12, 1738-1743
Deactivation of Pyrene Derivatives by Nitroxides in AOT Reverse Micelles. Dependence of Quenching Efficiency on the Probe and Quencher Location in the Microaggregates J. Alvarez, E. A. Lissi, and M. V. Encinas* Facultad de Quimica y Biologia, Universidad de Santiago de Chile, Casilla 307-2, Santiago, Chile Received March 27, 1995. In Final Form: December 15, 1995X The quenching of the fluorescence of pyrene derivatives with different hydrophobicity by a series of nitroxides has been measured in AOT reverse micelles. When both donor and quencher are predominantly located in the organic pseudophase (i.e., 1-methylpyrene and 2,2,6,6-tetramethylpiperidine-N-oxyl, TEMPO) the quenching process is not affected by the presence of the microaggregates. On the other hand, when one of the partners is located at the interface (pyrenesulfonate or 4-amino-2,2,6,6-tetramethylpiperidineN-oxyl, TEMPAMINE+), the rate of the process is slower than that in the bulk solvent, being determined by the rate of access to the interface. The restriction imposed by the interface changes with the water content of the micelle and the location of the bound species. When both species are bound to the interface, the quenching process presents static and dynamic components. The slow rate of the dynamic process indicates a highly restricted intra-interface mobility, particularly for quencher and/or donors that are counterions of the surfactant. The efficiency as quencher of 4-hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl, TEMPOL, decreases with the AOT concentration. From this dependence is derived the partition of TEMPOL between the bulk solvent, the interface, and the water pool.
Introduction Nitroxides are widely employed as EPR spin probes in studies concerning microheterogeneous media.1-3 These studies allow both the evaluation of the degree of incorporation of nitroxides to dispersed aggregates and the characterization of the microdomains where the probes are located. Furthermore, nitroxides can also be employed to characterize microdomains from absorption and fluorescence measurements. The effect of the microenvironment on the energy of the visible band of nitroxides has been employed to quantify their incorporation to micelles and the polarity of the micellar interface,4,5 and the fluorescence quenching of aromatic compounds by nitroxides has been employed to evaluate the partition constant and to characterize the microproperties of lipidic aggregates such as micelles, vesicles, or biological membranes.6-8 However, there is no study concerning the determination of the degree of incorporation, the location, and the mobility of nitroxides in reverse micelles. Reverse micelles comprise at least three well-differentiated regions, the dispersium solvent, the interface, and the water pool, allowing the location of solutes in widely different environments.9-11 Fluorescence quenching studies of probes associated to different locations can provide information regarding the distribution of the donor X
Abstract published in Advance ACS Abstracts, March 1, 1996.
(1) Haering, G.; Luisi, P. L.; Hauser, H. J. Phys. Chem. 1988, 92, 3574. (2) Anzai, K.; Higashi, K. I.; Kirino, Y. Biochim. Biophys. Acta 1988, 937, 73. (3) Heimburg, T.; Hideg, K.; Marsh, D. J. Phys. Chem. 1991, 95, 1950. (4) Ramachandran, C.; Pyter, R. A.; Mukerjee, P. J. Phys. Chem. 1982, 86, 3198. (5) Pyter, R. A.; Ramachandran, Mukerjee, P. J. Phys. Chem. 1982, 86, 3206. (6) Atik, S. S.; Kwan, C. L.; Singer, L. A. J. Am. Chem. Soc. 1979, 101, 5696. (7) Blatt, E.; Sawyer, W. H. Biochim. Biophys. Acta 1985, 822, 43. (8) Encinas, M. V.; Lissi, E. A.; Alvarez, J. J. Photobiol. 1994, 59, 30. (9) Wong, M.; Thomas, J. K.; Nowak, T. J. Am. Chem. Soc. 1977, 99, 4730. (10) Kumar Jain, T.; Varshney, M.; Maitra, A. J. Phys. Chem. 1989, 93, 7409. (11) Encinas, M. V.; Lissi, E. A. Chem. Phys. Lett. 1986, 132, 545.
and the quencher12-16 and/or the rate of access of the quencher to the different subregions of the microheterogeneous solution.17 Most of these works have been performed comprising donors and quenchers totally incorporated to the micellar pseudophase, and few studies have been carried out employing families of quenchers of different hydrophobicity and donors located in different environments.12,14 This type of study can provide information regarding the distribution and mobility of the quencher and/or the donor as a function of their structure and the properties of the micelles, determined by the water/ surfactant ratio. In the present work we have measured the fluorescence quenching of several aromatic hydrocarbon derivatives by nitroxides with widely different hydrophobicities (Figure 1), in order to determine the relative location of donor and quencher and their accessibility to different subregions in a system comprising AOT reverse micelles dispersed in n-heptane. Quenching of aromatic compounds by nitroxides is dominated by energy transfer18,19 and is diffusion controlled even in media of low polarity.8 This implies that the rate of the quenching process will depend only upon the local quencher concentration and its mobility, independently of any other properties of the micromedium (such as polarity) where the donorquencher encounters take place. Experimental Section Sodiumbis(2-ethylhexyl)sulfosuccinate (AOT) was purchased from Aldrich and was purified by the procedure previously described.20 Fluorescent probes 1-methylpyrene (MePy), 1-pyren(12) Costa, S. M. B.; Lopes, J. M. F. M.; Martins, M. J. T. J. Chem. Soc., Faraday Trans. 2 1986, 82, 2371. (13) Backer, C. A.; Whitten, D. G. J. Phys. Chem. 1987, 91, 865. (14) Encinas, M. V.; Lissi, E. A.; Previtali, C. M.; Cosa, J. J. Langmuir 1989, 5, 805. (15) Encinas, M. V.; Lissi, E. A.; Bertolotti, S. G.; Cosa, J. J.; Previtali, C. M. Photochem. Photobiol. 1990, 52, 981. (16) Verbeeck, A.; De Schryver, F. C. Langmuir 1987, 3, 494. (17) Saez, M.; Abuin, E. B.; Lissi, E. A. Langmuir 1989, 5, 942. (18) Karpiuk, J.; Grabowski, Z. R. Chem. Phys. Lett. 1989, 160, 451. (19) Green, S. A.; Simpson, D. J.; Zhou, G.; Ho, P. S.; Blough, N. V. J. Am. Chem. Soc. 1990, 112, 7337. (20) Maitra, A. N.; Eicke, H. F. J. Phys. Chem. 1981, 85, 2687.
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Deactivation of Pyrene Derivatives
Langmuir, Vol. 12, No. 7, 1996 1739 Table 1. Quenching Rate Constants by TEMPO Probe
R
MePy
n-heptane ethanol 4 10 27 ethanol 4 10 28 ethanol 4 10 27
PyMe+
Figure 1. Structure of nitroxides. emethanol (PyMeOH), 1-pyrenyl(methyl)trimethylammonium iodide (PyMe+), 1-pyrenyl(butyl)trimethylammonium bromide (PyBu+) 1-pyrenyl(undecyl)trimethylammonium iodide (PyUn+), pyrenesulfonic acid (sodium salt) (PySA), and 1,3,6,8-pyrenetetrasulfonic acid (tetrasodium salt) (PyTS) (Molecular Probes) were employed as received. Nitroxides (Aldrich) were purified by recrystallization or sublimation. Steady-state fluorescence measurements were performed with a Perkin-Elmer LS-5 spectrofluorometer. Quenching experiments were carried out at a single wavelength (337 and 390 nm for excitation and emission, respectively). The shape of the emission spectrum was independent on the quencher addition. Time-resolved fluorescence measurements were performed with excitation (337 nm, 5 ns pulse width) from a Laser Photonics nitrogen laser. The fluorescence decay was recorded at 390 nm (starting ca. 20 ns after excitation) with a Hewlett-Packard 54504A digitizing oscilloscope. In all systems, the decay covering more than five lifetimes and comprising more than 500 points was fitted to a monoexponential function without significant trend in the residuals. All measurements were carried out at 25 °C in heptane/AOT/ water solutions. The concentration of probes was lower than 10-5 M. Under these conditions the average number of probe molecules per micelle is considerably lower than 1. TEMPAMINE was incorporated as ammonium salt to water pools prepared with water at pH 3. Samples were deoxygenated by bubbling pure nitrogen.
Results and Discussion The kinetics of fluorescence quenching in reverse micellar solutions will heavily depend on the relative locations and mobilities of the donor and the quencher. Several limiting situations can be envisaged, depending on the location of the donor. If the donor is liposoluble and predominantly located in the dispersium medium (i.e., MePy), the quenching rate is better described in terms of the analytical concentration of the quencher or its concentration in the dispersium solvent. On the other hand, when the donor is predominantly (or exclusively) bound to the micellar interface (PyMe+) or the water pools (PyTS at a water-to-surfactant ratio, R, higher than 10) the best way to describe the quenching rate will depend not only on the quencher location but also on the quenching mechanism (static or dynamic) and the quencher mean occupation number (higher or lower than 1). Thus, for a probe located at the micellar interface, the quenching rate constant can be expressed in terms of the analytical quencher concentration, the concentration in the dispersium solvent (that requires to know its partition), in terms of the concentration at the interface (that requires to assume the “volume” of the interface), in terms of the number of quencher molecules per interface or in terms of the number of quencher molecules at the interface per surfactant head. In the following sections, we describe the results obtained in terms of the analytical quencher concentration ((kq)bulk), the quencher concentration in the dispersium medium ((kq)heptane), the mean occupation number of solute at the interface ((kq)Nint), or the mean number of solute molecules at the interface per surfactant head ((kq)Xint). Quencher in the Organic Phase. TEMPO is a liposoluble quencher, the measured partition constant
PyTS
[AOT], M
0.08-0.4 0.08-0.4 0.05-0.3 0.08-0.3 0.3 0.05-0.4 0.08-0.4 0.08-0.4 0.08-0.4
(kq)bulk, 109 M-1 s-1 15.5 7.4 13.7 13.2 14.7 6.8 5.3 3.8 4.0 6.5
