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Local Surface Environments and Their Effects on Molecular Encounter Rates at Silica/Solution Interfaces Studied by Quenching of Phosphorescence from a Silica-Immobilized Triplet-State Probe Stephanie R. Shield and Joel M. Harris* Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850 Received July 23, 2001. In Final Form: March 15, 2002 The triplet-state decay kinetics of erythrosin-ITC, chemically bound to aminated porous silica, provides information about the local environment of the liquid/solid interface and its influence on interfacial reaction kinetics. A dispersion in phosphorescence decay rates of the immobilized erythrosin was observed. From the pH response of the decay kinetics, two distinct populations appear to arise from domains of protonated amine and silanol sites on the silica surface. The decay rates of the excited-triplet populations are sufficiently slow to probe quenching encounters with solution-phase azulene, which can diffuse over relatively long distances during the lifetime of the excited-state. By variation of the solvent conditions to influence adsorption of azulene, it was found that adsorbed azulene does not participate in interfacial quenching. The quenching process, therefore, involves contact between solution-phase azulene and the immobilized probe. The rates of this process for the short-lived (solution-associated) erythrosin population were slower than free-solution rates by a factor of 4.3, which is expected from surface-immobilization of the probe. The results indicate that transport of azulene to this population is efficient and that the pore network through which the azulene diffuses is well connected over a distance scale of ∼0.5 µm. The quenching kinetics for the longerlived (surface-associated) erythrosin were somewhat slower, possibly due to steric hindrance or local exclusion of azulene, or less efficient molecular transport over longer distances within the porous silica.
Introduction While photochemistry of organic molecules in free solution has been investigated for decades, many recent studies have focused on interfacial photophysics and photochemical reactions. Photoactive organic molecules are typically adsorbed or chemically bound to the solid substrate. Photoinitiated kinetics of the immobilized molecules can provide valuable information about the physical and chemical characteristics of the surface or the surface reactive behavior of the adsorbed or bound species. The majority of research on surface photophysics and photochemistry has been at the gas/solid or vacuum/solid interface.1-13 Time-resolved diffuse reflectance and lu(1) Hara, K.; de Mayo, P.; Ware, W. R.; Weedon, A. C.; Wong, G. S. K.; Wu, K. C. Chem. Phys. Lett. 1980, 69, 105-108. (2) Bauer, R. K.; Borenstein, R.; de Mayo, P.; Okada, K.; Rafalska, M.; Ware, W. R.; Wu, K. K. J. Am. Chem. Soc. 1982, 104, 4635-4644. (3) Wilkinson, F.; Willsher, C. J. Chem. Phys. Lett. 1984, 104, 272276. (4) de Mayo, P.; Natarajan, L. V.; Ware, W. R. J. Phys. Chem. 1985, 89, 3526-3530. (5) Turro, N. J.; Zimmt, M. B.; Gould, I. R.; Mahler, W. J. Am. Chem. Soc. 1985, 107, 5826-5827. (6) Wellner, E.; Ottolenghi, M.; Avnir, D. Langmuir 1986, 2, 616619. (7) Drake, J. M.; Levitz, P.; Turro, N. J.; Nitsche, K. S.; Cassidy, K. F. J. Phys. Chem. 1988, 92, 4680-4684. (8) Krasnansky, R.; Koike, K.; Thomas, J. K. J. Phys. Chem. 1990, 94, 4521-4528. (9) Leheny, A. R.; Turro, N. J.; Drake, J. M. J. Phys. Chem. 1992, 96, 8498-8502. (10) Samuel, J.; Ottolenghi, M.; Avnir, D. J. Phys. Chem. 1992, 96, 6398-6405. (11) Marro, M. A. T.; Thomas, J. K. J. Photochem. Photobiol. A: Chem. 1993, 72, 251-259. (12) Katz, O.; Samuel, J.; Avnir, D.; Ottolenghi, M. J. Phys. Chem. 1995, 99, 14893-14902. (13) Worrall, D. R.; Williams, S. L.; Wilkinson, F. J. Phys. Chem. B 1997, 101, 4709-4716.
minescence techniques have been employed to study the surface photochemical kinetics.1-13 More recent studies have shifted to investigating photoinitiated reactions at the liquid/solid interface.14-26 Porous silica has been a commonly used solid substrate;1-22 its high surface area provides a large number of reactive molecules at the interface while its optical transparency from the nearUV through visible regions allows photoexcitation and spectroscopic monitoring. Quenching of short-lived fluorescent probes has been used to study fast encounter kinetics at the silica/solution interface.14-22 These studies are sensitive to transport over relatively short diffusional distances, xd ∼ 10 nm, that depend on the diffusion coefficient of the quencher, D, and the excited-state lifetime of the probe, τ, where xd ) (2Dτ)1/2.19 Over these distances, interfacial encounter rates have been found to be slower than free solution rates by small factors that could be (14) Wheeler, J.; Thomas, J. K. J. Phys. Chem. 1982, 86, 4540-4544. (15) Lochmu¨ller, C. H.; Colborn, A. S.; Hunnicutt, M. L.; Harris, J. M. J. Am. Chem. Soc. 1984, 106, 4077-4082. (16) Krasnansky, R.; Thomas, J. K. J. Photochem. Photobiol. A: Chem. 1991, 57, 81-96. (17) Samuel, J.; Ottolenghi, M.; Avnir, D. J. Phys. Chem. 1991, 95, 1890-1895. (18) Wong, A. L.; Harris, J. M. J. Phys. Chem. 1991, 95, 5895-5901. (19) Wong, A. L.; Hunnicutt, M. L.; Harris, J. M. J. Phys. Chem. 1991, 95, 4489-4495. (20) Wang, H.; Harris, J. M. J. Phys. Chem. 1995, 99, 16999-17009. (21) Levin, P. P.; Costa, S. M. B.; Ferreira, L. F. V. J. Phys. Chem. 1996, 100, 15171-15179. (22) Kavanagh, R. J.; Thomas, J. K. Langmuir 1998, 14, 352-362. (23) Shield, S. R.; Harris, J. M. J. Phys. Chem. B 2001, 104, 85278535. (24) McKay, G.; Otterburn, M. S.; Aga, J. A. J. Chem. Technol. Biotechnol. 1987, 34, 247-256. (25) Drake, J. M.; Klafter, J.; Levitz, P. In Dynamical Processes in Condensed Molecular Systems; Klafter, J., et al., Eds.; World Scientific: Singapore, 1989. (26) Hallmann, M.; Unger, K. K.; Appel, M.; Fleischer, G.; Ka¨rger, J. J. Phys. Chem. 1996, 100, 7729-7734.
10.1021/la011144+ CCC: $22.00 © 2002 American Chemical Society Published on Web 04/30/2002
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attributed to immobilization of the probe and exclusion of the solution volume by the solid support.16-19 When an additional population of quencher is brought to the interface by adsorption, significant rate enhancements can be observed compared to free-solution, due from surface-diffusion of the quencher to the excited-state immobilized to the surface in a Langmuir-Hinshelwood reaction mechanism.18 Similar rate enhancement by adsorption was reported for interfacial quenching of an excited-triplet state in free solution by a silica-immobilized quencher at a dispersed colloidal silica/solution interface.23 Long-range diffusive transport of molecules through high surface area, porous solids plays a key role in the efficiency of liquid chromatography, heterogeneous catalysis, and reagents immobilized in porous supports. Measurement of dye adsorption rates,24 forced-Rayleigh scattering,25 and NMR field-gradient techniques26 have shown that long-range (J10 µm) transport of molecules is 1 or 2 orders of magnitude slower through porous silica than through bulk liquids. To study interfacial encounter kinetics over intermediate distances (10 nm to 1 µm) through a pore network by excited-state methods, a longerlived triplet-state probe can be employed to study diffusion of quenchers over longer distances; for example, quenching of phosphorescence from a 100-µs-lived excited-state will be sensitive to molecular transport over diffusional distances up to ∼0.5 µm. In addition, the decay kinetics of excited triplet-state populations respond to their local environment and can provide information about the chemical heterogeneity of an interface averaged over longer time scales. Erythrosin B has been a useful probe for monitoring slow (microseconds to milliseconds) rotational mobility of proteins and biomolecules27-31 due to its long-lived room temperature phosphorescence. In the absence of oxygen, luminescence from the triplet state occurs through both the radiative relaxation to the ground state (phosphorescence) and activated, reverse-intersystem crossing to the excited-singlet state followed by radiative relaxation to the ground state (delayed fluorescence). Immobilization of erythrosin is easily accomplished using the isothiocyanate (ITC) form of the dye, which reacts at room temperature with surface-bound amines. In this study, we immobilized erythrosin ITC onto an aminated porous silica surface. The excited-triplet state of erythrosin was populated using a pulsed ultraviolet laser, and long-range encounter rates were determined by phosphorescence quenching using a solution-phase quencher, azulene. The efficient quenching of erythrosin by azulene occurs through energy transfer, driven by a significant difference in triplet energies, 45 kcal/mol for erythrosin (based on the phosphorescence emission spectrum) and 30.9 kcal/mol32 for azulene. The exchange mechanism for triplet-energy transfer requires molecular contact between the immobilized probe and quencher, which can be used to assess the rates of interfacial encounter between these molecules and their dependence on the local surface environment of the probe. (27) Bowers, P. G.; Porter, G. Proc. R. Soc. London, Ser. A 1967, 299, 348-353. (28) Garland, P. B.; Moore, C. H. Biochem. J. 1979, 183, 561-572. (29) Jovin, T. M..; Bartholdi, M.; Vaz, W. L. C.; Austin, R. H. Ann. N.Y. Acad. Sci. 1981, 366, 176-196. (30) Corin, A. F.; Blatt, E.; Jovin, T. M. Biochemistry. 1987, 26, 22072217. (31) Tilley, L.; Sawyer, W. H.; Morrison, J. R.; Fidge, N. H. J. Biol. Chem. 1988, 263, 17541-17547. (32) Murov, S. L. Handbook of Photochemistry; Marcel Dekker: New York, 1973; p 3.
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Experimental Section Instrumentation. For triplet-state decay kinetic measurements, a frequency-tripled Quanta Ray model GCR-11 Nd:YAG laser (λe ) 355 nm) was operated at 10 Hz, and the beam was blocked by a shutter to provide a repetition rate of 1.0 Hz. The 5 ns UV (120 µJ) excitation pulses were weakly focused to a spot size of 2.2 mm and used to photoexcite the sample. The suspensions were deoxygenated in a spectroscopic freeze-pumpthaw cell that was equipped with a 2 mm ID quartz capillary tube and the sample was allowed to settle for 15 min before excitation. Phosphorescence was collected at 90° from the excitation axis and filtered through a 1.0 cm path of a 5% aqueous solution of sodium nitrite and two glass filters (Schott KV 408 and RG 630). The filtered emission was detected by a red-sensitive Hamamatsu R976 photomultiplier tube, digitized with a LeCroy 9450 oscilloscope with a 50 Ω termination resistance, and averaged 50 times. For acquiring emission spectra of the immobilized dye, a continuous-wave argon ion laser (Lexel) was operated at 514.5 nm, and excitation was focused onto a bifurcated fiber optic probe (Fiberguide Industries) using a multimode fiber coupler (Newport Corporation) and a 10× microscope objective lens (Bausch and Lomb). The light-collection end of 44 fibers was arranged in a linear array to match the entrance slit of a F/7 single stage spectrograph (0.5 m, Spex, model 1870C). The radiation exciting this array was collected and collimated by an f/1.3 lens and focused on the entrance slit by a plano-convex lens (f ) 175 mm, Newport Corp.). Emission from the sample was detected with a 512 × 512 cooled CCD (EG&G PARC). Reagents. Erythrosin B (Sigma), erythrosin-ITC (Sigma), azulene (Aldrich, 99%), 3-aminopropyl)triethoxysilane (Gelest), toluene (spectrograde), methanol (HPLC grade), water (HPLC grade), sodium citrates (Aldrich), sodium borate (EM Science), boric acid (Aldrich, 99.999%), and 10 µm particle diameter, 60 Å porous silica (EM Science, LiChrosorb Si60, d90/d10 ) 1.5) were used without further purification. Oxygen was removed from the erythrosin/ silica suspensions by three freeze-pump-thaw cycles, pumped to a submilliTorr base pressure. Sample Preparation. The porous silica was surface derivatized with a propylamine silane reagent using the following procedure.33 The silica (1.2 g) was dried under vacuum for 24 h; 3-aminopropyl)triethoxysilane (1.0 mL) and toluene (150 mL) were then added. The mixture was stirred and refluxed at ≈ 90 °C under nitrogen for 6 h. The reaction mixture was cooled, filtered, and then washed with methanol. Elemental nitrogen analysis was performed on the aminated particles (M-H-W Laboratories, Phoenix, AZ) and found to contain 1.4% N. Using the weight percent of nitrogen and the reported particle surface area (550 m2/g), the amine coverage was determined to be 1.8 µmol/m2. Chemisorption of erythrosin was accomplished via an isothiocyanate-amine reaction (see Figure 1).34 Erythrosin-ITC was dissolved in dimethylformamide and diluted with a pH ) 9.0, sodium borate buffer. The dried, aminated particles were added to this solution and stirred for 1 h at room temperature. The particles were filtered to remove excess solution and physisorbed erythrosin and thoroughly rinsed with methanol. The optical absorbance of the filtrate solution at 530 nm was used to determine the coverage of erythrosin, 1.2 nmol/m2. The pH of the 14 mg/mL silica suspensions in methanol/water solutions was measured and varied to determine the role of pH on the kinetics. Buffers were prepared at 8 mM trisodium citrate and 18 mM disodium citrate for pH ) 5.9 suspensions and at 1 mM disodium citrate and 2 mM monosodium citrate for pH ) 7.5 suspensions. The activity of hydrogen ions in mixed solvents cannot be obtained directly from the measured pH value. To determine an accurate hydrogen ion activity in methanol/water solutions, a standardization procedure would need to be performed to ensure that the potential at the reference electrode liquid-junction remains constant for the various solvent com(33) Okabayashi, H.; Shimizu, I.; Nishio, E.; O’Connor, C. J. Colloid Polym. Sci. 1997, 275, 744-753. (34) Conjugation with Amine Reactive Probes; MP 0143; Molecular Probes: Eugene, OR, 1996.
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Figure 1. Chemisorption of erythrosin-ITC to an aminated silica surface. positions.35,36 The resulting corrections to the pH, however, are small (e0.15 pH units) and constant for a given mixed solvent composition. Chromatographic retention measurements were used to determine the fraction of azulene adsorbed onto aminated porous silica (4 × 250 mm column packed with aminopropylsilanefunctionalized LiChrosorb from Phenomenex) at pH ∼ 5-6. Uracil was unretained on this column and used as the dead volume marker. Data Analysis. Time-resolved phosphorescence transients from triplet-erythrosin covalently bound to porous silica were fitted to a discrete two-exponential decay model to determine rate constants for quenching by azulene. The data analysis program was compiled in Microsoft Fortran and utilized a Marquardt algorithm37 to optimize the kinetic parameters and population amplitudes by minimizing the χ2 statistic. Weighted least-squares minimization was used to account properly for the photon-shot noise in the data. The phosphorescence transients were also fitted to a continuous distribution of decay rates using a numerical Laplace inversion routine, CONTIN.38-39 Phosphorescence transients were acquired in triplicate and uncertainties in the quenching rate constants were estimated from a linear least-squares analysis of Stern-Volmer plots. The plotted error bars and the uncertainties in the rate constants are reported as (2 standard deviations.
Results and Discussion Decay of Excited-Triplet States of Erythrosin Immobilized on Silica. Erythrosin-derivatized porous silica was suspended in methanol/water solutions that were buffered to a pH of 5.9 and 7.5. The encounter rates between the surface probe and solution-phase molecules were determined by quenching the erythrosin phosphorescence with azulene. The unquenched and quenched phosphorescence transients from the surface-immobilized dye did not follow single-exponential decay kinetics; singleexponential fits of these transients exhibited structured residuals where the systematic deviations were 5 to 6 times greater than the peak-to-peak shot noise in the data. A two-exponential decay model reduced the sum of the weighted squared residuals (χ2) by a statistically signficant factor >30 and brought the residuals within the peakto-peak bounds of the photon-shot noise in the data. Further evidence for the validity of a two-exponential model to account for the triplet-state decay kinetics of erythrosin immoblized on silica was obtained from a (35) Bates, R. G.; Paabo, M.; Robinson, R. A. J. Phys. Chem. 1963, 67, 1833-1838. (36) Jensen, R. P.; Eyring, E. M.; Walsh, W. M. J. Phys. Chem. 1966, 70, 2264-2270. (37) Marquardt, D. W. J. Soc. Ind. Appl. Math. 1963, 11, 431-441. (38) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213-227. (39) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 229-242.
continuous rate distribution analysis.38-40 The results showed two major, well-separated peaks in the rate distribution; the decay rates of these peaks were somewhat faster but otherwise comparable to those determined from a discrete exponential analysis. These results indicate that the triplet excited-state decay is characterized by two populations having distinguishable decay rates. While there some further dispersion in the decay kinetics may exist within these two distinguishable populations, the signal-to-noise ratio for a reliable rate distribution analysis40 exceeds the level that could be achieved in the present experiments, as was confirmed by the analysis of synthetic data modeled with comparable levels of photon-shot noise. Neglecting additional dispersion within the two populations and analyzing their decay behavior using a discrete model yields more reliable results at the cost of more detailed information about a distribution of rates within the populations. The decay of the phosphorescence from silica-immobilized erythrosin is, therefore, modeled using a twoexponential decay model:
I(t) ) A exp(-t/τ1) + B exp(-t/τ2)
(1)
where A and B represent the amplitudes and τ1 and τ2 represent the triplet lifetimes of the two populations. An example of these results is shown in Figure 2, where the measured phosphorescence data and weighted leastsquares fits to eq 1 are plotted for immobilized erythrosin in contact with a 50/50 methanol/water solution at a pH ) 5.9; the solutions contained variable concentrations of a triplet quencher, azulene, ranging from 0 to 50 µM. Both the unquenched and quenched decay transients are well fitted by a two-exponential model. The two excited-state populations, represented by long-lived and short-lived components, exhibit different quenching rate constants (see below), which shows that both the local environment and quencher encounter rates differ for these populations. There are several possible origins of distinguishable erythrosin populations bound to the silica surface. There could be some difference between the outer particle surface and the interior pore environment. The outer surface of the 10 µm porous particles represents such a small fraction, however, of the overall surface area (
