12172
J. Phys. Chem. 1996, 100, 12172-12177
Time-Resolved Fluorescence of a Hemicyanine Dye: Dynamics of Rotamerism and Resolvation Carlheinz Ro1 cker, Andrea Heilemann, and Peter Fromherz* Department of Biophysics, UniVersity Ulm, D-89069 Ulm, Germany, and Department of Membrane and Neurophysics, Max-Planck-Institute for Biochemistry, D-82152 Martinsried/Mu¨ nchen, Germany ReceiVed: January 4, 1996; In Final Form: March 26, 1996X
We observed the time-resolved fluorescence spectra of a hemicyanine dye with no CC double bond and one CC single bond and of a homologous dye with blocked rotamerism of the CC single bond. Using the more rigid dye, we determined the dynamics of resolvation. Taking that reference, we analyzed the dynamics of rotamerism around the CC single bond. We found that the Franck-Condon geometry equilibrates with a polar-twisted rotamer which is deactivated by fast nonradiative decay. The rotamerism occurs during the relaxation of the solvation shell.
Introduction Amphiphilic hemicyanine dyes are used as molecular voltmeters in neuron membranes.1-3 The mechanism of their voltage-dependent fluorescence is unclear. One aspect which plays a role is the solvation of the dyes at the membrane-water interface. Depending on the external electrical field, it may affect the color and intensity of fluorescence.4 For comparison, the solvatochromism and the solventdependent yield of fluorescence were studied in bulk solvents by stationary spectroscopy.5-9 It was suggested that the solvatochromism is due to an intramolecular charge transfer6,9 and that the change of yield is due to a solvent-dependent twist around a CC single bond to a rotamer with fast nonradiative decay.5,7 In the present paper, we test the hypothesis of a solventdependent interplay of rotamerism and nonradiative decay by observing the time resolved fluorescence spectra. We use two dyes, one (BABP) with a free CC single bond and the other (BABAPH) with a covalent bridge such that the rotamerism is suppressed (Figure 1). Both dyes have no free ethylenic double bond which gives rise to complications due to isomerism.5 Both dyes bear the same dialkylamino group. Its rotamerism is not considered here. On the basis of the experimental data, we suggest a kinetic model for the dynamics of resolvation and of rotamerism around the CC single bond as well as of radiative and nonradiative decay. Materials and Methods Dyes. 2-(Sulfonatobutyl)-7-(dibutylamino)-2-azaphenanthrene (BABAPH, Figure 1). We started the synthesis with 3-aminobenzaldehyde dimethyl acetal.10 We introduced two butyl groups using butyl iodide and hydrolyzed the acetal by diluted sulfuric acid (yield 6%).11 Then, the aldehyde was reduced to the alcohol by formaldehyde (74%),12 and 3-(dibutylamino)benzyl chloride was obtained with phosphopentachloride (82%).13 We prepared the triphenylphosphonium salt (63%)13 and let it react in a Wittig reaction with 3-pyridylaldehyde to form 3-(dibutylamino)-3′-azastilbene (28%).14 The 7-(dibutylamino)-2-azaphenanthrene was obtained by photocyclization in 2-methyltetrahydrofuran at -30 °C within 45 min (48%).15 Using butane sultone, we prepared the final product * Corresponding author. X Abstract published in AdVance ACS Abstracts, July 1, 1996.
S0022-3654(96)00095-0 CCC: $12.00
Figure 1. Hemicyanine dyes without free ethylenic double bonds. 2-(Sulfonatobutyl)-7-(dibutylamino)-2-azaphenanthrene (BABAPH) and 1-(sulfonatobutyl)-4-[4′-(dibutylamino)phenyl]pyridine (BABP).
(24%).11 The dye and all precureors were identified by mass spectroscopy, infrared spectroscopy, and 1H NMR. 1-(Sulfonatobutyl)-4-[4-(dibutylamino)phenyl]pyridine (BABP, Figure 1). This dye was synthesized as described previously.6,8 The dyes were dissolved in 1-butanol and 1-octanol (2 µM) from 1 mM stock solutions in ethanol. Solvents of the highest available purity were used (Merck, Darmstadt, Fluka, Neu-Ulm). Octanol was further purified by rectification. The solutions were not deaerated. Measurements. Fluorescence decays were obtained by timecorrelated single-photon counting.16 The light source was a frequency-doubled mode-locked titanium sapphire laser (Spectra Physics) tuned to a wavelength of 420 nm. Light pulses of vertical polarization with a duration of 2 ps at a pulse-picked repetition rate of 4 MHz were focused onto the sample. The sample holder was inclined by 12° to avoid reflections from the back wall of the cuvette. The fluorescence was collected at magic angle conditions, focused onto a subtractive doublemonochromator (bandwidth 11 nm), and detected by a microchannel plate (Hamamatsu R3809U-01). Single-photon signals were amplified (Hewlett-Packard 8447F/8494B), discriminated (Tennelec TC 454), and fed into a time-to-amplitude converter (Ortec 567) together with the corresponding stop signal from a vacuum photodiode (ITL 1850). To avoid multiple-photon detection, the number of photons per laser pulse was kept below 0.005 using neutral density filters in the excitation beam. Events were accumulated in a multichannel analyzer (Ortec 921/ Maestro) up to 10 000 counts in the leading channel. The instrument response function (IRF) was measured with a scattering solution (Ludox HS40, 12 nm, DuPont). Its full width at half maximum (FWHM) was 24 ps. After deconvolution (see below), the time resolution of the system was around 5 ps. © 1996 American Chemical Society
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The steady-state fluorescence spectra used for normalization were recorded with a fluorometer (SPEX fluorolog II) under magic angle conditions at an excitation wavelength of 420 nm and an emission bandwidth of 8.5 nm. Quantum yield measurements were performed using acridine yellow (Aldrich, Steinheim) in ethanol as a standard at an excitation wavelength of 436 nm (quantum yield 0.47).17 All measurements were performed at 25 °C. Data Analysis. Time-resolved fluorescence spectra were reconstructed from wavelength-dependent fluorescence decays and parametrized by a line-shape function. Since this method is described in detail by Maroncelli and Fleming,18 we give only a short summary of our procedure. We recorded fluorescent decays across the band of fluorescence at a spacing of 500 cm-1. Each decay was fitted by three exponentials convoluted with the IRF. The deconvoluted decays were normalized relative to each other using the stationary fluorescence spectrum. Timedependent spectra were constructed from these normalized decays. They were fitted by a log-normal line-shape function g(νj)19
{( [
ln(R + 1) b 2b(νj - νjMAX) R) > -1 ∆
g(νj) ) g0 exp - ln 2
] )} 2
(1)
From the fit, we obtained the amplitude g0, the wavenumber νjMAX of the maximum, the spectral width Γ ) ∆(sinh b/b) (FWHM), and the asymmetry parameter b. By integration we evaluated the total intensity.
Figure 2. Normalized time-resolved fluorescence spectra in octanol at time delays of 20, 100, 300, and 2000 ps after excitation (from right to left). (a) BABAPH. (b) BABP. The data are fitted by log-normal line-shape functions.
Results and Discussion In the first part, we present the time resolved fluorescence of the more rigid dye BABAPH and of the more twistable dye BABP in octanol. We evaluate the data of BABAPH in terms of solvent reorganization and of electronic deactivation. To evaluate the data of BABP, we take into account explicitly the rotamerism around the CC single bond using BABAPH as a reference. In the second part, we consider the time-resolved spectra of both dyes in butanol and compare the results in two solvents. BABAPH in Octanol. Normalized fluorescence spectra of BABAPH in octanol are shown in Figure 2a for various time delays (20, 100, 300, and 2000 ps) after electronic excitation. The data are fitted by log-normal functions. The spectra are shifted to the red with an invariant shape. The dynamics of the spectral maxima are plotted in Figure 3a. The final fluorescence is emitted around 19 400 cm-1. We fitted the spectral relaxation by two exponentials. The dominating component (amplitude 1020 cm-1) has a time constant of 500 ps. In addition, there is a smaller exponential with 75 ps (amplitude 640 cm-1). The striking invariance of the spectral width at 2750 cm-1 is documented in Figure 3b. We also evaluated the total fluorescence intensity by integration of the time-dependent spectra. The result is plotted in Figure 3c. The decay can be fitted by a single exponential with a time constant τ ) 38 ns. The total quantum yield of fluorescence of BABAPH in octanol was ΦF ) 0.58. Quantumchemical MNDO computations20 of a homologue of BABAPH (with three methyl groups at the two nitrogen atoms) show that electronic excitation changes the distribution of electrons. The effect is described by the displacement of an elementary positive charge by 0.19 nm from the pyridinium moiety toward the amino group. The solvation shell has to follow this charge jump. The dynamical features of fluorescence (Figure 3) indicate a sequence of three relaxation processes
which are separated from each other by an order of magnitude of their time scale. The shift of the spectral maximum suggests a fast (75-ps) and a slow (500-ps) step of solvent reorganization. Thereafter, the excited state is deactivated electronically (3.8 ns). The slow component of solvent reorganization corresponds to the longitudinal relaxation time of octanol, τL ) 520 ps,21-23 which describes the response of a solvent to a macroscopic charge jump in a capacitor.24,25 Such a relation has been observed previously for the resolvation of coumarin dyes.26-28 The structural interpretation of the fast component is not straightforward. It may reflect a local reorganization of the polar moiety of octanol around the chromophore. The electronic deactivation is due to radiative and nonradiative processes. Their probabilities kF and kNR can be evaluated from the time constant τ and the quantum yield ΦF. We obtain kF ) ΦF/τ ) [6.5 ns]-1 and kNR ) (1 - ΦF)/τ ) [9.0 ns]-1. The nature of the nonradiative processes is not considered here. BABP in Octanol. Four fluorescence spectra of BABP in octanol are shown in Figure 2b 20, 100, 300, and 2000 ps after excitation. The first spectrum is very similar to that of BABAPH with respect to the position of its maximum and its shape. Also, here, the spectrum is shifted to the red as plotted in Figure 3a. The final fluorescence is emitted 500 cm-1 more to the red than in BABAPH. In striking contrast to BABAPH, we observe here a change of the spectral shape. There is a distinct broadening, and this broadening is restricted to the red flank, whereas the shape in the blue remains invariant (Figure 2b). The spectral width (FWHM) is plotted in Figure 3b and fitted with two exponentials. It increases up to a final width of 3750 cm-1. The asymmetry parameter changes from -0.26 to -0.46. Also, the fluorescence intensity differs significantly from BABAPH (Figure 3c). The decay is faster. We can fit it with two well-separated exponentials with time constants of 105
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Figure 4. Decomposition of time-resolved fluorescence of BABP in octanol into a primary fluorescence (full symbols) and a secondary fluorescence (open symbols). (a) Position of spectral maxima. (b) Relative spectral amplitudes. The fit of the data is discussed in the text.
Figure 3. Fluorescence dynamics in octanol of BABAPH (full symbols) and BABP (open symbols). (a) Position of spectral maximum. (b). Spectral width (full width at half-maximum). (c) Integral intensity normalized to its initial value. The fit of the data is discussed in the text.
ps (30%) and 70 ps (amplitude 70%). The total quantum yield of fluorescence is ΦF ) 0.15. Fluorescence and Rotamerism. We base the interpretation of the fluorescence data of BABP on two arguments: (i) The electronic structure of BABP is rather similar to BABAPH. The intramolecular charge shift upon excitation in the ground state has the same value of 0.19 nm. This result was obtained from an MNDO computation and refers to a twist angle of 21 °C of the CC single bond in the equilibrium geometry of the ground state. Thus, we expect a similar interaction with the solvent and similar features of spectral relaxation. We conclude that the enhancement of spectral width and of asymmetry of the timedependent fluorescence spectra of BABP cannot be the result of solvent reorganization. (ii) Despite of the similar electronic structure of both dyes, the electronic deactivation is distinctly faster in BABP than in BABAPH with a much lower quantum yield of fluorescence. We conclude that an additional channel of radiationless deactivation is opened in BABP. We suggest that the asymmetric spectral broadening and the enhanced nonradiative decay are both caused by the same processsby a rotamerism around the CC single bond. We propose the following scenario: The Franck-Condon transition of light absorption brings BABP into an excited state at the twisting angle of the ground state. At fixed geometry, this state would be similar to the excited BABAPH with respect to both
resolvation and to deactivation by fluorescence and nonradiative decay. Now a reversible rotamerism sets in toward a structure with enhanced twist. This reaction competes with the direct deactivation of the Franck-Condon state. After relaxation, the equilibrated rotamers are deactivated by radiative and nonradiative processes. Within this scheme the initial fast drop of fluorescence intensity (Figure 3c) reflects the relaxation of rotamerism. The concomitant broadening of fluorescence (Figure 3b) reflects the contribution of a red-shifted fluorescence of the twisted structure. The slow decay of the broad spectrum (Figure 3b,c) is due to electronic deactivation of the equilibrated rotamers. Two-State Model. We use a two-state model of rotamerism to evaluate the fluorescence of BABP quantitatively. We distinguish the geometry attained in the Franck-Condon transition from the ground state and another geometry with an enhanced twist. We suppose that the resolvation of both geometries is similar to that of BABAPH, i.e., that their spectra are shifted to the red at a constant shape. Thus, we decompose the observed fluorescence of BABP into two spectra of constant width and constant asymmetry, fitting the position and the amplitude of their spectral maxima using the log-normal function. For the width of the primary fluorescence, we take the value Γ1 ) 2750 cm-1 of BABAPH; for the width of the secondary fluorescence, we choose by trial and error Γ2 ) 4500 cm-1. For the asymmetry parameter of both spectra, we use the value of BABAPH (b ) -0.28). The positions of the spectral maxima of primary and secondary fluorescences are plotted in Figure 4a. The primary fluorescence relaxes to 19 250 cm-1. This value is similar to the final emission of BABAPH. The secondary fluorescence is shifted much more to the red with a final emission around 17 650 cm-1. The dynamics of both spectral relaxations is similar to that of BABAPH with a fast and a slow component.
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J. Phys. Chem., Vol. 100, No. 30, 1996 12175
The amplitudes of the spectral maxima of the two bands are shown in Figure 4b. The primary fluorescence decays in two phases; the secondary fluorescence increases during the fast phase and decays during the slow phase. We can fit the amplitudes of both spectral components with two exponentials with the same time constants τFAST ) 80 ps and τSLOW ) 800 ps. The relative amplitudes of the primary fluorescence are ) 0.37. The relative amplitude of the ) 0.63 and aSLOW aFAST 1 1 ) -aFAST ) 0.16. secondary transient is aSLOW 2 2 The result of the spectral decomposition is compatible with the proposed model: (i) The dynamics of the spectral shift of the primary fluorescence are similar to that observed for BABAPH. (ii) The spectral shift and the spectral width of the secondary fluorescence are enhanced, as is expected for a more polar species with stronger solvation. A connection of enhanced polarity with enhanced twist is suggested by quantum chemical MNDO computations. In fact, a large shift and large spectral width are observed for a homologue of BABP where an enhanced twist is induced by a methyl group adjacent to the twistable bond (Heilemann, A.; Fromherz, P. Unpublished results). (iii) The opposite fast dynamics of the fluorescence amplitudes are compatible with the decay of a fluorescing species and the concomitant formation of another fluorescing species. (iv) The identical slow dynamics of both emissions indicate a common decay of both species, i.e., an equilibration. (v) The slow dynamics are much faster than the decay of BABAPH. This difference suggests an enhanced radiationless deactivation from the secondary state. Rate Constants. For a further discussion of the two-state model, we introduce transition probabilities. For the sake of simplicity, we make the approximation that the rate constants of radiative and nonradiative deactivation of both rotamers kF1 as well as of rotamerism in both and kNR and kF2 and kNR 1 2 directions kROT and kTOR are independent of the actual state of the solvation shell. The rate constants of rotamerism define a relaxation time τROT and an equilibrium molar fraction jx1 of the primary rotamer according
τROT )
ROT
k
1 - jx1 jx1
)
1 + kTOR
(2)
kROT kTOR
(3)
The decrease of primary fluorescence and the increase of secondary fluorescence occur within τFAST ) 80 ps (Figure 4b). This time is much shorter than electronic deactivation, as suggested by the data of BABAPH (τ ) 3.8 ns). Thus, we may decouple the time scales of rotamerism and of electronic deactivation. In that case, we can identify the time constant τFAST with the relaxation time of rotamerism according to eq 4. and aFAST of the primary Furthermore, the amplitudes aSLOW 1 1 fluorescence indicate the remaining molar fraction of the primary rotamer and its loss due to formation of the secondary rotamer according to eq 5.
τFAST ) τROT aFAST 1
) SLOW
a1
kF1 (1 - jx1) kF1 jx1
(4) (5)
Using these relations together with eqs 2 and 3, we can evaluate the rate constants of rotamerism. We obtain kROT ) [125 ps]-1 and kTOR ) [220 ps]-1. The process of rotamerism is so fast that it occurs in a solvation shell which is not fully
Figure 5. Kinetics of excited BABP in octanol (a) and in butanol (b) with radiative (waves) and radiationless deactivation of the primary (left) and secondary (right) rotamer, with the reversible rotamerism and with the relaxation of the solvent (grey bands). The transitions within the primary rotamer are chosen to be those of the reference dye BABAPH. The numbers are characteristic times in picoseconds (reciprocal rate constants). Average relaxation times are given for the nonexponential resolvation.
relaxed. The equilibrated fraction of molecules in the primary rotamer is jx1 ) 0.36; i.e., about two-thirds of the molecules are in the twisted state. The slow time constant of both spectral components reflects electronic deactivation after equilibration. We can express it by the average of the intrinsic decay constants of fluorescence of the two rotamers according to
1 SLOW
)
τ
jx1 τ1
+
1 - jx1 τ2
(6)
With τSLOW ) 800 ps and jx1 ) 0.36, eq 6 provides a linear relation between the time constants of the two rotamers. If we attribute to the primary rotamer the time constant of BABAPH with τ1 ) 3.8 ns, we obtain τ2 ) 500 ps for the secondary rotamer. It decays faster by an order of magnitude. We can distinguish the contribution of radiative and nonradiative decay by evaluating the relative amplitudes of both fluorescence bands. We describe the intensity of both bands and aSLOW and their by the product of their amplitudes aSLOW 1 2 spectral widths Γ1 and Γ2. The ratio of the intensities reflects the ratio of the equilibrated molar fractions and of the rate constants of radiative decay according to
kF2 1 - jx1 ) aSLOW Γ1 kF1 jx1 1
Γ2 aSLOW 2
(7)
) 0.37 and aSLOW ) 0.16 with Γ1 ) 2750 Using aSLOW 1 2 -1 cm and Γ2 ) 4500 cm-1, we obtain for the ratio of radiative rate constants kF2 /kF1 ) 0.4 taking into account jx1 ) 0.36. The radiative decay constant kF1 of the Franck-Condon rotamer is similar to that of BABAPH as estimated from the absorption spectrum of BABP using the Strickler-Berg relation.7,29 Taking kF1 ) [6.5 ns]-1, the radiative rate constant of secondary fluorescence is kF2 ) [16 ns]-1. With that result, we can now also evaluate the nonradiative decay of the secondary rotamer. NR ) [570 ps]-1. The With [τ2]-1 ) kF2 + kNR 2 , we obtain k2 nonradiative decay is faster than in the primary state by a factor of 16. The quantum yield of secondary fluorescence is about ΦF2 ) 0.03. The result of the kinetic analysis is summarized in Figure 5a. It should be kept in mind that it relies (i) on the approximation of separated time scales and (ii) on the idea of a close relationship of the Franck-Condon rotamer and BABAPH. Fluorescence in Butanol. Several normalized spectra of BABAPH in butanol are shown in Figure 6a. The dynamics
12176 J. Phys. Chem., Vol. 100, No. 30, 1996
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Figure 6. Normalized time-resolved fluorescence spectra in butanol at time delays of 20, 50, 150, and 2000 ps after excitation (from right to left). (a) BABAPH. (b) BABP. The data are fitted by log-normal spectral-shape functions.
of the spectral maximum, of the spectral width, and of the integral intensity are shown in Figure 7. The final emission is somewhat more in the red than in octanol (Figure 7a). We observe a significant acceleration of solvent relaxation as compared to octanol: Within the window of our time resolution, it is dominated by a time constant of 70 ps (amplitude 1050 cm-1) with a minor slow contribution (460 ps at an amplitude of 165 cm-1). The spectral width is invariant (Figure 7b). The decay of the intensity is dominated by an exponential (Figure 7c) with a time constant τ ) 36 ns. In the initial phase, there is a minor, fast (20-ps) component. The overall quantum yield of fluorescence is ΦF ) 0.48. Qualitatively, the fluorescences of BABAPH in butanol and octanol are rather similar. The faster resolvation corresponds to the faster longitudinal relaxation time of butanol with τL ) 120 ps.21-23 The time scales of resolvation and of electronic deactivation are more separated in butanol; i.e., the fluorescence originates here almost completely from a fully relaxed state. Using an average lifetime jτ ) 3 ns, we obtain a transition probability of fluorescence kF ) [63 ns]-1 which is similar to that in octanol. The nonradiative decay is somewhat enhanced with kNR ) [5.8 ns]-1. Several time-resolved spectra of BABP in butanol are shown in Figure 6b. The dynamics of the spectral features are plotted in Figure 7. We observe a larger red shift than for BABAPH and a distinct spectral broadening in the red. The enhancement of the red shift and the change of spectral width are more pronounced than in octanol: The final fluorescence is more in the red by 1000 cm-1 (Figure 7a). The increase of the spectral width from 2800 up to 4700 cm-1 can be fitted with two exponentials (Figure 7b). We also observe a significant acceleration of the fluorescence decay (Figure 7c). It can be fitted with two exponentials with time constants of 50 and 270 ps of equal amplitude. In accordance, we observe a rather low quantum yield of fluorescence ΦF ) 0.035.
Figure 7. Fluorescence dynamics in butanol of BABAPH (full symbols) and BABP (open symbols) in butanol. (a) Position of spectral maximum. (b). Spectral width (full width at half-maximum). (c) Integral intensity normalized to its initial value. The fit of the data is discussed in the text.
Qualitatively, the differences of fluorescence for the two dyes in butanol are similar to those in octanol. However, they are more pronounced. To rationalize the data, we use the same two-state model as above. We partition the spectra in two components with constant spectral width. We use Γ1 ) 2750 cm-1 for the primary fluorescence (Figure 7b) and, for the secondary emission, again Γ2 ) 4500 cm-1. The result is shown in Figure 8a. The final emission of the primary rotamer occurs at 19 000 cm-1, whereas the secondary fluorescence is shifted to 16 900 cm-1. The more polar butanol leads to a stronger solvation of the twisted geometry with its enhanced intramolecular charge shift. The amplitude of the primary fluorescence disappears fast as shown in Figure 8b. We can fit it with two exponentials of ) 0.74) and τSLOW ) τFAST ) 20 ps (relative amplitude aFAST 1 SLOW ) 0.26). There is a con200 ps (relative amplitude a1 comitant fast increase of the secondary fluorescence. The decay of the secondary fluorescence can be fitted with the same time constant as that of the slow decay of primary emission (relative ) -aFAST ) 0.2). amplitude aSLOW 2 2 To evaluate the rate constants, we proceed as above. Using eqs 2-5, we estimate for the rate constants of rotamerism quantities around kROT ) [25 ps]-1 and kTOR ) [75 ps]-1, with a molar fraction of the primary state after equilibration of about jx1 ) 0.25. The dynamics are faster than in octanol, with the
Rotamerism and Resolvation of Hemicyanine
J. Phys. Chem., Vol. 100, No. 30, 1996 12177 rotamer of BABP in octanol is estimated to be ΦF2 ) 0.03. The deactivation pathway through this rotamer leads to a reduction of the quantum yield ΦF ) 0.58, as observed in BABAPH where rotamerism is suppressed to the overall quantum yield ΦF ) 0.15 of BABP. Analogous relations hold for butanol. However, we have to modify the original scenario: (i) The rotameric states involved are equilibrated within a short time. The rotamerism is no forward reaction. (ii) Rotamerism occurs in a nonrelaxed solvation shell. It is not adequate to use the static dielectric constant to evaluate the activation energy on the basis of Born’s equation. (iii) Polar solvents enhance not only the state of rotamerism but also the rate of nonradiative decay in the secondary rotamer. (iv) The stationary emission does not come solely from the primary fluorescent rotamer. There is a contribution from the secondary rotamer. We have studied here only the twist of the CC single bond. In both molecules BABAPH and BABP, there is a dialkylamino group which may undergo rotamerism, too. Twist of amino groups has been considered in the literature.30 Preliminary experiments with immobilized amino groups indicate that this process has a minor effect on the fluorescence of hemicyanines. Acknowledgment. We thank Uwe Theilen for his excellent technical assistance and Martin Leonhard for critical reading of the manuscript. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 239, Project E2).
Figure 8. Decomposition of time-resolved fluorescence of BABP in butanol into a primary fluorescence (full symbols) and a secondary fluorescence (open symbols). (a) Position of spectral maxima. (b) Relative spectral amplitudes. The fit of the data is discussed in the text.
equilibrium shifted more to the secondary rotamer. The radiative decay constant of the secondary rotamer is about kF2 ) [15 ns]-1. It agrees well with the radiative decay assigned in octanol. This coincidence confirms the consistency of our -1 approach. The nonradiative decay is about kNR 2 ) [150 ps] , i.e., it is accelerated in butanol by a factor of about 3 as compared to octanol. The quantum yield of secondary fluorescence is only about ΦF2 ) 0.01. The result of the kinetic analysis is summarized in Figure 5b. Octanol and Butanol. The fact that the same mechanistic scheme provides a consistent interpretation of the fluorescence data of BABAPH and BABP in butanol as well as in octanol supports our approach. In both solvents, a sequential process of fast resolvation and slow electronic deactivation occurs in BABAPH. In both solvents, the rotamerism of BABP equilibrates during resolvation and the deactivation of the equilibrated state is dominated by the fast nonradiative decay of a twisted geometry. There are three main differences (Figure 5): (i) The resolvation is faster in butanol in correspondence to the shorter longitudinal relaxation time. (ii) The rotamerism is accelerated in butanol. This effect may be due to the smaller viscosity or to the larger polarity. (iii) The rate of nonradiative decay of the twisted state is enhanced in butanol. We assign this effect to a smaller energy gap between the excited state and the ground state as caused by the larger reorganization energy of the polar twisted state in the polar butanol. Conclusions On the basis of stationary fluorescence, it was suggested that the low quantum yield of fluorescence of hemicyanine dyes in polar solvents is due to the formation of a polar rotamer with fast nonradiative decay.6 This hypothesis is confirmed by the present study. The fluorescence quantum yield of the secondary
References and Notes (1) Grinvald, A.; Hildesheim, R.; Farber, I. C.; Anglister, L. Biophys. J. 1982, 39, 301. (2) Loew, L. M.; Cohen, L. B.; Salzberg, B. M.; Obaid, A. L.; Bezanilla, F. Biophys. J. 1985, 47, 71. (3) Fromherz, P.; Vetter, T. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 2041. (4) Fromherz, P.; Schenk, O. Biochim. Biophys. Acta 1994, 1191, 299. (5) Ephardt, H.; Fromherz, P. J. Phys. Chem. 1989, 93, 7717. (6) Ephardt, H.; Fromherz, P. J. Phys. Chem. 1991, 95, 6792. (7) Fromherz, P.; Heilemann, A. J. Phys. Chem. 1992, 96, 6864. (8) Ephardt, H.; Fromherz, P. J. Phys. Chem. 1993, 97, 4540. (9) Fromherz, P. J. Phys. Chem. 1995, 99, 7188. (10) Cocker, W., Harris, J. O., Loach, J. V. J. Chem. Soc. 1938, 751. (11) Hassner, A.; Birnbaum, D.; Loew, L. M. J. Org. Chem. 1984, 49, 2546. (12) Stavrovskaya, V. I. J. Gen. Chem. USSR. 1951, 21, 1721. (13) Gloyna, D.; Alder, L.; Hennig, H. G.; Ko¨ppel, H.; Siegmund, M; Schleinitz, K. D. J. Prakt. Chem. 1980, 322, 237. (14) Aun, C. E.; Clarkson, T. J.; Happer, D. A. R. J. Chem. Soc. Perkins Trans. 2, 1990, 645. (15) Muszkat, K. A. Top. Curr. Chem. 1980, 88, 91. (16) O’Connor, D. V.; Philipps, D. Time correlated single photon counting; Academic: New York, 1984. (17) Olmsted, J., III. J. Phys. Chem. 1979, 83, 2581. (18) Maroncelli, M.; Fleming, G. R. J. Chem. Phys. 1987, 86, 6221. (19) Siano, D. B.; Metzler, D. E. J. Chem. Phys. 1969, 51, 1856. (20) Stewart, J.J. MOPAC Version 6, Frank J. Seiler Research Laboratory, U.S. Air Force Academy, CO 80890, 1990. (21) Garg, S. K.; Smyth, C. P. J. Phys. Chem. 1965, 69, 1294 (22) Simon, J. D. Acc. Chem. Res. 1988, 21, 128 (23) Onganer, Y.; Yin, M.; Bessire, D. R.; Quitevis, E. L. J. Phys. Chem. 1993, 97, 2344 (24) Fro¨hlich, H. Theory of Dielectrics; Clarendon: Oxford, 1968. (25) Mozumder, A. J. Chem.Phys. 1969, 50, 3153. (26) Barbara, P. F.; Jarzeba, W. AdV. Photochem. 1990, 15, 1. (27) Maroncelli, M. J. Mol. Liq. 1993, 57, 1. (28) Horng, M. L.; Gardecki, J. A.; Papazyan, A.; Maroncelli, M. J. Phys. Chem. 1995, 99, 17311. (29) Strickler, S. J.; Berg, R. A. J. Chem. Phys. 1962, 37, 814. (30) Lippert, E.; Rettig, W.; Bonacic-Koutecky, V.; Heisl, F.; Miehe´, J. A. AdVan. Chem. Phys. 1987, 68, 1.
JP960095K
