J. Phys. Chem. B 1999, 103, 10917-10923
10917
Temperature-Dependent Study of the Ultrafast Photophysics of All-Trans Retinal Erica J. Larson† and Carey K. Johnson* Department of Chemistry, UniVersity of Kansas, Lawrence, Kansas 66045 ReceiVed: May 4, 1999; In Final Form: October 13, 1999
Ultrafast transient absorption measurements are reported for all-trans retinal in ethanol, hexane, and 3-methylpentane at temperatures from 85 K to room temperature. Investigation of the temperature dependence allows the mechanism of electronic relaxation and the presence of multiple conformers to be investigated. Relaxation of the initially excited 1B+ u -like state of all-trans retinal in ethanol is detected by the decay of transient absorption at 550 nm and the decay of stimulated emission at 650 nm. The time constant for this component slows from 2 ps at room temperature to 420 ps at 85 K. The corresponding fluorescence quantum yields estimated from these decay times for the “1B+ u ” state are sufficient to explain the measured fluorescence yields, suggesting that the fluorescence observed for all-trans retinal in hydrogen-bonding solvents has its origin predominantly in the “1B+ u ” state. The relaxation rate follows Arrhenius behavior at high temperatures but decreases less steeply at low temperature. The temperature dependence is consistent with internal conversion in the strong coupling limit. Tunneling contributions become evident at low temperatures. In contrast, the rate of intersystem crossing detected by the appearance of triplet-triplet absorption at 450 nm is independent of temperature, occurring in about 30 ps. However, the amplitude of the triplet-triplet absorption signal increases at low temperatures. These results demonstrate the presence of at least two distinct populations of retinal, which follow different relaxation channels at low temperature. Similar behavior is observed in hexane and 3-methylpentane, although the amplitude of the slow internal-conversion component is small.
Introduction Retinal is the chromophore embedded in rhodopsin, a protein found in the rod cells of the eye, and in the bacterial rhodopsins. Because of its role in vision and proton pumping, retinal chromophore photochemistry and photophysics have been studied extensively.1-4 However, uncertainty still surrounds the fate of electronic excitation in retinal due to the presence of three low lying excited singlet states, whose ordering can change in different solvent environments. Previously, we reported results of a study of the photophysics of retinal in hexane and ethanol based on one- and two-photon femtosecond transient absorption experiments at room temperature.3 The motivation of that study was two-fold. First, we wanted to compare the photophysics of retinal in a hydrocarbon solvent to a polar and hydrogen-bonding solvent to determine how excited-state level shifts affect electronic relaxation in retinal. Second, we wished to compare the response of retinal following one-photon and two-photon excitation, which launch the photophysics from different excited states. In the present paper, we report the temperature dependence of both intersystem crossing (ISC) and internal conversion (IC) of all-trans retinal in ethanol and in the alkanes hexane and 3-methylpentane. There were two reasons for doing temperaturedependent experiments. First, we wanted to refine the previous model of retinal photophysics. Second, these measurements allowed us to study the temperature dependence of the electronic relaxation in retinal in order to analyze the mechanism of IC. * To whom correspondence should be addressed. E-mail: cjohnson@ eureka.chem.ukans.edu. † Current address: Chemical Sciences and Technology Division, Los Alamos National Laboratory, MS M888, Los Alamos, NM 87545.
Photophysical processes in retinal involve three excited singlet 1 + states, two ππ* states, “1Ag ” and “ Bu ” (where the quotation marks indicate that the C2h symmetry label is only an approximation for retinal) and an nπ* state. The “1B+ u ” state has a large oscillator strength (f ≈ 1.25) and is largely responsible for the strong 400 nm absorption of retinal. The “1Ag ” state lies to lower energy but has a smaller oscillator strength (f ≈ 0.075). In two-photon absorption, the relative absorptivities are reversed, with the “1Ag ” state absorbing more strongly than 5 The nπ* state, which is thought to be the the “1B+ ” state. u lowest excited singlet state in nonpolar solvents,5,6 is very weak in both one-photon and two-photon absorption. One-photon transient absorption decays of retinal in hexane have revealed three components.3,4,7,8 The fastest relaxation occurs in e300 fs. Because we also observed this component in two-photon transient absorption data,3 we assigned it to IC from the “1Ag ” state. Second, a 1-2 ps decay was observed at probe wavelengths of 450-650 nm and was attributed to IC from the “1B+ u ” state. Again, this assignment was based in part on comparison of the one-photon and two-photon transientabsorption responses.3 Finally, a component of about 30 ps was seen at wavelengths of 450-600 nm. Because the amplitude of the 30 ps process was the largest at the reported maximum of the triplet-triplet absorption spectrum, it was assigned to ISC.4,7-16 We found distinctly different behavior for retinal in ethanol. In this solvent, the one-photon-induced transients contain predominantly only two components.3 A 1-2 ps excitedstate absorption decay was observed at probe wavelengths of 450-550 nm. As in hexane, we assigned this component to IC from the “1B+ u ” state. In contrast to the behavior in hexane, this decay component also appeared at 650-750 nm as stimulated
10.1021/jp9914626 CCC: $18.00 © 1999 American Chemical Society Published on Web 11/19/1999
10918 J. Phys. Chem. B, Vol. 103, No. 49, 1999 emission. A 30 ps component assigned to ISC in ethanol was also observed. In the work reported here, the temperature dependence of electronic relaxation was measured in ethanol at temperatures from 85 K to room temperature. Transient absorption measurements demonstrate a marked temperature dependence for the decay times measured in transient absorption at 550 nm and stimulated emission at 650 nm. The temperature dependence of the relaxation rates is consistent with radiationless transitions between states with a large displacement between potential energy minima,17 where the IC rate at low temperatures points to tunneling from one state surface to the other. The slow decay of the “1B+ u ” state in ethanol at low temperature demonstrates that emission from the “1B+ u ” state is sufficient to explain the previously observed fluorescence quantum yield.18 In contrast to the dynamics observed at 550 and 650 nm, the appearance time constant for the triplet state at 450 nm remains virtually constant over the same temperature range. Evidently, a channel of efficient ISC remains open, even at low temperatures. These results demonstrate the presence of at least two distinct populations of retinal at low temperatures in both solvents. Methods and Materials The instrumentation used in the transient absorption experiments has been described previously.3 Briefly, a mode-locked Ti:sapphire laser19 was amplified with a Clark MXR regenerative amplifier. Pump pulses at 400 nm with a repetition rate of 1 kHz were generated by frequency doubling in BBO. Probe pulses were selected from a white-light continuum by interference filters. The intensities of the probe and a reference were monitored with photodiodes. The photodiode responses were integrated with a gated integrator (Stanford Research Systems). The difference of the integrated probe and reference signals was either input into a lock-in amplifier (Stanford Research Systems model SR510) or to a third gated integrator operated in toggle mode. All-trans retinal was purchased from Sigma Chemical Company and used without further purification. The sample was verified to be free of isomeric impurities by HPLC. The solvents hexane and 3-methylpentane were dried over calcium hydride and distilled. Samples at a concentration of 3.5 × 10-4 M were placed in a sample cell with either a 2 or 5 mm path length. Transient absorption data at different temperatures were obtained in a cryostat (CryoIndustries) and cooled with liquid nitrogen. The pump power at 400 nm was typically 10-20 mW and focused to a spot size of ∼1 mm. We estimate that less than 10% of sample molecules were excited by a laser pulse yielding a typical optical density change of 0.06 or less. Because the samples could not be flowed, the irradiated volume tended to bleach over time. To alleviate this problem, the cell was periodically moved to expose a fresh volume of sample. The shape of the transient absorption response did not change as scans were acquired, indicating that bleaching did not change the response or lead to a build up of other species detectable in the scans. Scans in which the signal at a particular delay time was monitored over time demonstrated that the bleaching process was slow compared to the time required to collect one scan. At each time delay, 15 laser shots were averaged, and 10-100 scans were averaged for each experiment. The sample cell was typically located about 2-3 in. away from the focus of the pump beam in order to avoid continuum generation in the cryostat windows or sample.
Larson and Johnson
Figure 1. Transient absorption scan of all-trans retinal in ethanol at 550 and 650 nm at 99 K. The solid lines show fits to the data. The excited-state absorption at 550 nm and the stimulated emission at 650 nm both decay in about 280 ps.
TABLE 1: Fitting Parameters for Transient Absorption Scans of All-Trans Retinal in Ethanol at 550 and 650 nm at Various Temperaturesa temperature (K) 86 95 97 100 110 125 142 150 155 170 201 240 248
550 nm τ1 (ps)
550 nm τ2 (ps)
1.0 ( 0.5 2.2 ( 0.4
400 310 270 260 ( 80
2.4 1.1 ( 0.3 2.3 1.6 ( 1.0
95 ( 45 84 48 54
1.8
7.7 ( 1.5 2.1
650 nm τ1 (ps)
650 nm τ2 (ps) 420
1.9
0.16
280 260 330 160 58 ( 6 42 13 ( 6 4.6 3.5
a
τ1 was obtained from 10 ps scans, while τ2 was obtained from longer scans. Errors are the estimated standard deviations in a series of measurements.
Results Transient absorption data of all-trans retinal in ethanol, hexane, and 3-methylpentane were collected at temperatures between 85 K and room temperature. Because hexane did not form satisfactory glasses in our sample cell, 3-methylpentane served as a hydrocarbon solvent at lower temperatures. The samples were pumped at 400 nm in order to populate the “1B+ u ” state. Transients of retinal in ethanol at 550 and 650 nm at 99 K are shown in Figure 1, and the fitting parameters for all of the temperatures studied are shown in Table 1. The dominant feature in both transient absorption at 550 nm and stimulated emission at 650 nm is a temperature-dependent component that lengthens from 2 to 4 ps at 248 K to 420 ps at 85 K. The room temperature decays of all-trans retinal in ethanol display a 1-2 ps excitedstate absorption at 550 nm and a 1-2 ps stimulated emission at 650 nm.3 This component was assigned to IC from the “1B+ u ” state based on the fact that this component is strong in the one-photon data, in which mostly the “1B+ u ” state is initially excited, but is very small or absent in the two-photon data in which the “1Ag ” state is primarily excited. Also, it is unlikely that the stimulated emission signal at 650 nm could
Ultrafast Photophysics of All-Trans Retinal
Figure 2. Transient absorption of all-trans retinal in ethanol at 450 nm at several temperatures. The rise in absorption corresponds to the formation of the triplet state from intersystem crossing. The amplitude of the signal increases as the temperature is lowered. However, the rise time remains nearly constant. Average rise times are 25 ( 7 ps at 291 K, 45 ( 15 ps at 201 K, and 41 ( 6 ps at 105 K. 1 arise from either the weak “1Ag ” state or the forbidden nπ* state. A 1-2 ps component is still present at 550 nm at lower temperatures, but its amplitude is smaller than at room temperature. The decays at low temperatures are instead dominated by a longer component whose lifetime is temperature dependent. The low-temperature behavior at 650 nm is comparable to that at 550 nm. At lower temperatures, the 1-2 ps component at 650 nm also seems to persist, but its presence is less clear than at 550 nm. A longer component also appears in stimulated emission at 650 nm with a temperature dependence that closely follows that of the 550 nm component. The temperature dependence of ISC was studied by measuring the rise time and amplitude of the absorption signal at a probe wavelength of 450 nm at different temperatures. The transient absorption of retinal in ethanol at 450 nm at several temperatures is shown in Figure 2. The rise time of the triplet-triplet absorption remains nearly constant to within (20 ps at all temperatures studied. However, the amplitude of the signal increases as the temperature was lowered. Spectral changes that retinal experiences at low temperatures may affect the amplitude of the triplet-triplet absorption signal. However, the following estimate shows that spectral changes can account for a change of at most 10% in the amplitude of the triplet-triplet absorption.20 The ground-state spectrum of retinal is known to red shift as the temperature is lowered.21-23 We have estimated that this shift increases the extinction coefficient of the sample at 400 nm by approximately 4%. Second, the max of retinal also increases by about 10% at 77 K compared to room temperature.21,22 The origin of both the red shift and the increase in max are thought to be caused by a change in retinal configuration. Retinal is twisted about a single bond at room temperature and becomes more planar at cold temperatures, enhancing the π conjugation and resulting in a red shift.21-23 Finally, the spectra of the excited states of retinal could experience similar changes at low temperatures. For example, it is likely that the max of the lowest excited triplet state increases at low temperature, and the spectrum may also shift toward 450 nm (the probe wavelength) as the temperature is decreased. All three of these factors could contribute to a larger triplet-triplet absorption signal. An estimate shows that the combined effect of these spectral changes results in an increase of 10% or less
J. Phys. Chem. B, Vol. 103, No. 49, 1999 10919
Figure 3. Transient absorption scan of all-trans retinal in 3-methylpentane at a probe wavelength of 550 nm and a temperature of 85 K. Two decay components are clearly discernible in this scan. The solid line shows a two-exponential fit to the data with time constants of 2.2 and 49 ps.
Figure 4. Transient absorption of all-trans retinal in hexane at 450 nm at two temperatures showing that the rise time associated with the creation of a triplet state is essentially constant, but the amplitude of the lower temperature transient is larger. The rise time is 28 ps at 291 K and 38 ps at 201 K.
in the 450 nm signal at low temperatures.20 Because our signal increases by much more than 10%, we conclude that the spectral changes cannot account for this phenomenon and consequently that the ISC yield increases at low temperatures. Data at 550 nm were also collected for retinal in hexane and 3-methylpentane, and representative data are shown in Figure 3. At 550 nm, there were two components at low temperatures. The first component was 1-3 ps at all temperatures. A slower component was also present, with a time constant that increased from 5 ps at 253 K to ∼55 ps at 86 K. However, greater scatter in the time constants for the slow component in 3-methylpentane than in ethanol precluded a more precise analysis of its temperature dependence. Data for retinal in hexane at a probe wavelength of 450 nm are shown in Figure 4. As in ethanol, the ISC time in hexane and 3-methylpentane is not strongly temperature dependent, but the triplet-triplet absorption signal increases as the temperature is decreased. Thus, as in ethanol, the ISC yield increases at low temperature, while the ISC rate remains essentially constant. We thought that the slow component observed in the 550 nm signal might correspond to
10920 J. Phys. Chem. B, Vol. 103, No. 49, 1999
Larson and Johnson
hydrogen-bonded retinal. Although care was taken to dry the hexane and 3-methylpentane solvents, the retinal sample itself could have contained water prior to dissolution. To test this possibility, the experiments were repeated with retinal that was dried under vacuum overnight. The solutions were prepared in a dry glovebox with dried and degassed 3-methylpentane. Even with these precautions, the small-amplitude temperature-dependent component was still observed. Therefore, the presence of a hydrogen-bonded species of retinal in the hydrocarbon solvents is unlikely. Discussion In a study of transient absorption of retinal at room temperature, we previously detected a 2 ps transient in ethanol following excitation at 400 nm.3 This decay component was observed in both stimulated emission at 650-750 nm and transient absorption at 450-550 nm and was assigned to decay of the “1B+ u ” state by internal conversion. This assignment follows from a comparison of the amplitude of this signal with one-photon and two-photon excitation.3 The observation of stimulated emission also suggests that the signal is associated with the large oscillator strength of the “1B+ u ” state. In contrast, the weak oscillator strengths of the neighboring “1Ag ” and nπ* states would probably preclude detection of stimulated emission. The presence of stimulated emission in the 650-750 nm region within 150 fs reveals fast intramolecular relaxation in the excited state. A good candidate for this relaxation is an increase in the CdC bond length in the excited state. Ab initio calculations of excited-state dynamics in the “1B+ u ” state of a retinal Schiff base analogue implicate such skeletal stretching as the initial relaxation from the Franck-Condon region,24 and resonance Raman measurements have provided evidence for a large Franck-Condon factor for this mode.25 Temperature Dependence of Internal Conversion. Further information about the mechanism of electronic relaxation in retinal can be garnered from the temperature dependence of the transients at 550 and 650 nm in ethanol (Figure 1). As the temperature is lowered, a longer component appears whose lifetime depends on temperature. This component is seen both as excited-state absorption at 550 nm and as stimulated emission at 650 nm, showing that it corresponds to the room temperature 1-2 ps relaxation of the “1B+ u ” state. A 1-2 ps decay seems to persist in the cold temperature data, but its presence is difficult to confirm at the lowest temperatures because its amplitude is small relative to the slower component. A plot of ln k versus 1/T for the 550 and 650 nm data is shown in Figure 5. The relaxation rate appears to display Arrhenius behavior at higher temperatures but decreases less steeply at lower temperatures. We do not believe that the nonArrhenius behavior is related to the glass transition. The glass transition temperature in ethanol is 90-96 K,26 significantly below the region where the temperature dependence becomes nonlinear in Figure 5. However, we cannot exclude the possibility that shifts in the excited-state energy levels with temperature lead to the non-Arrhenius temperature dependence. The data can be fit with an expression for IC derived by Engleman and Jortner for the strong coupling limit in which there is a large displacement between the two adiabatic potential surfaces involved.17 In this limit, the rate of IC, W, is given by
ln W )
-Ea ln T* + ln A kbT* 2
(1)
Figure 5. Temperature dependence of the rate of the slow component in the transient absorption of all-trans retinal in ethanol. Data from probe wavelengths of 550 and 650 nm are both included in the plot. The solid line shows a fit to the Engleman-Jortner expression (ref 17) for IC in the strong-coupling limit with a mean vibrational frequency of 260 cm-1 and an activation energy of 1960 cm-1.
where
T* )
( )
h〈ω〉 h〈ω〉 coth 2kb 2kbT
A)
C2x2π pxEMkB
(2) (3)
In eq 1, Ea is the activation barrier between the two potentials and T* is an effective temperature that accounts for tunneling through the activation barrier. In eq 2, 〈ω〉 is the mean vibrational frequency. In eq 3, C is the coupling matrix element between electronic states and EM is the molecular nuclear relaxation energy. (The reader is referred to ref 17 for additional details.) At high temperatures, the rate calculated with eq 1 approaches that predicted by the Arrhenius equation, whereas at low temperatures the rate predicted by eq 1 is faster than that predicted by the Arrhenius equation due to tunneling. The fit to our 550 and 650 nm data results in an activation energy of 1960 ( 316 cm-1 and a mean vibrational frequency of 260 ( 21 cm-1. In a previous study of the temperature dependence of fluorescence decay times of retinal in ethanol,15 fluorescence lifetimes were observed to increase from 17 ps at room temperature to 400 ps at 80 K. The data were fit to an Arrhenius activation energy of ∼340 cm-1. Similar results were found for retinal in carbon tetrachloride and for retinal powder. However, the 2 ps component could not be resolved in that study. The temperature-dependent results demonstrate the existence of an activation barrier along a coordinate associated with the temperature-dependent IC in one subpopulation of retinal in ethanol. The temperature dependence is consistent with strong coupling along this mode. The identity of the coordinate is not known, but one possibility is the C6-C7 single bond in retinal. Large displacements have been calculated between the 1nπ* 1 + surface and the “1Ag ” and “ Bu ” surfaces along this coordi27 nate, consistent with the strong-coupling limit. The large displacement between the potential energy surfaces along the C6-C7 coordinate could cause IC to slow dramatically as the temperature is lowered.
Ultrafast Photophysics of All-Trans Retinal Partitioning of Retinal Populations. The results also demonstrate the existence of a subpopulation of all-trans retinal in ethanol in which ISC occurs rapidly (in ∼30 ps). It is not possible to explain the observed ISC and IC times in ethanol unless we consider partitioning into at least two distinct populations of retinal. Even though IC slows down to 400 ps at the lowest temperatures studied, the data at 450 nm show that the rate of ISC does not change significantly with temperature. This finding is not consistent with slow IC to a triplet precursor, followed by ISC in ∼30 ps. On the other hand, if IC and ISC proceeded from the same state (for example the “1B+ u ” state), then virtually all of the molecules would relax by the more efficient ISC channel, making the IC component undetectable. The two populations follow different relaxation paths at low temperature. One of the populations at low temperature undergoes slow IC from the “1B+ u ” state and is responsible for the temperature-dependent decay at 550 and 650 nm. The other population relaxes from the “1B+ u ” state to the triplet precursor, which then crosses to the triplet manifold in 30 ps. At room temperature, interconversion between these populations may be too fast for them to be individually detected. Similarly, the data for retinal in hexane and 3-methylpentane at 450 and 550 nm also reveal the presence of two retinal populations. Although the identity of the two retinal populations in ethanol, hexane, and 3-methylpentane is not known, there are several possibilities that can be discussed. The two populations may correspond to two conformers of retinal. For example, two 6-scis conformations are possible. The energy difference between these two conformations, which are inequivalent because of nonplanarity of the cyclohexene ring, was calculated to be ∼150 cm-1 for the ground state of retinal, whereas the 6-s-trans conformation was predicted to lie ∼1100 cm-1 above the lowest conformation.28 Large displacements along this coordinate have 1 1 been calculated between the “1B+ u ” and the nπ* and “ Ag ” 27 surfaces for one of the 6-s-cis conformations. Different displacements along this mode between the excited-state surfaces in two conformations (e.g., the two 6-s-cis conformations or 6-s-cis and 6-s-trans) could explain the observed differences in electronic relaxation rates for these two populations. Another possibility is that the two retinal populations correspond to free retinal and retinal that is hydrogen-bonded to the solvent. The non-hydrogen-bonded species could be responsible for the observed ISC signal, while the hydrogen-bonded retinal species could be responsible for the temperaturedependent IC at 550 and 650 nm. The rate of the hydrogenbonding exchange may be slow enough at low temperature to trap a population of non-hydrogen-bonded retinal. One difficulty faced by this scenario is that it does not explain the presence of distinct populations of retinal in hexane and 3-methylpentane. However, the temperature-dependent component in 3-methylpentane is small, which suggests that only a small fraction of retinal belongs to this population. It is also possible that the photochemical formation of other isomers of retinal introduces sample heterogeneity. However, no qualitative change in the response is observed as the sample is scanned repeatedly, suggesting either that any photochemically generated species that could contribute to the signal did not build up or that the sample reached a photostationary state within the first scan. Such heterogeneity could contribute components to the observed signals. Near room temperature, the observed signals are similar to scans with a flowing sample,3 where the sample volume is replaced with every shot, suggesting that such contributions from photogenerated species are minor. The quantum yield for photoisomerization decreases at low temper-
J. Phys. Chem. B, Vol. 103, No. 49, 1999 10921
Figure 6. Possible excited-state level structures for all-trans retinal. Energy level scheme A with 1nπ* the lowest excited singlet state is consistent with the high triplet yield and low fluorescence yield observed for all-trans retinal in alkanes. Increasing polarity or hydrogen bonding is expected to bring the 1nπ* and “1Ag ” states closer together (scheme 1 B) and eventually to bring the “1Ag ” state below the nπ* state (scheme C). Coupled 1nπ* and “1A” states as in B are consistent g with the reduced triplet yield at room temperature compared to A due to competing internal conversion to the ground state. At lower temperatures, the triplet yield increases as the IC rate decreases. Because the observed fluorescence yields can be explained by “1B+ u ” fluorescence, the increased fluorescence yield in hydrogen-bonding solvents 1 does not necessarily show that the “1Ag ” state lies below the nπ* state as in C.
ature,29 so it seems less likely that photochemically formed isomeric species are responsible for the observed multiple populations at low temperature. Origin of Retinal Fluorescence. The temperature dependence of the decay of the “1B+ u ” state provides a new perspective on the fluorescence yield of all-trans retinal in ethanol. The observed decay times of the “1B+ u ” state show that ” state can explain the fluorescence emission from the “1B+ u quantum yield of retinal in ethanol and other hydrogen-bonding solvents. It had been suggested that the increased quantum yield of all-trans retinal in hydrogen-bonding solvents is a conse1 quence of a drop in the excited “1Ag ” level below the nπ* state, in contrast to dry alkane solvents where the absence of detectable fluorescence suggests a lowest lying 1nπ* excited singlet state.6,18,30,31 The idea was that the weakly emitting “1 5 Ag ” state (oscillator strength f ≈ 0.07 ) would lead to weak fluorescence in hydrogen-bonding solvents, whereas the 1nπ* state in alkane solvents would yield no detectable fluorescence. These two situations are illustrated by schemes A and C in Figure 6. It is now apparent that emission from the “1B+ u ” state is the major source of the observed fluorescence in hydrogen-bonding solvents. A simple estimate is sufficient to show this. The 5 oscillator strength of the “1B+ u ” state is f ≈ 1.2, corresponding 32 to a radiative decay time of about 8 ns. The room temperature “1B+ u ” lifetime of ∼2 ps then leads to an estimated fluorescence quantum yield of ∼2.5 × 10-4, compared to a reported fluorescence quantum yield of 7 × 10-5 for all-trans retinal in EPA (ether-isopentane-ethanol, 5:5:2).31 The actual quantum yield is probably higher in pure ethanol than in EPA, which would make the agreement with the predicted value even closer. At ∼80 K, the “1B+ u ” lifetime is ∼400 ps, providing an estimated fluorescence quantum yield of ∼5 × 10-2. This value is reasonably close to the fluorescence quantum yield of 2.5 × 10-2 reported for all-trans retinal in ethanol35 and 8 × 10-3 for all-trans retinal in EPA glass at 77 K.18 The quantum yield estimated from the radiative lifetime of course also needs to be multiplied by the fraction of all-trans retinal following the slow relaxation pathway at low temperature. In addition, coupling 1 between the “1B+ u ” and “ Ag ” states could increase the radia-
10922 J. Phys. Chem. B, Vol. 103, No. 49, 1999 tive lifetime.36 Consequently, the above estimated quantum yields based on the measured “1B+ u ” lifetimes may overestimate the actual fluorescence quantum yield. The extremely weak fluorescence of retinal in alkanes is consistent with fast relaxation from the “1B+ u ” state. We suggest that even at low temperature, most excited retinal molecules in alkanes quickly (j2 ps) reach the 1nπ* state, which has negligible oscillator strength, while a smaller fraction of retinal follows a slow relaxation path from the “1B+ u ” state. The dominant relaxation paths for all-trans retinal in alkanes are then ISC and IC to the ground state, consistent with scheme A in Figure 6. Given the measured lifetime and estimated oscillator strength of the “1B+ u ” state for retinal in ethanol, the predicted quantum yield for “1B+ u ” fluorescence is sufficient to explain the observed quantum yield. Therefore, the difference in fluorescence quantum yields in alkane and alcohol solVents does not in itself proVide information on the relatiVe positions of the 1 excited “1Ag ” and nπ* states. The fluorescence yield in ethanol increases at low temperature because of slower relaxation out of the “1B+ u ” state. This implies that, although the ” state may be populated either by direct excitation or by “1Ag 1A-” state makes only a minor IC from the “1B+ ” state, the “ u g contribution to the observed fluorescence in ethanol. It is possible that the “1Ag ” state lifetime is quite short, resulting in negligible “1A” fluorescence. We suggest, therefore, that the g ” state may not be the lowest lying state in ethanol or that “1Ag it is strongly mixed with the nπ* state (scheme B in Figure 6). Our basis for this suggestion is two-fold: (i) the fact that the fluorescence quantum yield can be accounted for by mainly “1B+ u ” state fluorescence suggests that the lowest excited singlet state is not fluorescing much; (ii) the fast intersystem crossing time is best explained by a lowest lying excited singlet having significant nπ* character (see below). Mechanism of Intersystem Crossing. We have found that whereas the ISC rate is essentially independent of temperature, the amplitude increases at lower temperatures. The increase in the ISC amplitude as the temperature is lowered could be due to two factors. First, the relative fractions of retinal in the two populations might change with temperature so that more retinal branches into the population that undergoes ISC at low temperatures. A more plausible possibility is that a competing IC process from the triplet precursor slows down as the temperature is lowered, allowing more molecules to intersystem cross. It is reasonable to suggest that IC to the ground state competes with ISC at room temperature because the sum of the quantum yields of fluorescence, ISC, and photoisomerization is only about 0.4-0.5 at room temperature.31 This means that the excitation energy must also be dissipated through an additional channel such as IC to the ground state. If IC to the ground state slows down as the temperature is lowered, the ISC yield should increase. The observation of enhanced triplet yields at lower temperatures is reminiscent of the proximity effect of close-lying nπ* and ππ* excited states.37 Vibronic coupling between such states can enhance IC to the ground-state via out-of-plane coupling modes. At lower temperatures the coupling is therefore diminished, reducing the rate of IC to the ground state. This picture is consistent with the increased amplitude of the triplet absorption we observe as the temperature is decreased and implies coupling of the 1nπ* and “1Ag ” states. One important result in our previous paper was the finding that the ISC time is essentially the same for retinal in the two
Larson and Johnson solvents, hexane and ethanol. The quantum yield of ISC in retinal had previously been known to be lower in polar and hydrogen-bonding solvents than in hydrocarbon solvents.9,12,31 The 1nπ* state is thought to be the lowest excited singlet state 1 + for retinal in hexane, with the “1Ag ” and “ Bu ” states lying 5,31 higher in energy (scheme A in Figure 6). In ethanol and other hydrogen-bonding solvents, the energy levels of the ππ* states 1 (“1B+ u ” and “ Ag ”) are shifted to the red. The lower ISC quantum yield in ethanol than in hexane would be consistent with a shift of the “1Ag ” ππ* state to lower energy in the polar solvent, ethanol (Scheme C in Figure 6). If the “1Ag ” state is the lowest excited singlet state in ethanol, then ISC to the 3Ag” state is expected to be inefficient compared to ISC from the 1nπ* state to the 3A-” state in nonpolar solvents such as g hexane.3 Therefore, one might have expected a slower ISC rate in ethanol compared to hexane. However, our room temperature results revealed that the ISC times were virtually identical in the two solvents, but with lower amplitude in ethanol.3 This indicates that the lower triplet yield in ethanol is not due to a decreased coupling to the triplet state, which would result in a slower ISC time. Rather, the fraction of molecules following this channel is decreased. This finding raised the question of the nature of ISC for retinal in polar solvents. There are several possible answers to this question. The present results allow us to re-evaluate these possibilities. We suggested previously3 that, even if it is not the lowest-lying excited singlet state, the 1nπ* state may be populated either by IC from the higher “1B+ u ” state or thermally, leading to ISC via the 1nπ* state. However, the fact that the amplitude of ISC increases at low temperature makes this possibility less likely. A more likely possibility hinges on the 1 existence of “1Ag ” and nπ* states close enough together to be coupled for retinal in ethanol (Scheme B in Figure 6), resulting in sufficient nπ* character in the lowest excited singlet state to account for ISC. At lower temperature, the increasing amplitude (but essentially constant rate) of ISC can be accounted for by a decrease in competing IC to the ground state, as discussed above. Conclusion. We have found that two populations of retinal follow two distinct relaxation pathways after photoexcitation into the “1B+ u ” state. This behavior was observed both in the polar solvent ethanol and in the nonpolar solvent 3-methylpentane at low temperatures. One relaxation process is temperaturedependent IC from the “1B+ u ” state, and the other is ISC with a rate of ∼(30 ps)-1 that is insensitive to temperature but which increases in amplitude at lower temperatures. The temperaturedependent IC process, detected by transient absorption (at 550 nm) and by stimulated emission (in ethanol at 650 nm), slows down from a time constant of 2 ps at room temperature to 420 ps at 85 K in ethanol. The corresponding fluorescence yield from the “1B+ u ” state accounts for the previously observed fluorescence quantum yield of all-trans retinal in hydrogenbonding solvents, identifying the origin of the observed fluorescence yield predominantly with the “1B+ u ” state. Thus, the increased fluorescence quantum yield for retinal in hydrogenbonding solVents is not necessarily eVidence for a change in the identity of the lowest-lying excited singlet state. The temperature dependence is consistent with the strong-coupling regime of radiationless transitions, suggesting that the relaxation is promoted by a large displacement between two excited-state surfaces along a mode with a vibrational frequency of ∼260 cm-1. Tunneling effects become evident at low temperature. Several possibilities for the identities of the two populations
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