Primary Photochemical Processes in Thymine in Concentrated

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J. Phys. Chem. 1996, 100, 5570-5577

Primary Photochemical Processes in Thymine in Concentrated Aqueous Solution Studied by Femtosecond UV Spectroscopy A. Reuther Physik Department der Technischen UniVersita¨ t Mu¨ nchen, D-85748 Garching, Germany

D. N. Nikogosyan Institute of Spectroscopy, Russian Academy of Sciences, Troitzk, Moscow Region, 142092 Russia

A. Laubereau* Physik Department der Technischen UniVersita¨ t Mu¨ nchen, D-85748 Garching, Germany ReceiVed: NoVember 7, 1995X

Using a 282 nm fs light source, we have investigated the primary photochemical processes in liquid aqueous solution of thymine (Thy), one of the DNA bases using probe pulses in the range 282-588 nm. The studied processes include two-step Thy photoionization with the formation of an electron-cation pair (Thy+,e-) followed by partial geminate recombination, the formation of primary photoproducts, energy transfer from the excited Thy molecule to surrounding water molecules as well as the S1 f S0 relaxation. The two-photon absorption of the solvent water and the resulting generation of photoproducts was also taken into account. By comparison of the numerical simulations of the model derived with the experimental results, we have estimated the absorption cross section of the species mentioned above and determined the involved time constants; e.g., the S1 lifetime τ1 ) (1.2 ( 0.2) ps. The theoretical model is supported by the measured intensity dependence.

1. Introduction Although the nucleic acid molecules such as DNA, RNA and their major constituents, nucleotides, nucleosides, and nucleobases, were widely investigated during the past four decades by many thousands of scientists, some of their photophysical parameters have not been measured accurately or are even yet unknown. For example, such an important photophysical parameter of nucleic acid components as the lifetime of the first singlet state in aqueous solution at room temperature is only known to be in the range 1-10 ps.1 This short-lifetime value is estimated from the low fluorescence quantum yield of all nucleic acid bases which is in the range 10-4-10-6.2 This circumstance complicates the measurement of photophysical parameters of nucleic acid molecules by conventional luminescence methods and is the reason for the low accuracy of the experimental results. For example, the S1-state lifetime of adenine, one of the nucleic acid bases, reported by different groups ranges from 1 to 8.9 ps.3-5 Twelve years ago the method of picosecond kinetic spectroscopy with excitation and probing at the same wavelength (266 nm) was applied for the measurement of the S1-state lifetime of some nucleic acid constituents.6 Unfortunately, due to rather long laser pulse duration (3.5 ps), the accuracy of the obtained lifetime values was low. To measure accurately the S1-state lifetime of nucleic acid constituents as well as DNA and RNA by pump-and-probe techniques, one has to apply a subpicosecond UV laser emitting in the spectral region between 250 and 280 nm. In the past decade femtosecond laser sources mainly based on colliding pulse mode-locked (CPM) dye lasers were developed and successfully used for studying ultrashort processes. The second harmonic of fundamental radiation of * To whom correspondence should be sent. X Abstract published in AdVance ACS Abstracts, March 1, 1996.

0022-3654/96/20100-5570$12.00/0

these lasers lies in the near ultraviolet range (307-313 nm), which allows for example to excite a water molecule by twophoton absorption7 and to study the subsequent primary processes such as photoionization, electron solvation, and geminate recombination with femtosecond time resolution.8-12 However, these laser systems are not suitable for the excitation of nucleic acids and their constituents. Using femtosecond pulses with energy of about ≈10 µJ, an intensity in the range 10-200 GW/cm2 can be easily achieved by focusing the laser beam. This is in the range of the saturation intensity for these molecules under femtosecond excitation.1,13 It means that the use of femtosecond pulses should lead to the realization of biphotonic (two-step) photoprocesses.14 The total energy of two sequentially absorbed UV light quanta is significantly higher than the ionization threshold of nucleic acid components in water solution (≈5-6 eV1); therefore under highintensity UV excitation the two-step ionization of nucleic acids and their components takes place.7,15 In the experiments discussed below we have employed a femtosecond UV light source with a wavelength of 282 nm. Using this light source, we studied with femtosecond time resolution the primary photochemical processes in water and in concentrated water solution of Thysone of the DNA bases for various probing wavelengths and excitation intensities. We have also determined the two-photon absorption coefficient of pure water at 282 nm and established the water and Thy contributions to the induced absorption kinetics recorded in the solution. Our analysis suggests that the cation radical of Thy is rapidly formed within several 10 fs and decays with a lifetime of approximately 120 fs by both geminate recombination and the formation of photoproducts. From the model derived we have determined the absorption cross section for H3O+, Thy in the S1 state, the electron-cation pair, and the photoproducts of Thy photolysis. We have also determined the lifetimes of the © 1996 American Chemical Society

Primary Photochemical Processes in Thymine

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processes involved, for example the S1-state lifetime of τ1 ) 1.2 ( 0.2 ps. The proper description of the measured intensity dependence by the numerical simulations supports the validity of the model derived. 2. Experimental Methods As a source of fundamental radiation a non-continuous-wave synchronously pumped dye laser was used.16 After the amplification and stretching-recompression procedure,17 the laser pulse was frequency-doubled to 282 nm wavelength in an ADP crystal.18 The energy conversion was about 25% yielding an output energy of typically 10 µJ. The UV pulse duration was measured by a novel correlation technique using two-photon absorption in water, allowing also in situ intensity measurements. The details of the method will be published elsewhere. Similar results were obtained for measurements by standard methods, e.g. two-photon absorption in diamond.19 The measured duration of the UV laser pulse was approximately 180 fs (fwhm). For the investigation of primary photophysical processes, we employed the standard approach of kinetic spectroscopy, the pump-and-probe technique. The pumping in all experiments was carried out at 282 nm. For the probing process pulses with different wavelengths (282, 292, 299, and 588 nm) were used and the transient sample transmission measured. The last three wavelength positions were generated via continuum generation from the remainder of the fundamental radiation at 565 nm. For probing at 292 and 299 nm the continuum pulse was frequency-doubled in a second ADP crystal. Frequency selection was performed using interference filters (fwhm ≈ 6 nm in the UV region and fwhm ≈ 10 nm at 588 nm). Using different lenses, the radius of the laser beam in the focal region was varied between 80 and 320 µm and measured by pinhole transmission. The corresponding peak intensity I0 of the pump pulse was 20-200 GW/cm2. The energy of the pulses was monitored by silicon photodiodes. The sensitivity of the probe detection scheme allowed us to measure induced absorbance as low as 5 × 10-4. The time resolution was better than 30 fs for probing with UV pulses and about 50 fs for probing in the visible spectral region. The laser repetition rate was 15 Hz. The peak power of the pump pulse exceeds the critical power of self-focusing.20 Because of the short sample (1 mm), the calculated intensity rise via (weak) large-scale self-focusing is less than 1% and therefore not noticeable. The effect of smallscale self-focusing is experimentally not seen. For the preparation of the solutions the bidistilled deionized water was used (pH ) 6.8, conductivity 3.3 µS). Thy (Sigma) was dissolved as delivered. The absorbance of the Thy solution in the 1 mm sample cell was about 1 at 282 nm corresponding to a concentration of approximately 3.2 × 10-3 M. The sample cell was continuously moved during the measurements providing necessary stirring of the irradiated solution. CaF2 was used for the cell windows (thickness 1 mm). For every experimental run the pump intensity was measured replacing the Thy sample by neat water and observing the two-photon absorption peak of this medium. The conventional spectroscopic measurements were performed with a Perkin-Elmer spectrophotometer Lambda 19. All experiments were carried out at room temperature (292 K). 3. Results and Discussion 3.1. Measurements in Pure Water. 3.1.1. Two-Photon Absorption (TPA). Figure 1 demonstrates the induced absorption of the probe pulse with pump and probe wavelength at 282 nm. The observed kinetics consist of the peak and long

Figure 1. Induced absorption kinetics of neat water at a pump and probe wavelength of 282 nm and excitation intensity of I0 ≈ 215 GW/ cm2, sample length 1 mm. The thick line is numerically calculated; the thin line represents the calculated signal contribution of the particles generated by the pump pulse.

Figure 2. Energy transmission of neat water (sample length 1 mm; CaF2 windows) versus peak intensity I0 of the femtosecond UV pulse (tp ) 180 fs, λ ) 282 nm). From calculated curves (see eq 1) the two-photon absorption coefficient of water is estimated to be β ) 0.19 ( 0.05 cm/GW.

tail absorption. As pure water is highly transparent at 282 nm the observed peak has to be attributed to nonlinear absorption. The intensity dependence shown in Figure 2 strongly suggests a two-photon process.7 The small positive signal at longer delay times in Figure 1 indicates the generation of photoproducts (thin line). It should be noted that the TPA peak is the convolution between pump and probe pulse and can therefore be used for the determination of the laser pulse duration. The thick line is the numerically calculated signal (best fit) indicating a pulse duration of 180 ( 20 fs. Using the separately measured TPA coefficient (see below) the intensity of the pump pulse is simultaneously determined by the signal amplitude. It is interesting to note that a coherent coupling artifact is not observed in our investigation; the phenomenon is often seen in pump-probe measurements with same frequency of the two laser pulses21-23 and leads to a nonlinear amplitude increase of the transmitted probe pulse, in contrast to the amplitude decrease (induced absorption) of our TPA data of water. The expected quadratic intensity dependence of the coherence peak is also at variance to the measured linear dependence of the two-photon peak on pump intensity. It is concluded that the process is negligible in the intensity range 27-215 GW/cm2 used in our investigation. We have measured the TPA coefficient β of pure water (Figure 2) from intensity-dependent transmission measurements.19 The theoretical lines were calculated using the

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Reuther et al. TABLE 1: Relative Number of Hydrated Electrons Escaping Geminate Recombination in Pure Liquid Water at 80 ps Time Delay after the Excitation Pulse

Figure 3. Induced absorption kinetics for excitation at 282 nm (tp ) 180 fs, I0 ≈ 80 GW/cm2) and probing at 588 nm; (a, b) pure water sample and (c, d) concentrated aqueous thymine solution. The geminate recombination of the eaq- is measured to be large in the water sample on the long time scale (Figure b) but small in the Thy sample (Figure d); note the different time intervals of a, c compared to b, d.

expression

T(I,β) )

(1 - R)I(r,t) 1-R ∞ ∫ 2πr∫-∞∞1 + βl(1 - R)I(r,t) dr dt (1) E0 -∞

The used pulse shape I(r,t) is supported by the time-dependent and the pinhole transmission measurements:

I(r,t) ) I0 sech2(1.763t/tp)e-2r /r0 2

2

(2)

Comparing the experimental points with the theoretical lines, the TPA coefficient is determined to be β(282 nm) ) 0.19 ( 0.05 cm/GW. This value agrees satisfactorily with earlier picosecond data (λ ) 266 nm, tp ) 36 ps).7 In all experiments discussed below the TPA of the H2O sample was used as an internal standard of the pump intensity. The observed absorption kinetics for probing at 292 and 299 nm were similar to Figure 1 with a smaller amplitude of the TPA peak due to a lower TPA coefficient (data not shown). 3.1.2. Hydrated Electron Kinetics. Absorbing two light quanta at λ ) 282 nm a water molecule aquires an energy of 8.8 eV. This is significantly higher than the ionization and dissociation threshold values of pure liquid water (6.36-6.41 eV24,25 and 6.41-6.76 eV,26 respectively). This means that the water molecules excited by a 282 nm laser pulse may ionize or dissociate and the formation of different radicals and ions is expected, such as hydrated electrons e-aq, hydroxyl radicals OH•, hydrogen atoms H, and hydronium ions H3O+ (for details see refs 7, 11, and 12). The induced absorption at long delay times (td > 1 ps) in Figure 1 can be attributed to the above-mentioned particles. From all these particles only the hydrated electrons possess strong absorption in the visible part of the spectrum.27 We have studied the kinetics of hydrated electron formation and decay in pure liquid water at an excitation wavelength of 282 nm. The signal transient shown in Figure 3a,b consists of rapid formation via electron localization (ewet-) and hydration (eaq-)9

λpump (nm)

2pω (eV)

∆OD80ps/∆ODmax (%)

ref

312 310 282 248

7.9 8.0 8.8 10

50 54 70 83

10 11 this work 28

and slow decay in the subsequent 50-100 ps due to geminate recombination.10 The full line is the result of the numerical simulations (best fit) and consists of two different contributions: the signal of the wet electron (ewet-, dotted line) and of the hydrated electron (eaq-, dashed line). It was found previously that the fraction of hydrated electrons escaping from geminate recombination rises with increasing photon energy.28 Our experimental data on the relative number of hydrated electrons at 80 ps time delay after the excitation pulse agree well with the data of other authors10,11,28 listed in Table 1. The numerical analysis of the pure water data was performed by a rate equation model, taking into account the decrease of the pump pulse during its propagation through the sample due to TPA, the geminate recombination, and the different group velocities of pump and probe pulse. A detailed analysis of the water data will be published elsewhere. Here only the results necessary for the interpretation of the Thy measurements will be briefly discussed: Using the determined TPA coefficient of pure liquid water β ) 0.19 cm/GW, the measured peak intensity of the pump pulse I0 ) 80 GW/cm2 and the molar extinction coefficient of the nm ) 11150 cm-1 M-1,29 one can hydrated electron 588 aq estimate the quantum yield of hydrated electrons to be ΦeHaq2O- ) 0.11 ( 0.03. Hence the probability of hydrated electron formation from the excited water molecule at 8.8 eV excitation energy is ≈22%. Returning to the induced absorption depicted in Figure 1 with probing wavelength of 282 nm, it should be noted that the induced absorption at long delay times is determined by the absorption of the eaq-, H3O+ and OH• (the H atom does not absorb at 282 nm30). The hydronium ion is produced like the hydrated electron via the ionization of water; therefore, both particles should be generated with the same quantum yield. The quantum yield of the hydroxyl radical at our excitation energy of 8.8 eV can be estimated taking the literature value for the H 2O /ΦeHaq2O- ) 1.87 at excitation energy of 9.3 eV.7 ratio of ΦOH Using this ratio of quantum yields and the literature values for the molar extinction coefficients at 282 nm of eaq- ) 655 cm-1 M-1 and OH ) 237 cm-1 M-1,29,30 it is possible to estimate the molar extinction coefficient of the hydronium ion to be in the range 102-103 cm-1 M-1. 3.2. Measurements of Thy Solution. 3.2.1. Hydrated Electron Kinetics. In all experiments described below, we have used a concentrated aqueous solution of Thy (c ) 3.2 × 10-3 M, 1 mm CaF2 cell). Nevertheless due to the high pump pulse intensities, the TPA of water cannot be neglected. Performing the numerical simulations described below, we have evaluated the fraction of incident energy respectively absorbed by Thy and water as well as the transmitted intensity. The computations show that for our experimental conditions water absorbs 5-30% of the totally absorbed energy of the solution, depending on intensity. More detailed values are given in Table 2. The numbers clearly show that the solvent contribution has to be taken into account for a determination of the dynamics of the solute Thy. We have studied the kinetics of hydrated electron formation and decay in concentrated aqueous solution of Thy using 588

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TABLE 2: Relative Energy Amounts Absorbed by Water, rH2O, by Thy, rThy, and Transmitted Energy rtr for High-Intensity 282 nm Irradiation of a Concentrated Aqueous Thy Solution (OD ≈ 1, Length 1 mm) I0 (GW/cm2)

RThy (%)

RH2O (%)

Rtr (%)

25 80 210

85 78 67

6 15 28

9 7 5

nm pulses as a probe. The measurement is shown in Figure 3c,d. The corresponding measurement for neat water is shown in Figure 3a,b for comparison. The thick lines are the results of the numerical calculations (best fit) described below. It consists of four different contributions to the induced absorbance resulting from the different particles involved: the wet electrons generated via the ionization of water (dotted line) or Thy (dashed-dotted line) as well as the hydrated electrons from water (dashed line) and from Thy (thin line). Analogous to the water measurement the kinetics of the solution also shows the fast absorbance rise due to localization and hydration of the generated electrons (Figure 3 a,c), whereas in contrast to the water signal (Figure 3b) only a small decay during the first 80 ps takes place in the solution (Figure 3d). As can be seen from the numerical simulation this decay is fully described by the geminate recombination of that fraction of electrons generated via the ionization of solvent molecules. The reactions of the electrons should be fairly independent of the generation process. Therefore, we suggest that an effective and fast screening of the Thy cation radical and/or of the fast generation of photoproducts takes place. So the electrons produced via Thy ionization simply have no gemini at longer delay times (td > 1 ps), and the geminate recombination in this time interval is reduced. Figure 4a displays the intensity dependence of the quantum yield of hydrated electron formation from Thy in concentrated water solution versus peak intensity of the 282 nm pump pulse. The points were obtained using experimental values of induced absorbance at 588 nm probing wavelength minus calculated values of induced absorbance due to two-photon water ionization, i.e., only the solute contributions are depicted. The thin line is the numerical result using the values and the model given in section 3.3. The measured rise of the quantum yield with increasing intensity supports the two-step (biphotonic) excitation mechanism, the saturation intensity suggested to be Is > 60 GW/ cm2. This is in good agreement with the theoretical value for the transient saturation intensity of the two level transition: Is ) pω/σtp ≈ 100 GW/cm2 with the pulse duration tp and absorption cross section σ of the first absorption step. It should be noted that the two-step excitation of Thy proceeds via the singlet channel: S0 f S1 f SN. The yield of intersystem crossing for Thy at the experimental conditions used is very low (Φisc ) 1.5 × 10-3 at λ ) 282 nm) and thus the population of the triplet states can be neglected. In Figure 4b the calculated quantum yield of the two ionization channels, Thy and water, versus pump intensity is plotted. Whereas at low intensities the absorption of Thy and the generation of electrons via the two-step channel (thin line) is dominant, at intensities above 500 GW/cm2 more electrons are generated via the TPA of water (dashed line), since the Thy channel saturates (depletion of S0 molecules). 3.2.2. Induced Absorption Kinetics Probed in the UV Range. Figure 5b demonstrates the induced absorption kinetics of the concentrated Thy solution for a pump and probe wavelength of 282 nm. The induced TPA peak of water at the same conditions is shown in Figure 5a and represents the convolution between pump and probe pulse. The accumulation of the hydrated

Figure 4. Intensity dependence of the hydrated electron quantum yield of Thy with excitation at 282 nm. (a) Quantum yield of the electrons generated via the thymine ionization (solute contribution; experimental points, calculated curve). (b) Calculated quantum yields of hydrated electrons generated via thymine ionization (thin line) and via two-photon absorption water ionization (dashed line) are shown in a larger intensity range. Above 500 GW/cm2 the two-photon absorption from water is expected to exceed solute ionization.

Figure 5. Induced absorption kinetics of water (a) and of the concentrated aqueous solution of Thy (b, c) at 282 nm excitation wavelength (sample length 1 mm; CaF2 windows). (a) I0 ≈ 215 GW/ cm2, λprobe ) 282 nm; (b) I0 ≈ 215 GW/cm2, λprobe ) 282 nm; (c) I0 ≈ 70 GW/cm2, λprobe ) 588 nm.

electrons in accordance with the time evolution of the pump laser pulse is depicted in Figure 5c (similar to 3c). The numerical calculation (best fit) is represented by the thick lines in Figure 5. It can be seen from the comparison of Figure 5a,b that the main peak of the Thy-induced absorption kinetics at 282 nm disappears with a fast time constant. As the ionization threshold of Thy is exceeded by more than 2 eV15 after the absorption of two pump light quanta, we assume that the main

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Figure 7. Energy level scheme of the model used for the numerical simulations of the Thy data. See text for further explanation.

Figure 6. Induced absorption kinetics of concentrated aqueous thymine solution (sample length 1 mm; CaF2 windows) at 282 nm excitation wavelength and various UV probing wavelengths: (a) I0 ) 215 GW/ cm2, λprobe ) 282 nm; (b) I0 ) 90 GW/cm2, λprobe ) 292 nm; (c) I0 ) 70 GW/cm2, λprobe ) 299 nm.

peak around zero delay is due to the absorption of occupied higher excited singlet states as well as due to the absorption from the generated electron-cation pair (Thy+;e-) and the water TPA. The fast decrease of the absorption peak can be explained by relaxation processes and/or the generation of photoproducts via the Thy cation radical. This assumption is supported by the missing geminate recombination of the electrons generated by Thy ionization (Figure 5c), discussed in context with Figure 3. It should be noted that the reactions of the Thy cation radical with surrounding water molecules leading to its deprotonation (Thy+ f Thy•(-H)) and/or hydration (Thy+ f Thy•(OH)) were discussed earlier in the literature (see for example ref 31). The change of induced absorption kinetics with increasing probe wavelength (282-299 nm) is depicted in Figure 6. The lines again represent the result of the numerical calculations (best fit). The decline of the maximum signal amplitude in Figure 5a-c is caused both by the lowered pump intensities and the smaller TPA coefficient of water at the longer wavelength.7 In contrast to the changing induced absorption kinetics with rising probe wavelength, the shape of the signal transient is nearly independent of the pump intensity (see Figure 8). 3.3. Numerical Simulations. Numerical simulations were performed based on the energy level scheme depicted in Figure 7. The details of the system of differential equations are given in ref 32; a shorter version of the description can be obtained on request. The absorption of the pump pulse leads to the population of the excited singlet states S1 and SN via two-step pω pω absorption (S0 98 S1 98 SN)6,15 (see also Figure 4a). The energy of the two absorbed photons (2pω ) 8.8 eV) exceeds the ionization threshold of Thy by about 2 eV,15 so it can be assumed that the molecule in the SN state will ionize with a high probability and a fast time constant τion. This assumption is supported by the detected solvated electrons (Figures 5c and 3c,d). The absorption of pump photons from the molecule in pω the SN state leads to a much higher excess energy (SN 98 SN′, not shown in Figure 7); therefore the created state SN′ will ionize

quasi-instantaneously, and no population of the intermediate state (SN′) will be seen. For particle number conservation the absorption of a pump photon by the SN state is numerically treated as an induced transition from the SN state to the electron-cation pair: (Thy+;e-). The branching ratio for the geminate recombination of the electron-cation pair is denoted by η, while the probability for the generation of photoproducts of thymine Thyphot+ is given by (1 - η). The lifetime of the (Thy+;e-) pair is named τpair. Related to the generation of Thy photoproducts is the production of solvated electrons eaq- via the intermediate state ewet- in analogy to the creation of solvated electrons in water.8-10,33,34 The hydration time of the wet electron is taken to be the same as measured in neat water, τhyd ) 540 ( 80 fs. In contrast to the behavior of the eaq- in water10,11 a geminate recombination of the eaq- generated via the ionization of Thy is experimentally not seen (see for example Figure 3), the reason is proposed to be the missing gemini at this time scale as discussed in context with Figure 3. In our model (Figure 7) the geminate recombination of the (Thy+;e-) pairs ends in the vibronic ground state of the S1. Intermediate states during this process (e.g., higher excited singlet states) are assumed to be short-lived and, therefore, not notably occupied. The S1 state relaxes with the lifetime τ1 to vibrational excited states Shot 0 of the electronic ground state S0. From here the hot molecules undergo an energy redistribution process with time constant τvib leading to a thermal equilibration of the vibrationally excited states. Our model does not differentiate intra- and intermolecular contributions to the cooling process. In the literature intramolecular redistribution times as fast as 1 or 2 ps are reported.35-37 Literature values for the time constants of the energy equilibration between excited molecules and the surrounding solvent are in the range of a few picoseconds.38 Due to the TPA of water7 discussed above, the contribution of the solvent dynamics to the signal must be taken into account. Before each measurement of the Thy solution a water sample was probed as a reference to determine the pump pulse intensity and pulse duration. The numerical description of water after UV excitation and the used parameters for the numerical simulation of the solvent will be published elsewhere. The different absorption mechanisms of water (TPA) and Thy (two-step absorption) require the proper evaluation of the amplitude of the pump pulse during its propagation through the sample; the different group velocities between pump and probe pulses are also taken into account in our computations. An important test of the model derived is the satisfactory description of the intensity dependence of the induced absorption. Figure 8 represents experimental data of the Thy solution with pump and probe pulses at the same wavelength of 282 nm. The intensity of the pump pulse was varied over nearly 1 order of magnitude from about 27 (a) to 215 GW/cm2 (c). The full lines are the results of the numerical simulations with the set of parameters given in Tables 3 and 4. It can be seen that

Primary Photochemical Processes in Thymine

Figure 8. Intensity-dependent measurements of thymine at pump and probe wavelength of 282 nm. The thick lines are the results of the numerical simulation with the parameter values given in Table 3 (relaxation times) and Table 4 (absorption cross sections). The signal shape is independent of the excitation intensity with the exception of the signal amplitude at long delay times (td > 8 ps); the latter is negative at low intensity (a) but positive at high intensity (c).

the typical form of the signal transient with a large peak at zero delay and a second peak at about 1.5 ps delay time is nearly independent of the pump pulse intensity. Attention should be paid to the signal at long delay times (td > 8 ps). The excitation with high pump pulse intensities leads to an induced absorption at long delay times (positive signal, Figure 8c), whereas at low pump intensity a reduced absorption is measured (negative signal, Figure 8a). The reason for this intensity behavior must have its origin in different excitation processes for the states involved: the state providing the reduced absorption of the sample and the state increasing the probe absorption. We propose that the heating of the system (thermal equilibration between the relaxing Thy molecule and the surrounding solvent) reduces the absorption. As will be seen later, nearly every photon absorbed by Thy leads to a heating of the surrounding solvent, so this process can be treated as linearly proportional to the pump intensity and dominates at low intensities. Consequently, the increased absorbance with rising pump intensity must result from higher order excitation, with the formation of photoproducts from the Thy ionization. The ionization involves the absorption of at least two pump photons and is therefore proportional to In with n g 2. The contributions of the different states described above to the measured induced absorption kinetics is shown in Figure 9 (same experimental data as in Figure 8c). A pump-and-probe wavelength of 282 nm is used. The thick line indicates the sum of the individual contributions of the different states (best fit). Figure 9a shows the signal around zero delay time. The dominant signal originates from the TPA of water (thin solid line). The small shift of the signal maximum is due to a stepwise occupation of the higher singlet state S1 (dasheddotted line) and SN (dotted line) as well as the fast generation of the electron-cation pair: (Thy+;e-) (dashed line). The time constant τion can be estimated to be less than 60 fs. The lifetime of the electron-cation pair we can determine from the rapid decay of the first peak to be τpair ) 120 ( 50 fs. The coefficient η can be estimated from different measurements probing with

J. Phys. Chem., Vol. 100, No. 13, 1996 5575

Figure 9. Different signal contributions of the Thy solution for a pump and probe wavelength of 282 nm and at high intensity I0 ) 215 GW/ cm2; experimental points, calculated curves: (a) around zero time delay; (b) for a larger time interval.

ultraviolet and visible pulses (see for example Figure 3), yielding η ) 0.73 ( 0.08. This value indicates that, although the absorbed photon energy exceeds the ionization threshold15 by more than 2 eV, the majority of the electron-cation pairs undergo geminate recombination. In Figure 9b the molecular dynamics are shown on a longer time scale. The thin line in Figure 9b indicates the signal contribution of the photoproducts from the water TPA. The occupation of the Thy S1 state (dashed-dotted line) gives a negative signal contribution; therefore, the effective cross section of the S1 state (absorption minus stimulated emission), σ1 ) σ1N - σ01, is less than that of the electronic ground state: σ0 ) σ01. In other words σ1N < 2σ01. The negative contribution resulting from the occupation of the S1-state is necessary for the description of the intensity-independent dip between the first and the second maximum. The state providing the second maximum must be occupied indirectly via the S1 state. We propose this state to be the hot S0 state (Shot 0 , dashed line). Assuming the opposite, i.e., a positive signal contribution of the S1 state as should be expected after ref 15: In this case the numerical simulations display a strong intensity dependence of the shape of the signal curve in contradiction to the experiments. This finding presents strong evidence that the S1 state occupation reduces the absorption of the sample, i.e., dominant bleaching of the ground-state transition compared to excited-state absorption. The next important point is the physical interpretation of these states: The excitation wavelength of 282 nm is in the range of the 00′ transition at about 290 nm,3 so only low-lying vibronic states of the S1 are excited and no vibrational redistribution of energy in the S1 should be seen. From the vibrational ground state of the S1, the internal conversion to high-lying vibrational excited states in the electronic ground state, designed as Shot 0 , will take place. Due to the low fluorescence quantum yield, which is in the range of 10-4,3,4,39 the probability for this transition is high. Because of the vibrational energy of Shot 0 the probe transition starting from Shot leads to higher terminating 0 states toward the maximum of the S0 f S1 absorption band. Therefore the occupation of these vibrational excited states is expected to generate induced absorption with a cross section

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TABLE 3: Time Constants (ps) following from the Model Discussed τ1

τvib

τheat

τion

τpair

1.2 ( 0.2

1.3 (0.2

≈2

0.03 ( 0.03

0.12 ( 0.05

σS0hot > σ0. The time constant τ1 for the transition from S1 to Shot 0 can be estimated from the signal transients to be 1.2 ( 0.2 ps. The temporal evolution of the Shot 0 -state population (dashed line) is dominated by two time constants of nearly the same magnitude and is mainly responsible for the second maximum at tD ≈ 1.5 ps. The rise is described by τ1, whereas the decrease is due to the vibrational energy redistribution time τvib ) 1.3 ( 0.2 ps. The dashed line shows the contribution of the photoproducts generated from Thy ionization (Thyphot+, ewet-, and eaq-). On the time scale of the experiments performed the products are stable. The equilibration of energy between Thy and the surrounding solvent is performed via an intermolecular energy transfer from the excited Thy molecule to water. This heating can cause a broadening of the absorption band. Measurements of the smallsignal spectrum of a hot Thy solution (360 K, measurement not shown) give evidence that this process leads to a reduced absorption of the sample at the probing wavelength of 282 nm. At wavelengths longer than 285 nm, an increased absorption is expected for the hot solution. As discussed above, this effect should depend approximately linearly on the number of photons absorbed by Thy, since the probability for ionization (1 - η) is rather low and so nearly all photons absorbed will give a heating of the surrounding. This energy transfer to water in principle can take place during all excited states of Thy as discussed by other authors.15,40 The time constant for the heating is about 2 ps (inscribed thin line in Figure 9b). The time constants derived from the fitting of the numerical simulation to the experimental data are listed in Table 3. Figure 10 shows measurements with probing at 292 nm (Figure 10a) and at 299 nm (Figure 10b). The thick line again represents the sum of the various contributions. The calculated signal contribution of the water TPA is not shown for clarity. Similar to 282 nm probing, the shape of the signal curve is independent of the pump intensity (measurements not shown). The peak at zero delay again is mainly caused by the water TPA. Corresponding to the decreasing TPA coefficient of water with increasing wavelength7 the maximum signal is reduced. The contributions of the SN state (dotted line) and of the electron-cation pair (dashed line) are only small due to the low intensity used in the measurements shown (≈80 GW/cm2) and smaller absorption cross sections. The S0 absorption strongly decreases with increasing wavelength and the condition for a net absorption increase resulting from the occupation of the S1 state (dashed-dotted line) is fulfilled: σ1 > σ0. Consequently the S1 state occupation leads to an induced absorption and the dip seen in the probe measurements at 282 nm disappears. The relative signal contribution of the S1 state increases with increasing wavelength and dominates the time evolution of the signal together with the occupation of the state (thin line). The signal at long delay time again Shot 0 results from the generated photoproducts of Thy (Thy+, longdashed line) and of water (not shown). The influence of the solvent heating as discussed above is expected to give a positive signal but is no more resolved at these probing wavelengths. The absorption cross sections of the states involved in the dynamics are listed in Table 4 for the different probing wavelengths. As a simplifying assumption for the orientational distribution the numberical calculations were performed for

Figure 10. Measurements of Probing Wavelength of 292 nm (a) and 299 nm (b) similar to Figure 6 but I0 ≈ 80 GW/cm2 (λpump ) 282 nm). The thick line represents the sum of the calculated contributions.

TABLE 4: Absorption Cross-Section Values (10-17 cm2) Determined by Comparison of Numerical Calculations with the Experimental Data (Parallel Orientation of the Thy Molecules) σ0 σ1 σN σ(Thy+;e-) σS0hot σThyPhot +

282 nm

292 nm

299 nm

3.6 3.5 ( 0.2 ≈70 ≈9.3 5 ( 0.4 5.6 ( 0.5

0.68 1.7 ( 0.5 ≈7 7 ( 1.5 2.8 ( 0.7 1.6 ( 0.2

0.18 1.7 ( 0.5 ≈7 2.5 ( 1 1.3 ( 0.4 ≈1

parallel orientation of the molecules; as a consequence the values of σ are 3 times the values of the randomly oriented sample obtained from the conventional (small-signal) spectrometer; correspondingly, the effective number density for the Thy molecules is reduced in the computation by a factor of 3 (1/3 oriented parallel, 2/3 oriented perpendicular ) dark molecules). At intensities in the range of or above the saturation intensity (Is ≈ 100 GW/cm2), these assumptions will influence especially the depletion of the states involved. As explained above for the description of the intensityindependent second maximum seen at the probing wavelength of 282 nm the indirect occupation of the state giving the induced absorption (as we assume the Shot 0 state) via the first excited state is necessary. As an alternative explanation a photoinduced tautomer of Thy was considered. There are different tautomers of Thy known in the literature41,42 and the reported time constants for photoinduced proton transfer are in the range of the time constant determined here for the S1 f Shot 0 transition.43-45 But the absorption spectrum of the tautomer is redshifted41 in contradiction to the spectrum of the species observed here, so that this model was not pursued any longer. 4. Conclusions We have investigated the primary photochemical processes in pure neat water and in concentrated aqueous solution of Thysone of the DNA basesswith femtosecond pulses as a function of excitation intensity and at various probing wavelengths. Studying water, we have measured its TPA coefficient and investigated the kinetics of hydrated electron formation and decay with probing at 282 and 588 nm. We have observed a

Primary Photochemical Processes in Thymine slow decay of eaq- due to geminate recombination in the first 80 ps after excitation with 8.8 eV energy, which agrees very well with other literature data. We have measured the quantum yield of eaq- formation and estimated the molar extinction coefficient of the hydronium ion H3O+ at 282 nm. The induced absorption kinetics of an aqueous Thy solution were studied after 180 fs high-intensity excitation at 282 nm and with probing at 282, 292, 299, and 588 nm. We have found that the main peak of the induced absorbance kinetics can be attributed to the occupation of higher excited singlet states and the generation of Thy electron-cation pairs as well as the TPA of water. The observed shoulder in the kinetics is mainly due to S1 f S0 relaxation with vibrational redistribution and energy equilibration processes involved. The transformations of the Thy cation radical as geminate recombination and the generation of primary photoproducts takes place on a very fast time scale,