Decomposition of Transient Absorption Spectra

A decomposition of the transient absorption signal for RF in DMSO and FAD in water (400 and 440 nm excitation, respectively) into excited-state absorption (ESA), bleach (BL), and stimulated emission (SE) is achieved with the information from fluorescence upconversion measurements; results are shown in Figure 6.17.The following assumptions are made:

(i) The evolution of the bleach can be monitored best at the transient absorption minimum (446 nm for FAD in water and 440 nm for RF in DMSO); normalized time traces are shown in Figure 6.17, right. Smooth curves without oscillations and coherent contribution are obtained from multi-exponential fits. Bleach spectra are modelled by multiplying the ground-state absorption spectrum with the amplitude of the previously fitted trace at each delay time.

(ii) Stimulated emission spectra σSE are taken from corresponding fluorescence up-conversion measurements. Figure 6.18 demonstrates that the fluorescence upcon-version traces reproduce the changes of induced absorption at wavelengths where ESA is small. Note that for RF in DMSO even the wavepacket oscillation with

∼100 cm⁻¹is observed in both experiments. The evolution of the stimulated emis-sion signal is approximated by a global exponential fit of all individual time traces.

For RF in DMSO a damped oscillation with ν = 102 cm⁻¹ and a damping time γ = 0.5 ps is included, see Figure 6.18. For FAD in water, the apparatus function was significantly longer for fluorescence upconversion (0.35 ps fwhm) compared to transient absorption (0.05 ps). In order to correct for this difference, the ob-tained fit was re-convoluted with a Gaussian of 0.05 ps width (fwhm), Figure 6.18, dashed red. The fit of the evolution was then used to correct transient absorption for the contribution of stimulated emission, apropriately scaled according to the assumptions in (iv).

(iii) Coupling of the nπ^∗ state to the S₁(ππ^∗) state is assumed to be negligible for FAD in water (see Figure 6.15 and the associated discussion). Then, the oscillator strengths for S₁ ←S₀ absorption and S₁ →S₀ emission are the same at zero delay time. The total transition probability is proportional to the band integral, i.e.

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6.5 Discussion

Figure 6.17: Left: Decomposition of the transient absorption spectra of Figure 6.4 into bleach BL, stimulated emission SE, and excited-state absorption ESA. Right: Band integrals separated for BL (black), SE (blue), and ESA (green) (normalized) from the panels to the left.

the integrated band-shape function σ(ν)/ν. Since S₀–S₁ absorption and initial fluorescence have approximately mirror symmetry, bleach and stimulated emission are scaled to equal peak band-shape values at zero delay time. For RF in DMSO, the influence of solvation on the oscillator strength should be taken into account.

According to the model suggested in Figure 6.16,ππ^∗–nπ^∗coupling occurs at early delay times but diminishes due to solvation with τ₁ = 0.22 and τ₂ = 2.7 ps. It is proposed that the excited-state population does not change during this evolution.

This assumption is supported by the bleach trace in Figure 6.17. Consequently, for RF in DMSO, the fluorescence evolution is scaled such that stimulated emission and bleach bandshapes are of equal amplitude after solvation has completed. The relative scaling of bleach and stimulated emission thus corresponds to the situation shown in Figure 6.15.

(iv) The pure ESA at each delay time is obtained by subtracting the set of bleach and stimulated-emission spectra from the previous steps, scaled by a constant factor.

6 Excited Flavin: A Femtosecond Stimulated Raman Study

Figure 6.18: a and b: time-dependent transient absorption (black) and scaled fluores-cence cross-section (green) signals at single wavelengths. In red: multi-exponential fit to the fluorescence. Dashed red in b: the exponential sum from the fit of the FAD fluo-rescence, convoluted with a Gaussian of 0.05 ps width to match the apparatus function of the transient absorption experiment.

The absolute scaling is adjusted to the minimum factor that assures non-negative ESA signals at all delay times.

Comparing the transient absorption spectra of RF and FAD in water, it is found that the adenine moiety only causes an overall red-shift of 200 cm⁻¹. When the solvent is changed from water to DMSO, the UV absorption band shifts to the red from 359 to 368 nm, whereas the visible band blue-shifts from 516 to 500 nm. A hump at 302 nm is only observed with RF/DMSO. Additional measurements with excitation at 475 nm show an ESA band around 780 nm, Figure 6.3. For future use the excited-state absorption spectra at 1 ps delay are fitted with lognormal functions, equation (6.1); the optimal parameters are collected in Table 6.7. Spectra are scaled so that the bleach contribution has -0.058 optical density at the first absorption peak (this information may be used to estimate the ESA extinction scale).

Band integrals revisited. Now integration over the range 290–690 can be performed separately for the ESA, bleach and stimulated emission spectra; normalized results are compared in 6.17 c. For RF/DMSO the bleach integral (black) shows only minor change during the first 30 ps. Therefore, the decay of the transient absorption band integral in 6.4 (black) should not be associated with population return to the electronic ground state (in contrast to FAD). The ESA band integral, green in Figure 6.17, reproduces the temporal evolution of the bleach trace. This supports the previous view that the population in the excited state does not change on the time scale of solvation. Moreover it is concluded that dynamic solvation and initialππ^∗-nπ^∗ coupling do not affect the total oscillator strength of the excited state absorption in the range investigated, although bands may shift. The decay of the transient absorption band integral with 2.9 ps is

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6.5 Discussion

Table 6.7: Log-Normal fit to the ESA spectra from the decomposition of the transient absorption spectra of RF/DMSO (400 nm) and FAD/water (440 nm) at 1 ps delay.^a,b

(a) RF/DMSO, 400 nm pump in mOD. bSpectra were scaled to equal amplitude of the bleach contribution (-58.3 mOD for the maximum of the first absorption band). The log-normal descriptions are valid strictly only in the spectral region 287–690 nm, corresponding to the spectra in Figure 6.17. The range was extended to 1000 nm with excitation at 475 nm. Brackets indicate bands which are centered outside the observation window.

therefore ascribed solely to the solvent-induced rise of the stimulated emission (blue), as discussed in the previous section.

For FAD/water, Figure 6.17 c shows a drop of the stimulated-emission band integral below the traces for bleach and ESA on the 1 ps timescale; this evolution is associated with the 1.6 ps time constant from the previous analysis. Stimulated emission is sen-sitive to the drain of population from the S₁ state and to electronic changes, whereas the bleach reports on the return of population to the room-temperature equilibrium in S₀. A difference between the two band integral traces suggests the existence of addi-tional evolution after actinic excitation. The following processes should be considered:

dynamic solvation, electron-transfer, back-electron-transfer with recovery of the ground state, and vibrational cooling of the hot ground state. The signal may also be affected by intersystem crossing to the triplet state. Previous infrared experiments found a 1 ps rise and 9 ps decay in the spectral region of adenine ring vibrations, which were assigned to electron-transfer and back-transfer.^[196] Characteristic changes of excited-state absorp-tion are not observed; an involvement of electronic transiabsorp-tions like electron transfer or triplet formation is therefore not apparent. This suggests that states may be involved that are dark with respect to the S₁ →S₀ transition. Evidence for ππ^∗-nπ^∗ vibronic coupling is not found in water, but dynamic changes of the adenine moiety may also influence the electronic structure of the isoalloxazine ring.

6 Excited Flavin: A Femtosecond Stimulated Raman Study