Femtosecond Stimulated Raman Spectra

Transient spectra R(ν) upon 776 nm Raman excitation are shown in Figures 6.6 a,b for RF/DMSO and FAD/H₂O. In both cases well-resolved, emissive, vibrational bands are seen, which decay with only minor spectral changes. The appearance of new bands is not observed. The time behavior is shown in Figure 6.6 c where the integral over the range 1100–1650 cm⁻¹ is considered. It is fitted by a sum of exponential functions;

optimal decay times and relative amplitudes are summarized in Table 6.1. It is noted

6 Excited Flavin: A Femtosecond Stimulated Raman Study

Table 6.1:Time constants from multi-exponential fits of the transient Raman evolution under different experimental conditions.^a

RF (DMSO) FAD (H₂O)

Raman excitation at 500 nm, 1.2 ps, 1 µJ 0.15 (0.34) 0.15^∗ (0.32) Raman excitation at 523 nm, 0.73 ps, 1 µJ

0.33 (0.16) 6.8^∗ (0.69) offset (0.15) Raman excitation at 776 nm, 2.7 ps, 3.7 µJ

0.84 (0.42) 1.2 (0.69)

7.8 (0.19) offset (0.58) offset (0.12)

aintegrated over the range 1100–1650 cm⁻¹. Time constants in ps, with the relative exponential amplitudes in brackets. Asterisks mark time constants that were fixed during the fit. Underlayed grey: evolution attributed to depletion by the Raman pulse. Raman pulse durations are given for transform-limited Gaussian pulses.

here already that the initial decay, until∼3 ps, is probably related to depletion of the S₁ state by the ps Raman pulse, as will be argued later.

Small spectral changes have molecular significance, though. Raman peaks shift slightly to lower frequencies on a 3 ps time scale, by∼1 cm⁻¹for RF/DMSO and∼2.5 cm⁻¹for FAD/water. A larger downshift is seen only for the 1579 cm⁻¹ band of FAD, albeit at low signal/noise. The shifts are accompanied by narrowing of the Raman bands, leading to better resolved spectra at longer delay times. For FAD/water this allows to identify peaks at 1328, 1377, 1406, and 1453 cm⁻¹, which are only recognized as shoulders in initial spectra.

Spectral comparison of different experiments (sample, solvents, actinic excitation wavelength) is best done at t=0, before each system relaxes along different pathways.

To generate the time-zero Raman spectrum global exponential analysis of the spectral evolution is employed. Two or three exponential functions were included, and the pa-rameters varied freely to obtain the best fit of the time-dependent spectra.

Time-zero spectra for measurements with 776 nm Raman excitation are shown in Fig-ure 6.7. Compared to RF/DMSO (black), the bands of FAD/H₂O (green) are observed

118

6.4 Experimental Results

Figure 6.6: (a,b) Excited-state stimulated Raman spectraR(ν) of flavin, from Raman pulses at 776 nm that are resonant with excited-state absorption only. (c) Time-traces of ^R R(ν)dν integrated over the range 1100–1650 cm⁻¹. Spectra were recorded with parallel polarizations, and nonresonant solvent signal has been subtracted. The asterisk in (a) indicates a dispersive solvent residuum at earliest delay times.

at higher frequencies by up to 21 cm⁻¹. Smaller shifts are observed for the bands at 1254 cm⁻¹, with a shoulder around 1280 and 1499 cm⁻¹. These changes are accompanied by a decrease in relative intensity for the bands at 1188, 1327, 1373, 1570, and 1637 cm⁻¹.

6 Excited Flavin: A Femtosecond Stimulated Raman Study

Figure 6.7: Excited-state stimulated Raman spectra R(ν) at zero delay time, from Raman pulses at 776 nm, as in Figure 6.6 a,b. Shown are extrapolations tot= 0 from a global multi-exponential fit of each evolution.

In heavy water (magenta) most of the FAD Raman signals are reproduced in position and relative amplitude. The strongest isotope effect is found for the 1206 cm⁻¹ band:

it splits in D₂O, giving rise to a weaker band with two maxima at 1135 and 1160 cm⁻¹ and a band at 1228 cm⁻¹. The position of the 1258 cm⁻¹ band is not affected by isotope substitution, but the band has slightly increased intensity in D₂O. Another change is found for the broad low-intensity band at 1640 cm⁻¹, which shifts to 1611 cm⁻¹in D₂O.

Ground-state spectraG(ν)upon 500 nm Raman excitation are shown in Figure 6.8 b.

Here Raman resonance with the first absorption band is used. Similar spectra are ob-tained upon pre-resonance at 523 nm (not shown). Compared to RF/DMSO (black), FAD/water (green) generally shows slightly up-shifted peak frequencies. Strong differ-ences are observed between 1100 and 1300 cm⁻¹: for FAD, bands around 1180 and 1230 cm⁻¹ have decreased intensity, and additional bands are found at 1256 and 1283 cm⁻¹. Transient spectraR(ν) upon 500–523 nm Raman excitationalso decay uniformly with only minor spectral changes, similar to what was observed above. Time-zero spectra are compared in Figure 6.8 a. They are dominated by negative features which resemble the ground-state Raman signal, and thus differ seriously from those which were observed in

120

6.4 Experimental Results

Figure 6.8: (a) Transient stimulated Raman spectra R(ν) of flavin after optical exci-tation, from Raman pulses at 500 and 523 nm. Now resonance originating from the S₁ state involves not only excited-state absorption but also stimulated emission. Shown are extrapolations to t=0 from a global multi-exponential fit of each evolution. Negative signal is largely due to the bleached S₀state. (b) Ground-state resonance Raman spectra G(ν). (c) Time-traces of^R |R(ν)|dν integrated over the range 1100-1650 cm⁻¹. Results for 523 nm Raman resonance have been scaled for better comparison.

6 Excited Flavin: A Femtosecond Stimulated Raman Study

the near-infrared. Positive (= emissive) contributions are found mainly around 540 cm⁻¹ and 1370 cm⁻¹. In contrast to excitation at 500 nm, the 523 nm Raman pulse is only pre-resonant with the S₁ ←S₀ transition. Spectra for FAD/H₂O under both resonance conditions are compared in Figure 6.8 a, and found to be identical within precision. The influence of various electronic transitions on the Raman signal was modelled in Section 2.4.2.

Time-dependent integrals are shown in Figure 6.8 c. SinceR(ν) now comprises positive and negative parts, the absolute values|R(ν)|are integregrated, as before over the range 1100–1650 cm⁻¹. Fort≥3 ps the results from transient absorption spectroscopy (Figure 6.4) are well reproduced. For example, with RF/DMSO only nanosecond decay of Raman intensity is found, whereas FAD exhibits again an additional loss on the 5–10 ps time scale.

6.5 Discussion

6.5.1 Assignment of FSRS Bands Based on Quantum Chemistry