Excitation spectra corrected

Montalti M, Credl A, Prod L, Gandolfi MT (2006), Handbook of photochemistry, 3rd edn. CRC Press, Boca Raton. An essential reference book containing data tables for a wide range of compounds, and a variety of reference materials including quantum yields, lifetimes, quenching rate constants, electrochemical potentials and solvent properties as well as information on standard procedures used in chemical actinometry, determination of emission and excitation spectra correction factors, and quantum yield measurements and also information on equipment such as lamps and filters. 

Usually, fluorescence spectra are uncorrected , i.e. they contain wavelength dependent sensitivities of monochromator and detector (emission spectra) or excitation source and monochromator (excitation spectra). Correction may not be necessary, if only relative changes of individual emission bands are measured. However, for getting correct spectral intensities 1f(A), a reference source with a well known spectrum S(A)... 

For excitation spectra, the accuracy of the correction procedure can be checked by comparing the corrected, normalized values with the absorbance spectrum (19) or determining If any peaks from the source are observed In the corrected excitation spectrum (23). 

Figure 1. Average corrected emission spectrum (- -) and excitation spectrum (- -) for quinine sulfate In 0.1 mol/L HC10 obtained during round-robin test with ten laboratories coefficient of variation at each wavelength (-t).

Since no experimental work is available to confront the theoretical model designed to describe C3H2 excited states correctly, test calculations had to be done in a preliminary step. For that purpose, we have chosen ethylene, for which extensive calculations of the vertical spectrum as well as experimental measures are available. It is well known indeed that a correct quantitative and even qualitative description of small rr-electronsystems, is still a challenge for theoretical chemistry. The difficulties are found at each step of the computational approach ... 

The values of ftot for various benzotriazole compounds in a range of solvents are listed in Table II. Values of the fluorescence quantum yield for TIN and TINS, corrected for the absorbance by their non-fluorescent, planar conformers at the excitation wavelength, are listed in Table III. In all the benzotriazole solutions examined, maximum fluorescence emission was observed at about 400 nm indicating that this emission originates from the non proton-transferred species. This was confirmed by examination of the fluorescence excitation spectrum which corresponds to the absorption spectrum of the non-planar form of the molecule. 

The variations in fluorescence intensity as a function of the excitation wavelength XE for a fixed observation wavelength represents the excitation spectrum. According to Eq. (3.20), these variations reflect the evolution of the product /0(FE)A(AE). If we can compensate for the wavelength dependence of the incident light (see Chapter 6), the sole term to be taken into consideration is A(AE), which represents the absorption spectrum. The corrected excitation spectrum is thus identical in... 

Fig. B5.2.1. Corrected excitation spectrum (broken line) and excitation polarization spectrum of indole in propylene glycol at -58 °C. The fluorescence is observed through a cut-off filter (Corning 7-39 filter) (reproduced with permission from Valeur and Weber3 ).

Steady-state method 2 comparison between the absorption spectrum and the excitation spectrum (through observation of the acceptor fluorescence) The corrected excitation spectrum is represented by... 

Figure 8.2 presents the fluorescence of pyrene on silica gel. The loading is low so that pyrene is predominantly adsorbed as nonaggregated monomers (Mi). The backward fluorescence spectrum Fb of this sample is very comparable to the spectrum in polar solvents and not distorted by reabsorption. However, the forward spectrum Ft is almost completely suppressed in the region of overlap with the o -transition and hot sidebands of the weak first absorption band Si. The absorption coefficients of the sample vary widely from k" = 0.1 cm 1 (Si-band, Aa = 350-370 nm) to k = 25 cm-1 (S2-band, 1 290-340 nm), and in a first approximation the excitation spectrum of Fh reflects this variation correctly (Figure 8.2, left). The Ff-excitation spectrum, however, has only little in common with the real absorption spectrum of the sample. 

The TRICKS-TDDFT intensities of [Fe(CN)6]3- are consistent with the experimental spectrum and the excitation energies are somewhat overestimated leading to a simulated MCD spectrum correct in form but blue shifted. 

Figure 23-13 (A) Corrected emission and excitation spectra of riboflavin tetrabutyrate in w-heptane. Concentration, about 0.4 mg I-1. Curve 1 excitation spectrum emission at 525 nm. Curve 2 emission spectrum excitation at 345 nm. FromKotaki and Yagi.128 (B) Indole in cyclohexane, T = 196 K. 1, Fluorescence excitation spectrum 2, fluorescence spectrum and 3, phosphorescence spectrum. From Konev.125...

Similarly, the instrumental excitation spectrum depends on the wavelength characteristics of the excitation monochromator and of the light source. Mercury arcs which emit line spectra are not suitable and xenon arcs are normally used. Correction is made against a reference such as a solution of Rhodamine which has a wavelength-independent fluorescence quantum yield. 

Figure 7.23 Absorption and excitation spectra of Rhodamine G in ethanol, (a) Corrected excitation (open circles) compared with absorption (full line), (b) Uncorrected excitation spectrum. Vertical axes, fluorescence intensity in arbitrary units...

The absolute excitation spectrum is then identical to the absorption spectrum. The correction factor can again be stored in a computer memory and used to multiply other instrumental excitation spectra (so long as the lamp is not changed). 

Instrumental excitation spectra are so badly distorted that they are practically useless. This results from the very sharp drop in light intensity of xenon arcs in the UV region (see Figure 7.23). The Figure shows the apparent (instrumental) excitation spectrum of Rhodamine compared with the absorption and corrected excitation spectra. 

In recent years, the first applications of DFT to excited electronic states of molecules have been reported. In the so-called time-dependent DFT (TDDFT) method, the excitation energies are obtained as the poles of the frequency-dependent polarizability tensor [29], Several applications of TDDFT with standard exchange correlation functionals have shown that this method can provide a qualitatively correct description of the electronic excitation spectrum, although errors of the order of 0.5 eV have to be expected for the vertical excitation energies. TDDFT generally fails for electronic states with pronounced charge transfer character. 

The excitation spectrum is technically perturbed by two problems the light intensity of the excitation lamp, which varies with the wavelength, and the intensity upon detection, which is also wavelength-dependent. Corrections can be performed using rhodamine B, dissolved in glycerol, as reference. In fact, radiation from rhodamine is proportional to the excitation intensity independently of the excitation wavelengths. Therefore, excitation of rhodamine will yield a fluorescence excitation spectrum that characterizes excitation lamp spectrum. In order to obtain the real fluorescence excitation spectrum of the studied fluorophore, the recorded excitation spectrum will be divided by the excitation spectrum obtained from rhodamine. This procedure is done automatically within the fluorometer. 

In general, when one wants to determine if global and/or local structural modifications have occurred within a protein, circular dichroism experiments are performed. Also, one can record the fluorescence excitation spectrum of the protein. If perturbations occur within the protein, one should observe excitation spectra that differ from one state to another. One should not forget to correct the recorded spectra for the inner filter effect. 

In the previous section we calculated the rearrangement of a molecular electron shell induced by / decay using the Hartree-Fock approximation, i.e., making no allowance for the electron correlation. In the present section we estimate the influence of electron correlation on the shape of the excitation spectrum. The account of the correlation by the Cl method allows one to make correct allowance for the double excitations, the calculation of which, within the one-determinant approximation, seems unreliable. The Cl method allows one to take into account all possible two-electron processes, such as those similar to the appearance of satellites in the X-ray photoelectron spectra. 

Excitation spectrum Plot of the spectral radiant exitance or of the spectral photon exitance against the frequency (or wavenumber, or wavelength) of excitation. When corrected for wavelength dependent variations in the excitation radiant power this is called a corrected excitation spectrum. 

Figure 20. Absorption (upper curve) and corrected fluorescence excitation (lower curve) spectra for carotenopyropheophorbide 27 in toluene. The spectra have been normalized over the 6(X)-630 nm region. The excitation spectrum is virtually identical in shape to the absorption spectrum of methyl pyropheophorbide-a, and singlet-singlet energy transfer from the carotenoid to the tetrapyrrole is therefore minimal (<7%).

The excitation spectrum(after corrected) of TBA has a shoulder near 34500 cm"1 which coincides with (n- Tt ) transition (Pig.8. As was shown by a few authors(3,9), the quantum yields of photocomposition of aromatic azide by the light of (n->Ti ) transition were lower than those by that of transition. The fact means that(n->7f) ... 

Hence maximum amplitude occurs when wt = 0 and zero when wt = ir with the phase varying from 0 to 7t/2. Since only hard pulses are used, all of the spectrum is equally excited however, there is a linear phase shift of the excitation spectrum. The amplitude modulation can be corrected for however, it results in the noise being unevenly scaled. ... 

Figure 2. Fluorescence spectra of PVCA cat and PCP. The emission intensities of polymer and dimeric model are not comparable the excitation spectrum is not corrected for equal quantum intensity excitation at 330 4 nm...

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