Small spectral shifts

The chromophore environment can affect the spectral position of the absorption and emission bands, the absorption and emission intensity (eM, r), and the fluorescence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices or hydrogen bonding. Changes in temperature typically result only in small spectral shifts, yet in considerable changes in the fluorescence quantum yield and lifetime. This sensitivity can be favorably exploited for the design of fluorescent sensors and probes [24, 51], though it can unfortunately also hamper quantification from simple measurements of fluorescence intensity [116], The latter can be, e.g., circumvented by ratiometric measurements [24, 115],... 

However for the tetraphenylporphyrin diacid the increase in conjugation resulting from interaction of the almost coplanar phenyl TT-systems with the porphyrin 7r-system results in a red shift of the visible bands ( ). For the individual atropisomers of H PF,THA the 4,0 isomer exhibits a net blue shift upon protonation while the diacid of the trans-2,2 isomer is significantly red shifted. Small spectral shifts are seen for the cis-2,2 and 3t1 isomers. This has in turn been interpreted to mean that in the excited singlet state "stabilities are shifted such that trans-2,2 > 3,1 > cis-2,2 > 4,0 for this diacid. A comparison of the spectral shifts observed upon protonation is given in Table IV. 

Simple transmission experiments to determine the spectral location of the LSPR with standard absorption spectrometers of random nanoparticles and ordered NP arrays suffer from a weak signal and a nonacceptable signal-to-noise ratio. This makes it very difficult to pick up the small spectral shifts of 10 0 nm in the LSPR spectral position, when the analyte binds to the recognition sites located on the nanoparticles [43]. Therefore, a combination of a spectrometer with a microscope is used to detect the LSPR in small NP arrays with a high signal-to-noise ration. 

In general, the absorption spectra of sensitizers bound to colloidal semiconductor films closely resemble those measured in fluid solution. In some cases small spectral shifts have been observed and attributed to Stark effects, acid-base chemistry or stabilization of the sensitizer excited states by the semiconductor surface. However, the effects are small, typically a few nanometers in the visible region. 

Recent progress in this field has been made by using high spectral resolution from light sources that rely on insertion devices, such as undulators. Small spectral shifts are found in the soft X-ray regime of molecular clusters that include vibrational resolution of excited valence states. 

More direct evidence for this postulate was secured by Givol et al. (135), who took advantage of a small spectral shift at 460 to 470 nm, which occurs in certain Dnp derivatives when bound to protein 315. They were able to compare the spectral shifts for covalently and noncovalently bound affinity labeling reagents because covalent attachment occurs very slowly at 4°C. Both for compounds BADE and BADL the spectral shift was virtually the same whether the binding was covalent or noncovalent. This was interpreted as indicating that the covalent bond could form with little if any displacement of hapten fi-om its normal position in the binding site of the protein. 

Under carefully controlled conditions, wavenumber measurements may be precise to 0.01 cm (discussed below), but usually only when a sample is left undisturbed in the sample compartment. Even if the sample is simply removed and reinserted between measurements, the repeatability is often worse than 0.01 cm . Several reasons can be advanced to explain why band shifts occur. First, the temperature of the sample may change between measurements, which leads to small spectral shifts. Second, it was noted in Section 2.6 that changes in the effective solid angle of the beam through the interferometer can lead to small wavenumber shifts. Because the cell may represent a field (Jacquinot) stop, if a cell is not placed in exactly the same position for successive measurements, bands will appear to shift from one measurement to the next. Furthermore, if the cell is slightly tilted and the angle changes appreciably from one measurement to the next, the beam may be refracted to a different position on the detector, which also shifts the wavenumber scale. Loose or insecure sample mounts should be avoided if users require the wavenumbers of absorption band maxima to be repeatable to better than 0.1 cm . ... 

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