Fluorescence spectrum state detection

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

Direct observation of fluorescence from higher singlet states of benzene and some methyl derivatives has recently been achieved by Hirayama, Gregory, and Lipsky (247). Using apparatus capable of detecting fluorescence yields as low as 10 they recorded the emission spectra from oxygenated solutions of pure benzene and other aromatics excited at 184.9 nm. Subtraction of the tail of the residual S, emission gives a fluorescence spectrum with approximately 235 nm and = 8 x 10 for... 

Luminescence titrimetry has been developed chiefly for acid-base titrations. Therefore, fluorescence pH-indicators are now widely used. Their application is based on changes of fluorescence spectrum upon the addition of a proton or its loss. At present, over 200 fluorescence pH-indicators are available the structural formulae of the most the widely applied indicators are given in Table 8. Some of them (No. 2, 8, 9, 12, 16, 17, 23, 25 and 29) and also, primuline, tripaflavine, and rhodamine 6G are widely used as adsorption fluorescence indicators. The titration end point can be detected in this case because of the differences in of the indicator in the adsorbed state and in solution. Redox fluorescence indicators including rhodamines B and 6 G, 3,6-dihydroxy-phthalic acids, complexes of Ru(II) with 2,2 -dipyridyl or 1,10-phenanthroline and other... 

Di(1-pyieny1)propane. Bauer et al. (32) observed an excimer-like emission in the fluorescence spectrum of 1Py(3)1Py on dry silica, which was attributed to intramolecular interactions in the ground state. The excimer emission disappeared on adsorption of 1-decanol to the surface. In contrast, strong intramolecular excimer fluorescence was found by Avnir et al. for 1Py(3)1Py adsorbed on a silica surface with up to a double-layer equivalent of 1-octanol (38), see Section 4.3.2. Excimer formation was also detected on reversed-phase Si-C g and on untreated silica, the ratio I /I depending on the amount of 1Py(3)1Py adsorbed (38), see Fig. 3. 

With a glassy solution of poly-1-vinylnaphthalene, the delayed emission spectrum has been shown to consist of an emission having a mean lifetime of approximately 80 ms at the normal fluorescence wavelength, in addition to the phosphorescence having a mean lifetime of about 2 s [159]. The delayed fluorescence did not appear in the spectrum of 1-ethylnaphthalene. With the polymer it was found to be inhibited by piperylene, a well-known triplet quencher. These results have been explained by mutual annihilation of two excited triplet states produced by the absorption of two photons by the same polymer molecule. They are considered as strong evidence for migration of the excited triplet state in poly-1-vinylnaphthalene. In polyacenaphthalene, however, which is chemically very similar to poly-1-vinylnaphthalene (see p. 409), no delayed fluorescence could be detected in the same experimental conditions [155]. 

It has been shown [155,171] that the dependence of excimer emission intensity on acceptor concentration obeys the Stern—Volmer equation whether M or D is the donor, whereas a second-order equation is obtained if both types of excited state simultaneously act as donor. It seems that in poly-1-vinylnaphthalene and polyacenaphthalene films at room temperature, energy transfer to benzophenone occurs from M, although normal fluorescence cannot be detected in the emission spectrum of the polymers in these conditions [155]. Decay time measurements have shown that the excimers in solid polyvinylcarbazole are traps rather than intermediates in the energy transfer process [148]. With polystyrene, however, it has been clearly demonstrated that energy transfer to tetraphenylbutadiene occurs from both excimer and isolated excited chromophore [171]. 

A considerable improvement can be expected from simultaneous detection of the fluorescence spectrum and the fluorescence lifetime. Fluorescence lifetime detection not only adds an additional separation parameter but also yields direct information about the metabolic state and the microenvironment of the fluorophores [306, 308, 398, 417]. Fluorescence lifetimes of endogenous fluorophores and their dependence on the microenvironment are given in [282, 339, 452, 517]. 

X-ray absorption spectra are usually recorded as fluorescence excitation spectra. The limited resolution (150 eV) of the X-ray detectors has precluded obtaining useful information from the fluorescence spectra per se. However, the resolution of the fluorescence spectrum can be improved to about 1 eV by using energy resolving optics. The K jS X-ray emission spectra (3p-l5) of Mn complexes have shown that the spectrum is sensitive to the oxidation state and spin state of the metal. The potential for site-and spin-selective detection of X-ray absorption spectra in bioinorganic chemistry is considerable. 

While collision-induced transitions in excited electronic states can be monitored through the satellite lines in the fluorescence spectrum (Sect. 8.2.2), inelastic collisional transfer in electronic ground states of molecules can be studied by changes in the absorption spectrum. This technique is particularly advantageous if the radiative lifetimes of the investigated rotational-vibrational levels are so long that fluorescence detection fails because of intensity problems. 

The Ti state (but not the Si state) of toluene sensitizes the photoisomerization of both ( )- and (Z)-2-heptene. Direct irradiation of either isomer of 1-phenyl-2-butene leads to photoisomerization. The fluorescence spectrum of l-phenyl-2-butene is simitar in shape to that of toluene but is slightly reduced in intensity. However, phosphorescence from 1-pheny 1-2-butene was not detected under conditions in which phosphoresence from toluene could readily be observed. Propose an explanation for the photoisomerization of l-phenyl-2-butene upon direct irradiation that is consistent with these observations. 

In order to detect the influence of the state of the PS II acceptor side on the properties of the fluorescence spectrum, we have compared the spectra of the constant (Fo) and variable (F. ) fluorescence during the fluorescence increase process in fully photoactivated leaves. The F<> spectrum was measured during 20 ms at time 0.16 s after the onset of... 

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