Fluorescence polarity dependence

To perform structural research on a food stuff into which a colorant is incorporated, special properties of fluorescing molecules are exploited fluorescence efficiency, fluorescence lifetime, fluorescence quenching, radiationless energy (Foerster) transfer, stationary or time-dependent fluorescence polarization and depolarization." Generally, if food colorants fluoresce, they allow very sensitive investigations which in most cases cannot be surpassed by other methods. 

Figure 1. Schematic diagram of polarization-dependent total-reflection fluorescence XAFS.

Karpovich DS, Blanchard GJ (1995) Relating the polarity dependent fluorescence response of pyrene to vibronic coupling. Achieving a fundamental understanding of the py polarity scale. J Phys Chem 99 3951-3958... 

Additionally, note that the polarity of the solvent significantly affects not only the positions of absorption and fluorescence spectra but also the fluorescence quantum yields. The largest difference in quantum yield is observed for G19 (eight times larger in toluene) [86]. The effect of solvent polarity on quantum yield and fluorescence lifetime was investigated in mixtures of toluene and ACN (polarity range 0.013-0.306). Polarity dependent quantum yield and lifetime measurements are presented in Fig. 22. 

Polarization effects The transmission efficiency of a monochromator depends on the polarization of light. This can easily be demonstrated by placing a polarizer between the sample and the emission monochromator it is observed that the position and shape of the fluorescence spectrum may significantly depend on the orientation of the polarizer. Consequently, the observed fluorescence intensity depends on the polarization of the emitted fluorescence, i.e. on the relative contribution of the vertically and horizontally polarized components. This problem can be circumvented in the following way. 

Let Ix, Iy and Iz be the intensity components of the fluorescence, respectively (Figure 6.3). If no polarizer is placed between the sample and the emission monochromator, the light intensity viewed by the monochromator is Iz + Iy, which is not proportional to the total fluorescence intensity (Ix + Iy + Iz). Moreover, the transmission efficiency of the monochromator depends on the polarization of the incident light and is thus not the same for Iz and Iy. To get a response proportional to the total fluorescence intensity, independently of the fluorescence polarization, polarizers must be used under magic angle conditions (see appendix, p. 196) a polarizer is introduced between the excitation monochromator and the sample and... 

Moreover, it is easy to show that, if the emission is observed without a polarizer, an excitation polarizer must be set at 0 = 35.3° (cos2 6 = 2/3). This arrangement is suitable when the fluorescence is detected through an optical filter (to reject scattering light) and not through a monochromator, because of the polarization dependence of the transmission efficiency of the latter. 

Fig. 7.8. Fluorescence spectra of pyrene in hexane, n-butanol, methanol and acetonitrile showing the polarity dependence of vibronic band intensities (excitation wavelength 310 nm) (reproduced with permission from Kalyanasun-daran and Thomas, 1977b).

The concept of polarity covers all types of solute-solvent interactions (including hydrogen bonding). Therefore, polarity cannot be characterized by a single parameter. Erroneous interpretation may arise from misunderstandings of basic phenomena. For example, a polarity-dependent probe does not unequivocally indicate a hydrophobic environment whenever a blue-shift of the fluorescence spectrum is observed. It should be emphasized again that solvent (or microenvironment) relaxation should be completed during the lifetime of the excited state for a correct interpretation of the shift in the fluorescence spectrum in terms of polarity. 

Karpovich D. S. and Blanchard G. J. (1995) Relating the Polarity-Dependent Fluorescence Response of Pyrene to Vibronic Coupling. Achieving a Fundamental Understanding of the Py Polarity Scale,... 

In conclusion, the method of intramolecular excimer formation is rapid and convenient, but the above discussion has shown that great care is needed for a reliable interpretation of the experimental results. In some cases it has been demonstrated that the results in terms of equivalent microviscosity are consistent with those obtained by the fluorescence polarization method (described in Section 8.5), but this is not a general rule. Nevertheless, the relative changes in fluidity and local dynamics upon an external perturbation are less dependent on the probe, and useful applications to the study of temperature or pressure effects have been reported. 

The choice of method depends on the system to be investigated. The methods of intermolecular quenching and intermolecular excimer formation are not recommended for probing fluidity of microheterogeneous media because of possible perturbation of the translational diffusion process. The methods of intramolecular excimer formation and molecular rotors are convenient and rapid, but the time-resolved fluorescence polarization technique provides much more detailed information, including the order of an anisotropic medium. 

MEGX is readily detected by HPLC and fluorescence polarization immunoassay techniques [14,21,25,40,41]. The test is simple, normally requiring a onetime blood sampling, and informative because it depends on the capacity of the hepatic enzymes to metabolize lidocaine. While the analysis of lidocaine metabolites is rapid, this method has not been adapted for continuous hepatic function monitoring, which may be possible with the radiolabeled analogues such as Tc-Sn-lidocaine iminodiacetic acid [42]. 

This problem does not exist with time-dependent fluorescence polarization measurements where the decay of the emission anisotropy r(t) is obtained by determining the decay of Iz and Ix according to eq 12. 

The fluorescence polarization immunoassay is used for routine, automated immunoassay of small molecules, such as drugs. It depends on the principle that a fluorophore attached to a macromolecule such as an antibody is not free to rotate in solution. If polarized light is used to stimulate the fluorophore to fluoresce, emission from the bound fluorophore (attached to the antibody, which is bound to a surface) will continue to be polarized, but polarization will be lost from free fluorophore (111). 

Early work on peridinin demonstrated that its structure leads to breaking of the idealized C2h symmetry resulting in relatively strong fluorescence from the Si state [16], Recent studies demonstrated that the intensity of the peridinin Si emission depends on solvent polarity [8,9], and time-resolved studies revealed that the polarity-dependent change in the Si emission yield... 

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