Polarization fluorometry

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

An indication of the potential sensitivity of fluorometry is that single-molecule detection has been based almost exclusively on the use of fluorescent labeled compounds. In addition, fluorometric determinations can combine several parameters simultaneously ( multidimensional techniques), such as excitation and emission wavelengths, fluorescence lifetime, and polarization, providing additional specificity and versatility to the analytical measurements. 

The absorption of light is proportional to the scalar product of the incident electric field and of a molecular vector named the transition moment. Thus, excitation of an isotropic population of fluorescent species by polarized light generally creates a temporary anisotropic population of excited molecules. Molecular motions progressively destroy this anisotropy, and affect the polarization of the reemitted fluorescence light which can be studied using pulse fluorometry techniques. 

Alumina (Chromarods A) was as good as silica gel (Chromarods S 111) for the separation of neutral lipids, but silica gel produced better separation and higher FID response for polar lipids Chromarods S-III (silica gel-coated quartz rods) were used to quantify neutral and polar lipids in cooked beef Nile red solution used for detection of lipids densitometry by reflectance fluorometry detection limit of assay was 25-100 ng for each lipid... 

Ishizaka, S., H. B. Kim, and N. Kitamura, Time-resolved total internal reflection fluorometry study on polarity at a Uquid/Uquid interface. Anal Chem, Vol. 73, (2001) p. 2421. 

Fluorescence measurements and detection can either be made under steady-state or time-resolved conditions. Some of the commonly used measurement techniques focus on changes in optical properties such as fluorescence intensity quenching, phase fluorometry (lifetime), polarization, surface plasmon resonance (SPR), and evanescent waves. Here, we will present detector systems based for (a) fluorescence intensity quenching and (b) phase fluorometry in detail. A few example references of integrated optical sensor systems based on the various optical measurement techniques are given in Table 1 and the reader is encourage to review those papers if more details are desired. 

This ratio is an indication of the structural order of the fluid matrix, and for an isotropic fluid it should be close to unity [4]. In order to differentiate between anisotropy of the medium and that of the molecular rotations, one should compare the V vs. To plots (determined from the steady-state anisotropy measurements at low temperatures in high density fluids at different excitation wavelengths) for a particular fluorophore embedded in an isotropic medium to a similar plot for the probe embedded in the test fluid. If, for example, a strong dependence of v on the value of ro is evident for both media and the ratio of in-plane to out-of-plane rotational rates is very high (>10), one can conclude that the rotations are anisotropic but the medium is isotropic. The technique of differential polarized phase fluorometry [8,12], which is beyond the scope of this chapter, has been successfully applied to study the types of rotations displayed by fluorophores embedded in different media. 

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