Fluorescence factors affecting

Precision When the analyte s concentration is well above the detection limit, the relative standard deviation for fluorescence is usually 0.5-2%. The limiting instrumental factor affecting precision is the stability of the excitation source. The precision for phosphorescence is often limited by reproducibility in preparing samples for analysis, with relative standard deviations of 5-10% being common. 

Factors such as dissociation, association, or solvation, which result in deviation from the Beer-Lambert law, can be expected to have a similar effect in fluorescence. Any material that causes the intensity of fluorescence to be less than the expected value given by equation (2) is known as a quencher, and the effect is termed quenching it is normally caused by the presence of foreign ions or molecules. Fluorescence is affected by the pH of the solution, by the nature of the solvent, the concentration of the reagent which is added in the determination of inorganic ions, and, in some cases, by temperature. The time taken to reach the maximum intensity of fluorescence varies considerably with the reaction. 

Hebert, G.A., Pittman, B., and Cherry, W.B. (1967) Factors affecting the degree of nonspecific staining given by fluorescent isothiocyanate labeled globulins./. Immunol. 98, 1204—1212. 

The optimal enhancement effect is observed when the localized surface plasmon resonance is tuned to the emission wavelength of a locally situated fluorophore [86]. This is consistent with the model suggesting a greatly increased efficiency for energy transfer from fluorophores to surface plasmons [78]. Since resonance energy transfer is involved, the important factors affecting the intensity of fluorescence emission must also be the orientation of the dye dipole moments relative to the... 

Fluorescence polarization is the subject of Chapter 5. Factors affecting the polarization of fluorescence are described and it is shown how the measurement of emission anisotropy can provide information on fluidity and order parameters. 

Examples, as described by Eqs. (4.41), (4.42) and (4.43) show what kind of information one may obtain directly by registering oscillations in the fluorescence decay. These are the lifetime r = T-1 of the state, the factors affecting it, the precession frequency uij/ and, consequently, the value of the Lande factor gj>, as well as its sign (by the initial phase of oscillations), and, finally, the degree of polarization V. A favorable condition for registration should be the validity of T = 2tt/(Qu>ji) [Pg.135]

Figure l6-2 Excitation and emission spectra of a 0.00005% solution of sodium fluorescein in KH2PO4-K2HPO4 buffer at pH 8. (Reprinted with permission from Romanchuk KG. Fluorescein. Physiochemical factors affecting its fluorescence. Surv Ophthalmol 1982 26 269-283.)... 

Romanchuk K. Eluorescein physiochemical factors affecting its fluorescence. Surv Ophthalmol 1982 26 269-283. 

Another factor affecting the mapping pattern is the source design. Short-arc (xenon and metal halide) lamps can be considered point sources, which generate bulls-eye patterns whereas long-arc lamps (xenon and fluorescent) will produce oblong patterns. Low-resolution mapping tends to obscure these differences, which are real. How pertinent these results will be to an individual study will depend on a number of factors, one of which will be the samples physical size. 

Baunsgaard D, Factors affecting 3-way modelling (PARAFAC) of fluorescence landscapes, Internal report, Dept. Dairy and Food Science, The Royal Veterinary and Agricultural University, Denmark, 1999. 

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15 -cA-spheroidene, were studied by absorption, fluorescence, CD, fast transient absorption and EPR spectroscopies in solution and after incorporation into the RC of Rb. sphaeroides R-26.1. High performance liquid chromatography (HPLC) purification of the synthetic molecule reveal the presence of several Ai-cis geometric isomers in addition to the mono-c/x isomer of locked-15,15 -c/x-spheroidene. In solution, the absorption spectrum of the purified mono-cA sample was red-shifted and showed a large c/x-peak at 351 nm compared to unlocked all-spheroidene. Spectroscopic studies of the purified locked-15,15 -mono-c/x molecule in solution revealed a more stable manifold of excited states compared to the unlocked spheroidene. Molecular modeling and semi-empirical calculations revealed that geometric isomerization and structural factors affect the room temperature spectra. RCs of Rb. sphaeroides R-26.1 in which the locked-15,15 -c/x-spheroidene was incorporated showed no difference in either the spectroscopic properties or photochemistry compared to RCs in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 RCs which naturally contain spheroidene. The data indicate that the natural selection of a c/x-isomer of spheroidene for incorporation into native RCs of Rb. sphaeroides wild type strain 2.4.1 was probably more determined by the structure or assembly of the RC protein than by any special quality of the c/x-isomer of the carotenoid that would affect its ability to accept triplet energy from the primary donor or to carry out photoprotection. 

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