Fluorescence indicator absorption

On emitting phases it is not possible to determine directly (in situ) the fluorescence and absorption spectra of compounds that absorb in the excitation range of luminescence indicators without distorting the measurement signal. 

Note In the case of HPTLC plates the detection limit for the visual recognition of the violet = 530 nm) colored chromatogram zones was 20 ng per chromatogram zone. With the exception of the two tetrahydrosteroids the cor-ticosteriods could be detected on TLC plates with fluorescent indicators by reason of fluorescence quenching (Fig. 1 A). Figure 2 illustrates the absorption scans of the separations illustrated in Figures 1A and 1B. 

Figure 1.1 The electiomagnetic spectrum, showing the different microscopic excitation sources and the spectroscopies related to the different spectral regions. XRF, X-Ray Fluorescence AEFS, Absorption Edge Fine Structure EXAFS, Extended X-ray Absorption Fine Structure NMR, Nuclear Magnetic Resonance EPR, Electron Paramagnetic Resonance. The shaded region indicates the optical range.

Visible and ultraviolet absorption and fluorescence spectra, obtained from alcoholic solutions of a commercially packed (out of the State of Florida) FCOJ, are presented in Fig. 9, 10, 11 and 12. Qualitatively, Fig. 9 reveals a lack of resolution in the visible absorption region and a well resolved peak at 280 nm. Comparison with Fig. 1 and 3 shows its absorption characteristics to be more similar to those of orange pulpwash in Fig. 3, indicating adulteration by pulpwash addition. Fluorescence excitation spectra (Fig. 10, 11) reveal well defined peaks at 270-75 nm, and Fig. 12 a shoulder at 270-75 nm. The spectra appear deformed when compared to Fig. 2 and 4 of pure orange juice. However, characteristics are similar to those obtained from pulpwash and very similar to those obtained from prepared model systems. Qualitatively both visible and ultraviolet absorption, and room temperature fluorescence indicate the presence of pulpwash in the FCOJ sample. The spectra are complementary. Absorption also did not indicate further adulteration by dilution which would have been denoted by weaker overall absorption and a shift at 227 nm to shorter wavelength. The sum of absorption at 443, 325 and 280 nm is 0.098 + 1.040 + 1.622 = 2.760 absorbance units. Florida State statute 20-64.07(l)(a) requires FCOJ to be 44.8° Brix which reconstituted to 12.8° Brix (16) and Federal standards, Section 52.2582(a), require 41.8° Brix, reconstituted to not less than 11.8° Brix (17). Therefore, the sum of absorbance is multiplied by the ratio of 12.8 to 11.8° Brix, with a corrected sum of 2.995. The sample absorbance ratio at 443/325 nm is 0.098/1.040 which is equal to 0.094. If the sum and natural log ratio values are substituted into the regression equation ... 

In bulk samples, X-ray yields need to be adjusted by the so-called ZAF correction. Here, Z represents the element number (heavier elements reduce the electron beam intensity more than lighter elements, because they are more efficient backscatterers), A indicates absorption (different elements have different cross-sections for X-ray absorption), and F indicates secondary fluorescence (the effect described above). Corrections are much less important when the sample is a film with a thickness of 1 pm or less, because secondary effects are largely reduced. The detection limit is set by the accuracy with which a signal can be distinguished from the Bremsstrahlung background. In practice, this corresponds to about 100 ppm for elements heavier than Mg. 

If no direct measurement of the fluorescence lifetime is available the relations between the radiative lifetime and the fluorescence and absorption spectra can be used in conjunction with the quantum yield to obtain an indication of the fluorescence lifetime. Birks and Munro (1967) have reviewed the methods of calculating the radiative lifetime. In general these methods are limited to specific groups of compounds. For example, Favaro et al. (1973) applied Stickler and Berg s (1962) formula to the spectral data obtained from an excited state acid-base study of some styrylpyridines and found a lack of quantitative agreement between the measured and calculated lifetimes. 

Quantum Yield Efficiency of fluorescence percentage of incident energy emitted after absorption. The higher the quantum yield, the greater the intensity of the fluorescence, luminescence, or phosphorescence. See Papp, S. and Vanderkooi, J.M., Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics, Photochem. Photobiol. 49, 775-784, 1989 Plasek, J. and Sigler, K Slow fluorescent indicators of membrane potential a survey of different approaches to probe response analysis, J. Photochem. Photobiol. 33, 101-124, 1996 Vladimirov, Y.A., Free radicals in primary photobiological processes, Membr. Cell Biol. 12, 645-663, 1998 Maeda, M., New label enzymes for bioluminescent enzyme immunoassay, J. Pharm. Biomed. Anal. 30, 1725-1734, 2003 Imahori, H., Porphyrin-fullerene linked systems as artificial photosynthetic mimics, Org. Biomol. Chem. 2, 1425-1433, 2004 Katerinopoulos, H.E., The coumarin moiety as chromophore of fluorescent ion indicators in biological systems, Curr. Pharm. Des. 10, 3835-3852, 2004. 

Acoustic levitation has also been used for microtitration with absorptive and fluorescent indicators. The addition of titrant was efficiently controlled via a piezoelectric micropump. This application testifies to the possibility of using this technique in routine laboratories where sample and (or) reagent availability may be a limiting factor [90]. 

It has been shown recently by Kapturkiewicz and co-workers [14] that the analysis of the CT absorption CT <— So and the radiative and radiationless charge recombination processes CT So (Figure 4) in selected D-A n-n interacting systems sterically hindered to coplanarity (such as 9-anthryl and 9-acridyl derivatives of aromatic amines [14a,b], carbazol-9-yl derivatives of aromatic nitriles [14c] and ketones [14d] and D-A derivatives of indoles [14e] or phenoxazines and phe-nothiazines [14f]) in terms of the theory of photoinduced ET processes in absorption [52, 53] and emission [53-55] and Mulliken and Murrell models of molecular CT complexes [56, 57] leads to the determination of the quantities relevant for the rate of the radiative ET processes (exemplified by the CT absorption and emission) and to the estimation of the electronic structure and molecular conformation of the states involved in the photoinduced ET. A similar approach can be applied to describe the properties of the fluorescent singlet CT states and phosphorescent triplet CT states [58]. It should be pointed out that the relatively large values of the electronic transition dipole moments of the CT fluorescence indicate a non-... 

In ethanol, the 31,850 cm maximum (A) is reduced to a shoulder near 33,000 cm. Whereas in heptane a normal "Weller shift" of fluorescence indicating Intramolecular proton transfer is observed, in alcohols we note a fluorescence band at higher frequency with a maximum at 23,800 cm. The latter is due to the anion (D) and corresponds to the absorption maximum of D at 29,400 cm The intensity of this emission increases with increasing pH and remains constant above pH = 9 (in ethanol). The anilide of 2-methoxy benzoic acid shows a ultraviolet fluorescence that was too weak to be recorded (21). Low quantum efficiency may, therefore, be the reason that the fluorescence of C has not been detected. The only evidence of the presence of C is the close similarity of the absorption spectra of SAN and the methoxy derivative in ethanol (21). 

Fluorescence of radical cation gives important information during various reaction processes. For example, distance between the ion pair can be estimated based on the Forster-type ENT theory under the condition where a pair of radical ions generated from the photoinduced ELT are fluorescent and there is a good spectral overlap of fluorescence and absorption for the pair. Here, we introduce an example in which two-color two-laser flash photolysis was employed to estimate the distance between TMB + and the radical anion of 1,4-dicyanonaphtha-lene (DCN-) [121], since TMB + shows fluorescence as indicated earlier [26]. 

W14. Weber, G., and Laurence, D. J. R., Fluorescent indicators of absorption in aqueous solution and on the solid phase. Biochem. J. 66, xxxi (1954). 

The dependence of fluorescence spectra and intensity of fluorescence of ionizable compounds on the pH of the solution enables them to he used as fluorescence indicators. The naphthyl sulphonic acids are typical examples. In the case of hydroxy and amino pyrene sulphonic acids, sharp changes in fluorescence do not occur at the same pH values as changes in absorption. This has been explained by Forster30 by considering the attainment of the ionic equilibrium appropriate to the excited state within its life time. The fluorescence of some purines and pyrimidines also depends on the acidity or alkalinity of the medium31. 

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