Fluorescent quenching

Photodegradation as well as fluorescence quenching has been observed in chlorophyll monolayers [302,316]. Whitten [317] observed a substantial decrease in the area of mixed films of tripalmitin and a ci5-thioindigo dye as isomerization to the trti 5-thioindigo dye occurred on irradiation with UV light. 

There are two approaches to estimation of AG fThe first is an empirical approach (36) based on dynamics of fluorescence quenching of aromatic hydrocarbons ia acetonitrile solution. Accordingly,... 

NEW FLUORESCENCE QUENCHING METHOD FOR DETERMINATION OF COPPER (II) IN WATER... 

Fluorescence quenching methods wits ai omatic complexing reagents are often recommended for copper (II) determination in water. 

Physical methods Physical methods include photometric absorption and fluorescence and phosphorescence inhibition, which is wrongly referred to as fluorescence quenching [1], and the detection of radioactively labelled substances by means of autoradiographic techniques, scintillation procedures or other radiometric methods. These methods are nondestructive (Chapt. 2). 

Fig. 4 Explanation of the fluorescence-quenching effect [2]. — (A) chromatograms of the same quantities of saccharin and dulcin observed under UV 254 light, (B) schematic representation of fluorescence quenching, (C) spectral reflectance curves of saccharin and dulcin.

Short-wavelength UV radiation (A = 254 nm) is employed for excitation. This allows aromatic organic compounds, in particular, to be detected by fluorescence quenching. Uranylacetate may also be excited at A = 366 nm. 

Fig. 22 Optical trains of the commercially available scanners. (A) absorption, (B) fluorescence quenching and true fluorescence.

However, the optical train illustrated in Figure 22B allows the determination of fluorescence quenching. The interfering effect described above now becomes the major effect and determines the result obtained. For this purpose the deuterium lamp is replaced by a mercury vapor lamp, whose short-wavelength emission line (2 = 254 nm) excites the luminescence indicator in the layer. Since the radiation intensity is now much greater than was the case for the deuterium lamp, the fluorescence emitted by the indicator is also much more intense and is, thus, readily measured. 

Fig. 25 Calibration curve for the determination of dulcin by fluorescence quenching and absorption [2],...

Color reproduction of the chromatograms can be achieved by color photography — the best, but also the most expensive method of documenting thin-layer chromatograms. It can be used not only to produce true-color reproductions of colored zones but also — with the aid of a Reprostar (Fig. 64) or a UVIS analysis lamp (Fig. 6) — of fluorescent or fluorescence-quenched zones. When photograph-... 

The required exposure times are difficult to estimate. They are best found by trial and error. Documentation of fluorescence quenching at A = 254 nm usually only requires one trial. The exposure time found to be adequate here is normally suitable for all following exposures of fluorescence quenching if the exposure conditions are maintained constant (camera type, film type, distance of objective and lamp, aperture etc.). The exposure time required for fluorescent chromatograms is primarily dependent on the intensity of the fluorescence and, therefore, has to be optimized for each chromatogram. It is best to operate with a range of exposure times, e.g. aperture 8 with exposures of 15,30,60,120 and 240 seconds. Experience has shown that one exposure is always optimal. 

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. 

Acid Color Fluorescence quenching (>l = 254 nm) Sensitivity (pg/zone) TLC HPTLC ... 

Note The alternative fast blue salt BB produced the most intensely colored chromatogram zones for visual analysis in daylight, while fluorescence quenching in UV light (A = 254 nm) was greater with fast blue salt B and fast blue salt RR (Figs. 1 and 2). 

The intensity and colour of the fluorescence of many substances depend upon the pH of the solution indeed, some substances are so sensitive to pH that they can be used as pH indicators. These are termed fluorescent or luminescent indicators. Those substances which fluoresce in ultraviolet light and change in colour or have their fluorescence quenched with change in pH can be used as fluorescent indicators in acid-base titrations. The merit of such indicators is that they can be employed in the titration of coloured (and sometimes of intensely coloured) solutions in which the colour changes of the usual indicators would... 

Forward Electron Transfer as Studied by Fluorescence Quenching 69... 

Figure 10 illustrates Stern-Volmer plots for the fluorescence quenching of APh-x by MV2+ and SPV in aqueous solution [74]. With MV2+, the quenching is so effective that it occurs at very low quencher concentrations (in the range of 10 6 M), whereas with SPV, it proceeds to about the same extent at two-orders of magnitude higher quencher concentration (in the range of 10 4 M). 

Table 2. Rate constants for the fluorescence quenching of APh-8 (8) by SPV (14) and MV2 + (13) in aqueous solution [74]...

The fluorescence quenching depends on the content of the Phen units (the x values) in APh-x. An aqueous solution of APh-9 contained as many charged groups (SOJ) as about 10 times that of APh-50, when compared at the same molar concentration of the Phen residues. When AMPS homopolymer (PAMPS) was added to a solution of APh-50 so that the SOJ residue concentration was equal to that for APh-9, the kq value for the APh-50 quenching by MV2 + decreased from 2.1 x 1012 to 4.2 x 1011 M-1 s 1, which is close to the kq value for APh-9 (Table 2). From these facts the lower kq values for APh-x with lower x (higher... 

Figure 11 shows Stern-Volmer plots for fluorescence quenching of the amphiphilic cationic copolymer QPh-x [74]. The quenching of QPh-x with MV2+ is expected to be much less effective than that of APh-x. The quenching data for the QPh-x system are presented in Table 3. For comparison, the data for a related... 

More recently, several groups have investigated electrostatic effects on the fluorescence quenching of hydrophobic chromophores covalently attached to various polyanions. The photophysics of the chromophores incorporated in the polyeletrolytes at small mole fractions is relatively simple, because no interaction is expected to occur between the incorporated chromophores. For this reason, most of the studies have focused on amphiphilic polyeletrolytes loaded with a low amount of hydrophobic chromophores. 

Similar data were reported by Turro et al., [62,63] who synthesized a copolymer of AA with 1.5 mol% of 2-[4-(l-pyrene)butanoyl]aminopropenoic acid, 19 and studied the fluorescence quenching with Tl +, Cu2+, and 1 ions in aqueous solution. 

Webber et al. [60, 78] also studied the fluorescence quenching of diphenylan-thracene (DPA) covalently bound to poly(methacrylic acid), PMAvDPA (23) [60], and to sodium poly(styrenesulfonate), PSSvDPA (24 )[78]. The fluorescence quenching of the excited DPA moiety by MV2+ and Cu2+ was also highly efficient. For example, with PMAvDPA of 0.073 mol% DPA content, the kq values at pH... 

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