Detection fluorescence quenching

Detection Fluorescence quenching under 254-nm UV light 200 ng sensitivity. 

Fig. 102. Separation of some phthalides from coumarin. Layer silica gel GF254 detection fluorescence quenching in short wave UV light [258]. 1 phthalide 2 coumarin 3 isobutylidene-3a, 4-dihydrophthalide 4 phenylphthalide 5 butyl-phthalide 6 benzalphthalide 7 ligustilide blue fluorescence in 365 nm light 8 butylidene phthalide. G mixture 1—8...

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). 

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. 

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. 

Thus, for the investigation of buried polymer interfaces, several techniques with molecular resolution are also available. Recently NMR spin diffusion experiments [92] have also been applied to the analysis of a transition zone in polymer blends or crystals and even the diffusion and mobility of chains within this layer may be analyzed. There are still several other techniques used, such as radioactive tracer detection, forced Rayleigh scattering or fluorescence quenching, which also yield valuable information on specific aspects of buried interfaces. They all depend very critically on sample preparation and quality, and we will discuss this important aspect in the next section. 

Detection and result When viewed under short-wavelength UV light X = 254 nm) dark zones were visible due to fluorescence quenching. 

Detection is primarily based on the principle of fluorescence quenching by substances absorbing UV light. It is also possible to detect certain substances whose absorption wavelengths interfere with the uranyl cation [1]. 

The LIF technique is extremely versatile. The determination of absolute intermediate species concentrations, however, needs either an independent calibration or knowledge of the fluorescence quantum yield, i.e., the ratio of radiative events (detectable fluorescence light) over the sum of all decay processes from the excited quantum state—including predissociation, col-lisional quenching, and energy transfer. This fraction may be quite small (some tenths of a percent, e.g., for the detection of the OH radical in a flame at ambient pressure) and will depend on the local flame composition, pressure, and temperature as well as on the excited electronic state and ro-vibronic level. Short-pulse techniques with picosecond lasers enable direct determination of the quantum yield [14] and permit study of the relevant energy transfer processes [17-20]. 

In Chapter 7, approaches for visualization of zones in chromatograms are discussed, including use of nondestructive and destructive dyeing reagents, fluorescence quenching on layers with a fluorescent indicator, and densitometry. In Chapter 8, additional detection methods, such as those used for biologically active and radioactive zones, as well as the recovery of separated, detected zones by scraping and elution techniques are covered. 

Vo-Dinh T, Pal, T. 1992. Development of a fluorescence quenching technique to detect permeation of chemical agent simulants through protective clothing materials. Appl Spectrosc 46(4) 677-681. 

St /G AG-0.14 V fluorescence quenching stilbene radical anion detected in transient absorption no Gs near injection site or in intervening sequence k = 1012-108 s 1 for 0-4 intervening A-T base-pairs (-3.4-17 A) exponential distance dependence of CT rate constant (3 0.6-0.7 A"1 small variations in k depending on whether G is in the A or T arm of the hairpin... 

It has been important to determine if the neoxanthin distortion signature could be detected during the nonphotochemical quenching in vivo. Resonance Raman measurements on leaves and chlo-roplasts of various Arabidopsis mutants have revealed a small increase in the 950 cm 1 region. The relationship between the amplitude of this transition and the amount of NPQ suggests that the LHCII aggregation may be the sole cause of the protective chlorophyll fluorescence quenching in vivo (Ruban et al., 2007). 

Morrison, L.E. (1992) Detection of energy transfer and fluorescence quenching. In Nonisotopic DNA Probe Techniques (L.J. Kricka, ed.), pp. 311-352. Academic Press, New York. 

T. Ohyashiki, M. Nunomura, and T. Katoh, Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran. Biochim. Biophys. Acta. 1421, 131-139 (1999). 

Brown et al.68 have developed a cellulose plate with a fluorescent indicator. Compounds are developed in 3.0% (w/v) NHfc.Cl and detected by fluorescence quenching. These authors also use 0.5% mercaptoethanol in their mobile phase, but this is only to prevent oxidation of the labile reduced pteridines, which are not adequately protected by substitution at the N5 position. Since neutral or alkaline solutions of leucovorin are relatively stable in air, this precaution may not be required for routine assay. 

---