Detection in fluorescence

Flourescence spectrometers can be divided into either lifetime or steady-state instruments, depending on whether they resolve the temporal behaviour of the emission (or more correctly the excited state), or not, respectively. In both cases there are strong similarities with single beam absorption instruments. Thus, much of the preceding sections is equally relevant to them. However, the levels of photons detected in fluorescence (or equally phosphorescence) are typically much lower than those in absorbance in the former one is detecting the few photons that are emitted by the sample, in the latter one is detecting those of the light source attenuated by the number absorbed by the sample. As a consequence certain features are optimized differently for fluorescence. 

Goodwin P M, Ambrose W P and Keller R A 1996 Single-molecule detection in liquids by laser-induced fluorescence Acc. Chem. Res. 29 607-13... 

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

The limits of lifetime detection and resolution in on-the-flight fluorescence lifetime detection in hplc were evaluated for simple, binary systems of polycycHc hydrocarbons (70). Peak homogeneity owing to coelution was clearly indicated for two compounds having fluorescence lifetime ratios as small as 1.2 and the individual peaks could be recovered using predeterrnined lifetimes of the compounds. Limits of lifetime detection were deterrnined to be 6 and 0.3 pmol for benzo[b]fluoranthene and benzo[k]fluoranthene, respectively. 

Since the avermectins exhibit unprecedented potency, they are used at unusually low doses of 6 —300 )-lg/kg, which makes the detection and isolation of residues and metaboUtes from animal tissue a new challenge. For this reason a sensitive analytical assay requires a derivative suitable for detection at concentrations down to 1/10 or 1/100 of one ppm. Ivermectin and avermectin B are therefore converted into an aromatic derivative which allows detection by fluorescence absorbance. To achieve this derivatization, avermectin B, ivermectin, or their derivatives are heated with acetic anhydride in pyridine at 100°C for 24 h (30). The reaction time can be reduced to 1 h by using /V-methylimidazole as a catalyst (31). The resultant... 

Fast concentration and sample injection are considered with the use of a theory of vibrational relaxation. A possibility to reduce a detection limit for trinitrotoluene to 10 g/cnf in less than 1 min is shown. Such a detection limit can by obtained using selective ionization combined with ion drift spectrometry. The time of detection in this case is 1- 3 s. A detection technique based on fluorescent reinforcing polymers, when the target molecules strongly quench fluorescence, holds much promise for developing fast detectors. 

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. 

Acids can also be converted to fluorescent dansyl derivatives The reaction of Cg to C24 fatty acids with dansyl semipiperazide or semicadavende provides an excellent example (Fig 34) [87] Odd-numbered and unsaturated fatty acids [88] and propionic, sorbic and benzoic acid [89] can be detected in the same manner... 

Under long-wavelength UV light (A = 365 nm) the chromatogram zones fluoresce yellow to yellow-green, they are sometimes detectable in daylight as colored zones, too. 

A T Rhys Williams, Fluorescence Detection in Liquid Chromatography, Perkin-Elmer, Beaconsfield, 1980... 

The ultimate in fluorescence detection is a detector that uses a monochromator to select the excitation wavelength and a second monochromator to select the wavelength of the fluorescent light. This instrument is ideal, giving the maximum versatility and allowing the... 

The derivatization process (5) is accomplished in aqueous media at basic pH (pH 7-10) in a matter of approximately 15 min to yield a 2-cyanobenz[f]isoindole (CBI), which is stable for 10 to 12 hr in solution. As shown in Figure 1, the absorption characteristics of the CBI adducts are also readily accessible for assay by standard fluorescence or ultraviolet detection. In addition to the absorption between 200 and 300 nm, there are two maxima in the visible spectrum at approximately 420 and 440 nm accessible for fluorescence or ultraviolet detection. A probable mechanism (5,11) for the CBI formation is illustrated in Scheme 1. 

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