Fluorometric response

As mentioned above, weak UV irradiation (325-427 nm) is frequently used for excitation. When far-UV radiation is used, however, increased fluorometric response and more information on structural properties of dissolved organic compounds can be obtained, since organic molecules found in natural waters have a greater absorbance at shorter wavelengths. Stabel (personal communication) attempted to differentiate between probable sources of DOM in lakes using fluorescence spectra of DOM from various inland waters, such as softwater, hardwater, and saline systems. With exci-... 

One otlier common source of nonlinear response, singlet-triplet annihilation, is often tire reason for a discrepancy between fluorometric and absorjDtion kinetic measurements [27, 28 and 29]. 

Fig. 11 (a) Chemical structure left, 9 90°) and cation response right) of virtually decoupled probe 30 for Hg2+ and Ag+. Absorption and emission spectra of 30 in the absence (black, dotted line = fit of the CT emission LE = fluorophore-localized emission band) and presence (at full complexation) of Hg2+ red) and Ag+ blue) in MeCN fluorometric titrations of 1 with Hg2+ and Ag+ shown in the inset FEF (LE) determined from the integrated fluorescence intensity of the LE band, (b) Chemical structures of other virtually decoupled probes for Na+ (31), Pb2+ (32), and Ni2+ (33). For color code, see Fig. 3. (Adapted in part from [115], Copyright 2000 American Chemical Society)...

Because of their high sensitivity, fluorescence detectors are particularly useful in trace analysis when either tire sample size is small or the analyte concentration is extremely low. Although fluorescence detectors can become markedly nonlinear at concentrations where absorption detectors are still linear in response, their linear dynamic range is more than adequate for most trace analysis applications. Unfortunately, fluorometric detectors are often susceptible to background fluorescence and quenching effects that can plague all fluorescence measurements. 

Bioassay procedures for the determination of gibberellic acid have been developed (2, 5), but more recent chemical fluorometric assay methods are equally specific. However, both assay methods show a low response with samples containing less than 10 /x/xg. of the gibberellins. Consequently, in determining residual amounts within the part per billion (p.p.b.) range, relatively large samples must be extracted and extracts partially purified to satisfy the assay conditions. These operations are usually accompanied by some material losses or degradation, which impair quantitative interpretation of the results. Natural inhibitors can influence the results in the bioassay method (2), and fluorescent contaminants can interfere with the spectrophotometric analysis. 

Two disparate translation methods are investigated for the measurement of sulfur dioxide. Both involve interaction with an aqueous solution. In the first, collected S(IV) is translated by the enzyme sulfite oxidase to which is then measured by an enzymatic fluorometric method. The method is susceptible to interference from i CWg) efforts to minimize this interference is discussed. The second method involves the translation of SO2 into elemental Hg by reaction with aqueous mercurous nitrate at an air/liquid interface held in the pores of hydrophobic membrane tubes. The liberated mercury is measured by a conductometric gold film sensor. Both methods exhibit detection limits of 100 pptv with response times under two minutes. Ambient air measurements for air parcels containing sub-ppbv levels of SO2 show good correlation between the two methods. 

Light, fluorometric, electronic emission, and confocal absorption microscopy techniques or their combinations with immunometric techniques are applicable to analysis of allergenic compounds regarding the structure of a product—its matrix). Cells and metabolites produced by the immune system cells involved in the allergenic response (specific types of lymphocytes) can be examined by flow cytometry techniques in addition to traditional microscopic techniques. 

FIGURE 2-13. High performance liquid chromatographic separation of unknown TV-nitroso compounds. The colorimetric, photometric, and fluorometric detection responses are shown. Column /uBondapak C]8 3.9 mm ID x 30 cm. Mobile-phase composition was programmed from 20 to 80% acetonitrile in water (pH = 4, phosphoric acid) over 20 min, was held isocratic (constant mobile phase composition) for 22 min, and then was programmed from 80 to 100% acetonitrile in 1 min and held at 100% acetonitrile for 20 min. The flow rate was 1.0 mL/min. (Reprinted from reference 10 with permission. 

Several action spectra have been constructed for phototaxis (and photophobic responses) in Eu lena. They are distinctly different from those measured, e.g., in Chlamydomonas and have been suggested to represent the involvement of flavins and pterins. Fluorometric analysis has indicated that pterins, absorbing in the UV-A range of the spectrum with a maximum near 360 nm, function as antenna pigments. Pterins emit at about 450 nm, which corresponds with one of the maxima for flavin absorption. The fluorescence emission of the flavins can be detected at 520 nm (82). Therefore the final photoreceptor is thought to be a flavin. 

Table 14. Responses of microsomal cytochrome P-450, NADPH-cytochrome c (P-450) reductase and benzo[a]pyrene hydroxylase activities (radiometric and fluorometric) to experimental exposure of molluscs to xenobiotics ... 

Many small molecules of clinical interest can be detected by fluorescence. For example, porphyrins, which are intermediates in the metabolism of iron-containing heme prosthetic groups, can be detected and quantitated in urine by reversed-phase chromatography followed by fluorometric detection, exploiting the natural fluorescence of porphyrin compounds [18]. An example of reversed-phase chromatography for a mixture of carboxylated porphyrin precursors is demonstrated in Figure 21.2. The relative abundances of different porphyrin compounds can aid in diagnosing porphyria, which is a term that describes the collection of disorders caused by defects in the enzymes responsible for porphyrin metabolism. 

This reaction was exploited by Tsukatani and Matsumoto (2000) in a stopped-flow FIA method. An immobilized D-malate dehydrogenase enzyme reactor was employed and the reduced enzymatic cofactor NADH that was formed was monitored fluorometrically (Xex = 340 nm = 460 nm). Due to the slow reaction rate, the flow was stopped with the sample in the reactor to increase reaction time. The intrinsic sample fluorescence was also assessed using a parallel blank reactor without immobilized enzyme. The method was validated through the analysis of red and white wine samples. The enzyme reactor stability was also evaluated and it was found that the sensitivity (evaluated as amplitude of response at a constant concentration of the analyte) gradually decreased to 60% within a week but then remained stable for a month. As D-malate cannot be present in naturally fermented wines (except for fraudulent addition), the interference of this primary substrate can be considered negligible. 

An automatic method for the separation and determination of RF vitamin in food samples (chicken liver, tablet, and powder milk) is proposed by Zougagh and Rios [2], The method is based on the online coupling of supercritical fluid extraction (SFE) with a continuous flow-CE system with guided optical fiber fluorometric detection (CE-CE-ED). The whole SFE-CF-CE-FD arrangement allowed the automatic treatment of food samples (cleanup of the sample followed by the extraction of the analytes), and the direct introduction of a small volume of the extracted material to the CE-ED system for the determination of RF vitamins. Fluorescence detection introduced an acceptable sensitivity and contributed to avoidance of interferences by nonfluorescent polar compounds coming from the matrix samples in the extracted material. Electrophoretic responses were linear within the 0.05-1 pg/g range, whereas the detection limits of RE vitamins were in the 0.036-0.042 pg/g range. 

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