Fluorescent Compound

For fluorescent compounds and for times in die range of a tenth of a nanosecond to a hundred microseconds, two very successftd teclmiques have been used. One is die phase-shift teclmique. In this method the fluorescence is excited by light whose intensity is modulated sinusoidally at a frequency / chosen so its period is not too different from die expected lifetime. The fluorescent light is then also modulated at the same frequency but with a time delay. If the fluorescence decays exponentially, its phase is shifted by an angle A([) which is related to the mean life, i, of the excited state. The relationship is... 

Description of Method. Quinine is an alkaloid used in treating malaria (it also is found in tonic water). It is a strongly fluorescent compound in dilute solutions of H2SO4 (f = 0.55). The excitation spectrum of quinine shows two absorption bands at 250 nm and 350 nm, and the emission spectrum shows a single emission band at 450 nm. Quinine is rapidly excreted from the body in urine and is easily determined by fluorescence following its extraction from the urine sample. 

Many chemical compounds have been described in the Hterature as fluorescent, and since the 1950s intensive research has yielded many fluorescent compounds that provide a suitable whitening effect however, only a small number of these compounds have found practical uses. Collectively these materials are aromatic or heterocycHc compounds many of them contain condensed ring systems. An important feature of these compounds is the presence of an unintermpted chain of conjugated double bonds, the number of which is dependent on substituents as well as the planarity of the fluorescent part of the molecule. Almost all of these compounds ate derivatives of stilbene [588-59-0] or 4,4 -diaminostilbene biphenyl 5-membeted heterocycles such as triazoles, oxazoles, imidazoles, etc or 6-membeted heterocycles, eg, coumarins, naphthaUmide, t-triazine, etc. 

Fluorescence Microscope. A useful light microscope utilizes UV light to induce fluorescence in microscopic samples (40). Because fluorescence is often the result of trace components in a given sample rather than intrinsic fluorescence of the principal component, it is useful in the crime laboratory for the comparison of particles and fibers from suspect and crime scene. Particles of the same substance from different sources almost certainly show a different group of trace elements. It is also very useful in biology where fluorescent compounds can be absorbed on (and therefore locate and identify) components of a tissue section. 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

In the case of nicotinamide, the color yield is often low. This problem can be circumvented by either hydrolysis to nicotinic acid or by conversion of the amide to a fluorescent compound. Treatment of nicotinamide with methyl iodide yields the quaternary ammonium salt, /V-methyl nicotinamide (5). Reaction of this compound with acetophenone yields a fluorescent adduct (49). Other carbonyl compounds have also been used (50—54). 

Fig. 5. (a) Antibody 2 is added to test solution where some or all of becomes complexed with target, T. (b) A fiber-optic probe containing covalently attached target is insetted into the solution. Unbound binds to probe, (c) The probe is inserted into solution containing en2yme-modified detector antibody which binds to probe if any is attached, (d) The probe is inserted into a solution producing a fluorescent compound, which is then... 

Isolation of F and P. The first attempt to isolate and purify the substances responsible for the light emission of M. norvegica was made by Shimomura and Johnson (1967). They isolated two substances, a protein (P) and a fluorescent compound (F), which produce a blue light... 

Fig. 3.2.2 Influence of pH on the initial light intensity of euphausiid luminescence when the fluorescent compound F and protein P are mixed in 25 mM sodium phosphate buffers of various pH values, each containing 1M NaCl, at near 0°C. Both F and P were obtained from Meganyctiphanes norvegica. From Shimomura and Johnson, 1967, with permission from the American Chemical Society.

The fluorescent compound F, a luciferin, emits blue light (Amax 476 nm Fig. 3.2.4) in the presence of molecular oxygen and the protein P, a luciferase. In the luminescence reaction, F is changed into an oxidized form (structure 8, Fig. 3.2.6). The luminescence reaction is highly sensitive to pH, with a narrow optimal range around pH 7.8 (Fig. 3.2.2) the optimum salt concentration is 0.15 M for NaCl... 

Oxyluciferin. During the luminescence reaction catalyzed by luciferase, luciferin is converted into a fluorescent compound, oxyluciferin, accompanied by the emission of greenish-blue light that spectrally matches the fluorescence of oxyluciferin (Fig. 7.2.6). The absorption spectrum of oxyluciferin is shown in Figs. 7.2.1 and 7.2.2. 

Mild chromic acid oxidation of luciferin (CrOs/KHSC /HiO, room temperature) yielded 3-methyl-4-vinylmaleimide (1, Fig. 8.7), 3-methyl-4-ethylmaleimide (2), and an aldehyde (3), whereas vigorous chromic acid oxidation (CrOs/2N H2SO4, 90°C) gave hema-tinic acid (4) (Dunlap et al., 1981). These results closely resemble the results of the chromic acid oxidation of the fluorescent compound F of euphausiid (p. 76), indicating a structural similarity between dinoflagellate luciferin and the compound F. 

Both compounds were non-fluorescent. Compound KM-1 was similar to PMs in the absorption maximum (A.max 488 nm) but not in the spectral shape, whereas KM-2 was similar to PMs in the spectral shape but not in the absorption maximum at 515 nm (Fig. 9.10). The chemical structures of KM-1 and KM-2 have been determined by high-resolution mass spectrometry and NMR spectrometry (Fig. 9.11 Stojanovic, 1995). 

Kuse et al. (2001) isolated a fluorescent compound from L. sequoiae (fluorescence emission 7max 505 nm upon excitation at... 

Storage under vacuum in a sealed tube (Method II). Substances that are extremely oxygen-sensitive, such as the fluorescent compound F of euphausiids and dinoflagellate luciferin, have to be stored in an evacuated sealed container at a low temperature. For long-term storage, they must be fuse-sealed in an evacuated glass vial using the method outlined below. 

Ebrahimzadeh, M. H., and Haddadchie, G. R. (1993). Intrinsic fluorescent compounds in Armillaria mellea hyphae and rhizomorph./. Set. Islamic Repub. Iran 4 241-246. 

Quantula (Dyakia), 180, 334 Quantum yield, xvi, 361, 362 aequorin, 104, 106, 110 aldehydes in bacterial bioluminescence, 36, 41 Chaetopterus photoprotein, 224 coelenterazine, 85, 143, 149 Cypridina luciferin, 69-71 definition, xvi, 361 Diplocardia bioluminescence, 242 firefly luciferin, 12 fluorescent compound F, 73 Latia luciferin, 190 pholasin, 197 PMs, 286... 

A preliminary test for the biodegradability of the 3-phenyl- and 3-carbamoyl-2(lH)pyridones was conducted in a barnyard humus suspension. The analysis by HPLC showed some loss, and the fluorescent compounds seemed to be adsorbed onto the solid. The 3-carbamoyl-2(lH)pyridone (II) also hydrolyzed to 3-carboxylic acid-2(lH)pyridone both in the slurry test and in water solutions that had been left standing 1-2 weeks. In preliminary tests both the 3-phenyl- and the 3-carbamoyl-2(lH)pyridones apparently adsorbed to some extent on silica sand columns. In addition, the solubility of both 1-H compounds was somewhat low, 1.3 x 10 M for II, and 1.0 x 10 M for IV. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Substructures commonly found in dyes (chromophores), fluorescent compounds, pesticides (polyhalogenated alkanes, cycloalkanes). 

Fluorescence spectroscopy offers several inherent advantages for the characterization of molecular interactions and reactions. First, it is 100-1000 times more sensitive than spectrophotometric techniques. Second, fluorescent compounds are extremely sensitive to their environment. Tryptophan residues that are buried in the hydrophobic interior of a... 

Quenching Impurities in the mobile phase, particularly oxygen, may entirely quench the signal from low concentrations of fluorescent compounds (see solvent degassing). 

An assay result in which a sample known to be inactive produces a signal or response above the activity threshold. FALSE POSITIVES can occur when an assay lacks appropriate discriminatory power, when the threshold is inappropriately set, as a result of certain physical properties of the substance (e.g., a fluorescent compound in a fluorescence intensity assay, aggregation), or as a result of mistaken identity of the substance. 

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