Chemiluminescence severity

As can be seen in Table 3, a wide range of analytes derivatized with different labels have been detected using the POCL reaction. Most of these applications have employed flow injection or liquid chromatographic techniques. An area of growing interest is the combination of capillary electrophoresis with chemiluminescence. Several strategies have been used to detect analytes with fluorescent... 

Chemiluminescence has been studied extensively (2) for several reasons (/) chemiexcitation relates to fundamental molecular interactions and transformations and its study provides access to basic elements of reaction mechanisms and molecular properties (2) efficient chemiluminescence can provide an emergency or portable light source (J) chemiluminescence provides means to detect and measure trace elements and pollutants for environmental control, or clinically important substances (eg, metaboHtes, specific proteins, cancer markers, hormones, DNA) and (4) classification of the hioluminescent relationship between different organisms defines their biological relationship and pattern of evolution. 

Several new oxalates have been developed for use ia analytical appHcations. Bis(2,6-difluorophenyl) oxalate (72) and bis(4-nitro-2-(3,6,9-trioxadecylcarbonyl)phenyl) oxalate (97) have been used ia flow iajection and high performance Hquid chromatography (hplc) as activators for chemiluminescence detectors. These oxalates are generally more stable and show better water solubiUty ia mixed aqueous solvents yet retain the higher efficiencies ( ) of the traditional oxalates employed for chemiluminescence. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Siloxene is fluorescent and red chemiluminescence results from oxidation with ceric sulfate, chromic acid, potassium permanganate, nitric acid, and several other strong oxidants. The chemiluminescence spectmm peaks at 600 nm and has been reported (199) to give a maximum brightness of 3.43 cd/m (1 footlambert). 

Chemiluminescence is also obtained by anionic autooxidation of (41) with oxygen ia alkaline dimethyl sulfoxide (DMSO) (216). Qc has been reported to be 10% and ketone (43) and CO2 are obtained. Several analogues of luciferin have been prepared that are also chemiluminescent when they react with oxygen ia alkaline DMSO (62). 

Analytical Applications. Chemiluminescence and bioluminescence are useful in analysis for several reasons. (/) Modem low noise phototubes when properly instmmented can detect light fluxes as weak as 100 photons/s (1.7 x 10 eins/s). Thus luminescent reactions in which intensity depends on the concentration of a reactant of analytical interest can be used to determine attomole—2eptomole amounts (10 to 10 mol). This is especially useful for biochemical, trace metal, and pollution control analyses (93,260—266) (see Trace and residue analysis). (2) Light measurement is easily automated for routine measurements as, for example, in clinical analysis. 

Several other biosensors have been developed usiag this oxygen-quenched fluorescence approach. Target species iaclude ethanol [64-17-5] hydrogen peroxide [7722-84-17, H2O2, lactate, and xanthine [69-89-6] C H4N402, usiag alcohol oxidase, catalase [9001-05-2] lactate oxidase, and xanthine oxidase, respectively. An additional technique for biocatalytic biosensors iavolves the firefly chemiluminescent reaction (17) ... 

The UV spectra of pyrido[2,3-d]pyridazine-l,4-diones have been recorded (68MI21501), whilst several of the latter were also used in chemiluminescence studies. Quinolinic and cinchomeronic hydrazides showed no chemiluminescence (60NKK173, 37JPR(148)135), but it was observed in the 8-hydroxy derivative (305) (72YZ703). 

The methods of investigation of metal species in natural waters must possess by well dividing ability and high sensitivity and selectivity to determination of several metal forms. The catalytic including chemiluminescent (CL) techniques and anodic stripping voltammetry (ASV) are the most useful to determination of trace metals and their forms. The methods considered ai e characterized by a low detection limits. Moreover, they allow detection of the most toxic form of metals, that is, metal free ions and labile complexes. 

Solubility and stability of coelenterazine. Coelenterazine is very poorly soluble in neutral aqueous buffer solutions, and the solutions are unstable in air. It can be easily dissolved in water in the presence of alkali, but the resulting solution is extremely unstable under aerobic conditions. Coelenterazine is soluble in methanol, and the solution is relatively stable. The stability is enhanced by the addition of a trace of HCl. A methanolic solution of coelenterazine can be stored for several days at — 20°C, and a methanolic solution containing 1-2 mM HCl can be stored for several months at — 70°C under aerobic conditions without significant oxidation. In many other organic solvents, coelenterazine is less stable, and spontaneously auto-oxidized at significant rates. In dimethylformamide and DMSO, it is rapidly decomposed accompanied by the emission of chemiluminescence. e-Coelenterazines are generally less stable than coelenterazines. 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

We have also investigated other oxalate esters as a potential means to improve the efficiency. The most commonly used oxalates are the 2,4,6-trichlorophenyl (TCPO) and 2,4-dinitrophenyl (DNPO) oxalates. Both have severe drawbacks namely, their low solubility in aqueous and mixed aqueous solvents and quenching of the acceptor fluorescence. To achieve better solubility and avoid the quenching features of the esters and their phenolic products, we turned to difluorophenyl oxalate (DFPO) derivatives 5 and 6 (Figure 14). Both the 2,4- and the 2,6-difluoro esters were readily synthesized and were shown to be active precursors to DPA chemiluminescence. In fact, the overall efficiency of the 2,6-difluorophenyl oxalate 5 is higher than for TCPO in the chemical excitation of DPA under the conditions outlined earlier. Several other symmetrical and unsymmet-rical esters were also synthesized, but all were less efficient than either TCPO or 2,6-DFPO (Figure 14). 

Equation (13) appears to be a good approximation for describing isothermal chemiluminescence kinetics for homogeneous systems where oxidation takes place uniformly. However, as has been shown by several authors [53-58], the different sections of a polymer sample may oxidize with its autonomous kinetics determined by different rates of primary initiation. A chemiluminescence imaging technique revealed that the light emission may be spread from some sites of the polymer film and the isothermal chemiluminescence vs. time runs are then modified, particularly in the stage of an advanced oxidation reaction [59]. 

Chemiluminescence reactions are currently exploited mainly either for analyte concentration measurements or for immunoanalysis and nucleic acid detection. In the latter case, a compound involved in the light emitting reaction is used as a label for immunoassays or for nucleic acid probes. In the former case, the analyte of interest directly participates in a chemiluminescence reaction or undergoes a chemical or an enzymatic transformation in such a way that one of the reaction products is a coreactant of a chemiluminescence reaction. In this respect, chemiluminescent systems that require H2O2 for the light emission are of particular interest in biochemical analysis. Hydrogen peroxide is in fact a product of several enzymatic reactions, which can be then coupled to a chemiluminescent detection. 

Lucigenin 71 chemiluminescence is more complicated than that of luminol and related compounds due to the presence in the reaction mixture of several species capable of emission 135>. N-Methylacridone 72, however, has been established as the primary excited product (for references see 0 p. 90) from which energy transfer occurs to the other species 3>. 

Hetero-excimer chemiluminescence yields were measured by A. Weller and K. Zachariasse 214) the system dimethylanthracene anion radical/tri-p-tolylaminium perchlorate in tetrahydrofurane exhibits particularly strong chemiluminescence with quantum yields of about 7.5 x 10-2 215>. A. J. Bard and coworkers 216> very thoroughly investigated the influence of several parameters, e.g. supporting electrolyte concentration, on the efficiency of electrogenerated chemiluminescence. 

Many sophisticated analytical techniques have been used to deal with these complex mixtures.5,45,46 A detailed description is not possible here, but it can be noted that GLC, often coupled with mass spectrometry (MS), is a major workhorse. Several other GLC detectors are available for use with sulfur compounds including flame photometer detector (FPD), sulfur chemiluminescence detector (SCD), and atomic emission detector (AED).47 Multidimensional GLC (MDGC) with SCD detection has been used48 as has HPLC.49 In some cases, sniffer ports are provided for the human nose on GLC equipment. 

CL emissions can be characterized by four parameters, including color, intensity, rate of production, and decay of intensity. The properties of several organic, chemiluminescent reactions known to produce emissions of light are shown in Table 1. 

The unsubstituted phthalic acid hydrazide and several nonaromatic cyclic hydrazides such as maleic acid hydrazide or succinic acid hydrazide are either nonchemiluminescent or show extremely weak CL. However, the 6-amino isomer of luminol, which is called isoluminol, is chemiluminescent to about the same extent as is luminol. Isoluminol has been used in many chemiluminescent studies, and because the amino group is less sterically hindered than that of luminol, it is probably derivatized for chemiluminescent labeling far more often than is luminol (Fig. 3). 

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