Luminol Oxidation

Quantum yields from (3.100) are only around 1%, limiting its sensitivity and applicability. Replacing the amino group with a nitro or chloro group leads to a decrease in the luminescence intensity, whilst alkylation of the amino gronp renders it non-luminescent. 

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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). 

CHEMILUMINESCENT ANALYSIS BASED ON THE LUMINOL OXIDATION REACTION IN SPECIFIC REGIMES... 

The overall reaction scheme of the luminol chemiluminescence in an aqueous medium is shown in Figure 1. The luminol oxidation leads to the formation of an aminophthalate ion in an excited state, which then emits light on return to the ground state. The quantum yield of the reaction is low ( 0.01) compared with bioluminescence reactions and the emission spectrum shows a maximum1 at 425 nm. 

The hydrophobicity of the solvent and solution composition [as example, the (])cl of luminol oxidized in dimethylsulfoxide (DMSO) is 0.05 compared with 0.01 in water, the colors being blue-violet (425 nm) and blue-green (480-502 nm), respectively]... 

The oxidative behaviour of glycolaldehyde towards hexacyanoferrate(III) in alkaline media has been investigated and a mechanism proposed, which involves an intermediate alkoxide ion. Reactions of tetranitromethane with the luminol and luminol-peroxide radical anions have been shown to contribute substantially to the tetranitromethane reduction in luminol oxidation with hexacyanoferrate(III) in aerated aqueous alkali solutions. The retarding effect of crown ethers on the oxidation of triethylamine by hexacyanoferrate(III) ion has been noted. The influence of ionic strength on the rate constant of oxidation of ascorbic acid by hexacyanofer-rate(III) in acidic media has been investigated. The oxidations of CH2=CHX (where X = CN, CONH2, and C02 ) by alkaline hexacyanoferrate(III) to diols have been studied. ... 

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

In this part of the chapter we will give a more detailed description of some highly efficient organic chemiluminescence systems, which occur with the involvement of peroxide intermediates. We have chosen to begin the subject with the well-known and widely applied luminol oxidation and will show that, even though this reaction has been exhaustively studied, several critical points in its mechanism remain unclear and are still the subject of... 

The chemiluminescence emission resulting from the oxidation of luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) has been extensively studied since its discovery by Albrecht in 1928. Although luminol oxidation is one of the most commonly applied chemiluminescent reactions, to date no definitive mechanism is known . Efficient chemiluminescence emission is only observed when luminol (25) is oxidized under alkaline conditions. Depending on the medium, co-oxidants are required in addition to molecular oxygen for the observation of light emission, but under any condition, 3-aminophthalate (3-AP) and molecular nitrogen are the main reaction products (equation 10). 

In the first proposal of a mechanism for chemiluminescent luminol oxidation, Albrecht postulates a bicyclic endoperoxide as the high-energy intermediate. The endoperoxide is presumably formed by nucleophilic attack of hydrogen peroxide monoanion on one of the diazaquinone 27 carbonylic groups to form 28, followed, after deprotonation to 29, by ring closure to 30 (Scheme 21) . 

Scheme 28) Quantum yields of lucigenin oxidation by hydrogen peroxide in alkaline media are comparable with the values obtained in luminol oxidation (1.24 x 10 E mol ) ° . However, the use of other peroxides, such as tcrt-butyl hydroperoxide, results in a decrease of chemiluminescence quantum yields of two orders of magnitude, confuming the hypothesis that a 1,2-dioxetane is the HEI, since its formation would be impossible with alkyl peroxides . 

Ai -Aminooxymethylcarbonylhydrazino-D-biotin, DNA oxidative damage, 736, 634 3-Aminophthalate, luminol oxidation, 643, 644, 1239-41, 1244-6 7-Amino-4-trifluoromethylcoumarin, hydrogen peroxide determination, 649 Ammonium ion complex, mass spectrometry, 703, 704... 

Bacillus subtilis defense mechanism, 610 bovine semm albumin y-radiation, 614 generation inhibition, 612 hydroperoxide synthesis, 315, 320 ludgenin oxidation, 645, 1250-1 luminol oxidation, 643, 644, 1242-4 organic sulfur compounds, 1032-9 ozone water disinfection, 606 peroxynitrite generation, 10, 611-12 Superoxide dismutase (SOD)... 

N,N, N, iV -Tetrakis(2-benzimidazolylmethyl)-l,3-diaminopropan-2-ol, hydrogen peroxide determination, 637 A,A,A, iV -Tetrakis[2 (l -ethylbenzimidazol-yl)-l, 3-diamino-2-hydroxypropane], transition metal peroxides, 1066 meiO-Tetrakis(4-sulfonatophenyl)porphine, luminol oxidation, 646... 

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