3-Aminophthalate, luminol oxidation

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... 

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 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). 

Firstly, in order to obtain an understanding of the mechanism of the sensitized luminol-based CL reaction, the CL emission spectrum was obtained with a F-4500 fluorescence spectrophotometer. The results showed the maximum emission wavelength in the presence of ofloxacin was the same as that in the absence of ofloxacin and the maximum light emission was at about 425 nm. The emitter was 3-aminophthalate, the oxidation product of luminol. 

Since the emission spectra of enhanced and unenhanced luminol oxidations are similar, it seems clear that emission is from the excited-state aminophthalate derived from luminol itself and not from the enhancer. The enhancement is also specific for peroxidase i.e., it does not occur under conditions in which the heme would dissociate from the enzyme. Furthermore, luminol chemiluminescence triggered by heme-containing compounds, such as hemoglobin or cytochrome c, is actually reduced by enhancers such as p-iodophenol (T7). This latter phenomenon explains the potential usefulness of enhanced chemiluminescence, i.e., an amplified signal combined with a reduced background and reduced interference from the reagents as well as from endogenous heme compounds. 

Albrecht [10] was the first to report the CL of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), in 1928. The chemiluminescent reaction involves the oxidation of luminol (usually by H202) and often occurs in the presence of a catalyst (or co-oxidant) such as Fc(CN)6 3, Cu(II), or Co(II)) (Fig. 2). The light emission, which is blue in water and yellow-green in DMSO, is identical with the fluorescence of the 3-aminophthalate oxidation product [11],... 

The source of chemiluminescence in the oxidation of luminol was explored by Merenyi and co-workers in detail (153). The oxidation of luminol yields aminophthalate as a final product and the reaction proceeds via a series of electron transfer steps. The primary oxidation product is the luminol radical which is transformed into either diazaquinone or the a-hydroxide-hydroperoxide intermediate (a-HHP). The latter oxidation step occurs between the deprotonated form of the luminol radical and O -. The chemiluminescence is due to the decomposition of the mono-anionic form of a-HHP into the final products ... 

Fig. 24 (a) Chemiluminescence reaction of luminol (63) to 3-aminophthalic acid (64). (b) Oxidation of diphenyloxalate (65) to produce the high-energy dioxetandione as a sensitizer for an organic dye fluorophore... 

In aprotic media, only molecular oxygen and a strong base are needed to produce chemiluminescence from luminol. In such media, an important intermediate in the reaction is the dianion of luminol, which can be oxidized by oxygen, resulting in the formation of the diazaquinone 27 and deprotonated hydrogen peroxide. The subsequent nucleophilic attack by hydrogen peroxide dianion to one of the diazaquinone carbonyls gives rise to the formation of a metastable peroxidic intermediate that, by several steps, results in chemiexcited 3-aminophthalate (Scheme 16)123,174, iso ... 

It is generally agreed that the CL obtained with luminol (124) is based on the series of transformations shown in Scheme 3, where the analyte (oxidant) as such or in combination with a catalyst produces a free radical (125), which in turn captures a superoxide anion to yield an endoperoxide (126), which on elimination of N2 produces an excited intermediate (127), which finally settles down to the 3-aminophthalate ion (128) on emission of a photon. A linear correlation may be established between the intensity of the CL emission and the concentration of the analyte334. 

Several other chromophores have been used in the development of sensors based upon ECL. For example, the luminol reaction is a conventional chemi-luminence reaction that has been studied in detail and it is believed that the mechanism of the ECL reaction is similar, if not identical, to that of the chemiluminescence. As shown in Fig. 2, the luminol ion undergoes a one-electron oxidation to yield a diazaquinone, which then reacts with peroxide or superoxide ( OOH) to give the excited 3-aminophthalate which has an emission maximum of 425 nm. This reaction is particularly versatile and has been utilized in a variety of ECL assays, many of which have been previously summarized by Knight [1], The luminol ECL reaction can be used for the determination of any species labeled with luminol derivatives, hydrogen peroxide, and other peroxides or enzymatic reactions that produce peroxides. A couple of examples are described later. 

The oxidative reactions of luminol and isoluminol derivatives at high pH result in the formation of 3-aminophthalate or 4-aminophthalate and nitrogen via an electronically excited state. The transition from the excited to the ground state induces the emission of light having a wavelength maximum of 425 nm. Quantitation is possible at picomolar or even attomolar levels of the aminophthalhydrazide. 

Of the artificial chemiluminescent compounds, luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) is the most popular, and has been applied not only in analytical chemistry but also in other fields. The luminol chemiluminescence is based on the light emission from an excited 3-aminophthalate ion generated by oxidation with hydrogen peroxide or atmospheric oxygen in the presence of bases and catalysts. This peculiar chemiluminescence property and its industrial value have attracted continuous interest and prompted many chemists to investigate its reaction in detail... 

The luminescence exhibited by luminol was one of the earliest cases of chemiluminescence to be studied. Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) is an aminophthalic hydrazide that is able to exist in several tautomeric forms. Other significant derivatives of luminol include isoluminol, aminobutyl-ethylnaphthalhydrazide, and diazoluminol (Fig. 7). Luminol and its derivatives are generally oxidized in alkaline media to form an excited aminophthalate derivative, which then releases the energy as light. In protic media, a catalyst and a source of oxygen are required. 

Fio. 15. Postulated mechanism for the chemiluminescence of luminol, taken from McCapra and Beheshti (M2I). In this sequence, ferricyanide acts as a one-electron oxidant. The products are nitrogen and an excited-state aminophthalate dianion that decays to the ground state with concomittant light emission. 

Luminol (3-aminophthalhydrazide) is used in a commercially available portable device called the Luminox that measures minute concentrations (parts per billion) of the pollutant nitrogen dioxide in air. Luminol is also used ftequently in laboratory demonstrations of the chemiluminescence phenomenon. Luminol-mediated chemiluminescence is the result of an oxidation reaction. The oxidation proceeds in two steps, which ultimately lead to the production of the aminophthalate anion in an excited state and the elimination of water and molecular nitrogen. The formation of the strong triple bond (N=N) is a major factor in the release of energy in the form of light. 

The chemiluminescent nature of 5-amino-2,3-dihy-dro-l,4-phthalazinedione, historically termed luminol, was first reported by H.O. Albrecht in 1928. The characterization of the reaction kinetics and the emitting species required more than three decades of investigations. Luminol is almost quantitatively oxidized to the 3-aminophthalate ion in both protic and aprotic solvents (Scheme 4). 

Several methods are used for the determination of the "active" zones in a vessel. These include optical (light diffraction), chemical, electrical, or mechanical (metal foil perforation), the measurement of local temperature increase (silicone-coated thermocouple), microdiffusion sensor, microvibration thermal sensor, microphones, or sonoluminescence.45 The principle of the latter consists of the oxidative degradation of luminol to aminophthalate under the action of the hydroxyl radical produced by water sonolysis. Aminophthalate is produced in an... 

In an aqueous medium, luminol reacts with a potent oxidizing agent in the presence of a catalyst (generally a metal, a metal-containing compound or an enzyme) in alkaline solution to yield 3-aminophthalate in an excited electronic state, which returns to ground state with the production of light. Metal ions such as Co(ii), Cu(n) and Fe(ii) or enzymes like HRP and microperoxidase catalyse the luminol reaction efficiently (Figure 27.3). The CL reaction can be summarized as follows ... 

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