3-aminophthalate excited

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 requirement of hydrogen peroxide or oxygen in diazaquinone chemiluminescence appears to be another argument against the hypothesis of Albrecht 128> or of Kautsky and coworkers 132> that the diazaquinone is simply hydrolyzed to yield aminophthalate and diimine which in turn reduces other diazaquinone molecules to luminol in an excited state so that it then emits. 

The reaction L" + 02" —> LHOCT leads, after some intermediate stages, to generation of electronically excited compound AP (aminophthalate anion) [23, 24], In these reactions, let kj = 31k2, but at pH = 9.2 both proceed equally fast. 

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

The emitting species was found to be the singlet-excited state of 3-aminophthlate ion in both protic and aprotic solvents. This identification was made based on the equivalence of the chemiluminescence spectrum of luminol and the fluorescence spectrum of 3-AP ion . In different reaction media, slightly different maximum chemiluminescence wavelengths are observed (Table 2). The spectral shift observed when the system changes from aqueous media to DMSO or other aprotic solvents can be ascribed to a quinoidal form of 3-aminophthalate (26) formed in aprotic solvents (Scheme 15). ... 

Steinfatt proposed an alternative mechanism for the formation of excited aminophth-alate, based on the concept of dioxirane-carbene mediated chemiexcitation, which is also attributed to other chemiluminescent systems ° °. After the attack of hydrogen peroxide on the diazaquinone 27 carbonyl carbon, a perhydrolysis step is postulated to result in the intramolecular dioxirane-carbene system (32) in the excited state ° ° . This species presumably rearranges to 3-aminophthalate dianion while still in the singlet-excited state (Scheme 23). Although this is a very interesting mechanistic proposal, it is based on experimental evidence obtained with indirect phthaloyl peroxide chemiluminescence and no further evidence corroborates this proposal. 

A CIEEL approach can also be used to explain chemiexcitation in luminol chemilumi-nescence . Two possibilities arise (i) an electron transfer from the amino group to the peroxidic moiety in the antiaromatic peroxide 33, resulting in bond cleavage followed by intramolecular back-electron transfer and formation of excited 3-aminophthalate (Scheme 24) (ii) the equilibrium between the peroxycarboxylic aldehyde 34, formed after elimination of nitrogen, and the cyclic peroxy semiacetal 35 is shifted in the direction of 35, as the result of an electron transfer from the amino group to the cyclic peroxide moiety, followed by 0—0 bond cleavage . Back-electron transfer would result in chemiexcitation (Scheme 25). 

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. 

It appears that there may be several paths leading to eventual generation of electronically excited state products from luminol. In non-protic solvent, in particularly DMSO, the reaction apparently proceeds through the intermediate luminol dianion [33]. Reaction of the dianion with oxygen results in the formation of 3-aminophthalate in the excited state (42). White and Roswell (1970) have shown that under these conditions the chemiluminescence is due to emission from the excited phthalate. 

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. 

Figure 7.6 Reaction between hydrogen peroxide and luminol in the presence of peroxidase producing the excited 3-aminophthalate and the emitted light at a wavelength of 430 nm...

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. 

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. 

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 tautomerism is driven by the increased basic strength of 3-aminophthalate in the aprotic solvent with the resultant structural change decreasing the energy of lowest excited singlet state. The kinetics of the reaction in dimethylsulfoxide are first order with respect to the concentrations of hydroxide, oxygen, and luminol. An important analytical feature is the catalytic effect of certain transition metal cations upon the reaction shown in Scheme 4. The luminescence intensity is proportional to the concentration of species including cobalt(II), copper(II), chromium(III), iron(II), and nickel(II) at levels down to 10 moll . ... 

---