The Mechanism of Luminol Chemiluminescence

The starting point for luminol oxidation is clearly an anion, since base is an essential catalyst. Luminol monoanion (37) is present in aqueous alkaline solutions, almost as the sole ionic luminol species. Luminol dianion (38) on the other hand, has been shown to exist in aprotic solutions (DMSO or DMF, e. g.) with tert.butoxide as base [6, 29, 48]. 

In mixed aqueous/DMSO solutions, both the mono- and dianion are observed [29]. Table 7 lists the absorption and fluorescence of luminol and its ions [29]. 

It is therefore possible that the first steps of the chemiluminescence mechanism are different in these two classes of solvents. 

In the aprotic systems, molecular oxygen can attack the dianion to form a peroxide and additional catalysis is not required for chemiluminescence. 

This is almost certainly the result of the greater stability of the intermediate radical anion as opposed to the radical 

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The study of both bioluminescence and chemiluminescence which led to our present understanding was inaugurated by Harvey [8] in 1953 with the publication of Bioluminescence . Also about this time White [9] made the first thorough and convincing study of the mechanism of luminol chemiluminescence. A review of the chemistry of the hy dr azides, with special reference to their chemiluminescence [10] also added to the foundations. 

Although diazaquinones could be considered in a separate class of chemiluminescent compounds their importance lies in their association with the mechanism of luminol chemiluminescence. It is not yet certain whether they are intermediates under all reaction conditions, but they are clearly implicated. 

Hydrazide chemiluminescence has been investigated very intensively during recent years (for reviews, see 1>, p. 63, 2>, 90>). Main topics in this field are synthesis of highly chemiluminescent cyclic diacyl hydrazides derived from aromatic hydrocarbons, relations between chemiluminescence quantum yield and fluorescence efficiency of the dicarboxylates produced in the reaction, studies concerning the mechanism of luminol type chemiluminescence, and energy-transfer problems. 

E. H. White and coworkers 2> have proposed a mechanism of luminol (Lum) chemiluminescence in aprotic media in which the luminol dianion (Lum2<-)) is a key intermediate ... 

Concerning the mechanism of the chemiluminescent reaction between luminol and molecular oxygen in DMSO M. T. Beck and F. Joo 209> performed kinetic experiments leading them to the conclusion that the formation of an oxygen containing intermediate (see p. 109) is a reversible step, in contrast to the opinion of E. H. White and M. M. Rauhut, D. M. Hercules and their coworkers, and others 2 3 109). 

Independent of the precise mechanism of the chemiluminescence-producing reaction, the process of luminol-dependent PCL can be subdivided into two stages ... 

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). 

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. 

Haapakka, K., and Kankare, J.J., 1982. The mechanism of the electrogenerated chemiluminescence of luminol in aqueous alkaline solution. Analytica Chi-... 

Chemiluminescence generally arises from chemical reactions in solution in which an oxidation occurs that involves either molecular oxygen or hydrogen peroxide (H2O2). The quantum yield in solution is generally very low because certain derivatives inhibit luminescence, notably oxygen. There are some exceptions such as luminol, acridinium salts, and some oxalic esters. Although the mechanism is poorly understood, the reaction of luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) can be written schematically ... 

With a few exceptions, monoacyl hydrazides chemiluminesce with efficiencies some 100 fold lower than the cyclic diacylhydrazides. With the assumption that the mechanism of the luminol type chemiluminescence involves an o-xylene peroxide (p. 97), this behaviour of the linear hydrazides would be easily understood such an intermediate is not possible in the Unear case. 

DPA) in dimethylphthalate at about 70°, yields a relatively strong blue Umax =435 nm) chemiluminescence the quantum yield is about 7% that of luminol 64>. The emission spectrum matches that of DPA fluorescence so that the available excitation energy is more than 70 kcal/mole. Energy transfer was observed on other fluorescers, e.g. rubrene and fluorescein. The mechansim of the phthaloyl peroxide/fluorescer chemiluminescence reaction very probably involves radicals. Luminol also chemiluminesces when heated with phthaloyl peroxide but only in the presence of base, which suggests another mechanism. The products of phthaloyl peroxide thermolysis are carbon dioxide, benzoic acid, phthalic anhydride, o-phenyl benzoic acid and some other compounds 65>66>. It is not yet known which of them is the key intermediate which transfers its excitation energy to the fluorescer. 

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 effect of the hydroxyl radical (HO ) on luminol chemiluminescence has also been intensively studied178 187 198 199. Although detailed mechanisms for the reaction of hydroxyl radicals with hydrazides remain unknown, two different processes are assumed to be involved oxidation of the hydrazide group and addition of hydroxyl radical to the aromatic ring (Scheme 19)180 1". 

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