Luminol structure

Luminol (5-aminophthalazin-l,4-dione) [521-31-3] M 177.2, m 329-332°, pK] 3.37, pK2 6.35. Dissolved in KOH soln, treated with Norit (charcoal), filtered and ppted with cone HCl. [Hardy, Sietz and Hercules Talanta 24 297 1977.] Stored in the dark in an inert atmosphere, because its structure changes during its luminescence. It has been recrystd from O.IM KOH [Merenyi et al. J Am Chem Soc 108 77716 1986]. 

However, the formation of these products does not appear to play a critical role in the decision as to whether the 425 nm and 480 nm maxima are due to different states of the same molecule or to different compounds. It was reported that special care was taken to ensure the purity of luminol and of 3-aminophthalate 109>. In commercially available 3-amino-phthalic acid a yellowish impurity exhibiting brilliant green fluorescence was detected 109> this substance also formed in neutral solutions of pure 3-amino phthalic acid and crystallized from these solutions in yellow crystals. The structure of this substance was determined to be 53 its absorption spectrum has a maximum at 388 nm the fluorescence maximum is at 475 nm, with a fluorescence quantum yield of about 0.75 in DMF i 9). 

Figure 4 Chemical structure of (A) luminol (5-amino-2,3- dihydro-l,4-phthalazinedi-one) and (B) isoluminol.

Burdo and Seitz reported in 1975 the mechanism of the formation of a cobalt peroxide complex as the important intermediate leading to luminescence in the cobalt catalysis of the luminol CL reaction [116]. Delumyea and Hartkopf reported metal catalysis of the luminol reaction in chromatographic solvent systems in 1976 [117], while Yurow and Sass [118] reported on the structure-CL correlation for various organic compounds in the luminol-peroxide reaction. 

The structures of luminol derivatives used for HPLC-CL detection are shown in Figure 7A. Analytes labeled with luminol derivatives can be detected using hydrogen peroxide and potassium hexacyanoferrate(III) under alkaline conditions after HPLC separation (Table 1). For example, ibuprofen in saliva [34], saturated... 

As in the luminol case, the main role of the enhancer (EnH) seems to be related to turnover of the enzyme, generating enhancer radicals (En rad) in the process that are capable of oxidizing the acridan ester (AcH). The structure of the enhancer obviously is very important. To accelerate HRP turnover, the enhancer must on the one hand be able to rapidly react with the reactive HRP intermediates Cl and especially CII (k2 and k3 large). On the other hand, the oxidized enhancer intermediate (radical or radical cation) must be able to oxidize the acridan ester (light-generating step). This last reaction also depends on the structure of the acridan ester in a very unfavorable case, adding an enhancer for enzyme turnover could actually diminish the light production if k 4 > fct (Fig. 5), i.e., if the enhancer radical would not be able to oxidize the acridan ester. 

Although substituted phenols (e.g., para-iodophenol, para-phenylphenol, firefly luciferin, coumaric acid) are popular enhancers, in both luminol and acridan ester oxidation, enhancers with other functional groups [24], e.g., phe-nylboronic acids [25-28], phenothiazines [29], are also useful. As an example the structure of the phenothiazine enhancer used in the Supersignal substrate family is shown in Figure 6. 

Commercially available horseradish peroxidase (crystalline) will substitute for luriferase in the foregoing reaction. In addition, a compound of known structure. 5-amino-2, 3-dihvdro-l, 4-phthalazinedione (also known as luminol), will substitute for luciferin. The mechanisms appear to be the same regardless of the way in which the crosses are made. Thus, a model bioluminescent system is available and can be used as a sensitivity assay for H2O2 at neutral pH. The identification of luciferase as a peroxidase is of interest since this represents the only demonstration of a bioluminescent system in which the catalytic nature of a luciferase molecule has been defined. 

The chapter has been structured into several sections first, there is a brief introduction, including a description of the concept and general principles of CL and how it is used in the field of explosives. The second section describes CL applications in the field of explosives and focuses in particular on the thermal energy analyser (TEA) because of its important role in the trace detection of explosives. The recent applications of luminol CL and electrochemiluminescence (ECL) to explosive detection are also described. Finally, because much of the research into explosive detectors has been directed towards civilian safety, a third section describes how CL is used as a security measure to detect explosives. 

Smith and Schuster (1978) have reported the observation of chemiluminescence from endoperoxide [36] which is structurally related to [35], the proposed luminol intermediate (44). This observation suggests yet another possibility for the structure of the key chemiluminescent intermediate from luminol, the o-xylylene peroxide analogous to [37]. 

There are several chemical systems that have been observed to generate light and whose mechanisms have not been clearly defined. Many of these reactions suffer from the same difficulty as does luminol that is, the key intermediate has not been isolated, hence its structure is not known, and its properties must be inferred from indirect experimental results. In this section we will introduce some of these systems and review them briefly. 

Leong, M. M. L, Fox, G. R, and Hayward, J. S (1988) A photodetection devise for luminol-based immunodot and western blotung assays. AnaL Btochem. 168,107-114. Kaufmann, S. H. (1989) Additional members of the rat liver lamin polypepude family Structural and immunological characterization./ BtoL Otem 264,13946-13955... 

Fig. 7 Chemical structures of luminol derivatives exhibiting chemiluminescence.

Fig. 13. Chemical structures of the chemiluminescent cyclic hydrazides luminol and isoluminol.

While luminol and isoluminol require an oxidant plus a catalyst for initiation of the chemiluminescent reaction, esters derived from A -methyl acridinium carboxylic acid require only alkaline hydrogen peroxide (W4). Acridinium esters were first introduced by McCapra s group (M23, M25, S32), based on earlier work on the bioluminescence of the lucigenin/luciferase system (G18), and reviewed in McCapra and Beheshti (M21). From Fig. 19, one can see the structural similarity between lucigenin and a typical acridinium ester. 

Determine the molecular formula for luminol and draw its Lewis structure. 

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