Oxalate esters chemiluminescence sensitivity

Table VI. RP-HPLC Chemiluminescence Sensitivity in Previous Studies with Oxalate Esters...

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

Fig. 46. Possible mechanism for chemiluminescence of oxalate esters. Attack by peroxide ions leads to a peroxyoxalate intermediate which, in turn, breaks down to an unstable excited-state dio-xetaneone. In the presence of an appropriate fluorophore, energy transfer from the dioxetaneone leads to luminescence from the fluorescent sensitizer.

While the main field of application of chemiluminescence (CL) reactions is catalytic processes (e.g., oxidation of liuninol in the presence of metal ions), some noncatalytic reactions are also of great interest on accormt of the high sensitivity they provide. One of the most prominent of such reactions is that involving the hydrogen peroxide-induced oxidation of aryl oxalate esters in the presence of a suitable fluorescent species (Scheme 2). The most widely used aryl oxalates for this purpose are bis(2,4, 6-trichlorophenyl)oxalate (TCPO) and bis(2,4-dinitro-phenyl)oxalate. These CL reactions have been used... 

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