Chemiluminescence detection in capillary electrophoresis using an ultra-fast co-catalyzed peroxyoxalate chemiluminescent reaction and electrokinetic reagent delivery

Despite the impressive limits of detection and inherent selectivity afforde d by peroxyoxalate chemiluminescence (POCL) detection, efficient coupling o f POCL to capillary electrophoresis (CE) remains limited by the relatively slow kinetics of the reactions that drive imidazole-catalyzed chemiluminesc ence. Moreover; oxalate esters, used in POCL are sparingly soluble in polar solvents and hydrolyze rapidly, presenting an additional challenge with re spect to detection following aqueous phase separations. In this paper, a no vel method for coupling an ultrafast POCL reaction to CE is presented. Post separation electrokinetic delivery of the POCL reagent bis(2,4,6-trichloro phenyl)oxalate (TCPO) was accomplished using a commercially available micro tee. Electrokinetic addition of TCPO allowed for precise control of the ra tio of TCPO to the chemiluminescence (CI) reagents 1,2,2,6,6-pentamethylpip eridine (PMP) and 1,2,4-triazole (triazole), spiked into the running buffer . This novel method for CL reagent delivery avoided the problems and costs associated with using pressure or mechanical pumps to deliver reagents post separation. Use of this dual-component system (PMP and triazole) resulted in intense CL with half-lives of less than 2 seconds. Optimum conditions fo r CE-POCL detection were investigated using stopped-flow kinetics. The dete ction limit for 3-aminofluoranthene, following separation by CE, was <0.95 nM.