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

Summary Despite the impressive limits of detection and inherent selectivity afforded by peroxyoxalate chemiluminescence (POCL) detection, efficient coupling of POCL to capillary electrophoresis (CE) remains limited by the relatively slow kinetics of the reactions that drive imidazole-catalyzed chemiluminescence. Moreover, oxalate esters, used in POCL, are sparingly soluble in polar solvents and hydrolyze rapidly, presenting an additional challenge with respect to detection following aqueous phase separations. In this paper, a novel method for coupling an ultrafast POCL reaction to CE is presented. Post separation electrokinetic delivery of the POCL reagent bis(2,4,6-trichlorophenyl)oxalate (TCPO) was accomplished using a commercially available micro tee. Electrokinetic addition of TCPO allowed for precise control of the ratio of TCPO to the chemiluminescence (CL) reagents 1,2,2,6,6-pentamethylpiperidine (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 for CE-POCL detection were investigated using stopped-flow kinetics. The detection limit for 3-aminofluoranthene, following separation by CE, was<0.95 nM.