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

Abstract

SummaryDespite the impressive Jimits of detection and inherent seJectivily afforded by peroxyoxaJate 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, usecl in POCL, are sparingly soluble in polar solvents ancl hydrolyze rapidly, presenting an additional challenge with respect to detection following aqueous phase separations. In this paper, a novel method forcouphng 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) ancl 1,2,4-triazole (triazole), spiked into the running buffer. This novel method for CL reagent delivery avoided the problems ancl costs associated with using pressure or mechanical pumps to deliver reagents post separation. Use of this dual-component system (PMPand 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 byCE, was <0.95 nM.

Advances and analytical applications in chemiluminescence coupled to capillary electrophoresis

“…Kuyper et al [157] presented a novel method for coupling an ultrafast PO-CL reaction to CE based on the use of the ancillary base 1,2,2,6,6-pentamethylpiperidine (PMP) to assist the nucleophilic reagent 1,2,4-triazole (triazole). Under certain conditions, this PO-CL reaction exhibits a half-life of less than 2 s with light yields that are two times larger than the more well-known imidazole-catalyzed PO-CL reaction.…”

“…The method avoids problems associated with tedious reactor configurations and expensive syringe pumps. In relation to its previous study [157], a significant improvement in separation efficiency employing electrokinetic reagent delivery is realized through the use of a post-column co-catalyzed CL configuration. Using separation buffers containing small percentages of water (e.g.…”

Post-column ultra-fast co-catalyzed peroxyoxalate chemiluminescence detection in capillary electrophoresis and capillary electrochromatography

“…The ideal aryl oxalate used in CL detection in CE would react rapidly (half life of 500 milliseconds or less), efficiently (quantum yield approaching 20%) and be soluble in buffers typically employed in CE or CEC separations. Recently, we discovered that ultrafast co-catalyzed POCL [39] is well suited for detection in capillary dimensions [36]. However, our prototype CE-CL instrument was based on the post separation electrokinetic delivery of the POCL reagent TCPO using a commercially available micro tee.…”

“…More importantly, a significant amount of light may be lost if the time over which the CL emission occurs is much greater than the bandwidth for each analyte. Recently, chemical band narrowing, accomplished by altering the kinetics of the POCL reaction, was used in an on-column CE-CL detection scheme [36]. Unfortunately, because a micro-tee was used to couple a separation capillary with a CL reagent capillary, a significant amount of band broadening was observed (theoretical plates-1000 2000).…”

Electrochemical and optical detectors for capillary and chip separations

“…As a result, CL serves as a good candidate for detection in capillary and chip separations [40,41,[67][68][69][70][71][72][73][74]. Several CL reagents have been used (e.g., luminol l [71][72][73], peroxyoxalate [68,70], and tris(2,2′-bipyridyl)ruthenium (II) [67,74]). …”