Immobilized Luminol as a Chemiluminescent Reagent

218 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.Work presented in this thesis investigates the first use of immobilized luminol chemiluminescence (CL) reagents in the flow injection analysis of hydrogen peroxide. The research reported here has progressed in two principal areas: first, the method development and characterization of immobilized luminol CL reagents and second, the application of the immobilized reagents to the flow injection determination of peroxide with emphasis on the development of a totally solid-state detection scheme for peroxide based on the luminol reaction.The largest luminol loadings (approximately 90 $\mu$mol/g support) have been achieved with a glutaraldehyde linkage to silica particles which have been silanized with an aminoalkylsilane. The luminol CL catalyst horseradish peroxidase has also been immobilized to silica-based supports with the most stable preparation utilizing a cyanoborohydride reduced glutaraldehyde linkage.Luminol is found to be released from the silica support via alkaline hydrolysis. Flow injection schemes have been devised which allow the light measurement to be made: (I) In the presence of the immobilized luminol and solution phase catalyst, (II) In a flow cell with released luminol and solution phase catalyst, and (III) In a flow cell which contains an immobilized catalyst (solution phase or electrode) and released luminol. Peroxide detection limits of 100-200 pmol were obtained with the silica-immobilized luminol in the various flow injection systems. Because silica was shown to degrade overall performance (via consumption of considerable amounts of base), an alternate support (Ambersorb) was examined for luminol immobilization. The best peroxide determinations were achieved using luminol adsorbed to Ambersorb with aqueous hemin as the CL catalyst. The log-log working curve shows linearity over a peroxide concentration range of 0.2-800 $\mu$M with a detection limit (15 pmol) comparable to that obtained with aqueous luminol.The use of a packed bed of solid luminol was also examined for the on-line generation of a luminol solution in the flow injection determination of peroxide. This approach resulted in slightly poorer performance for peroxide determination than that obtained with the Ambersorb-bound luminol.Possible extension of the use of immobilized luminol CL reagents is presented for the flow injection determination of analytes which can enzymatically yield peroxide. (Abstract shortened with permission of author.)U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD