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|Title:||Investigation of the Luminol Chemiluminescence Reaction as a Post-Column Reaction Detector for HPLC|
|Author(s):||Koerner, Philip James, Jr|
|Department / Program:||Chemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The luminol chemiluminescence (CL) reaction has been used as a post-column reaction detector for high performance liquid chromatography (HPLC). The research reported here has progressed in two principle areas: first, the development of a method for the determination of amino acids and other complexing species based on suppression of the Cu(II)-catalyzed luminol CL reaction and second, the development of a method for the determination of $\beta$-D-glucosides using immobilized enzymes and luminol CL detection of the enzymatically generated hydrogen peroxide.
Free Cu(II) catalyzes the luminol CL reaction and the CL emission increases with increasing Cu(II) concentration. Amino acids were then detected by their complexation of Cu(II), resulting in negative signals (decreases in the observed CL emission) as the amount of free Cu(II) was decreased. Detection limits of 1 pmol-2 nmol were obtained using a flow injection system, and the method was demonstrated to be applicable to the determination of amino acids separated via ion-exchange chromatography. This method was extended to the determination of other species (such as amines, aminoglycosides, catecholamines, and cyanide) which also complex Cu(II), and detection limits in the low pmol range were obtained for several species. This method was also demonstrated to be applicable for the determination of catecholamines separated via reverse phase HPLC, and for the determination of the gentamicin C components in gentamicin sulfate separated via ion-pair chromatography.
The determination of $\beta$-D-glucosides utilized sequential immobilized enzyme reactors (IMER) to first hydrolyze $\beta$-D-glucosides to $\beta$-D-glucose (using $\beta$-glucosidase) and then to produce hydrogen peroxide from the $\beta$-D-glucose (using glucose oxidase); the hydrogen peroxide was then detected with luminol CL (the emitted light intensity is proportional to peroxide concentration). This method was used for the determination of individual $\beta$-D-glucosides (phenyl, p-nitrophenyl, salicin, and metsulfuron methyl) via flow injection analysis and was extended to the determination of a mixture of glucosides following their separation via reverse phase HPLC. (Abstract shortened with permission of author.)
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
|Date Available in IDEALS:||2014-12-15|