Development of a detection technique for liquid chromatography and microbore liquid chromatography using tris(2,2'-bipyridyl)ruthenium(II) electrogenerated chemiluminescence.

摘要

This research has involved the development of a detection technique for liquid chromatography (LC) and microbore LC using tris(2,2{dollar}spprime{dollar}-bipyridyl)ruthenium(II) (Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar}) electrogenerated chemiluminescence (ECL). This detection technique has proven to be valuable in detecting a wide variety of amine-containing compounds without the need for derivatization. Many of these compounds are difficult to detect with other LC detection techniques such as UV/visible absorbance, fluorescence, and electrochemical detection. Because of the biogenic properties of many amines, their determination is of importance in a variety of pharmaceutical, clinical, and environmental studies. Because of the very specific selectivity of Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} ECL, it is very useful for detection of amine-containing compounds in complex samples, including biological fluids and environmental samples. The Ru(bpy){dollar}sb3sp{lcub}3+{rcub}{dollar} species which reacts with the analyte molecule to give light emission is electrochemically generated in the electrochemical/optical flow cell used for detection. This technique has proven to be more reproducible and allow better detection limits than pumping externally generated Ru(bpy){dollar}sb3sp{lcub}3+{rcub}{dollar} into the flow cell. Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} can also be immobilized on an electrode surface in the flow cell, eliminating the need for Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} to be added post column or to the mobile phase. Oxalate, a biologically important compound responsible for several clinical disorders, was separated and quantitated from both urine and blood plasma samples. This was done using an ion-pair reverse-phase separation with Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} being added postcolumn for ECL detection. Results for the quantitation of oxalate in urine were compared to commercially available enzymatic test kits, with results differing by only 1%. Indole compounds were discovered to be interferants which decrease the CL intensity. The detection range for oxalate using Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} ECL detection easily covered the normal clinical range in both urine and blood plasma and was superior to the enzymatic tests which cannot be used at the low oxalate levels in blood plasma. Oxalate was also separated and detected by putting Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} directly in the mobile phase to decrease band broadening caused by post-column addition and to reduce the amount of instrumentation necessary. These experiments showed that adding Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} to the mobile phase did not significantly change the separation. Detection limits were improved due to the decrease in band broadening and sample dilution. Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} ECL was also applied to detection in microbore LC. Custom electrochemical/optical flow cells were designed for this application. Electrode orientations in the cell were redesigned to obtain optimum electrochemical performance. Cell designs for flow injection analysis (FIA), LC, and microbore LC are compared and their characteristics are discussed, as well as the reasons new cells were designed. Fiber optics were also incorporated into microbore LC cells, showing several advantages over conventional flow cells. Fiber optic cells allowed greater collection of light emission, eliminate the need for a dark box, and isolate the mobile phase from the detector electronics. Oxalate and erythromycin were separated and detected using Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} ECL with microbore LC. An evaluation of potential pharmaceutical, biological, and environmental applications for Ru(bpy){dollar}sb3sp{lcub}2+{rcub}{dollar} ECL detection with microbore LC was done.