The development of a detection method based on the electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+], immobilized in a Nafion film coated on an electrode is discussed. Control of the electrode potential controls creation of the reactive reagent Ru(bpy)3(3+) which reacts with certain analytes to yield chemiluminescence emission of intensity proportional to the analyte concentration. The reaction results in Ru(bpy)3(3+) being converted to Ru(bpy)3(2+), which then is recycled to Ru(bpy)3(3+) again at the electrode. This sensor has been used in flow injection to determine oxalate, alkylamines, and NADH. Detection limits are 1 microM, 10 nM, and 1 microM, respectively, with working ranges extending over 4 decades in concentration. Sensitivity is constant over the wide pH range from 3 to 10. With oxalate, and to a small extent with amines, emission intensities increase with increasing ionic strength; this was shown to be a phenomenon related to the Nafion film and not to the chemiluminescence reaction. Emission intensities increase with temperature. The sensor remains stable for several days with suitable storage conditions. Significant amounts of Ru(bpy)3(3+) are shown to be capable of storage within the film.

Three approaches are comparatively evaluated for the use of tris(2,2'-bipyridyl)ruthemum(II), Ru(bpy)33+, as a chemiluminescent reagent in flowing streams: (1) external generation of the reactive Ru(bpy)33+ oxidation state followed by contact with the analyte, (2) in situ generation of the Ru(bpy)33+ species from a solution mixture of the analyte and the Ru(bpy)32+ species as it passes through the reaction/observation cell, and (3) in situ generation of the Ru(bpy)33+ species from the Ru(bpy)32+ species immobilized within the observation cell. Oxalate and proline were used as representative analytes for comparison of these three modes with respect to the influence of experimental variables (reagent concentration, flow rate, pH) and resulting analytical performance (detection limit, working range, measurement precision). Additionally, a comparison was made of the relative ECL intensities obtained for a variety of analytes including oxalate, amino acids, aliphatic amines, peptides, and NADH. We find that each approach has its unique set of strengths and weaknesses. The external generation mode yields the most intense emission, especially for simple aliphatic amines, but working curves have poor linearity, and emission intensities have a large dependence on solution flow rate. The in situ immobilized approach results in lower intensities but yields the widest linear dynamic ranges, is most conservative of reagent, and has a particular sensitivity advantage for proline and NADH determinations. The in situ solution mode is superior for the detection of amino acids such as tryptophan, 5-hydroxytryptophan, and histidine and has time, convenience, and reliability advantages.In recent years, tris(2,2'-bipyridyl)ruthenium(III) electrogenerated chemiluminescence (ECL) has received considerable attention in chemical analysis because of its utility for detection of alkylamines, amino acids, and NADH without derivatization.Several research groups have been actively investigating the ECL mechanism of Ru(bpy)32+ and its analytical applications. Bard and co-workers have reported a series of investigations on the

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The effect of hydrodynamics on chemiluminescence (CL) emission is investigated for the tris(2,2‘-bipyridine)ruthenium(II/III) (Ru(bpy)3 2+/3+) electrogenerated chemiluminescence (ECL) system in solution phase and immobilized in Nafion. The maximum CL emission is obtained at low linear velocities for slow reactions, at intermediate linear velocities for reactions with intermediate kinetics, and at high linear velocities for fast reactions. Relative kinetic information for a variety of analytes is acquired by comparing charge-coupled device (CCD) images to stopped-flow and flow injection analysis data obtained with a photomultiplier tube (PMT). CCD images are also utilized to observe reproducibility and homogeneity of different Nafion film preparations. The spatial resolution provided by the CCD allows a more comprehensive analysis of the effects of flow on observed CL reaction kinetics than is possible with a PMT. These studies yield important guidelines for application and optimization of CL detection in flow injection, liquid chromatography, and capillary electrophoresis.