Evaluation of Use of Tris(2, 2'-Bipyridyl)Ruthenium(III) as a Chemiluminescent Reagent for Quantitation in Flowing Streams

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.