Electrogenerated chemiluminescence of isoluminol and acridinium esters in flow injection analysis and HPLC

Electrogenerated chemiluminescence (ECL) is the process in which electrochemically generated reactants undergo high energy electron transfer reactions in solution to generate excited state molecules which relax to the ground state with emission of light. Such reactions offer several advantages in analysis. Specifically, in situ reagent generation eliminates band-broadening and dilution effects associated with the solution addition of reagents. In addition precise control over the spatial and temporal characteristics of the light emission is gained. Motivated by these advantages, this research focused on the study and application of the electrogenerated chemiluminescence reactions of isoluminol and acridinium esters.;Initial research involved the ECL reaction of an isoluminol derivative, 4-isothiocyanatophthalhydrazide (ILITC). The derivative was synthesized and characterized prior to use as a label for amino acid determination. In analysis, chromatographic separation of ILITC-amino acids preceded the ECL detection. A drastic reduction in ECL efficiency was observed upon coupling of ILITC to the amino acid analytes. Experiments suggested that although an ILITC-amino acid coupling reaction occurs, the resultant product has lost CL activity. Additional studies illustrated a correlation between CL activity and the conditions used to couple ILITC to the analyte. In spite of difficulties associated with loss of CL activity, the ECL determination of ILITC-amino acids (1 nmol) and the feasibility of on-line analyte derivatization were demonstrated.;The ECL reaction of acridinium esters was studied and shown to proceed via the electrochemical generation of hydrogen peroxide (a reagent necessary for the CL reaction). In further fundamental studies of the acridinium ester decomposition reactions, kinetic parameters associated with this decay were determined and applied in a model for pH optimization. The use of ECL for the determination of acridinium ester-labeled species was demonstrated via the successful quantitation (to 50 fmol) of lysine labeled with a hydroxysuccinimide acridinium ester. Instrumental configurations for use of ECL detection in immunoassay were validated; however, loss of ECL signal in the presence of BSA and human TSH antibodies hindered application of acridinium ester ECL detection to model immunoassay reactions.