HPLC Band Shape Analysis for Postcolumn Chemiluminescence Detection Obtained by Finite-Difference Simulation:  The Chemical Band Narrowing Effect

Algorithms for the finite-difference digital simulation of partition chromatography with chemiluminescence (CL) detection have been developed for a proposed postcolumn CL reaction mechanism for acridinium esters reacting with peroxide and base. These algorithms use classical plate theory to establish the theoretical chromatographic band shape; subsequently, statistical moment analysis is employed to evaluate the retention time and the variance for the “spectrophotometric” peak obtained prior to postcolumn reaction with peroxide and base and for the CL peak shape detected downstream. The resulting model can be used to compare the two band shapes and to optimize postcolumn CL detection conditions such as the concentration of peroxide and base and the length of tubing connecting reaction tee and detector. The simulation assesses the effect of each photochemical reaction involved in the CL mechanism on detection level and chromatographic efficiency. As such, this work provides fundamental insight into the efficiency-enhancing phenomenon known as the “chemical band narrowing effect”. For one of the cases considered herein, a competitive dark reaction resulting in acridinium pseudobase formation is shown to contribute to this effect. The theoretical model predicts chromatographic behavior similar to the experimental chromatograms of 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methylacridinium-9-carboxylate fluorosulfonate reported herein.