Laser-induced quenched phosphorescence detection in capillary electrophoresis.

Abstract

The feasibility of laser-based excitation for quenched phosphorescence detection in capillary electrophoresis (CE) was explored for the first time by using a small-size, quadrupled Nd-YAG laser emitting 266 nm pulses (duration, 0.4 ns) at a repetition rate of 7.8 kHz. To provide a continuous phosphorescence background, the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) was added to the separation buffer. Both experiments and theory show that in laser-induced phosphorescence (LIP) - in contrast with lamp-excited phosphorescence - one normally deals with such high triplet-state phosphorophore concentrations that triplet-triplet annihilation is the major deactivation pathway. This results in a lower quantum yield of the analyte-induced bimolecular quenching interaction and, thus, the observed quenching signal. The situation can be improved by using a cylindrical lens for excitation in order to reduce the irradiance. In this case limits of detection (LODs) similar to those obtained using lamp excitation (1x10(-8) M) were achieved, while the width of the detection window was reduced from about 4 mm to 1 mm. Even under exclusion of triplet-triplet annihilation, i.e., under conditions of low irradiance, for our setup the quenching yields in LIP were smaller than in lamp-based phosphorescence detection. This is due to the repetition rate of the laser (7.8 kHz), which is too high in view of the phosphorescence lifetime (ca. 300 micros at low irradiance). Theory shows that this disadvantageous effect will be fully eliminated if the repetition rate is decreased to 1 kHz.