Identification of stereoisomeric benzo[a]pyrene tetrols by reversed-phase liquid chromatography coupled semi-ondashline to fluorescence line-narrowing spectroscopy

A low-temperature laser-based fluorescence technique, i.e., fluorescence line-narrowing (FLN) was used for the identification of four stereoisomeric benzo[a]pyrene tetrols, which are the hydrolysis products of benzo[a]pyrene-DNA adducts. Coupling of micro-liquid chromatography in the reversed-phase mode (eluent: methanol-water, 60:40, v/v) and thindashlayer chromatography utilizing silica plates was involved to obtain an “immobilized” liquid chromatogram. The chromatographic peaks thus obtained could be identified by FLN spectroscopy. By FLN the vibrational levels of the first excited electronic state can be determined. Some of these vibrations differ significantly for the stereoisomers concerned, so that unambiguous spectroscopic identification is possible. A Nd:YAG pumped dye laser was frequency doubled to obtain excitation wavelengths in the 360–368 nm range. The fluorescence was monitored by an intensified linear diode-array detector mounted on a monochromator with high resolving power. Detection limits of the complete separation and identification system are in the 100 femtomole range.