Fluorescence line-narrowing spectroscopy of analytes deposited on thin-layer chromatography plates Original Research Article

The sensitivity and potential robustness of fluorescence line-narrowing spectroscopy (FLNS) coupled with column liquid chromatography (LC) in a semi-on-line mode by depositing the analytes on a thin-layer chromatography (TLC) plate was studied with emphasis on analyte identification. Parameters considered were the type of TLC material, the liquid used to ensure heat conduction in the sample holder of the closed-cycle cryostat, and the spectral acquisition time involved in recording the FLN spectrum. One of the four isomeric benzo[a]pyrene tetrols formed upon hydrolysis of benzo[a]pyrene-DNA adducts was used as a test compound. The separation of these tetrols with reversed-phase LC requires an eluent with high water content. Optimal results were obtained with deposition on silica TLC plates and the use of glycerol in the sample holder with a spectral acquisition time of 1 min. Under these conditions the detection limit is 30 fmol at 355 nm excitation. To improve the between-days fluctuation of the FLNS signal intensities, several compounds with a pyrene chromophore were tested as reference compounds. The use of 1-methoxypyrene as internal reference reduced the standard deviations 2–3-fold, viz. to less than 15% in FLN peak area. This indicates that the fluctuations are caused by experimental parameters, and not by the FLN process itself.