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

The sensitivity and potential robustness of fluorescence Line-narrowing spe ctroscopy (FLNS) coupled with column liquid chromatography (LC) in a semi-o n-line mode by depositing the analytes on a thin-layer chromatography (TLC) plate was studied with emphasis on analyte identification. Parameters cons idered were the type of TLC material, the liquid used to ensure heat conduc tion in the sample holder of the closed-cycle cryostat, and the spectral ac quisition time involved in recording the FLN spectrum. One of the four isom eric benzo[a]pyrene tetrols formed upon hydrolysis of benzo[a]pyrene-DNA ad ducts was used as a test compound. The separation of these tetrols with rev ersed-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 compo unds 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 flu ctuations are caused by experimental parameters, and not by the FLN process itself. (C) 1999 Elsevier Science B.V. All rights reserved.