Ss. Kok et al., Fluorescence line-narrowing spectroscopy of analytes deposited on thin-layer chromatography plates, ANALYT CHIM, 389(1-3), 1999, pp. 77-83
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.