Page 226 - Modern Derivatization Methods for Separation Sciences

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methods used in HPLC and CE, fluorescence detection has been frequently utilized for the
determination of trace levels of bioactive compounds in complicated matrices such as biological and
environmental samples, owing to its high selectivity and sensitivity. However, most biologically and
environmentally important substances are weakly fluorescent or nonfluorescent. Thus, various reagents
have been developed for the fluorescence detection of their substances in HPLC and CE.

The reagents utilized for fluorescence detection are divided into two groups: 'fluorogenic reagent' and
'fluorescence labeling reagent'. The fluorogenic reagents are generally non-fluorescent and react with
target compounds to form conjugatedring molecules, resulting in the generation of fluorescence. The
fluorescence labeling reagents are composed of a highly fluorescent aromatic moiety (fluorophore) and
a reactive group, and the reactive group attaches to an analyte to give a fluorescent derivative.

There are two approaches to derivatization for fluorescence detection in HPLC and CE: precolumn
(pre-capillary) derivatization and postcolumn (post-capillary) derivatization. The former and latter are
based on the fluorescence derivatization of analytes before and after separation by HPLC or CE,
respectively. The pre-column derivatization should produce, individually, the different derivatives from
the analytes. However, the optimum reaction conditions are not limited by the HPLC separation
conditions. On the other hand, post-column derivatization should proceed rapidly because a prolonged
reaction time causes peak broadening in chromatography. The reagents for post-column fluorescence
derivatization need to be non-fluorescent or markedly different from the derivatives in their
fluorescence excitation and/or emission spectra in the mobile phase; they are allowed to give multiple
fluorescent derivatives, provided that their reactions are reproducible. In general, the fluorogenic
reagents have the possibility of being used for both pre and post-column derivatization methods,
although fluorescence labeling reagents are used only for precolumn derivatization.

Determination of sample components at extremely low concentrations (ppb-ppt level) are

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