Postcolumn fluorescence-quenching

The choice of the mobile phase is very important, as fluorescence is sensitive to fluorescence quenchers. Highly polar solvents, buffers, and halide ions quench fluorescence. The pH of the mobile phase is also important to fluorescence efficiency for example, quinine and quinidine only display fluorescence in strongly acidic conditions, whereas oxybarbiturates are only fluorescent in a strongly alkaline solution [67,68]. Due to the stability of the chromatographic sorbents, the use of very acidic or basic mobile phase may not be possible. One alternative is to alter the effluent pH postcolumn. Postcolumn addition of sulfuric acid has been used for the assay of ethynodiol diacetate and mestranol in tablets [69]. Another example is the determination of tetracycline antibiotics in capsules and syrup where EDTA and calcium chloride were added to enhance fluorescence [70]. 

Aflatoxins are naturally strongly fluorescent compounds, so the HPLC identification of these molecules is most often achieved by fluorescent detection. Reverse-phase eluents quench the fluorescence of AFBl and AFGl [60] for this reason, to enhance the response of these two analytes, chemical derivatization is commonly required, using pre- or postcolumn derivatization with suitable fluorophore, improving detectability. 

A few methods based on fluorescence have been described for biotin and avidin determinations. A first one is based on the quenching of the avidin tryptophan fluorescence by biotin upon binding (71). A second one involves the increase of the fluorescence of avidin labeled with fluorescein isothiocyanate upon binding of biotin (72). The latter technique has been applied to HPLC postcolumn detection of biotin (see below). The sensitivities are, respectively, 20 and 0.5 ng. Another method is based on the variation of the fluorescence polarization of a biotin-fluorescein derivative upon interaction with avidin (73). Minimal detectable concentrations reported were 5 ng for avidin and 0.1 ng for biotin (73). Mock et al. reported another technique relying on the displacement by biotin of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) (Fig. 10) when bound to avidin (74). The advantage of this method is obviously the large increase of fluorescence of 2,6-ANS when bound to avidin as compared to the unbound form in water solution. The detection limit was around 1 ng. This technique has also been applied to postcolumn detection of biotin (see below). 

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