Fluorescent derivatizing reagents development

A wide range of fluorescent derivatization reagents and techniques have been developed to utilize the analytical advantages that fluorescence offers. These derivatization reagents are usually specific to functional groups (e.g., amine, hydroxy, thiol), and their specificity offers yet another opportunity for discriminating against interferents. For excellent reviews, the reader is referred to references [38] and [39]. 

The inability to detect precludes the ability to develop a separation. The selected technique is defined by the required limit of detection. If low-pg/mL levels are needed, it is fruitless to use a UV/visible absorbance detector. Laser-induced fluorescence (LIF) is usually appropriate, provided derivatization reagents are available if the solute does not have significant native fluorescence [2], Limits of detection of 10 10 M are easily achieved using LIF, provided the solute absorbs at a laser emission wavelength and has a reasonable fluorescence quantum yield. 

If the materials that are separated do not naturally fluoresce, then the plate, after development and drying, must be treated with a suitable derivatizing reagent to form appropriate fluorescent products. 

Metal complexation reactions are very convenient for fluorescence derivatization as they are fast and can be driven to completion by adding excess of reagent. These characteristics made them especially suitable for postcolumn derivatization in chromatographic separations, HPLC, and capillary electrophoresis (CE). An example of the use of complexation reaction is a CE postcolumn method developed for the determination of catecholamines and related compounds. The method is based on the ternary complex formed with Tb + ions, ethylenediaminetetraacetic acid and the catecholic quencher concentration exceeds 10 " moll... 

Many chiral derivatization reagents have been developed for the enantioseparation of amino acids wherein ultraviolet-visible or fluorescence tags are introduced. The fluorescence derivatization is more effective for the determination of amino acid enantiomers in complex matrices in terms of sensitivity and/or selectivity. Table 1 shows the chiral derivatization reagents, whose structures are shown in Figure 2, used for the enantioseparation of amino acids. 

Fluorescamine was developed by Weigele et al. in 1972 [8], based on the fact that strongly fluorescent pyrrolinones were formed by the reaction of ninhydrin, phenylacetaldehyde, and primary amines. The reagent, 4-phenylspiro[furan-2(3H),l -phthalan]-3,3 -dione (fluorescamine), is nonfluorescent, and it reacts with primary amines, amino acids, and peptides under aqueous conditions in a few minutes at room temperature to form intensely fluorescent substances (Figure 6.1). On the other hand, nonfluorescent derivatives are formed by the reaction of fluorescamine and secondary amino compoimds. Therefore, fluorescamine can be used for the selective determination of primary amino compounds, and the fluorophore produced by the reaction is the expected pyrrolinone. Because the reaction is sufficiently rapid and the hydrolysis products are nonfluorescent, the fluorescamine reaction is applicable for the postcolumn fluorescence derivatization of primary amino compounds [9]. The amino acids are separated by a cation-exchange column similar to the ninhydrin method, and the column effluent is mixed with an alkaline-buffered solution and fluorescamine reagent. The fluorescent derivatives are detected at 480 nm with excitation at 390 nm. 

Note The developed chromatogram must be completely freed from nonpolar solvents before derivatization, otherwise an intense fluorescence will be stimulated over the whole plate. The fluorescence intensity of the chromatogram zones remains stable for ca. 40 min it decreases slowly as the layer dries out and can be returned to its original intensity by renewed immersion in the reagent solution or in water. 

After removal of the mobile phase from the developed plate by heating, zones are detected on the layer by their natural color, natural fluorescence, quenching of fluorescence, or as colored, UV-absorbing, or fluorescent zones after reaction with a reagent (postchroma-tographic derivatization). Zones with fluorescence or quench fluorescence are viewed in cabinets that incorporate shortwave (254 nm) and longwave (366 nm) UV lamps. 

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