Amino acids separation fluorescamine, postcolumn

For many years, automated amino acid analyzer using lEC and postcolumn derivatization with nin-hydrin (or less frequently with fluorescamine) has been the most popular technique for amino acid determination. Amino acids are separated in their free form by employing stepwise elution with sodium- or lithium-based buffers. 

Sample Derivatization. For HPLC analyses, many analytes are chemically derivatized before or after chromatographic separation to increase their ability to be detected. For example, in automated amino acid analyzers, eluted amino acids are reacted with ninhydrin in a postcolumn reactor (see Chapter 20). The resulting chromogenic species are then detected with a photometer. Other examples include labeling amino acids or other primary amines with dansyl or fluorescamine tags either before or after the chromatographic step. 

Fluorescamine has found its main application as a postcolumn reagent in the estimation of amines, amino acids or peptides separated by column chromatography, especially in atuomated amino acid analysis, where the reagent (0.15 mg ml of fluorescamine in acetone) is continuously added to the column effluent buffered with sodium borate (0.16 M pH 9.6) [234]. It can also be used for the estimation of proteins [233, 235] and their visualization in gel electrophoretograms [236]. 

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. 

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