Derivatization increasing fluorescamine

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

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. 

For increased sensitivity, reagents that yield fluorescent products are employed. These include OPA, fluorescamine, and 4-fluoro-7-nitro-benzo-2-oxa-l, 3-diazole (NBD-F). Derivatization with OPA provides the best limits of detection for most amino acids. Although the products of OPA derivatization are unstable and decompose fairly rapidly to produce nonfluorescent 2,3-dihydro-lH-isoindol-l-ones, this is not a problem with postcolumn derivatization since the products are detected almost instantaneously. 

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