Fluorescamine sodium, reagent

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

The specific detection of aromatic nitro compounds is a second example. These can be converted by reduction to primary amines, which are then diazotized and coupled to yield azo dyes (cf. reagent sequence Titanium(III) chloride — Bratton-Marshall reagent ). Sodium nitrite —naphthol reagent, diazotized sulfanilic acid and other reagents specific for amino groups (e.g. ninhydrin, fluorescamine, DOOB, NBD chloride [9]) can also be used in the second stage of the reaction (Fig. 21). 

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]. 

Some of the innumerable reagents used in TEC are based on reactions with more or less well-established mechanism. Eor example, sodium iodobismuthate (Dragendorff reagent) is widely used among others for alkaloids and quaternary ammonium compounds, 4-dimethylaminobenzaldehyde for primary amines and amino acids, 2,4-dinitrophenylhydrazine for aldehydes and ketones, ninhydrin for amino acids and some antibiotics, fluorescamine for primary and secondary amines, phosphomolybdic acid for lipids, various steroids, and other compounds, chlorine vapor followed by Kl/starch for amines and amides. More complex is the mechanism of the reactions with some other reagents, containing high concentrations of sulfuric acid, vanillin/sulfuric acid, phosphoric acid, aluminum chloride, antimony(III)... 

Detector UV 270 F ex 395 em 495 following post-column reaction. The column effluent mixed with ice-cold reagent pumped at 0.3 mL/min and this mixture flowed through a 2.3 m X 0.5 mm ID coil in a cooled ultrasonic bath to the detector. (Prepare reagent by dissolving 25 mg fluorescamine in 25 mL MeCN and adding 75 mL buffer and 200 -L mercaptoethanol. The buffer was 20 mM sodium dihydrogen phosphate adjusted to pH 3 with 1 M phosphoric acid.)... 

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