Estimation of Amines

Those amines which, when treated as above, give diazoamino compounds or dyes and also those whose diazonium compounds blacken starch-iodide cannot be estimated directly. They may be estimated either by (1) coupling with a standard diazonium solution, or (2) by adding excess of nitrous acid and coupling with alkaline /3-naphthol of known strength. 

The above outline is general, but is subject to variation for the particular amine under estimation. For instance, the amount of sodium carbonate—the essential point is to have the mixture alkaline during the coupling—depends on the acidity of the diazonium solution, and the presence of acid groups, such as sulphonic. When the coupling is carried out in acetic acid solution, sodium acetate is added in place of sodium carbonate and in three times the quantity. 

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The estimation of amines, amino acids, peptides and proteins is carried out by essentially the same procedure 2S2, 233]. Three volumes of a solution of a primary amine, peptide or protein in 0.05 M phosphate buffer, pH ft-9.5 (or a 0.05 M sodium borate buffer, pH 8-9.5) are mixed under vigorous stirring with 1 volume of fluorescamine solution (0.28 mg ml in acetone or 0.56 mg mlin dioxane). Fluorescence measurement is carried out 5—30 min after mixing. Yields of the fluorophore are 80—90%. There is a linear relationship betv/een the concentration of the amine and the observed fluorescence up to 25—30 nmol ml... 

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]. 

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