Qualitative Tests for Amino Acids There are a number of qualitative tests to detect the presence of amino acids These are largely dependent on the nature of R-group.

Exp. 1 Ninhydrin Reaction A color reaction given by amino acids and peptides on heating with the chemical ninhydrin. The technique is widely used for the detection and quantitation (measurement) of amino acids and peptides. Ninhydrin is a powerful oxidizing agent which reacts with all amino acids between pH 4-8 to produce a purple colored-compound. The reaction is also given by primary amines and ammonia but without the liberation of Co2 The amino acids proline and hydroxyproline also reacts but produce a yellow color.

Exp. 1 Ninhydrin Reaction ■ Method: 1 ml AA + 1 ml NH heat in boiling WB for 5min. Purple color.

Exp. 1 Ninhydrin Reaction ■ Method: α-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex (purple) + 3H2O In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method.

Exp. 2 Xanthoproteic Reaction This reaction involves the nitration of benzene nucleus in alkaline medium. As a result AAs that contain aromatic nucleus undergo this reaction. Aromatic AAs form yellow nitro derivative on heating with conc. nitric acid, the salts of these derivatives are orange. Phenylalanine Tryptophan Tyrosine

Exp. 2 Xanthoproteic Reaction ■ Method: 1 ml AA + 1 ml conc. HNO3 heat the mixture in WB for 30s cool add drop-wise 40% NaOH to render the solution alkaline Yellow to orange color.

Exp. 2 Xanthoproteic Reaction Tryptophan Tyrosine (a)Nitrated tyrosine and tryptophan (b)

Exp. 3 Millon Reaction This reaction is used to detect the presence of phenol (hydroxybenzene) which reacts with Millon's reagent to form red complexes. The only phenolic AA is tyrosine. Tyrosine

Exp. 3 Millon Reaction 1 ml AA + 5 drops of Millon reagent ■ Method: 1 ml AA + 5 drops of Millon reagent heat the mixture in BWB for 10min cool too room temp add 5 drops of NaNO2 Brick red color.

Exp. 4 Hopkin-cole Reaction This reaction is used to detect the presence of indol group The indol group of tryptophan reacts with glyoxalic acid in the presence of conc. H2SO4 to give purple color. Tryptophan

Exp. 4 Hopkin-cole Reaction ■ Method: 1 ml AA + 1 ml Hopkin-cole reagent mix well Carefully pour conc. H2SO4 down the side of the tube so as to form two layers Purple ring at the interface.

Exp. 5 Sulfur Reaction This reaction is specific to detect the presence of sulfur. The sulfur of cystein and cystine is converted to inorganic sulfide with conc. NaOH. Lead acetate is added and a ppt of black lead sulfide indicates a +ve reaction. Cystein

Exp. 5 Sulfur Reaction 2 ml AA + 1 ml 40% NaOH + 1-3 drops of lead acetate solution heat the mixture in WB for 3min Cool observe any change Black ppt.

Exp. 6 Sakaguchi Reaction This reaction is used to detect the presence of guanidine group. The only AA that contains guanidine group is arginine which reacts with α-naphthol and an oxidizing agent such as bromide water to give a red color. Arginine

Exp. 6 Sakaguchi Reaction 2 ml AA + 1 ml 2M NaOH + 1 ml ethanolic 0.02% α-naphthol mix well cool in ice add 1 ml of alkaline hypochlorite solution Red color