Qualitative Tests for Amino Acids

1. 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.

2. Exp. 1Ninhydrin 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.

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

4. Exp. 1Ninhydrin 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.

5. Exp. 2Xanthoproteic 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 areorange. Phenylalanine Tryptophan Tyrosine

6. Exp. 2Xanthoproteic 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.

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

8. Exp. 3Millon 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

9. Exp. 3Millon Reaction ■ 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.

10. Exp. 4Hopkin-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

11. Exp. 4Hopkin-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.

12. Exp. 5Sulfur 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

13. Exp. 5Sulfur 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.

14. Exp. 6SakaguchiReaction • 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

15. Exp. 6SakaguchiReaction • 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