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

■ 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