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. 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.

3
Exp. 1 Ninhydrin Reaction

• Method
• 1 ml AA 1 ml NH
• heat in boiling WB for 5min.
• Purple color.

4
Exp. 1 Ninhydrin Reaction

• Method
• a-amino acid 2 ninhydrin ---gt
• 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. 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
6
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.

7
Exp. 2 Xanthoproteic Reaction
Tryptophan
Tyrosine
(a)Nitrated tyrosine and tryptophan (b)
8
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
9
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.

10
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
11
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.

12
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
13
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.

14
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 a-naphthol and an
  oxidizing agent such as bromide water to give a
  red color.

Arginine
15
Exp. 6 Sakaguchi Reaction

• 2 ml AA 1 ml 2M NaOH 1 ml ethanolic 0.02
  a-naphthol
• mix well
• cool in ice
• add 1 ml of alkaline hypochlorite solution
• Red color