Transcript Qualitative Tests for Amino Acids

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:
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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:
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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