Qualitative Tests for Amino Acids
There are a number of qualitative tests to detect the presence of amino acids and these are
largely dependent on the nature of R-group.
Experiment-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 Co 2
-The amino acids proline and hydroxyproline also reacts but produce a yellow color.
Method:
1 ml AA + 1 ml NH---- heat in boiling WB for 5min-----Purple color.
Alpha-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.
Experiment-2:
Xanthoproteic Reaction:
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- 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
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.
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Nitrated tyrosine (a) and tryptophan (b)
Experiment-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
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.
Experiment-4:
Hopkin-cole Reaction:
- This reaction is used to detect the presence of indol group
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Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi
- The indol group of tryptophan reacts with glyoxalic acid in the presence of conc. H2SO4 to
give purple color.
- The reagent is glyoxalic acid in conc. H2SO4 (Glacial acetic acid which has been exposed to
light contains glyoxalic acid).
Tryptophan
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.
Experiment-5:
Sulfur Test:
- 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
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Method:
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.
Experiment-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
Method:
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
References:
www.wikipedia.org
www.chemtopics.com
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RESULTS & LAB REPORT
- You are supplied with samples of different amino acids, identify them.
-Present your results in a good and full lab report.
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Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi