1. 4. Qualitative identification of various amino acids.
Amino acids are molecules containing an amine group, a carboxylic acid group and a side
chain that varies between different amino acids. All amino acid (expect glycine) exhibit optical
activity due to the presence of an asymmetric α-carbon atom. The principals and procedure for
qualitative test usually employed to detect the presence of amino acid in the sample
Ninhydrin test:
Ninhydrin is a powerful oxidizing agent used for the detection -amino acid as well as for
the free amino acids groups on proteins except one (proline), In the pH range of 4-8. When few
drops of 0.1% solution of is taken In test tube ninhydrin mix with amino acid solution.. We get a
purple color after heating the tube for 5 mints. The amino acid proline also reacts with ninhydrin,
but give yellow color .as fingerprints contain small amounts of amino acids so Ninhydrin is also
used for crime scenes to detect fingerprints . derivative of ninhydrin used which have contrast
with fingerprint amino acids.
Procedure:
Prepare 0.1% solution of known amino acid in isopropane and 0.2% ninhydrin in acetone.
Take 1ml of amino acid solution in attest tube and adjust pH at 7.0. Add 5 drops of ninhydrin
solution in amino acid solution which we have already taken in attest tube. Boil it for 5 minutes
in boiling water bath.
Result:
Purple color is appeared.
Xanthoproteeic Test
This test is for amino acid contain aromatic ring.in the presence of HNO2 acid
Ring niterated to give yellow color.
Procedure.
0.5 ml amino acid mixed with 0.5 nitric acid acid cooled it yellow color appear then added
NaOH color changed into orange.
Result:
No color changed but when we added NaOH yellow color appear.
2. Paulys Test .
Procedure:
Cool 2ml of test solution added 1ml sulfonic acid followed by NaNO2 solution and cold it
for 3 mint. Add 2 ml of Na2CO3 and noted the color changes.
Nitroprusside test:
The nitroprusside test is specific for cysteine, the only amino acid containing
sulfhydryl group (-SH). This group reacts with nitroprusside in the presence of excess ammonia.
Procedure:
Added 0.5ml nitroprusside solution into 2ml of amino acid solution in test tube and shake
thoroughly. Add 0.5ml ammonium hydroxide. Heat the test tube in water bath for 10-12 min and
observe the change in color.
Result
No Red color is appeared I think nitroprusside solution is not accurate.
Lead acetate test:
When sulphur containing amino acid is boiled with NaOH, some of sulfur in its
structure is converted to sodium sulfide .The Na2S can be detected by using sodium plumbate
solution which causes the precipitation of PbS from an alkaline solution.
Procedure:
Take 2ml amino acid solution added few drops of lead acetate and boil it with a 1 ml of 40%
NaOH for 2 min.. A black color or precipitate is a positive test for sulfides.
Result:
Black precipitate appeared which shows positive result.
Reference:
Lab manual in biochemistry by Arti Nigam.
