Tests For Amino Acids and Proteins A. Biuret Test: 1. Principle

Tests for amino acids and proteins
A. Biuret test
1. Principle.
This test is used to detect the presence of protein because this

test is specific for the peptide bond therefore substances containing not
less than two peptide linkages give this test. When proteins are treated
with an alkaline solution of dilute copper sulfate a complex with violet
color is formed. The intensity of the color formed is directly proportional
to the amount of protein present in the sample therefore this test is
used to determine the concentration of proteins quantitatively.
2. Procedure.
1. In clean dry test tube add 2 ml of 5% albumin solution (protein).

2. In the second test tube add 2 ml of 5% argenine solution (amino
acid).
3. For each test tube add 1 ml of biuret reagent and mix well.
4. Observe the formation of violet color for protein (positive result)
and no color change for amino acid (negative result).
B. Ninhydrin test
1. Principle.
This test is a general test used to identify the presence of proteins

and all products of protein hydrolysis including amino acid. This test
depends on the presence of alpha amino acids and proteins that contain
free amino groups, therefore proline and hydroxyl prolin imino acids lack
an amino group yield yellow color product with ninhydrin while the
other amino acids yield blue-violet color complex. The mechanism of this
test is the reaction between amino group of amino acid and ninhydrin
which is a powerful oxidizing agent oxidize amino acid to liberate
ammonia, carbon dioxide and the corresponding aldehyde, ninhydrin
will be converted to the reduced form hydrindantin. The liberated NH3
reacts with hydrindantin with another molecule of ninhydrin to produce
a blue-violet complex which is called Ruhemanns complex. In case of
imino acid like proline and hydroxyproline, a different product having a
bright yellow color is formed. Asparagine, which has a free amide group,
reacts to give a brown colored product.
2. Procedure.
1. In clean dry test tube add 1 ml of 1% certain amino acid.

2. Add 5 drops of 2% ninhydrin solution and mix well.
3. Incubate the test tube in boiling water bath for three minutes.
4. Allow the tube to cool and observe the formation of blue color.
Warning
Avoid spilling ninhydrin solutions on your skin, as the resulting stains are
difficult to remove. (Ninhydrin is the most commonly used method to
detect fingerprints, as the terminal amines or lysine residues in peptides
and proteins sloughed off in fingerprints react with ninhydrin) therefore
it used in crime scene.
C. Xanthoproteic test
1. Principle.
This test is used to detect the presence of amino acids containing

an aromatic ring like tyrosine, tryptophan and phenylalanine. The
benzene ring in these amino acids can be nitrated by the addition of
concentrated nitric acid and heating to form yellow colored nitro
derivatives. Phenylalanine gives negative or weakly positive reaction
despite of this amino acid contains aromatic ring because it is difficult to
nitrate under normal condition. On adding alkali to these nitro
derivative salts, the color change from yellow to orange.
2. Procedure.
1. In clean dry test tube add 1 ml of 1% tyrosine.

2. Add 1 ml of conc. nitric acid and mix well.
3. Heat the test tube over a flame for two minutes.
4. Observe the color changing to yellow.
5. Cool the tube under the tap water and add 2 ml of 40% NaOH.
6. Observe the changing of color from yellow to orange.
D. Rosenheim test
1. Principle.
This test is also used to detect the presence of tryptophan in a

protein solution because tryptophan is the only amino acid that contain
indol ring in its structure. The test depends on the presence of
formaldehyde which breaks the bonds to differentiate amino acids,
ferric chloride which gives the color to the solution and sulfuric acid
which forms two layers in the solution. The protein sample when
treating with these chemical compounds a brown ring appears in the
junction of the two layers which indicates the presence of indol ring.
2. Procedure.
1. In clean dry test tube add 1 ml of 1% tryptophan solution or 1 ml

of tryptophan containing protein like albumin.
2. Add 10 drops of Rosenheim reagent and mix well.
3. Add 2 ml of sulfuric acid drop by drop on the internal wall of
slopping tube and don’t mix.
4. Observe the appearance of brown ring in the junction between
two layers.
E. Lead sulfide test
1. Principle.
This test is used to detect the presence of cysteine, when cysteine

is heated with strong alkali like NaOH, some of the sulphur is converted
to sodium sulphide (Na2S) which can be detected by precipitation as a
black precipitate of lead sulphide (PbS) from alkaline solution.
Cysteine + 40% NaOH heating Na2S
Na2S + Pb (CH3COO)2 PbS + 2CH3COONa
2. Procedure.
1. In clean dry test tube add 1 ml of 1% cystein solution.

2. Add 2 ml of 40% NaOH and mix well.
3. Boil the mixture over Bunsen burner for more than one minute.
4. Add 1 ml of 25% lead acetate and mix well again.
5. Observe the formation of black or brown precipitate of lead
sulfide.

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Tests for amino acids and proteins

This test is used to detect the presence of protein because this

1. In clean dry test tube add 2 ml of 5% albumin solution (protein).

This test is a general test used to identify the presence of proteins

1. In clean dry test tube add 1 ml of 1% certain amino acid.

This test is used to detect the presence of amino acids containing

1. In clean dry test tube add 1 ml of 1% tyrosine.

This test is also used to detect the presence of tryptophan in a

1. In clean dry test tube add 1 ml of 1% tryptophan solution or 1 ml

This test is used to detect the presence of cysteine, when cysteine

Cysteine + 40% NaOH heating Na2S

Na2S + Pb (CH3COO)2 PbS + 2CH3COONa

1. In clean dry test tube add 1 ml of 1% cystein solution.

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