Amino Acid + Amino Acid + Amino Acid

Peptide linkage
Proteins (many peptide linkages)

Proteins

Amino Acids

Casein

Cysteine

Gelatin

Arginine

Albumin

Tryptophan

Peptone

Tyrosine

Cysteine

Tyrosine

Arginine

Tryptophan

Tests to be done:

Ninhydrins test (specific to all Proteins & Amino acids)

Biurets test (specific to Proteins)

Sakaguchis test (specific to Arginine)

Nitroprusides test (specific to Cysteine)

Millons test (specific to Tyrosine)

Hopkins-Coles test (specific to Tryptophan)

Ninhydrins
test
Ninhydrin reaction
Principle:
Ninhydrin is Specific for Amino Acids & Proteins to differentiate between
Carbohydrates (-ve) and Amino Acids & Proteins (+ve).
Ninhydrin Reacts with -amino acids (NH2) in proteins giving a purple colored
complex, except Proline and hydroxy proline gives yellow color(no NH2).

Ninhydrin is most commonly used as a forensic chemical to detect

fingerprints, as amines left over from proteins sloughed off in
fingerprints react with ninhydrin giving a characteristic purple color.
Procedure & observation:
To 1 mL amino acid solution in a test tube, add 2 drops of ninhydrin reagent.
On cold and observe the formation of a purple color, OR
- Put in a boiling water bath and observe the formation of a purple color.

Ninhydrins
test
Few drops of B

Mix

Mix

Heat
2-3 min

1ml of protein
solution

Mix
D
Result

Unknown

Proteins or Amino Acids & Carbohydrates

Reagent

Drops of Ninhydrin reagent

Observation

Dark purple colour on heat

Proteins (free NH2)

Purple-Violet colour or ppt on cold

Amino Acids(free NH2)

No reaction

Carbohydrates

Biurets test
Principle:
Biuret test is Specific for Proteins To differentiate between Proteins (+ve) and
Amino Acids (-ve). The biuret reagent (copper sulfate in a strong base) reacts with
peptide bonds in proteins to form a blue to violet complex known as the Biuret
complex.
Two peptide bonds at least are required for the formation of this complex.

Procedure & observation:

To 1 mL of protein solution in a test tube, add 2 mL of 10% sodium
hydroxide solution and 2 drops of 1% copper sulfate solution.
Mix well; a violet color is obtained with albumin, casein & gelatin and a pinkish
violet color with peptone.

Biurets test
2 drops of B

1ml of NaOH

Mix

Mix

1ml of protein
solution

Result
Unknown

Proteins or Amino Acids

Reagent

2 ml NaOH, then 2 drops of

CuSO4 (1%)

Observation

Violet colour

Proteins (reacts with Di-peptide bonds)

No change in colour (Blue)

Amino Acids (No di-peptide linkage)

Sakaguchis test
Principle:
Sakaguchi test is Specific for Arginine
Sakaguchi's test is positive for the amino acid containing the guanidine
group in Arginine. Guanidine group present in the amino acid reacts with
-Naphthol and alkaline hypobromite to give red-coloured complex.

Procedure & observation:

To 1 mL of protein solution in a test tube, add 40%NaOH (2-4 drop) +
Ethanolic a-Naphthol (2 drops) and Bromine water (5-10 drops)
Mix well; a red-colour complex will be formed with Arginine or protein
containing Arginine.

Sakaguchis
test
Few drops of B

Mix

Mix

1ml of protein
solution

C
Result

Unknown

Proteins or Amino Acids

Reagent

2 drops NaOH + 2 drops Alcoholic naphthol. Mix then add Bromine water
(for safety, use Chlorine water)

Observation

Red colour

Proteins containing Arginine

Red colour

Arginine (Guanidine gp)

Nitroprussides test
Principle:
It is specific for Proteins containing sulfur , -SH (in cysteine & cystine)
give a red-purple colour called Mrner test.

Procedure & observation:

To 1 mL of protein solution containing Cysteine/Cystine in a test tube,
add 3 drops of a 5% solution of sodium nitroprusside
- Mix well and add few drops of ammonia solution, a deep red-purple
color appears; called also Mrner test.

Nitroprussides
test
Few drops of B

Mix

Mix

1ml of protein
solution

C
Result

Unknown

Cystine or Cysteine in
Proteins or Amino Acids

Reagent

3 drops Sodiun nitropruside

Then, 1 ml Ammonia soln.

Observation

Red purple colour

Amino Acid: Cystine or Cysteine (-SH gp)

Red purple colour

Proteins containing Cystine or Cysteine (-SH gp)

Millons test
Principle:
It is specific for Tyrosine. Millon's reagent (Hg/HNO3) gives positive
results with proteins containing the phenolic amino acid tyrosine.

Procedure & observation:

To 1 mL of protein solution in a test tube, add 1ml of Millons reagent
[Hg(SO4) + HNO3] then add NaNO2
- Heat up for few min
A red pink colour appears

Millons test
Few drops of B

Mix

Heat

Mix

2-3 min
1ml of protein
solution

Result
Unknown

Tyrosin in Proteins or Amino

Acids

Reagent

1 ml Millon reagent [Hg(SO4) +

HNO3] then heat

Observation

Red-Pink colour

Amino Acid: Tyrosin (-Ph-OH gp)

Red-Pink colour

Proteins containing Tyrosin (-Ph-OH gp)

Aldehyde test
Principle:
It is Specific for Tryptophan. Sulphuric acid in presence of mercuric sulphate
oxidizes the indole nucleus of tryptophan. The product formed reacts with
aldehydes to form violet colored complex.

Procedure & observation:

To 1 ml of protein solution in a test tube, add 2 drops of 0.2 of Formalin + 1
drop of 10% HgSO4] and mix well.
Incline the test tube and slowly add 2 ml of concentrated H2SO4 on the inner
wall of the test tube to form violet-purple ring at the junction of the 2 layers.
A reddish violet ring is formed at the junction between the 2 layers with albumin
and casein; gelatin gives negative results.

Aldehyde test
Few drops of B
2 mL of H2SO4
On the side wall

Mix

Tryptophan

Mix

1ml of protein
solution

C H2SO4
Result

Unknown

Tryptophan in Proteins or
Amino Acids

Reagent

[Formalin + HgSO4] then add

conc H2SO4

Observation

Violet/Purple colour ring

Amino Acid: Tryptophan (Indole ring)

Violet/Purple colour ring

Proteins containing Tryptophan (Indole ring)

Some additional tests for qualitative

identification of proteins
A) Biuret Test:
Reagents: 1) 1 % CuSO4
2) 5% NaOH
Procedure: To 1 ml of proteins solution, add 2ml of 5% NaOH
and 2 drops of 1% CuSO4 solution, mix well. A violet color
forms, which indicates the presence of peptide bonds in the
molecule.
B) Heat coagulation test
Place about 5 ml of egg-white solution (albumin solution) in a
test tube and heat the top part of the solution only. Note that it
gradually becomes cloudy and a flocculent precipitate of
coagulated protein is produced.

C) Xanthoproteic Test:
Reagents: a- Concentrated HNO3
b- 40% NaOH
Procedure:
a. To 3 ml of protein solution, add 1 ml of conc. HNO3. Mix and boil for 1min.
Cool under tap water. Yellow color forms.
b. Add 2 ml of 40% NaOH to make the solution alkaline. The color changes to
orange indicating the presence of aromatic amino acids.
Principle:
This test is specific to the amino acids containing the benzene ring (aromatic
amino acids).
Phenylalanine, tyrosine and tryptophan reacts with conc. HNO3 at high
temperature to form nitro-compounds which are yellow in color, it turns to
orange color in an alkaline medium.
D) Picric acid test:
To 3 ml of gelatin solution in a test tube, add 2ml of saturated picric acid
solution, a yellow gelatinous precipitate is formed.

Identification of unknown protein solution

Biuret Test

Violet Color
Solution is protein

No Violet Color
Solution is not protein
(carbohydrates or amino acids)

Heat coagulation test

Cloudness at top part
of the solution
(Albumin)

No Cloudness at top part

of the solution
(Casein-Gelatin-Peptone)

Reaction to litmus paper

Neutral
Soln. is (peptone or Gelatin)

Alkaline
Soln. is (Casein)

Picric acid Test

No yellow gelatinous ppt.
Soln. is (Peptone)

Yellow gelatinous ppt.

Soln. is (Gelatin)
(Peptone gives pink color with biuret)