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:
Ninhydrin’s test (specific to all Proteins & Amino acids)
Biuret’s test (specific to Proteins)
Sakaguchi’s test (specific to Arginine)
Nitropruside’s test (specific to Cysteine)
Millon’s test (specific to Tyrosine)
Hopkins-Cole’s test (specific to Tryptophan)
Ninhydrin’s
test
Ninhydrin reaction
Principle:
Ninhydrin is Specific for Amino Acids & Proteins – to differentiate between
Carbohydrates (-ve) and Amino Acids & Proteins (+ve).
Ninhydrin Reacts with α-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.
Ninhydrin’s
test
Few drops of B
Mix
Mix
Heat
2-3 min
1ml of protein
solution
C
Mix
D
Result
Unknown
A
Proteins or Amino Acids & Carbohydrates
Reagent
B
Drops of Ninhydrin reagent
Observation
C
Dark purple colour on heat
Proteins (free –NH2)
D
Purple-Violet colour or ppt on cold
Amino Acids(free –NH2)
D
No reaction
Carbohydrates
Biuret’s 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.
Biuret’s test
2 drops of B
1ml of NaOH
Mix
Mix
1ml of protein
solution
C
Result
Unknown
A
Proteins or Amino Acids
Reagent
B
2 ml NaOH, then 2 drops of
CuSO4 (1%)
Observation
C
Violet colour
Proteins (reacts with Di-peptide bonds)
C
No change in colour (Blue)
Amino Acids (No di-peptide linkage)
Sakaguchi’s 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
α-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.
Sakaguchi’s
test
Few drops of B
Mix
Mix
1ml of protein
solution
C
Result
Unknown
A
Proteins or Amino Acids
Reagent
B
2 drops NaOH + 2 drops Alcoholic αnaphthol. Mix then add Bromine water
(for safety, use Chlorine water)
Observation
C
Red colour
Proteins containing Arginine
C
Red colour
Arginine (Guanidine gp)
Nitroprusside’s test
Principle:
It is specific for Proteins containing sulfur , -SH (in cysteine & cystine)
give a red-purple colour called “Mörner 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 Mörner test.
l
Nitroprusside’s
test
Few drops of B
Mix
Mix
1ml of protein
solution
C
Result
Unknown
A
Cystine or Cysteine in
Proteins or Amino Acids
Reagent
B
3 drops Sodiun nitropruside
Then, 1 ml Ammonia soln.
Observation
C
Red – purple colour
Amino Acid: Cystine or Cysteine (-SH gp)
C
Red – purple colour
Proteins containing Cystine or Cysteine (-SH gp)
Millon’s 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 Millon’s reagent
[Hg(SO4) + HNO3] then add NaNO2
- Heat up for few min
− A red – pink colour appears
Millon’s test
Few drops of B
Mix
Heat
Mix
2-3 min
1ml of protein
solution
C
Result
Unknown
A
Tyrosin in Proteins or Amino
Acids
Reagent
B
1 ml Millon reagent [Hg(SO4) +
HNO3] then heat
Observation
C
Red-Pink colour
Amino Acid: Tyrosin (-Ph-OH gp)
C
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
A
Tryptophan in Proteins or
Amino Acids
Reagent
B
[Formalin + HgSO4] then add
conc H2SO4
Observation
C
Violet/Purple colour ring
Amino Acid: Tryptophan (Indole ring)
C
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)