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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 2-3 drops of ninhydrin reagent.
- Put in a boiling water bath and observe the formation of a purple color.

Ninhydrins
test
2-3 drops of B

Mix

Mix

Heat
2-3 min

1ml of
solution A

Result
Unknown

Proteins or Amino Acids & Carbohydrates

Reagent

Drops of Ninhydrin reagent

Observation

Dark purple colour on heat

Proteins or Amino Acids


(free NH2)

No reaction (no change in colour)

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 violet complex known as the Biuret
complex. Two peptide bonds are at least required for the formation of this
complex , this is why amino acids give negative results with Biuret test.

Procedure & observation:


To 1 mL of protein solution (Albumin Casein Gelatin Peptone) in a test
tube, add 1 mL of 10% sodium
hydroxide solution and 2-3 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
2-3 drops of CuSO4

1ml of NaOH

Mix

Mix

1ml of
solution A

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 free Arginine or proteins containing
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 (2-3 drops) +
Ethanolic a-Naphthol (2-3 drops) and Bromine water (2-3 drops)
Mix well; a red-colour complex will be formed with Arginine or protein
containing Arginine.

Sakaguchis
test
B

Mix

Mix

1ml of
solution A

C
Result

Unknown

A Proteins or Amino Acids

Reagent

B 2-3 drops 40%NaOH, then 2-3 drops alcoholic


-Naphthol, mix then
add 2-3 drops Bromine water or
(for safety, use Chlorine water)

Observatio
n

C Red colour

Proteins containing Arginine

C Red colour

Arginine (Guanidine gp)

Nitroprussides test
Principle:
It is specific for Amino Acids or Proteins containing sulfur , -SH (in
cysteine & cystine) gives 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
B

Mix

Mix

1ml of
solution A

C
Result

Unknown

Cystine or Cysteine in
Proteins or Amino Acids

Reagent

3 drops Sodiun nitropruside,


Then, 3-5 drops Ammonia
soln.

Observation

Deep Red purple colour

Amino Acid: Cystine or Cysteine (-SH gp)

Deep 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 (red pink colour appears) 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]
- Heat up for few min
A red pink colour appears

Millons test
1 mL of B

Mix

Heat

Mix

2-3 min
1ml of
solution A

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
Hopkin-Cole
Principle:
It is Specific for Tryptophan amino acid, whether it is free or in proteins.
Sulphuric acid in presence of mercuric sulphate oxidizes the indole nucleus of
tryptophan. The product formed reacts with aldehydes to form violet-purple
colored complex (ring).

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
Hopkin-Cole
2 drops of [Formalin + HgSO4]

2 mL of H2SO4
On the side wall

Mix

Tryptophan

Mix
Wait 1 min

1ml of
solution A

C H2SO4
Result

Unknown

Tryptophan in Proteins or
Amino Acids

Reagent

2 drops [Formalin + HgSO4]


then add 2 mL 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) 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.

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.

Proteins & Amino Acids


Procedure

Observation

Unknown

1- Ninhydrin test
Sample solution (l ml) + Ninhydrin reagent (2-3 drops). Observe on
Cold or if not, Boil in a water bath for (5 min) and Cool

Purple colour is
formed

All A.A. having NH2


except proline &
hydroxyproline

2- Biuret test
Sample solution (1 ml) + Biuret reagent (1 ml)
(Better to warm it)

Violet colour is formed

All Proteins (not


A.A.) as it needs at
least 2 peptide
bonds

3- Millon's test
Sample solution (1 ml) + 1 mL Millon reagent [HgS04 reagent 0.5 ml) +
0.5 ml 2M HNO3]
Boil in water bath for (2 min) and Cool it.

Red colour/precipitate
is formed

Tyrosin
contains a phenolic
group

4- Nitroprusside's test (Mrner test)


Sample solution (1 ml) of protein/Amino acid containing Cysteine
Add (3) drops of sodium nitroprusside
Add (3-5) drops of ammonia solution

Deep dark purple red


colour called also

Cysteine or Cystine
Contain SH group

5- Sakaguchi's test
Sample solution (1 ml) + 2-3 drops of Sakaguchi reagent [40%NaOH (4
drop) + Ethanolic a-Naphthol]
Add Bromine water (1-2) drops [for safety, use chlorine water]

Red colour is formed

Arginine
Contains guanidine
group

6- Hopkin-Cole test (Aldehyde-test)


Sample solution (1 ml) + 2-3 drops of Hopkin-Cole reagent [Diluted
Formalin (2 drops) + 10% Mercuric sulphate (4 drops)] Mix well
Add Conc. H2S04 on the side of test tube (1-2) ml

A violet or purplecoloured ring appears


at the junction of two
layers

Tryptophan
Contains Indole
group

Mrner test

Identification of unknown protein solution


Biurets 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)

Identification of unknown Amino Acids


Biuret Test

Not Violet Color


Solution is not protein
(carbohydrates or amino acids)

Violet Color
Solution is protein

Molischs test

Violet ring
Carbohydrate

Nitroprussides
test

Cysteine

No violet ring
Amino Acids
(Cysteine Tryptophan Tyrosin - Arginine)

Hopkins-Coles
test

Tryptophan

Millons
test

Tyrosin

Sakaguchis
test

Arginine