POST-LABORATORY

REPORT
(BMLS -2A GROUP IV)
QUALITATIVE TESTS FOR
PROTEINS
 INTRODUCTION

Proteins are diverse molecules and the most important

biomolecule in the body. It is a complex nitrogenous organic
compounds which when hydrolyzed produces α-amino acids.
 I. BURNING TEST FOR PROTEINS
PROCEDURE
Procedure: Place a small amount of egg albumin on an Evaporating Dish and
apply heat gently.

Materials: Evaporating Dish, Test Tube Holder, and Alcohol Burner

Results: Changes (specifically odor)

 II. Preparation of Egg Albumin Sample
Procedure: Separate white from the yolk of an egg. Suspend the egg white
in a 150ml distilled water.

Materials: Beaker, not sure han iba. Bain may knows kamo

Results: Changes (specifically odor)

 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
A. BIURET TEST
Principle: This test detects the presence of peptide bonds. Thus, this test is specific for
proteins.
Procedure: Prepare 3 Test Tubes.
(1): 2ml egg albumin sol’n
(2): 2ml 0.2 % Urea
(3): 2ml 0.2 % Glycine

 Add 2ml of 10% NaOH

 Add 2 drops of 10% CuSO4
 Shake! Observe for color change.
III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
A. BIURET TEST
TABLE 1: RESULTS FOR BIURET TEST
SAMPLE OBSERVATIONS
Egg Albumin Violet-colored solution (PASENSYA NA DIK SURE HIT PICTURE)
0.2% Urea Light-blue-colored solution
0.2% Glycine Light-blue colored solution
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
A. NINHYDRIN TEST
Principle: Alpha Amino groups of proteins at N-terminal are responsible for the positive test
with proteins. This test detects the presence of free amin group. Procedure: Prepare 3 Test
Tubes.
(1): 2ml EAS
(2): 2ml 0.2 % Urea
(3): 2ml 0.2 % Glycine

 Add 0.5ml of 0.1% Ninhydrin Reagent

 in water bath. (5 mins)
 Observe for color change.
III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
A. NINHYDRIN TEST

TABLE 2: RESULTS FOR NINHYDRIN TEST

SAMPLE OBSERVATIONS
Egg Albumin
0.2% Urea
0.2% Glycine
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
C. XANTHOPROTEIC TEST
Principle: On heating with conc. HNO3 proteins containing aromatic amino acids form yellow
colour due to the nitration of the benzene ring. Detects the presence of aromatic amino acids,
tyrosine and tryptophan.
Procedure: Prepare 3 Test Tubes.
(1): 1ml EAS
(2): 1ml 5% Gelatin
(3): 2ml 0.2% Glycine

 Add 5 drops of conc. HNO3

 Observe for Coagulation
 Apply Heat & Observe for color change.
 Cool & add 2ml NH4OH
 Observe for changes.
III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
A. XANTHOPROTEIC TEST

TABLE 3: RESULTS FOR NINHYDRIN TEST

SAMPLE OBSERVATIONS
Egg Albumin
5% Gelatin
0.2% Glycine
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
D. MILLON’S TEST
Principle: Millon’s test is used for the detection of tyrosine-containing proteins in a given sample. Millon's test is based on the
principle of nitrification of the phenol group in tyrosine. The nitrated tyrosine then combines with the mercury ions in the
solution to form a red-colored precipitate or solution.
Procedure: Prepare 3 Test Tubes.
(1): 0.5ml EAS
(2): 0.5ml 5% Gelatin
(3): 0.5ml 5% Phenol

 Add 5 drops of Millon’s Reagent

 in water bath. (10mins)
 Cool in running water.
 Add 4 drops 0.1 % NaNO2
 Warm for 1min.
III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
A. MILLON’S TEST

TABLE 4: RESULTS FOR MILLON’S TEST

SAMPLE OBSERVATIONS
Egg Albumin
5% Gelatin
5% Phenol
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
E. HOPKIN’S-COLE TEST
Principle: The indole ring of tryptophan combines with aldehydes to form violet coloured compound. This test is
specific for tryptophan which contains the indole ring.
Procedure: Prepare 2 Test Tubes.
(1): 1ml EAS
(2): 1ml 5% Gelatin

 Add 5 drops of HCR

 Incline the tube (45°). Carefully add at the side 1ml conc. H2SO4
 Observe the junction of the two liquids. DO NOT SHAKE.
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS

E. HOPKIN’-COLE TEST

TABLE 5: RESULTS FOR HOPKIN’S COLE TEST

SAMPLE OBSERVATIONS
Egg Albumin
5% Gelatin
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS
F. LEAD-ACETATE TEST
Principle: The sulfur-containing amino acid such as cysteine, cysteine, and methionine (sulfhydryl/thiol group)
reacts with lead acetate under alkaline conditions to form a brown precipitate. These sulfur-containing amino
acids are degraded in strongly alkaline media to release sulfide ion (S2-) in the form of H2S (hydrogen sulfide).
The sulfide ions can react with lead (II) acetate to form a brownish-black precipitate.
Procedure: Prepare 2 Test Tubes.
(1): 1ml EAS
(2): 1ml 5% Gelatin

 Add 5 drops of 10% NaOH.

 Add 5 drops of Lead Acetate.
 Shake! And heat in water bath. (5mins)
 Obseve for color change.
 III. COLOR TESTS FOR AMINO ACIDS AND PROTEINS

F. LEAD-ACETATE TEST

TABLE 6: RESULTS FOR LEAD-ACETATE TEST

SAMPLE OBSERVATIONS
Egg Albumin
5% Gelatin
 IV. PROTEIN DENATURATION TEST

Denaturation is the process of unfolding and rearrangement of the secondary and tertiary structure of protein
causing to loose its biological function.

A. COAGULATION BY HEAT, ACIDS AND ALCOHOL

1. HEAT
Procedure: Prepare 1 Test tube.

 Place 2ml of EAS in a test tube.

 Heat directly in flame.
 Observe the changes occured.
 IV. PROTEIN DENATURATION TEST
A. COAGULATION BY HEAT

RESULTS: ________________________________________________________
 IV. PROTEIN DENATURATION TEST
A.COAGULATION BY HEAT, ACIDS AND ALCOHOL

2. A. INORGANIC ACIDS
Procedure: Prepare 2 Test tubes
(1): 1ml EAS (conc. HCL)
(2): 1ml EAS (conc. H2SO4)

 Add conc. HCL dropwise until ppt is observed.

 Add excess HCL, note what happened to the ppt.
 Repeat the procedure w/conc. H2SO4
 IV. PROTEIN DENATURATION TEST
A.COAGULATION BY HEAT, ACIDS AND ALCOHOL
2. a. INORGANIC ACIDS

TABLE 7: RESULTS FOR INORGANIC ACIDS

SAMPLE OBSERVATIONS
Test Tube 1
Test Tube 2
 IV. PROTEIN DENATURATION TEST
A.COAGULATION BY HEAT, ACIDS AND ALCOHOL
2.B. HELLER’S RING TEST
Principle: Heller's test is a chemical test that shows that strong acids cause the denaturation of precipitated
proteins. Concentrated nitric acid is added to a protein solution from the side of the test tube to form two layers.
A white ring appears between the two layers if the test is positive.
Procedure: Prepare 1 Test tube.
 Prepare 1ml EAS
 Incline the tube @45° angle.
 Add 3ml conc. H2SO4 to the side
 Observe the fluffy zone.
 IV.PROTEIN DENATURATION TEST
A.COAGULATION BY HEAT, ACIDS AND ALCOHOL
2. A. HELLER’S RING TEST

RESULTS: ________________________________________________________
 IV. PROTEIN DENATURATION TEST
A.COAGULATION BY HEAT, ACIDS AND ALCOHOL
3. ALCOHOLS

TABLE 8: RESULTS FOR ALCOHOLS

SAMPLE OBSERVATIONS SOLUBILITY OF PPT
EAS
5% Gelatin
 B. PRECIPITATION OF PROTEINS
1. BY HEAVY METALS
Principle: Heavy metals such as Ag+, Pb2+, Hg2+, etc form a complex with the alkaline proteins and precipitate.
Observe the extent of precipitate in each experiment. Organic acids carry a large negative charges which
neutralize positively charged protein to form an insoluble salt.
Procedure: Prepare 2 Test tubes
(1): 2ml EAS
(2): 2ml 5% Gelatin

 Add 1% HgCl2 dropwise until ppt is observed.

 Add excess reagent.
 Note what will happen to the ppt.
 B. PRECIPITATION OF PROTEINS
3. HEAVY METALS

TABLE 8: RESULTS FOR HEAVY METALS

SAMPLE No. of Drops OBSERVATION
EAS
5% Gelatin
 B. PRECIPITATION OF PROTEINS
1. ORGANIC ACIDS
Principle: Heavy metals such as Ag+, Pb2+, Hg2+, etc form a complex with the alkaline proteins and precipitate.
Observe the extent of precipitate in each experiment. Organic acids carry a large negative charges which
neutralize positively charged protein to form an insoluble salt.
Procedure: Prepare 1 Test tube.
 Place 2ml EAS in a test tube.
 Add 2ml Tannic Acid
 Describe the solution.
 B. PRECIPITATION OF PROTEINS
2. ORGANIC ACIDS

RESULTS: ________________________________________________________
 POST-LABORATORY
REPORT
(GROUP IV)
QUALITATIVE TESTS FOR
PROTEINS