EXPERIMENT 1
QUALITATIVE TESTS OF PROTEINS AND AMINO ACIDS
Protein is the most abundant substance in the cell next to water, comprising 15% of its
over-all mass. This may be attributed to protein’s many functions in the body. It can be
structural like collagen that provides tensile strength and elasticity to connective tissues;
catalytic like pepsin that breakdowns protein in our food; or transport of molecules like the
lipoproteins that carries cholesterol to and from target cells.
Protein is composed of amino acids as its building block. Different proteins are
composed of different amino acids. It is linked together with peptide bonds with a positive
charged nitrogen-containing group at one end and a negatively charged carboxyl-group. Along
the chain is a series of different side chains from different amino acids. Some side chains are
nonpolar like glycine, or neutral polar like serine, some are acidic like glutamic acid, and some
are basic like arginine.
Below is insulin, an example of protein and its amino acid composition.
Credits: Libretexts
The different tests in this experiment will help you identify the presence of protein and
different types of amino acids in a protein sample.
OBJECTIVES
• To perform qualitative test for different types of proteins.
• To identify amino acids based on the different tests performed.
• Relate the test results to the chemical structure of each protein or amino acid.
EXPERIMENT 1
PRE-LABORATORY ASSIGNMENT
Name: _________________________________________ Date: ________________
Yr. & Sec.: ______________________ Group No. ____ Rating: _______________
PROTEINS
EXPERIMENT 1
Familiarize the different tests that you will encounter in this experiment by completing the
table below:
TEST PURPOSE POSITIVE
INDICATOR
A. Biuret Test
B. Ninhydrin Test
C. Hopkins-Cole Test
D. Sakaguchi Test
E. Xanthoproteic Test
WHAT TO BRING
Aspartame or Equal (1 sachet)
Glutathione (2 capsules)
1 fresh chicken egg Other protein samples (optional): milk
MATERIALS
Graduated cylinder Test tube holder
Beaker Test tube brush
Stirring rod Hot plate
Test tubes Water bath
Test tube rack Dropper
Watch glass Wash bottle
Red Litmus paper
REAGENTS
Distilled water Hopkins-Cole reagent
10% NaOH Concentrated H2SO4
50% NaOH 0.2% α-naphthol solution
1% CuSO4 solution Sodium hypochlorite
2% Ninhydrin Reagent Concentrated HNO3
PROCEDURES
I. SAMPLE PREPARATION
A. Aspartame or Equal – Dissolve 1 sachet in 10 ml water.
B. Glutathione capsule – Dissolve 2 capsules in 10 ml water.
C. Egg Albumin Solution – Mix 5 ml of egg white in 45 ml water.
D. Milk (Evaporated Milk) – Mix 5 ml of evaporated milk with 45 ml water
II. QUALITATIVE TESTS
A. BIURET TEST
1. Add 1.0 ml each of protein samples in different test tubes.
2. To each test tube, add 2 ml of 10% sodium hydroxide solution and mix
thoroughly.
3. Add 2-3 drops of 1% copper sulfate solution to each test tube, mix well and
record your observation.
B. NINHYDRIN TEST
1. Add 2.0 ml each of protein samples in different test tubes.
2. Add 1ml of 2% ninhydrin solution to each sample. Mix thoroughly.
3. For samples with no color change, heat the test tubes in boiling water bath
until a color change is observed.
C. HOPKINS-COLE TEST
1. Add 2.0 ml each of protein samples in different test tubes.
2. To each test tube, add 2.0 ml of Hopkins-Cole reagent and mix thoroughly.
3. Tilt the test tube and carefully pour along one side the tube 1.0 ml of
concentrated sulfuric acid.
4. Hold the test tube in an upright position without disturbing and observe the
color of the ring formed at the interface of the two liquids. If no ring is visible,
gently agitate the tube to cause a very slight mixing at the surface.
5. Record your observations.
D. SAKAGUCHI TEST (Strictly observe the order of addition of reagents)
1. Add 2.0 ml each of the protein samples in different test tubes.
2. To each test tube, add 2-3 drops of 40% NaOH then 2-3 drops of alcoholic
αnaphthol. Mix well.
3. Then add 2-3 drops of chlorine water.
4. Record observations.
E. XANTHOPROTEIC TEST
1. Add 1.0 ml each of the protein samples in different test tubes.
2. Add 1.0 ml of concentrated nitric acid to each sample and mix with a stirring
rod.
3. Note the color of precipitate.
4. Warm in water bath for 5 minutes.
5. Cool the contents then make it basic by adding 50% sodium hydroxide.
6. Record observations.
EXPERIMENT 1
DATA SHEET
Name: _________________________________________ Date: ________________
Yr. & Sec.: ______________________ Group No. ____ Rating: _______________
PROTEINS
EXPERIMENT 1
Instruction: Write the colors observed in each sample and write positive (+) or negative (-) to
the test results.
TESTS ASPARTAME GLUTATHIONE EGG ALBUMIN
Observations
BIURET
Results
Observations
NINHYDRIN
Results
Observations
HOPKINS-COLE
Results
Observations
SAKAGUCHI
Results
Observations
XANTHOPROTEIC
Results
EXPERIMENT 1
POST-LABORATORY ANALYSIS
Name: _________________________________________ Date: ________________
Yr. & Sec.: ______________________ Group No. ____ Rating: _______________
PROTEINS
EXPERIMENT 1
1. What is the principle behind each test?
A. Biuret Test _________________________________________________________________
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B. Ninhydrin Test ______________________________________________________________
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C. Hopkins-Cole Test ___________________________________________________________
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D. Sakaguchi Test ______________________________________________________________
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E. Xanthoproteic Test __________________________________________________________
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2. Explain the results of your sample why did it show positive or negative. Base your answers in the
structure of your sample. If it does not coincide with your expected results, cite some possible
errors during the experiment.
A. Aspartame _________________________________________________________________
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B. Glutathione ________________________________________________________________
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C. Egg Albumin _______________________________________________________________
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D. Milk Casein ________________________________________________________________
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