EXPERIMENT NO.

Isolation of Casein from Milk and Analysis of Amino Acids

I. BACKGROUND

Proteins are polymers composed of sub-units called amino acids that are linked together
by a peptide bond (amide bond). They serve as primary structural components of muscles,
connective tissues, hairs, nails, and other parts of a living organism. They also serve as catalyst
in several biochemical reactions, material transport, and metabolism regulators in the body, as
well as in providing defenses for the body (McKee & McKee, 2003).

There are two major fractions of proteins in cow’s milk and these are casein and whey
protein. Caseins are phosphate-containing proteins that occur as micelles in the native form, and
precipitate at pH 4.6. Milk soluble proteins remain in solution at pH 4.6 and constitute a
heterogeneous group of proteins (-lactoglobulin, -lactalbumin, serum albumin,
immunoglobulins, lactoferrin, and other minor fractions). Caseins and milk soluble proteins
greatly differ regarding their amino acid (AA) composition. The latter contains higher
concentrations of total sulfur amino acids (methionine and cysteine), lysine, threonine, and
tryptophan (Lacroix et al., 2006).

II. OBJECTIVES

At the end of this activity, the students should be able to:

1. Isolate casein from milk by isoelectric precipitation
2. Characterize the amino acid present in casein using qualitative chemical tests.

DAY 1: ISOLATION OF CASEIN FROM MILK

MATERIALS
The students need to bring the following:
100-mL non-fat milk
cheesecloth or old handkerchief

Reagents
glacial acetic acid ethanol
dichloromethane

Materials
(2) 250-mL beaker
(1) graduated cylinder
(1) stirring rod
thermometer
filter paper
pH paper

Equipment
pH meter
hot plate
water bath
vacuum filtration setup

IV. SAFETY

 Wear lab gown and protective goggles at all times.

 Place highly volatile samples under fume hood.
 Wash hands thoroughly with soap and water before leaving the laboratory.
PROCEDURE

1. Place 100.0 mL of non-fat milk into a pre-weighed empty 250-mL beaker. Determine and
record the mass of the 100.0 mL milk.
2. Place it in a water bath.
3. Heat the water bath while constantly stirring the milk inside the beaker.
4. Monitor the temperature of the milk sample using a thermometer.
5. Stop heating when the temperature reaches 40°C and remove the beaker from the water
bath.
6. Add 10 drops of glacial acetic acid while constantly stirring the warm milk solution.
7. Note your observations. Check for the formation of any precipitate.
8. Measure the pH of the mixture after A.8 using the pH meter. The pH should be 4.65 to 4.70.
If the reading exceeds 4.70, add more glacial acetic acid (approximately 5 drops).
9. Filter the heterogeneous mixture using a cheesecloth into another 250-mL beaker.
10. Squeeze the cheesecloth gently to remove more traces of liquid on the precipitate.
11. Discard the filtrate in the beaker.
12. Collect the precipitate from the cheesecloth and transfer it into a 250-mL beaker.
13. Add 50 mL of 95% ethanol.
14. Stir the mixture for 5 minutes. Avoid hitting the walls of the beaker.
15. Allow the precipitate to settle to the bottom of the beaker.
16. Decant the mixture into a 250-mL beaker.
17. Discard the decantate.
18. Add 50 mL of dichloromethane-ethanol (1:1, v/v) to the residue.
19. Stir the mixture for 5 minutes. Avoid hitting the walls of the beaker.
20. Collect the solids using vacuum filtration.
21. Discard the filtrate.
22. Collect the casein precipitate on a dry pre-weighed filter paper.
23. Weigh the filter paper with the casein and determine the mass of the casein.
24. Calculate for the percent casein present in 50.0 g of milk using the following formula:
mass of casein precipitate
%casein= ´ 100
mass of 100-mL milk

Do step 25 on Day 2 of the experiment. Cover your casein and keep it in your locker.

25. Prepare a casein test solution by dissolving the precipitate in distilled water.
DAY 2: ANALYSIS OF AMINO ACIDS
MATERIALS

The students need to bring the following:

Isolated casein from Day 1

Reagents
L-glycine L-tryptophan
L-serine L-arginine
albumin L-tyrosine
casein (from Day 1)

3 M NaOH ninhydrin in ethanol

5% CuSO4 concentrated nitric acid
0.02% 1-naphthol bromine water
Hopkins-Cole reagent concentrated sulfuric acid

Materials
(2) 250-mL beaker
(1) graduated cylinder
(1) stirring rod
thermometer
pH paper

Equipment
hot plate
water bath

IV. SAFETY

 Wear lab gown and protective goggles at all times.

 Place highly volatile samples under fume hood.
PROCEDURE

A. Analysis of Amino Acids and Proteins

1. Preliminary Preparations for the Analysis of Proteins
a. Label seven (8) test tubes with glycine, serine, arginine, tryptophan, tyrosine, albumin,
casein, and blank.
b. Place 15 drops of each sample into their respective, properly labelled test tubes.
c. Be sure to wash all the test tubes after each test before proceeding to the next.

2. Biuret Test
a. Add five drops of 3 M NaOH solution into each sample.
b. Add two drops of 5% CuSO4 solution then swirl gently.
c. Note your observations.

3. Ninhydrin Test
a. Add 16 drops of ninhydrin in ethanol solution into each sample.
b. Heat the test tube in a boiling water bath.
c. Note your observations.

4. Xanthoproteic Test
a. Add 10 drops of concentrated nitric acid into each sample. Shake the test tubes after
each drop.
b. Heat the test tubes in a water bath.
c. Note your observations.

5. Sakaguchi Test
a. Add three drops of 3 M NaOH into each sample.
b. Add three drops of 0.02 % -naphthol solution then swirl gently.
c. Allow the test tubes to stand for 3 minutes.
d. Add three drops of freshly-prepared bromine water. Check if the bromine water is still
color orange. Ask your instructor for replacement if the reagent is colorless.
e. Note your observations.

6. Hopkins-Cole Test
a. Add 2.0 mL of Hopkins-Cole reagent into each sample then swirl gently.
b. Tilt the test tubes and allow 40 drops of concentrated H2SO4 to slide slowly down the
walls until it reaches the sample. Do not mix the solution.
c. Note your observations.
 PRELAB QUESTIONS (DAY 1) – EXPERIMENT NO. 4
Isolation of Casein from Milk and Analysis of Amino Acids

GROUP NO.: ________

NAME: _____________________________ CLASS NO. _______

1. What is the isoelectric point of casein?

2. Explain why a protein precipitates at its isoelectric point.

Research each test and write the expected positive and negative results.
Chemical Test Test for Positive Result Negative Result

Biuret Test

Ninhydrin Test

Xanthoproteic Test

Sakaguchi Test

Hopkins-Cole Test
DATA REPORT SHEET (DAY 1) – EXPERIMENT NO. 4
Isolation of Casein from Milk and Analysis of Amino Acids

GROUP NO.: ________

NAME: _____________________________CLASS NO. ____ SECTION: _________
DATE PERFORMED: ___________ INSTRUCTOR: ___________
DATE COMPLETED: ___________

A. Isolation of Casein from Milk

A.1. Mass of empty 250-mL beaker

Mass of 250-mL beaker + 100-mL milk
Mass of 100-mL milk

A.7 Describe the sample after the addition of glacial acetic acid.
____________

A.8 pH of the sample

A.22 Mass of the filter paper

Mass of the filter paper + casein
Mass of casein

A.24 % casein present in milk. Show your calculations.

 DATA REPORT SHEET (DAY 2) – EXPERIMENT NO. 4
Isolation of Casein from Milk and Analysis of Amino Acids

GROUP NO.: ________

NAME: ___________________________CLASS NO. _______ SECTION: _________
DATE PERFORMED: ___________ INSTRUCTOR: ___________
DATE COMPLETED: ___________

OBSERVATIONS
Write (+) for positive results and (-) for negative results.
Observations
Sample Biuret Ninhydrin Xanthoproteic Sakaguchi Hopkins-
Test Test Test Test Cole Test

Glycine

Serine

Arginine

Tryptophan

Tyrosine

Albumin

Casein

Blank
 POST-LAB QUESTIONS – EXPERIMENT NO. 4
Isolation of Casein from Milk and Analysis of Amino Acids

GROUP NO.: ________

NAME: _____________________________ CLASS NO. _______

1. Why was it necessary to use a non-fat milk in the isolation of casein?

2. In Day 1 Step #5, why were you asked to stop heating at 40°C?

3. In Day 1 Step #6, what was the purpose of adding glacial acetic acid?

4. In Day 1 Step #8, why was it important to have a pH of 4.6 to 4.7?

5. How does Biuret reagent react with proteins? What product is formed that causes the
purple color?
6. How does Xanthoproteic test detect aromatic amino acids? What chemical reaction is
involved and what products are formed?

7. Egg whites (a source of albumin) and milk are often used a first-aid treatment to heavy
metal poisoning. Explain why this treatment is possible.