Leighton Williams 1902815

Date: 18/11/22
Title of Experiment: Isolation and identification of Casein
Aim: To isolate casein from milk through the process of acidifying the milk.
Method:
 50 grams of milk was added to a 250 ml conical flask and heated in a water bath. The
solution was stirred constantly with a stirring rod. When the bath temperature reached
about 50 C, the flask was removed from the water bath and added about 10 drops of
glacial acetic acid while stirring. The formation of a precipitate was observed.
 The mixture was filtered into a 100 ml beaker by pouring it through a strainer over the
mouth of the beaker. Most of the water was removed from the precipitate by rubbing
cloth. The filtrate was discarded. Using a spatula, the solid was scraped from the strainer
into an empty flask.
 25 ml of 95% ethanol was added to a flask. After stirring the mixture for five minutes, the
solid was allowed to settle. The decant was carefully poured off into a beaker. The liquid
was discarded.
 25 ml of ether/ethanol was added to the residue. After stirring, the resultant mixture for 5
minutes, the solid was collected by vacuum filtration.
 The casein was spread on a paper towel and dried. The dried casein was weighed and the
percentage of casein in milk was calculated.

% casein = Weight of casein / 50g milk * 100

% casein = 1.046 / 50 * 100
% casein = 2.9%

Chemical Analysis of Proteins:

 Biuret test. 15 drops of each of the following solutions were placed in 4 clean test tubes
and labelled:
a. 2% alanine
b. 2% gelatin
c. 2% albumin
d. Casein solution provided

To each of the test tubes, 5 drops of 10% NaOH solution and two drops of dilute CuSO4
solution was added while swirling. The development of purplish violet color indicated the
presence of a protein. Results were recorded.
Leighton Williams 1902815

 The Ninhydrin test. 15 drops of each of the following solutions were placed in 4 clean
labelled test tubes:
a. 2% alanine
b. 2% gelatin
c. 2% albumin
d. Casein solution provided

To each of the test tubes, 5 drops of Ninhydrin reagent was added. The tubes were heated
in a boiling water bath for about 5 minutes. Results were recorded.

 Heavy Metal ions test. 2 ml of milk as placed in 3 clean labelled test tubes. A few drops
of each of the following metal ions was added to the corresponding test tubes as
indicated:
a. Pb2+ as Pb(NO3)2
b. Hg2+ as HgCl2
c. Na as NaNO3
Results were recorded.

Data Sheet:
Test 1
Alanine Remained Blue
Gelatin Changed to purple
Albumin Changed to purple
Casein Changed to purple

Table showing the change in color when NaOH solution was added

Test 2
Alanine Changed to purple before heat was added
Gelatin Changed to purple upon heating
Albumin Changed to purple upon heating
Casein Changed to purple upon heating

Table showing the change in color when Ninhydrin reagent was added
Leighton Williams 1902815

Test 3
Pb2+ as Pb(NO3)2 Coagulation
Hg2+ as HgCl2 Coagulation
Na as NaNO3 Coagulation

Table showing the results of heavy metal ion added to milk.

Questions:
1. Casein has an isoelectric point at pH 4.6. What kind of charges will be on the casein
in its native environment in milk?
Answer: Casein will have a negative charge in milk.

2. What are the three most toxic heavy metals?

Answer: Mercury, Cadmium and Lead.

3. In the isolation of casein following the acidification, you strained the precipitate. If
you did not let all the liquid run off, would your yield of casein be different? Explain.
Answer: The yield would be different because the casein would begin to redissolve
into the milk, yielding less casein.

4. The xanthoprotein test gives a yellow color with proteins and amino acids containing
phenyl groups. Name another amino acid in proteins besides tyrosine which would
give a positive xanthoprotein test.
Answer: Tryptophan

5. Why is milk or raw egg used as an antidote in heavy metal poisoning?

Answer: Egg and milk are used as an antidote for heavy metal poisoning because they
contain proteins which have sulfhydryl (-SH) groups in their structures, which can
bind heavy metal ions and rid the body of them. Egg whites absorb this metal, and
milk neutralizes the stomach acid
Leighton Williams 1902815

Discussion:
The goal of this experiment was to isolate and identify casein in milk. This objective was
completed, but many sources of error occurred during the experiment. Upon adding more than 15
drops of Acetic acid, we weren’t getting much coagulation, which resulted in us getting a small
yield, therefore affection ours results. In the end, we obtained 1.046 g of casein from the 50 g
milk sample provided which is about 2.9% of the total content.
When identifying the proteins with the corresponding tests, the expected color changes occurred
with no abnormalities.

Conclusion:
Casein was isolated and identified in the milk sample provided and the proteins were identified.