MINDANAO UNIVERSITY OF SCIENCE AND TECHNOLOGY

C.M. Recto Ave., Lapasan, Cagayan de Oro City

Laboratory Report
in
Isolation and Purification of Proteins
(Experiment 1)

Submittted by:
John Mark C. Saburao
BS Chem- 3H1

July 06, 2015

1. INTRODUCTION
1.1Theories and Principles
One of the biological fluids in which the protein casein is found is milk. According to Brieger
(1969), milk is a particularly instructive mixture of natural products, having the protein casein
(3%) as one of its major constituents. Moreover, casein is the principal protein found in milk and
comprises about 80% of all milk protein (Eaton, 1979).
Casein exists in milk as a colloidal suspension in the form of a water insoluble calcium salt.
It can easily be isolated since the other proteins found in milk remain water soluble in acidic
solutions. Casein is precipitated in acids according to the equation:
Ca-Caseinate + 2 H+ Casein + Ca2+
Milks are usually basic in nature thus, to precipitate casein, it must be acidified to pH 4.55.
This pH is known as the isoelectric point of casein which means that this is pH is the minimum
solubility of casein (Brieger, 1969). Casein is insoluble in water alcohol and ether but dissolves in
alkaline solution and in some acidic solutions.
Egg is one of the major sources of proteins. One of the proteins found in egg is albumin
which is particularly contained in the egg whites. According to Carpio (2011), egg white is the
common name for the clear liquid contained within an egg. It is formed from the layers of
secretions of the anterior section of the hen's oviduct during the passage of the egg. Egg albumin
which is a phosphorylated glycoprotein, is the major protein constituent of egg whites. In fact,
Stadelman & Cotterill (2001) cited that 54% of the egg whites is ovalbumin (also called albumin).
Saturated ammonium sulfate with acidic condition was used to separate egg white proteins in
early 1900s (Hopkins, 1900; Chick and Martin, 1913).
In this experiment, casein is isolated from evaporated milk and the albumin is isolated
from the egg whites.

1.2 Objectives
This experiment is designed (1) to isolate proteins casein and albumin from evaporated
milk and egg whites, respectively; (2) to purify the proteins extracted from the biological fluids
and; (3) to determine the quantity of the proteins extracted and calculate their respective yields.

2. METHODOLOGY
2.1 Materials, Chemicals and Equipment
evaporated milk

distilled water

eggs

thermometer

cheesecloth

10% HCl

tap water

1% AgNO3

beaker

diluted HNO3

glass rod

95% alcohol

centrifuge

acetone

test tubes

1 N acetic acid

watch glass

buffered saturated (NH4)2SO4

filter paper

2.2 Procedure
The procedure of this experiment is divided into two parts. The first part involves the first
part involves the isolation of casein from milk. The brand of the milk sample used in this
experiment was Alaska Evaporada. A volume of 12.5 mL of the milk sample was diluted with 12.5
mL of tap water to make a 25 mL solution. It was heated to 40C and added with drops of 10%
HCl then stirred thoroughly. This was done for 10 minutes until a flocculent precipitate was
formed. The pH of the mixture was decreased to 4.8 and it was stirred for 5 minutes. It was
centrifuged and the whey was decanted. The curd was resuspended in 15 mL distilled water with
vigorous stirring and then filtered. The residue was transferred to a beaker and again washed
with 10 mL distilled water with HNO3. For the second time, it was filtered and the curd was dried
on the filter paper. This washing procedure was repeated using 10 mL 95% alcohol and then 10
mL acetone. The filter paper with the residue on it was opened and placed in the pre-weighed
watch glass. It was allowed to dry. The watch glass with dried filter paper and residue on it was
weighed. The weight of the dried crude extract was determined and casein yield (in g/mL) was
calculated.

The second part involves the isolation of egg albumin. The egg whites, including the
chalazas, were separated from two fresh eggs and transferred to a 250 mL beaker. The volume
was then measured. It was added with 0.1 mL 1 N CH3COOH and stirred gently. Then it was
filtered through cheesecloth. The filtrate was placed in a 250 mL beaker and added with buffered
saturated (NH4)2SO4 equivalent to the volume of egg whites used. After 30 minutes, the solution
was transferred to test tubes which was then centrifuged. The centrifugate was transferred to a
beaker and added again with (NH4)2SO4. It was stirred until the precipitate ceases to dissolve.
Then it was refrigerated for 2-5 days. On the other hand, test tubes were placed inside a beaker
and pre-weighed. Then the refrigerated mixture was transferred to the pre-weighed test tubes
and then centrifuged. The supernate was discarded and the test tubes were placed on a beaker
and was weighed. The total weight of albumin isolated was determined and albumin yield was
calculated.

3. RESULTS AND DISCUSSIONS

Brand of milk

Alaska Evaporada

Volume of milk

12.5 mL

Mass of protein isolated

1.0058 g

Casein yield (g/mL)

0.0805 g/mL

Table 1.1 Isolation of Casein from Milk

Volume of egg whites

71.0 mL

Mass of protein isolated

7.8326 g

Albumin yield (g/mL)

0.1103 g/mL

Table 1.2 Isolation of Egg Albumin

Table 1.1 shows the data on the isolation of the protein casein form a milk sample. The
brand of the milk that was used in the experiment was Alaska Evaporada which is an evaporated
milk. It can be observed from the table that the mass of protein (casein) isolated from 12.5 mL
of the milk sample was 1.0058 g. This resulted a yield of 0.0805 g/mL indicating that 0.0805 g of
casein is contained in per volume of the milk sample. However, this yield may not agree with the
theoretical yield reported in the references because the pH of the milk sample was 3.5 which is
less than that of the required pH for casein to be precipitated. Casein, as cited, exists in milk as
calcium caseinate, an insoluble calcium salt. Pavia, Lampman and Kriz (1976) stated that calcium
caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, if the pH of the milk was not
decreased to 4.8, as the procedure of the experiment requires, casein is most likely insoluble. In
that case, the casein that was extracted in this experiment may be less than that of the reported
yield.
Other than failure to decrease the pH to about 4.8, one of the factors which may have
affected the yield of casein is the use of evaporated milk. Unlike skim milk, the former is not a
non-fat milk, which may not be suitable for the procedure since the fat that is contained in the
evaporated milk may have attenuated the quantity of casein to be extracted. Moreover, the use
of strong acids such as the hydrochloric acid (HCl) could have also affected the solubility of casein
in the milk sample. Thus, in the experiment, glacial acetic acid (CH3COOH) was used instead of
HCl.
On the other hand, Table 1.2 presents the data and results on the isolation of egg albumin.
With the values presented in the table, it is seen that 7.8326 g of albumin was produced from
71.0 mL of egg whites. It can be deduced from this result that per volume of the egg whites
used, 0.1103 g constitutes the protein albumin.

4. CONCLUSION
Based on the experiment conducted, it is concluded that biological fluids such as milk and
egg whites constitute the proteins casein and albumin, respectively. The isolation and purification
process yielded 0.0805 g of casein per volume of evaporated milk and 0.1103 g of albumin per
volume of egg whites. Furthermore, proteins may be precipitated through the use of heat, alcohol
and other reagents.

5. REFERENCES
Eaton, D. (1979). The Isolation of Lactose and Casein from Milk. In The World of Organic

Chemistry: A Laboratory Approach (p. 225). New York, USA: Mc-Graw Hill.
Brieger, G. (1969). Preparation and Properties of Natural Products. In A Laboratory Manual for

Organic Chemistry (p. 139). New York: Harper & Row,

Pavia, D., Lampman, G., Kriz, G. (1976). Chemistry of Milk. In Introduction to Organic

Laboratory Techniques: A Contemporary Approach. USA: W.B. Saunders Company.

Carpio, P. (2011, March 21). Isolation and Characterization of Egg white Proteins.
Retrieved July 5, 2015 from http://www.scribd.com/doc/51216821/Isolation-andCharacterization-of-Egg-white-Proteins#scribd
Stadelman, W. J., & Cotterill, O. J., (2001). Egg Science and Technology. 4th ed. AVI Publishing,
Corporation, Westport, CT.
Chick H., Martin C. J. (1913). The precipitation of egg albumin by ammonium sulfate. A
contribution to the theory of the salting out of proteins. Biochem. J.7:380398.