PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT

Group Number 5 Date Performed: 14 March 2017

Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

ISOLATION OF CASEIN FROM MILK AND TESTS FOR

PROTEINS

Introduction

Milk is made of several nutrients including the major biomolecules, sugar, protein,
fats, vitamins and minerals. The amounts of these nutrients present in different types of
milk differ greatly, however.
There are three kinds of proteins in milk: ​caseins, lactalbumins, ​and ​lactoglobulins​.
Casein, the main protein in milk, is a phosphoprotein, meaning that phosphate
groups are attached to hydroxyl groups of some of the amino acid side-chains. Casein
exists in milk as the calcium salt, ​calcium caseinate​.
Casein can be isolated from milk by acidification in order to convert casein to its
isoelectric point. At their isoelectric point, casein tends to aggregate and become less
soluble in water. Calcium caseinate has an isoelectric point of pH 4.6. Therefore, it is
insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore, casein
has a negative charge at this pH and is very soluble in water. If acid is added to milk, the
negative charges on the outer surface of the casein are neutralized an the neutral protein
precipitates. A natural example of this process occurs when milk sours. During souring,
lactic acid is produced and results in the lowering of the pH of the milk, the milk clots
when it sours due to the precipitation of casein.
In this experiment, we will isolate casein from milk and carry out a few chemical
tests for proteins. Casein is precipitated by simply adjusting the pH of the milk to be
sufficiently acidic that the protein is insoluble, taking care not to acidify too much so that
the other components of milk does not hydrolyze.
Casein is used commercially and industrially as a sticky substance in glues, the
coating of paper, and the binding of colors in paints and wallpaper. It is also used as a
coating for fine leather, and is cured with rennet to produce a plastic material used for
buttons. When isolated under sanitary conditions and dissolved in alkaline solutions,
casein is also employed in the manufacture of pharmaceutical and nutritional products.

Methods
I. Materials

● Diethyl ether
● Ethanol
● Milk
● Glacial acetic acid
● Concentrated nitric acid
● 2% albumin
● 2% gelatin
● 2% glycine
● 0.1 M lead (II) nitrate
● 0.1 M mercury (II) nitrate
● 0.1 M copper sulfate
● 3 M NaOH
● 1% tyrosine
PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

● 0.1 M NaNO​3
● Buchner funnel
● Hot plate
● Suction flask
● Beakers
● Droppers

II. Procedures

Isolation of Casein. ​Twenty mL of non-fat fresh milk was poured into a

100-mL beaker and the beaker was placed on a hot plate. A thermometer was left in
contact with the milk as it was being heated and the beaker was retrieved from the hot
plate when the reading on the thermometer showed 45​o​C.

Figure 1. Beakers with 20 mL milk being heated on hot plate

Drops of glacial acetic acid were then gradually added to the milk with
constant stirring. The addition of glacial acetic acid was stopped when the liquid
appeared clear and no more additional casein was precipitating.
PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

Figure 2. Glacial acetic acid being added to milk as it is being stirred

Figure 3. Amorphous mass formed after addition of glacial acetic acid in milk

A funnel was placed on top of a 125-mL erlenmeyer flask and a filter paper
was placed inside the funnel. The contents of the 100-mL beaker was poured into the
filter paper; the lactose filtrate was set aside to be tested for proteins while the casein
that did not pass through the filter paper was placed in another 100-mL beaker and
washed with 25 mL ethanol.
PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

Figure 4. Lactose being filtered out and casein is isolated

The casein and ethanol mixture was stirred for 15 seconds and was filtered in
the same way as how the lactose was retrieved. The ethanol filtrate was disposed and
the casein residue was once again placed into a separate 100-mL beaker and 10 mL of
a 1:1 mixture of diethyl ether-ethanol was added to it. The mixture was stirred for 15
seconds and was filtered; the filtrate was disposed and the casein residue was
removed from the filter paper using a spatula and placed on another filter paper which
was folded into four regions. The regions of the filter paper were pressed together for
10 seconds with the casein residue in between to dry it. The casein was then made to
stand in the air for 10 minutes.

Figure 5. final product, casein residue isolated from non-fat milk

PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

The Biuret Test.​ Six test tubes were prepared with each test tube containing
15 drops of: (a) a pinch of the isolated casein dissolved in 10 drops of water, (b)
dissolved standard casein, (c) egg white, (d) cysteine, (e) Asparagine, and (f) gelatin.
The test tubes were labelled accordingly. Five drops of 3 M NaOH solution were
added to each test tube and another 2 drops of dilute 0.10 M CuSO​4​ was added while
the test tube was being swirled. The color of the solutions were recorded.

Heavy Metal Ion Test​. Six test tubes were prepared, each containing the same
solutions used in the Biuret Test and were labelled. Five drops of mercury (II)
chloride HgCl​2​, sodium nitrate (NaNO​3​), and lead (II) nitrate (Pb (NO​3​)​2​) were added
to each batch of solutions. One type of heavy metal compound was added to every test
tube per batch and then their resulting colors were recorded before being disposed to
reuse the test tubes for another batch.

The Xanthoprotein Test​. Six test tubes were prepared, each containing the
same solutions used in the Biuret Test and were labelled. Ten drops of concentrated
nitric acid (HNO​3​) were added to each test tube and then they were heated in a warm
water bath for 5 minutes. This was done by heating 150 mL of tap water in a 250-mL
beaker on a hot plate until it reaches 80​o​C (which the hot plate was set to not exceed)
and then the test tubes were placed in the hot bath. The color of the solutions were
then recorded.

Results & Discussion

I. Biuret Test

Solution Resulting Color

Isolated casein purple

Standard casein purple

Egg white (albumin) purple

Cysteine light blue

Asparagine blue

Gelatin purple
Table 1. Qualitative data retrieved from biuret test

The Biuret test is used to detect the presence of peptide bonds in a solution.
The NaOH solution is added before the CuSO​4​ to raise the pH of the solution to
alkaline levels. This is because the Biuret test is based on the ability of the copper (II)
ions to bond with the nitrogen atoms in peptide bonds under alkaline conditions. In
the presence of a copper (II) ion, lone electron pairs from 4 nitrogen atoms in the
peptide bonds coordinate with the ion to form a chelate complex. A chelate complex
absorbs light at 540 nm, appearing violet. Thus, a solution that changes color from
PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

blue to violet indicates the presence of a peptide bond.

Figure 6. The formation of chelate complex with 4 nitrogen atoms coordinating with a copper (II) ion

In this experiment, the caseins, albumin, and gelatin solution turned purple
when a biuret test was performed. Cysteine and asparagine remained blue. This is
because the compounds that turned purple are either protein or comprise proteins (e.g.
gelatin and albumin) and thus, have peptide bonds. Cysteine and asparagine on the
other hand, are amino acids and amino acids are the molecules that should form
peptide bonds in between them.

Figure 7. Skeletal model of asparagine Figure 8. Skeletal model of cysteine

II. Heavy Metal Ion Test

Solution Resulting Color when Resulting Color when Resulting Color when
Pb (NO​3​)​2 is
​ added HgCl​2 is
​ added NaNO​3 is
​ added

Isolated casein unchanged unchanged unchanged

Standard casein lightly clouded unchanged unchanged

Albumin heavily clouded heavily clouded cloudy

Cysteine unchanged unchanged unchanged

Asparagine unchanged unchanged unchanged

Gelatin lightly clouded unchanged unchanged

Table 1. Qualitative data retrieved from heavy metal ion test
PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

A heavy metal ion test is employed to detect the presence of any protein and
the mechanism that allows for its detection is almost similar to that of the Biuret test.
When a heavy metal ion is present, it precipitates the proteins by cross-linking free
amino groups and carboxyl groups.

Figure 9. Reaction mechanism of heavy metal ion precipitating proteins

In the addition of lead (II) nitrate, all known proteins except the casein isolated
from milk precipitated, as indicated by the cloudy appearance of the mixture. It is
unsure why the isolated casein did not react with lead (II) nitrate the same way as
other proteins when in the biuret test that was performed prior, it tested positive for
peptide chains. The presence of peptide chains must indicate the presence of protein
molecules, so the reason why the casein behaved as it did in the heavy metal ion test
cannot be determined. Subsequently, all amino acids and proteins except for the
albumin tested negative for the heavy metal ion test when mercury (II) chloride and
sodium nitrate were added to them. The reason for the unusual reaction of the proteins
to the addition of the compounds were undetermined as well.

III. Xanthoprotein Test

Solution Resulting Color

Isolated casein unchanged

Standard casein unchanged

Egg white (albumin) yellow

Cysteine unchanged

Asparagine unchanged

Gelatin unchanged
Table 1. Qualitative data retrieved from xanthoprotein test

A xanthoprotein test is performed to determine the presence of an aromatic

amino acid such as phenyl alanine, tyrosine, and tryptophan. When a concentrated
nitric acid is added to a solution containing these amino acids, it forms xanthoproteic
acid which is a yellow-colored compound formed by the nitration of certain amino
PSHSWVC SY 2016-2017 CHEMISTRY CORE LABORATORY REPORT
Group Number 5 Date Performed: 14 March 2017
Aguirre, Ambag, Malan, Peregrino, Valencia Date Submitted: 18 April 2017

acids such as the groups named. In the experiment, only the albumin was tested
positive, indicating that the protein has component aromatic amino acids.

​Figure __. Nitration of aromatic amino acids that forms xanthoproteic acid