Experiment No.

- 02

SCT 222 – 2
Biochemistry
Isolation and Identification of Casein

UWU/SCT/20/035
Rameesh Ishak
2022.10.24
Date - 2022.10.17

Experiment No. – 02

Experiment – Isolation and identification of casein

Theory -

Casein is a protein in milk. Casein exists in milk as the calcium salt, calcium caseinate. The white color of milk
is due to this salt as well as to emulsified lipids. Most proteins show minimum solubility at their isoelectric
point and this principle is used to isolate the casein by adjusting the PH of milk to (4.5-4.8) its isoelectric point.
Casein is also insoluble in ethanol and this property is used to remove unwanted fat from the preparation.
Milk is present at a high PH than the isoelectric point of casein, so to precipitate the casein acetic acid is
added drop by drop until the isoelectric point is reached (acidification of casein). Then we are going to prove
the precipitated milk is a protein by the following chemical tests,

 Biuret test
 Ninhydrin test
 Heavy metal ion test
 Xanthoprotein test

Material –

 Milk
 Beaker
 Glass stirrer
 Pipette
 95% Ethanol
 2% Glycine
 2% Gelatin
 1% Tyrosine
 Distilled water
 Ninhydrin reagent
 Biuret reagent
 Lead Nitrate
 Mercury Nitrate
 Sodium Nitrate
 Con.HNO3
 Thermometer
 Acetic acid
 Diethyl ether ethanol
 Cheeses cloth
 Conical flask
Procedure –
a. Preparation of casein

50.0 g of milk was put into a conical flask, and it was immersed in a 200ml of water bath. It was heated up to
40oC and while it was getting heated it was continuously stirred. Then the flask was removed from the water
bath and to it drops of acetic acid was added until the formation of precipitation was observed. The
precipitate was filtered by a cheeses cloth and the filtered precipitate was displaced to another beaker by a
spatula and 25 ml of 95% ethanol was added to it and it was stirred continuously for 5 minutes. Then it was
again filtered and to the filtered solid 25ml of 1:1 mixture of diethyl ether-ethanol was added. The resulting
mixture was stirred continuously for 5 minutes, and it was filtered by a filter paper. Before the last filtration
the weight of the filter paper was measured and finally the filtered casein with the filter paper was left to dry
completely and its weight was measured after. By calculation the percentage of casein in milk was
determined.

b. Chemical analysis of protein

I. Biuret test
15 drops of the following solutions were taken to 5 test tubes, and they were labeled.
a) 2% glycine
b) 2% gelatin
c) 2% albumin
d) ¼ of spatula of prepared casein mixed with 15 drops of distilled water.
e) 1% tyrosine
For each of these prepared test tubes 15 drops of biuret reagent was added and the color change observed
was recorded.
II. Ninhydrin test
15 drops of the following solutions were taken to 5 test tubes, and they were labeled.
a) 2% glycine
b) 2% gelatin
c) 2% albumin
d) ¼ of spatula of prepared casein mixed with 15 drops of distilled water.
e) 1% tyrosine
15 drops of ninhydrin reagent were added to each prepared test tubes and they were heated in a water bath
for about 5 minutes. The observed changes were recorded.
III. Xanthoprotein test
15 drops of the following solutions were taken to 5 test tubes, and they were labeled.

a) 2% glycine
b) 2% gelatin
c) 2% albumin
d) ¼ of spatula of prepared casein mixed with 15 drops of distilled water.
 e) 1% tyrosine
To each of these 5 test tubes 10 drops of conc.HNO3 was added, and the test tubes were heated in a water
bath for about 5 minutes. The observed color change was recorded.
IV. Heavy metal ion test

Three test tubes were taken and 2ml of milk was added to each and they were labeled. Few drops of
Pb(NO3)2, Hg(NO3)2 and NaNO3 were added separately to the three test tubes respectively and the observed
changes were recorded.

Observations –
I. Biuret test

Solution Observation
2% glycine No color change was observed.
2% gelatin The solution was turned pink/purple color.
2% albumin The color of the solution changed pink/purple
color.
¼ of spatula of prepared casein mixed with 15 drops The milk white solution was turned to pink/purple
of distilled water. in color.
1% tyrosine No color change was observed.

Figure 01

II. Ninhydrin test

Solution Observation
2% glycine The solution was turned blue in color.
2% gelatin No color change was observed.
2% albumin No color change was observed.
¼ of spatula of prepared casein mixed with 15 drops No color change was observed.
of distilled water.
1% tyrosine No color change was observed.
 Figure 02
III. Xanthoprotein test

Solution Observation
2% glycine No color change was observed.
2% gelatin No color change was observed.
2% albumin After the addition of xanthoprotein reagent the
solution was turned yellow in color.
¼ of spatula of prepared casein mixed with 15 drops After the addition of xanthoprotein reagent the
of distilled water. solution was turned yellow in color.
1% tyrosine After the addition of xanthoprotein reagent and
heating the solution was turned yellow in color.

Figure 03

IV. Heavy metal ion test

Solution Observation
2ml milk + Pb(NO3) Formation of white precipitate was observed.
2ml milk + Hg(NO3) Formation of white precipitate was observed.
2ml of milk + NaNO3 No change was observed in color or in composition.
 Figure 04
Calculation –
The weight of the filter paper = 0.9 g
The weight of the filter paper + dried extracted casein = 2.6 g
The percentage of casein present in milk = ((2.6 – 0.9)/50) × 100%

= 3.4%
Discussion –
In this practical before the separation of the casein, milk was heated to 40oC. During this process it is
essential to maintain the heat at that temperature due to the casein protein might get damaged in high
temperature. Also, when adding the acetic acid to the heated milk to precipitate (acidification of casein) if
the concentration of the acetic acid is low the maximum number of precipitates might not be able to
obtained and excess amount of acetic acid might be needed for the formation of precipitate. In the
filtration process the glass wares used might contain the precipitate in the process of transferring the turbid
solution to the filtrate, then the acetic acid can be used to displaced them to the required filtration paper or
the cheeses cloth. Casein makes up most of this filtered precipitate, however there may also be some extra
fat. Diethyl ether-ethanol is used to will dissolve the trapped remaining fat. Since casein is insoluble in
diethyl ether-ethanol, it is possible to employ this feature to eliminate undesirable fat from the precipitate.
But due to these phenomenon and procedures the resulting/ calculated casein amount may differ from the
actual casein amount present in the milk used for the experiment.

In biuret test it is checked whether the protein tested have a peptide bond or not. In this experiment casein
dissolved in water, albumin and gelatin showed positive results by the pink color change of the solutions
observed. Due to the Cu2+ ions present in the biuret reagent and the formation of the copper complex by
reacting the copper ions present in the solution reacting with proteins with peptide bonds, the positive
resulting solutions shows pink color change while other doesn’t show any color change. In this test tyrosine
and glycine doesn’t show any color change due to lack of peptide bonds.
In ninhydrin test it is checked whether the proteins reacting is containing or is amino acid with free amine
group. The protein which gives a positive result for this test was the glycine protein. Due to glycine being an
amino acid with free amine group it reacted with ninhydrin reagent to form a complex which is blue in
color. Tyrosine also must provide a positive result for this test due to it being an enzyme with free amino
group it should also react with ninhydrin reagent but due to not enough addition of the reagent or the
protein or not supplying enough heat it did not give a positive result for the test.
In xanthoprotein test it is checked whether the protein contains an aromatic group/ phenyl group. In his
test conc. HNO3 is added to react with the phenyl group to form nitro compound with an aromatic yellow
color presence. In this test albumin, casein dissolved in distilled water and tyrosine react to give a yellow
color solution. This proves that these protein has a phenyl in them.
In heavy ion test protein precipitation from the solution is shown. Heavy metals like lead, mercury, copper,
silver, zinc etc. metal containing solutions react with solutions and precipitate proteins contain in it. In this
practical solutions with heavy metals (lead, mercury) showed that the proteins present in milk are
precipitated by them while metals like sodium which are not heavy metals does not show any reaction with
milk. The precipitate occurs due to proteins become cross-linked with heavy metals.

 The pH level at which the protein is least soluble is known as the protein's isoelectric point at that
position. The pH level at which acid casein precipitates is around 4.6, which is also the casein's
isoelectric point. Milk, which has a pH of about 6.6, contains casein micelles that have a net negative
charge and are generally stable.
 The casein micelles and fat globules exhibit distinct behaviors; they impede milk filtering and hinder
common separation methods. The typical first step is centrifuging the milk to separate the casein
micelles from the fat using a low pH or precipitating agents. A peptide bond can be detected using
the biuret test. Typically, a positive result from a biuret test.
 The three heavy metal ions that are most hazardous are lead, mercury, and cadmium.
 With an increase in acetic acid concentration, casein output rises. Acetic acid will hydrolyze protein
bonds into simpler peptide bonds. This alteration causes more simple proteins to dissolve in water
and increases casein production.
 Our quoted yield off the casing will be incorrect if we do not squeeze the cheesecloth and remove all
the liquid or all the water. because liquid in some quantity will be doing it.
 The mineral acid nitric acid is quite corrosive.
Conclusion –
The biuret test revealed a pink color for casein, albumin, and gelatin, indicating the presence of peptide
bonds. Glycine is the only amino acid that tested positively for Ninhydrin, meaning that it possesses free
amino groups. For the xanthoprotein test, only albumin, casein, and tyrosine produce positive results. As a
result, only these three contain phenyl groups. Also, heavy metals such as Pb2+ and Hg2+ can precipitate
proteins containing in a solution.
By these chemical reactions we can roughly determine the structure of casein, which is a protein with
peptide bonds, have a phenyl group in it and doesn’t contain free amino groups.
References –

 [email protected] (2018) Isolation of casein from milk, Bioscience Notes. Available at:
http://www.biosciencenotes.com/isolation-of-casein-from-milk/ (Accessed: October 23, 2022).
 CHEM2O06 - 1997/98 - experiment 11. Available at:
https://www.chemistry.mcmaster.ca/~chem2o6/labmanual/expt11/2o6exp11.html (Accessed:
October 23, 2022).