How get glutein and caseine sa milk ---- methodology

Colors colorsmethodology
Explanation ng colors-----results
Isolation and qualitative ng intact protein---yung naseparate na walang ginawa

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CHEM 200L Expt #2 2F MT- Group #1
ISOLATION OF CASEIN FORM SKIMMED MILK
Aligan, J*., Alvarez, J., Ang, E., Avendano, A., Balbieran, P., Carlos, Kamille P.
Department of Medical Technology, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT
Proteins are naturally occurring polymer in which the monomer units are amino acids, which are
linked together by peptide bonds. These proteins provide most of the molecular machinery of the cells
while others play structural or mechanical roles. Casein is one of the most abundant component found in
milk. In isolating casein, 10% of Acetic acid was added to 40 of the mixture of 20.0 g of powdered,
non-fat milk and 50.0 mL of water to adjust the pH to 4.6, which is the isoelectric pH of casein, wherein it
is generally least soluble. The experiment aims to isolate casein, the protein found in milk, by adding
acetic acid and identify the amino acids present in casein. Color reactions were performed and due to
experimental errors, the experiment only gave a positive result in: Biuret test, Xanthoproteic test,
Hopkins-Cole test, Fohls test, Amide test, and Paulys test.

INTRODUCTION while in basic groups, both amino and

Proteins are large, complex molecules
that provide most of the molecular machinery of
the cells. Many are enzymes, which catalyze
chemical reactions by lowering the activation
energy. Some function as hormones- which are
the chemical messengers in the body- for
transport, protection by making antibodies-
and storage. Other proteins play structural or
mechanical roles. Proteins are naturally
occurring polymer, which is composed of amino
acid as its monomer. These monomers are
connected by peptide bonds. Peptide bonds are
amide bonds formed by the -carboxyl group of
one amino acid and the -amino group of
another amino acid, which is linked together by
condensation reaction. The solubility of proteins
largely depend on the pH of its environment.
Subjecting them to acidic environment
protonates both the carboxylic and amino groups
carboxylic groups are protonated.
Casein is one of the most abundant
organic components in milk. It is composed of
several similar proteins that forms casein
micelle, which is multi-molecular, has a granular
structure, and contains water and salts. Casein
exists in milk as a calcium salt called calcium
caseinate. The micellar structure of casein is the
basis in separating the components of cows
milk. Casein alone are slightly not soluble in the
aqueous environment of milk while casein
micelle granules are maintained as a colloidal
suspension in milk. To isolate casein, acetic acid
is used to adjust the pH to 4.6, which is the
isoelectric pH of casein. Calcium caseinate is
then produced from skimmed milk in result of
adding an acid thus causing it to coagulate,
where it can be filtered to separate the curds
from the whey (Whitford, 2013).
 The objective of this experiment is to
isolate the protein, Casein, found in milk, by
adding acetic acid and to identify the amino
acids present in casein.
METHODOLOGY
Isolation
The sample, powdered milk and water,
Figure 2.
is heated to 40 (Fig. 1). If the temperature
goes beyond the required, casein will
Checking the pH of the sample using a pH
disintegrate. When the desired temperature is
meter.
reached, 10% of Acetic acid is added dropwise
and the solution is mixed until the pH reaches
4.6, which is the isoelectric pH of casein, or
until curd forms (Fig. 2). The casein is then
filtered off by gravity filtration and then dried
(F ig.
3).

Figure 3. Filtration of casein.

Qualitative Tests

Two sets of 10 test tubes were prepared

to perform the qualitative tests. The first 10 sets

Figure contains the intact protein solution (0.5 g of the

1. Checking protein in 1 mL of distilled water) and the

the second set of test tubes contain 0.5 mL of

hydrolyzed sample. The following tests are
conducted on each set of sample:

Biuret Test

Twenty drops of 2.5 M NaOH was

temperature of the sample while heating. added and is then mixed well to the samples.
Then, 2 to 3 drops of .1 M CuSO 4 solution is
introduced into the sample. The sample was then
mixed and the color of the solution was
recorded.
Ninhydrin Test
In this test, 6 to 10 drops of .1%
ninhydrin solution was introduced into the
diluted samples. The sample was heated in a
water bath and the color of the solution was
recorded.
Xanthoproteic Test
Ten drops of concentrated HNO3
solution was slowly added to the diluted
samples. The sample was mixed and 10 drops of
concentrated NaOH was then added, and the
color of the solution was noted.
Millons Test
Five drops of Millons reagent was
added into the sample and the change in color of
the solution was noted.
Hopkins-Cole Test
In this test, 20 drops of Hopkins-Cole
reagent was introduced into the sample. The
sample was then mixed well, and 20 drops of
concentrated H2SO4 was slowly added and the
color of the interface was recorded.
Sakaguchi Test
Ten drops of 10% NaOH and .02%
naphthol solution was added to the sample. The
sample was then mixed and after 3 minutes, 3
drops of 2% NaOBr was added. The solution
was then mixed and the color produced was
recorded.
Nitroprusside Test
In this test, 0.5 mL of 3 M NaOH
solution was introduced to 0.5 mL of the sample.
Then, 0.25 ml of 2% nitroprusside solution was
added and the color of the solution was noted.
Fohls Test
Five drops of 30% NaOH solution and 2
drops of 5% Pb(CH3COO)2 were added to the
sample. The sample was then subjected to
heating and the appearance of dark (or black or
brown) sediments was the noted.
Test of Amides
In this test, 1 mL of 20% NaOH was
added to 10 drops of the sample. The sample
was then heated and the evolution of gas during
heating was observed by placing a moistened
red litmus paper over the sample. The result was
then recorded.
Paulys Test
Diazo reagent was prepared by mixing 3
to 5 drops of 1% sulfanilic acid with 3 drops of
5% NaNO2 solution. Then, 5 drops of sample
and 3 to 5 drops of 10% Na 2CO3 were Fohls Test Black brown
introduced to the diazo reagent and the precipitate
appearance of a red coloration was recorded. Test for Amides Red to blue litmus
paper
RESULTS AND DISCUSSION Paulys Test Red orange solution

Isolation of Proteins
Different tests were performed to the
Casein was isolated from milk through samples in order to confirm the different
the addition of 10% Acetic acid (CH 3COOH). A properties of casein. The reactions of the intact
curd-like white precipitate formed when it proteins that occurred to the each test greatly
reached its isoelectric pH, thus indicating the depend on their characteristics.
presence of casein. The principle involved in
Biuret test is a test to detect the presence
this process is called isoelectric precipitation.
of peptide bonds or linkages. Positive results
Isoelectric precipitation is the precipitation of
must produce a blue-violet solution. The
protein at its isoelectric point, at which it is
experiment done yielded a positive result thus
generally least soluble. For casein, its isoelectric
indicating the presence of a peptide bond since it
point is 4.6 thus, a precipitate formed when the
has not yet broken through hydrolysis.
solution reached this pH.
Ninhydrin test is a test to indicate the
Qualitative Tests
presence of free -amino groups. The presence
Different tests were performed in the of free -amino groups is indicated by a deep
sample and the following results are listed in the blue/ violet solution. The more vivid the
table below: resultant color, the higher the amino acids that
Table 1. Results of Color Reactions are present. The intact protein is expected to
yield a positive result, however, due to
Color Reactions Results
experimental errors, it yielded a light yellow
Biuret Test Blue-violet solution
Ninhydrin Test Light yellow solution solution thus indicating absence of free -amino
Xanthoproteic Test Yellow orange
groups. (Experimental errors done are stated at
solution
the end of the results and discussion.)
Millons Test No color change
(Milky white with Xanthoproteic test is a test to determine
ppt) the presence of aromatic groups particularly of
Hopkins-Cole Test Purple color of the
Tyrosine, Tryptophan, and Phenylalanine. The
interface
result yielded a positive outcome thus indicating
Sakaguchi Test Clear solution
Nitroprusside Test Yellow solution presence of aromatic groups.
 Millons test is used to indicate the
presence of Tyrosine. Its principle involve the
complexation reaction between the phenolic
group and the mercury of the Millons reagent.
Complexation reaction is a reaction that takes
place between a metal ion and a ligand, which
contains at least one atom with an unshared pair
of electrons. The result must yield a positive
outcome; however, due to personal errors, it
yielded a negative result thus producing a milky
white solution with a precipitate. A positive
indication of this test would produce a reddish
brown precipitate.
Hopkins-Cole test indicates the presence
of Tryptohan. The experiment yielded a positive
result thus producing a purple color interface.
This involves the principle of condensation
reaction of the indole group with glyoxylic acid
and sulfuric acid. Condensation reaction occurs
when two amino acids combine to form a
peptide bond with expulsion of water in the
process thus forming a larger molecule.
Sakaguchi test is a test to detect the
presence of Arginine. It involves the principle
wherein guanidino group reacts with -Naphthol
and alkaline hypobromite to give a red-colored
complex. The intact protein gave a clear solution
thus yielding a negative result; however, the
result must give a positive result, thus indicating
errors in performing the experiment, which
affected the result.
complexation reaction. The gathered result gave
a negative outcome thus yielding a yellow
solution; however, the expected result should
give a positive outcome, which will give a red
solution. It can therefore be concluded that
errors were done in performing the experiment.
Fohls test is used to determine the
presence of sulfur-containing amino acid
particularly of Methionine. Positive result was
obtained giving a black brown precipitate.
Test for Amides is used to indicate the
presence of primary, secondary, and tertiary
amides and nitrites. Positive result will give a
change in color of the litmus paper from red to
blue and the principle involved is basic
hydrolysis. Hydrolysis is a reaction involving
the breaking of a bond in a molecule with the
use of water. The intact protein gave a change in
color of the litmus paper from red to blue thus
yielding a positive result.
Paulys test is used in determining the
presence of Tyrosine and Histidine. The basic
principle in this test is diazotized. Sulfanilic acid
is diazotized with the addition of sodium nitrite
and sodium carbonate, which will form
diazonium component. The intact protein gave a
red orange solution, which indicates the
presence of Tyrosine and Histidine.
The results of each color reaction may
be seen in the figure presented below:

Nitroprusside test is used to detect the

presence of Cysteine. The principle involved is

Figure 4. Results of color reaction with
the intact protein.
Here are some of the experimental errors done
during the experiment:
Personal errors
Inaccurate temperature of the water bath
Some water may have entered the test
tube while heating the sample in the
water bath
Wrong interpretation of the color results
Samples may have spilled while
performing the experiment
CONCLUSION
The white curd that formed when the
isoelectric pH was reached indicated the
presence of casein. In order to confirm the
different properties of casein in the skimmed
milk sample, qualitative tests were performed
using color reactions. Peptide bonds, -amino
groups, Tyrosine, Tryptophan, Phenylalanine,
Arginine, Methionine, Cysteine, Amides, and
Histidine were expected to be present in the
intact protein; however, due to experimental
errors stated above, only Peptide bonds,
Tyrosine , Tryptophan, Phenylalanine,
Methionine, Amides, and Histidine were found
to be present.
REFERENCES
Amino Acids, Peptides and
Proteins in Organic Chemistry,
Analysis and Function of Amino Acids
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Retrieved March 17, 2017, from
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