Date: September 20, 2022

Group Number : 9
Group Members : Falsis, Hannah Ishbel
Ganuhay, Charmy
Magallanes, Rhyanne
Nacionales, Lexi Evonne
Parcon, Jazmine Angela

Module 4 - Qualitative Test for Proteins

Results and Discussion

Part A
Expected Results and
Test Corresponding Discussion
Interpretation

If the color of the solution is

The Ninhydrin Test is used to see if the sample has
blue, it indicates the presence
ammonia, primary/secondary amines, or amino acids
of alpha amino acids.
present. The blue color is the result of ammonia reacting
Ninhydrin Test
with ninhydrin molecules to form diketo hydrin while the
If the color of the solution is
yellow color is the result of the analyte containing amino
yellow, it indicates the
acids.
presence of imino acids.

After adding 40% NaOH, the
color of the solution changes
Xanthoproteic Acid
to orange which indicates the
Test
presence of aromatic amino
acids.
Aromatic groups can be nitrated when heating with
concentrated nitric acid. This results in a yellowish color.
When alkali is added, this will turn the residue orange
because of the salt of the tautomeric form of the nitro
compound.

The appearance of red color

The Pauly’s Diazo Test is used to uncover the presence of
during the addition of sodium
tyrosine and histidine. The sulfanilic acid undergoes
Pauly’s - Diazo Test carbonate solution indicates
diazotization with sodium nitrate and hydrochloric acid.
the presence of histidine and
This will result in diazonium salt.
tyrosine.

The color of the solution in the

Milons’s test is used to detect tyrosine in proteins of a
test tube changes to red which
Millon’s Test given sample. Tyrosine forms complexes with heavy
indicates the presence of
metals like mercury. The tyrosine molecule is nitrated by
tyrosine.

The color of the solution in the
test tube changes to blue
Histidine Test
indicates the presence of
histidine.
Purple-violet rings appear in
Hopkins Cole Test the test tube indicating the
presence of tryptophan.
The color of the solution in the
test tube changes to red
Sackaguchi Test
indicating the presence of
arginine.
A black precipitate appears in
Lead Sulphide Test the test tube indicating the
presence of cysteine.
Follins McCarthy Red color indicates the
Sullivan’s Test presence of methionine.
A blue colored spot on the
Isatin Test filter paper indicates the
presence of imino acid.
HN𝑂3 which is present in Milon’s reagent. This then reacts
with the mercury ions and forms a red-colored precipitate.
The histidine test involves the bromination of histidine in an
acid solution. This is then followed by neutralization of the
acid with excess of ammonia. When the alkaline solution is
heated, this then develops a blue or violet coloration.
The purple ring is due to the indole group of the tryptophan
molecule, which is converted into a colored compound by
oxidation brought about by the aldehyde group of glyoxylic
acid.
The reaction results in the formation of a red-colored
complex due to the formation of an indole-like structure
Cysteine releases sulfur when interacting with strong
alkaline conditions at a high temperature. The sulfur will
combine with the NaOH to form N𝑎2This then reacts with
lead acetate to form lead sulfide, resulting in the black
color.
The red color is obtained when the sodium nitroprusside is
added to an alkaline solution of methionine and the
acidification of the reaction.
The combination of isatin and proline will produce a
colored product which will indicate the presence of amino
acids.
 Part B
Test Sample Observation
Biuret test Egg Albumin Bluish violet color is formed
Gelatin Bluish violet color is formed
The addition of nitric acid to
Xanthoproteic the Egg Albumin solution,
Egg Albumin
Test followed by heating, resulted
in a yellow precipitate.
Similarly to the Egg Albumin
solution, adding nitric acid to
Gelatin the Gelatin Dispersion and
heating it afterwards resulted
in a yellow precipitate.
The reaction of adding
ninhydrin solution to the test
Ninhydrin Test Egg Albumin tube containing the egg
albumin resulted in an
intense blue coloured solution
The addition of adding
ninhydrin solution to the test
tube containing the gelatin
Gelatin
dispersion and then heated
resulted an intense blue
coloured solution
White precipitate which
Millon's Test Egg Albumin changes to brick red on
boiling
Gelatin No characteristic change
Interpretation/Discussion
Egg Albumin is positive in Biuret Test. It
demonstrates a positive result, indicating
the presence of protein.
Gelatine is positive in the Biuret Test. It
contains an amide group that bends over
the copper ion, emitting a bluish violet
color to the visual eye.
The xanthoproteic acid is what causes
the yellow precipitate. It is created when
some proteins' amino acids, like tyrosine
and tryptophan, are nitrated.
This test is mainly used for the detection
of amino acids and proteins with free‾
‾N𝐻2 group. When such an ‾N𝐻2 group
reacts with ninhydrin, an intense blue
coloured imino derivative is formed.
Among the two samples, both the egg
albumin and gelatin have the same
results which illustrates an intense blue
coloured imino derivative-meaning, both
samples has ‾N𝐻2 group.
Egg Albumin is positive in the Millon’s
Test. A red solution or precipitate that
forms is heating is an indication that
tyrosine is present.
Gelatin is negative in the Millon's Test.
Proteins without tyrosine residues cannot
be detected using this test
Screenshots:
 Part C
Amino Acid
Member Test/s
Observed Results in the Structure
# Conducted
Unknown

(1) After adding the

unknown solution and
the Ninhydrin in one test
tube, I placed it in a
water bath. After 5
minutes the solution
became yellow and it
indicates the presence of
proline.After adding the
unknown solution and
the Ninhydrin in one test
tube, I placed it in a
water bath. After 5 (1) proline
minutes the solution
Ninhydrin
1 became yellow and it (2) alpha
Test
indicates the presence of amino acid
proline.

(2) After 5 minutes in the

water bath with the
unknown solution and
ninhydrin, the color of the
solution changed to
blue.After 5 minutes in
the water bath with the
unknown solution and
ninhydrin, the color of the
solution changed to
blue.
 (1) After adding the
unknown solution and
the conc.HNO3 the
solution became yellow.
After using the burner
and after adding NaOH
the solution resulted in
orange which indicates
the presence of aromatic
(1) aromatic
acid.
amino acid
2 Xanthoproteic
(2) After adding the
Acid Test (2) absence
unknown solution and
of amino acid
the conc. HNO3 the
solution became yellow.
However, after using the
burner and after adding
NaOH there is no
change in color which
indicates the absence of
aromatic amino acid

After adding NaOH and

the unknown solution to
test tube, heating it in the
Lead burner, and adding Lead
3 Sulphide Test acetate to the mixture cysteine
formed a black
precipitate which
indicates the presence of
cysteine.
 (1) After adding
Sulphanilic Acid, NaNO 2,
Amino acid, and Na2CO3
the solution produced a
red color which
indicates the presence of
tyrosine or histidine. (1) tyrosine or
histidine
Pauly’s Diazo
4 (2) Though after doing
Test
the same procedure like (2) tryptophan
number, the experiment
yielded a different result
where no changes in
the color of the mixture
was observed indicating
the presence of the
amino acid, tryptophan.

Adding the Millon's

reagent and the
unknown solution
together in the test tube,
heating it in the burner,
5 Millon's Test tyrosine
and adding conc. HNO3
to the mixture resulted a
red precipitate
indicating the presence
of tyrosine

After adding a few drops

of Acetic Acid-Glyoxylic
Acid and H2SO4 in the
Hopkins Cole
6 test tube with an tryptophan
Test
unknown solution, there
was a purple violet ring
in the solution.

Note: If 2 or more members got the same Amino Acid, others must repeat the activity. No
duplication within groups.
Conclusion (relate to the Learning Outcomes)

Proteins and their repeating unit, the amino acid, make up the majority of the cell.
Proteins serve as biological catalysts or enzymes, oxygen transporters, and hormones. Proteins
are crucial molecules in cells. Proteins constitute the majority of the dry weight of cells by
weight. Amino acids can be identified by the R-groups attached to the α-carbon that reacts with
specific chemicals. However, since proteins contain various amino acids, one test will not be
enough to identify the amino acids. It would be best to perform numerous tests before
concluding the components of a protein.

The Ninhydrin Test is a qualitative test for proteins performed in order to detect the
presence of alpha amino acids. With the presence of ammonia, primary, secondary and tertiary
amines, a deep blue color is obtained with the exception for proline which specially yields yellow
and brown, respectively.

Xanthoproteic Test is used to identify amino acids like tyrosine and tryptophan that
contain phenol or indolic groups. The test is also sometimes called the Yellow Protein Test. For
amino acids they contain benzene rings and other aromatic groups, the test yields a positive
result. The method of this qualitative test is based on the nitration of aromatic amino acid groups
by heating them with intense HNO4, which results in a yellow colored nitro derivative.

The Pauly's Diazo Test introduced the diazotization process, in which sulphanilic acid
generated a diazonium molecule that only forms at low temperatures. The diazonium salt is
combined with either histidine or tyrosine in a coupling reaction to make an Azo dye, a red color.

Millon’s Test, this biochemical test presents the concept of nitration wherein the
introduction of a nitro group to an organic compound is elaborated. Millon’s test is heavily based
on the principle of the nitration of the phenol group in tyrosine with the help of itric acid. The
nitrated tyrosine formed a complex with mercury ions which produced a red-color solution.

Hopkins Cole Test This test is used to identify the presence of tryptophan, one and only
one amino acid, in a given solution. Because it uses glyoxylic acid, this test is also known as the
glyoxylic acid reaction. By reducing oxalic acid with magnesium powder, glyoxylic acid is
created. The basis for this test's operation is the capacity of the amino acid tryptophan's indole
group to interact with glyoxylic acid when sulfuric acid (H2SO4) is present. This results in the
formation of a purple ring, which indicates that the test is positive.
 Lead Sulfide Test is a qualitative test for proteins that is performed to detect the
presence of sulfur containing amino acids such as cysteine. The principle behind this test lies in
the nature of amino acids such a cysteine to contain sulfur in their R groups which can react
with lead acetate when exposed to alkaline conditions resulting in a brown precipitate. The
sulfur containing amino acids are degraded in alkaline media to release sulfide ions in the form
of hydrogen sulfide. The sulfide then reacts with lead acetate to form a black precipitate.

Given the laboratory experiment we conducted in part B, we were able to identify the
proteins in the samples egg albumin and gelatin. We conducted different tests such as the
Biuret test, Xanthoproteic Test, Ninhydrin Test, and Millon's Test. The results in the Biuret test
shows Egg Albumin and gelatin turning into bluish violet color which gives a positive result in
Biuret Test, indicating the presence of protein in Egg albumin, and a gelatin containing an amide
group that bends over the copper ion, emitting a bluish violet color to the visual eye. Next is the
Xanthoproteic Test which both samples resulted in a yellow precipitate. The addition of nitric
acid to the Egg Albumin solution, followed by heating, resulted in a yellow precipitate which is
due to some proteins' amino acids, like tyrosine and tryptophan, being nitrate. Third is the
Ninhydrin Test which both samples yield the same result-an intense blue coloured solution
which shows the detection of amino acids and proteins with ‾NH2 group. Lastly is the Millon's
Test where the sample Egg Albumin is positive in the Millon’s Test. As a red solution precipitate
formed when the sample was heated, indicating that tyrosine is present. Gelatin on the other
hand had no changes which yielded a negative result in the Millon's Test as proteins without
tyrosine residues cannot be detected using this test.
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