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.

Milons’s test is used to detect tyrosine in proteins of a

The color of the solution in the
given sample. Tyrosine forms complexes with heavy
test tube changes to red which
Millon’s Test metals like mercury. The tyrosine molecule is nitrated by
indicates the presence of
HNO3 which is present in Milon’s reagent. This then reacts
tyrosine.
with the mercury ions and forms a red-colored precipitate.
 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.
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 Na2, this 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

After adding the

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

After 5 minutes in
the water bath with
the unknown alpha amino
Ninhydrin
2 solution and acid
Test
ninhydrin, the color
of the solution
changed to blue.

After adding the

unknown solution
and the conc.
HNO3 the solution
became yellow.
Xanthoproteic After using the
aromatic
3 Acid Test burner and after
amino acid
adding NaOH the
solution resulted in
orange which
indicates the
presence of
aromatic acid.
 After adding
Sulphanilic Acid,
NaNO2, Amino
acid, and Na2CO3
the solution
Pauly’s Diazo tyrosine or
4 produced a red
Test histidine
color which
indicates the
presence of
tyrosine or
histidine.

After adding a few

drops of Acetic
Acid-Glyoxylic Acid
and H2SO4 in the
Hopkins Cole
5 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 HNO3, 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.

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.

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.

The last experiment conducted also taught us how unknown amino acids can be
identified through the different tests available in identifying the unknown solution. Ninhydrin Test
identified proline after adding the unknown solution and the Ninhydrin in one test tube, the
solution became yellow and it indicated the presence of proline. The Ninhydrin Test was also
conducted in identifying the alpha amino acid. After 5 minutes in the water bath with the
unknown solution and ninhydrin, the color of the solution changed to blue. Xanthoproteic Acid
Test also detected an aromatic amino acid After adding the unknown solution and the conc.
HNO3. After using the burner and after adding NaOH the solution resulted in orange which
indicates the presence of aromatic acid. Pauly’s Diazo Test also identified tyrosine or histidine to
the unknown solution after adding Sulphanilic Acid, NaNO2, Amino acid, and Na2CO3 the
solution producing a red color which indicates the presence of tyrosine or histidine. Lastly, a final
test was conducted. The Hopkins Cole Test detected tryptophan after adding a few drops of
Acetic Acid-Glyoxylic Acid and H2SO4 in the test tube with an unknown solution, indicating the
presence of tryptophan when a purple violet ring was found in the solution. These different tests
play a significant part in identifying the different amino acids and proteins that could be present
in an unknown solution and eventually, one test to the other, it would play a huge role in
identifying the unknown sample or solution.