MODULE 4: QUALITATIVE TESTS FOR
PROTEINS
MLS2E - GROUP 4
CABASAN, Edward Theodore
DEOCAMPO, Jerone Rey
KANAAN, Anne Therese
PARIÑAL, Christian Haj
PASCUAL, Rengel
SIONOSA, Gene Peter
I. INTRODUCTION
Proteins are macromolecules in that they are
polymers of amino acids, whether branched or
unbranched, which are essentially made up of
carbon, hydrogen, nitrogen, oxygen, and sulfur.
While amino acids are the building blocks of
proteins, proteins are the building blocks of muscle
mass in living organisms. To synthesize proteins,
about 20 different amino acids are uniquely
combined and utilized. As such, these uniquely
sequenced amino acids that collectively form into
proteins dictate the overall shape and functional
properties of the specific protein they code for. A
typical protein contains about 200-300 amino acids,
but much smaller proteins contain less and are
called peptides. Larger proteins may contain up to
more than 20,000 amino acids. The largest to date
called the titin, is found in cardiac and skeletal
muscles. A single chain of titin contains about
27,000 amino acids!
While proteins are massively present
throughout the cells in the body, there are nine
essential amino acids that cannot be synthesized by
the body and therefore need to be included in one’s
diet to supply the body with those needed nutrients,
namely: histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan,
and valine. Although, occasionally, cysteine can
slightly substitute for methionine due to its sulfuric
contents, and tyrosine may substitute for
phenylalanine, it is still important to include
adequate amounts of these essential amino acids in
one’s diet.
Proteins have four different levels of
structure: a) primary, b) secondary, c) tertiary, and
d) quaternary.
a) Primary structures refer to the entire
make-up of proteins composed of
single covalently bonded amino
acids.
b) Secondary structures have either an
α-helix that contains one strand of
amino acid bonded by intermolecular
hydrogen bond or β-sheets that
contain two chains of amino acids
linked by hydrogen bonds.
c) Tertiary structures are combinations
of either pure α-helices, pure β-
sheets, or both. Interactions in the
tertiary structure can either be
stabilized by salt linkages, hydrogen
bonds, disulfide linkages, or
hydrophobic interactions.
Due to the presence of long sequences of
amino acids, several color tests are conducted to
identify the R-groups attached to the α-carbon in
amino acids that give specific reactions to specific
chemicals. As such, the following tests are
performed to conclude the components of a protein:
a. Biuret Test
b. Ninhydrin Test
c. Xanthoproteic Test
d. Millon’s Test
e. Hopkin’s Cole Test
f. Sakaguchi Test
g. Lead Acetate Test /
Reduced Sulfur Test
Proteins, having large and complex
molecules, carry out specific functions in the body
that support structure, function, and regulation of the
body’s tissues and organs. Cells in living organisms
depend on proteins for biological catalysis and
enzymatic reactions to occur within the body.
Proteins function for motion and locomotion of cells,
transport oxygen within and among cells and
tissues, regulate the secretion of hormones, and are
overall, highly essential molecules in cells.
Module 4: Qualitative Tests for Proteins
was geared towards distinguishing different protein
samples and identifying the amino acid sequences
present in each sample. In the conducted laboratory
simulators and online experiments, the students
were able to determine the different underlying test
results for proteins as well as rationalize the
implications of the qualitative tests performed on
proteins.
Page 1 of 9
II. METHODOLOGY
1. TESTS FOR PROTEINS
i.) Ninhydrin Test
A few drops of unknown
solution were transferred to an empty
test tube using a dropper. A few
drops of Ninhydrin reagent were then
added to the same test tube. For five
minutes, the test tube was placed in
a boiling water bath. After such, the
test tube was taken out of the water
bath and the students observed the
color of the solution.
ii.) Xanthoproteic acid Test
1 mL of unknown solution was
poured into an empty test tube using
a dropper. A few drops of
concentrated Nitric acid solution were
added to the same test tube. To avoid
bumping in the solution while boiling,
marble chips were placed into the
solution in the test tube. The test tube
was heated unto a Bunsen burner
and was later cooled by placing it
under running tap water. Then, a few
drops of 40% Sodium hydroxide
(NaOH) solution were added. The
students observed the color of the
solution.
iii.) Pauly’s - Diazo Test
The test was begun by first
transferring a small amount of an
unknown sample to an empty test
tube. The test tube was placed in a
small ice bucket and set aside. Just
briefly after, a few drops of pre-chilled
Sulphanilic acid, Sodium nitrite, and
Sodium carbonate were also added
to the test tube. The students
observed the reaction.
iv.) Hopkins cole Test
1 mL of unknown solution was
poured into a test tube using a
dropper. The same volume of acetic
acid-glyoxylic acid solution was also
added to the same test tube. The
students mixed the solution gently by
shaking the test tube. Lastly, a few
drops of concentrated Sulfuric acid
were added to the solution. The
students observed the color of the
solution.
v.) Lead sulphide Test
1 mL of unknown amino acid
solution was poured into an empty
test tube using a dropper. On the
same test tube, a few drops of 40%
NaOH solution were added. Marble
chips were added to avoid bumping
in the solution while heating. The
solution was heated unto the Bunsen
burner for five to ten minutes using a
test tube holder. The test tube was
then subjected to running tap water to
cool the contents of the solution.
Afterwhich, a few drops of 10% Lead
acetate solution were added to the
solution. The students observed the
color of the formed precipitate in the
solution.
vi.) Isatin Test
A drop of unknown amino
acid solution was applied to a filter
paper strip using a dropper. To speed
up the process, a hair dryer was used
to dry the spot of the dropped
unknown solution. A drop of Isatin
reagent was then added to the dried
spot. The drying procedure was
repeated using the hair dryer. The
students observed the color of the
spot on the filter paper to determine
the presence or absence of an amino
acid.
Page 2 of 9
vii.) Folins McCarthy Sullivan’s
Test
A few drops of 40% NaOH
solution, Glycine, and Sodium
nitroprusside solution were added to
a test tube containing 1 mL of
unknown amino acid solution. The
test tube was placed in a hot bath for
15 minutes with the temperature
maintained at 40°C. After the test
tube was taken from the hot bath, 0.5
mL of 6N Hydrochloric (HCl) acid was
added. The students observed the
change in color of the solution.
viii.) Sakaguchi Test
A few drops of 40% NaOH,
alpha-Naphthol solution, 5% urea,
and hypobromite solution were
respectively added to a test tube
containing a few drops of prechilled
unknown amino acid solution. The
students observed the change in
color of the solution.
ix.) Histidine Test
A few drops of 5% Bromine in
33% acetic acid solution were added
to a test tube containing 2 mL of
unknown amino acid solution. The
test tube was kept at room
temperature for ten minutes.
Afterwhich, 2 mL of Ammonium
carbonate solution was added, and
the test tube was kept in a boiling
water bath for five minutes. The
students determined the presence of
histidine by observing the color
change in the solution.
x.) Millon’s Test
A few drops of Millon’s
reagent were added to a test tube
containing 1 mL amino acid solution
and was mixed by shaking the test
tube. The same test tube was then
heated under a Bunsen burner for
three to five minutes and was later
cooled under running tap water. A
few drops of concentrated Nitric acid
solution were carefully added into the
same test tube. The students
determined the presence of Tyrosine
by observing the color change in the
solution.
B. QUALITATIVE TEST FOR
PROTEINS
The students conducted the
experiment using a lab simulator at
home through an educational
software developed by Amrita
University. They were able to perform
several qualitative tests for proteins:
Biuret Test, Xanthoproteic Test,
Ninhydrin Test, and Millon’s Test.
A similar set-up was prepared
for all four tests in which two test
tubes were labelled: “A” and “B”
respectively. Test tube A contained
egg albumin solution while test tube
B contained gelatin dispersion
solution. The preparations varied
merely in the reagents used for each
of the four qualitative tests for
proteins. To perform them
accordingly: the Biuret test utilized
Sodium hydroxide (NaOH) solution
and 1% copper sulphate (CuSO₄)
solution; Xanthoproteic test utilized
concentrated nitric acid; Ninhydrin
test utilized Ninhydrin solution; and
Millon’s test utilized Millon’s reagent.
A dropper was used to
carefully and equally add the specific
reagents of each corresponding test
to test tubes A and B, respectively.
The contents of both test tubes were
then heated under a Bunsen burner.
An Inference icon denoted by
the symbol ‘I’ found in the lab
simulator was clicked to see the
implications of each of the conducted
tests.
Page 3 of 9
 A Reset button found at the Finally, to conclude the series of tests
bottom left corner of the lab simulator performed by the MLS students through the
was selected intervally for each of the lab simulator, the Hopkins-Cole Test was
four tests to redo the procedure and performed.
ensure that the same results were
replicated. For the Hopkins-Cole Test, 1 mL of
unknown solution was mixed with 1 mL of
Acetic acid-glyoxylic acid reagent in a test
C. DETERMINATION OF THE tube. After mixing gently, concentrated
UNKNOWN Sulphuric (H₂SO₄) acid was poured along the
side of the test tube, ensuring that the test
tube was in an inclined position.
The students conducted the
experiment using a lab simulator at home A Messages tab found at the right
through an educational software developed pane of the lab simulator was used as a
by Amrita University. To determine an reference for the implications of each
unknown solution, the MLS students were performed test. The students observed the
able to perform a series of tests, namely: changes in the solutions and collected the
Ninhydrin Test, Xanthoproteic acid Test, notes stated for each of the four conducted
Pauly’s Diazo Test, and Hopkins-Cole tests.
Test. The tests were performed one after the
other in the same order as written.
III. RESULTS
For the Ninhydrin Test, the students
transferred 1 mL of the unknown solution to
an empty test tube. A few drops of Ninhydrin Part A. TESTS FOR PROTEINS
reagent were added to the same test tube.
The solution was mixed gently and then Test Result Interpretation
placed in a boiling water bath at 100°C for
five minutes. The solution was then cooled at
room temperature. Blue Presence of
Ninhydrin alpha amino acid
For Xanthoproteic acid Test, 1 mL
of the unknown solution was poured to an
empty test tube. Afterwhich, a few drops of Yellow Presence of
Concentrated Nitric (HNO₃) acid were added Imino acid
to the same test tube. The test tube was (Proline)
heated using a Bunsen burner for three to
five minutes. After the heating process, the Xanthoproteic Orange Presence of
test tube was subjected under tap water to aromatic amino
cool its contents. Lastly, a few drops of acid
Sodium hydroxide (NaOH) were added.
Pauly’s-Diazo Red Presence of
For the Pauly's Diazo Test, 1 mL of Histidine &
Sulphanilic acid reagent was poured to an Tyrosine
empty test tube after which the test tube was
chilled in a small ice bucket. A few drops of Millon’s Red Presence of
prechilled Sodium nitrate (NaNO₂) solution Tyrosine
were added. Another few drops of prechilled
amino acid solution were immediately added
to the solution, followed by the addition of a Hopkin’s Cole Purple Presence of
few drops of Sodium carbonate (Na₂CO₃) Violet Ring Tryptophan
solution to the same test tube.

Page 4 of 9
 PART C. DETERMINATION OF THE UNKNOWN
Sakaguchi Red Presence of
Arginine
Amino Test Number Structure
Acid conducted of
Lead Sulphide Black Presence of attempts
Precipitate Cysteine
Alpha Ninhydrin 1
amino acid
Isatin Blue Presence of
colored Imino acid
spot (Proline)
Aromatic Xanthoproteic 1
Amino acid
Folins Red Presence of
McCarthy Methionine
Sullivan’s

Tryptophan Pauly’s Diazo 1

Part B. QUALITATIVE TEST FOR PROTEINS

Test Sample Result Interpretation Tryptophan Hopkins Cole 1

Egg Violet Peptide bonds

Albumin present in
Biuret Test protein

Gelatin Violet Peptide bonds IV. DISCUSSION

present in
protein A. TESTS FOR PROTEINS

Egg Yellow Presence of i.) Ninhydrin Test

Albumin aromatic
Xanthoproteic amino acids The Ninhydrin Test is a qualitative
Test test for proteins performed in order to detect
Gelatin Yellow Presence of the presence of amines or ɑ-amino acids.
aromatic With the presence of ammonia, primary,
amino acids secondary, and tertiary amines, a deep blue
color is obtained with the exception for
Egg Blue Presence of ɑ- proline and asparagine which specially yield
Albumin amino acid yellow and brown, respectively.
Ninhydrin
Test Gelatin Blue Presence of ɑ- The mechanism of the test involves
amino acid the use of Ninhydrin. The amino group
belonging to a free amino acid undergoes a
Egg Red Nitration of chemical reaction with Ninhydrin which acts
Millon’s Test Albumin Tyrosine as an oxidizing agent. This reaction results in
the amino acid undergoing oxidative
Gelatin None Tyrosine is not
deamination liberating CO2, NH3, and an
present
aldehyde along with Hydrindantin which is a
reduced form of Ninhydrin. The ammonia
then reacts with another molecule of
Page 5 of 9
Ninhydrin producing Diketohydrin. This
complex is responsible for the deep blue
color. In the case of proline, no ammonia is
produced during its reaction with Ninhydrin
because of its ring structure. The reaction
can be summarized in the diagram:
ii.) Xanthoproteic Acid Test
The Xanthoproteic Test is a chemical
test for amino acids containing phenol or
indolic groups like tyrosine and tryptophan.
The test is named as such due to the
formation of yellow precipitates of
Xanthoproteic acid when performed.
“Xantho” refers to yellow, so the test is also
interchangeably termed as the Yellow
Protein Test. The test gives a positive result
for amino acids containing benzene rings
and other aromatic groups. This test can be
used to differentiate aromatic amino acids
from non-aromatic amino acids.
The mechanism of this qualitative
test revolves around the aromatic groups of
amino acids being nitrated through heating
with concentrated HNO3 to produce a yellow-
colored nitro derivative. The reaction can be
summarized in the diagram:
iii.) Pauly’s - Diazo Test
The process of diazotization was
introduced in this experiment, wherein
Sulphanilic acid formed a diazonium
compound which only occurs in low
temperatures. A coupling reaction occurred
in which the diazonium salt formed couples
with histidine or tyrosine which produced a
red color, called an Azo dye.
iv.) Hopkins Cole Test
This test is used to detect the
presence of only one amino acid in a given
solution - tryptophan. This test is also called
Glyoxylic acid reaction because it utilizes
Glyoxylic acid. Glyoxylic acid is prepared by
the reduction of Oxalic acid with magnesium
powder. The principle behind this test lies on
the ability of the indole group of the amino
acid, tryptophan, to react with glyoxylic acid
in the presence of Sulfuric acid (H2SO4)
which then gives the purple-colored ring that
is affirmative to the test. The reaction can be
summarized in the diagram:
v.) Lead Sulfide Test
This qualitative test for proteins is
performed to detect the presence of sulfur-
containing amino acids such as cysteine and
methionine in each solution. The principle
behind this test lies in the nature of amino
acids such as cysteine and methionine 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 brown-black precipitate
Page 6 of 9
which is affirmative to the test. The reaction
can be summarized in the diagram:
vi.) Isatin Test
The Isatin test is a biochemical test
specifically performed to test for the
presence of amino acids - proline and
hydroxyproline. Isatin reagent is a visualizing
agent that creates different colors when in
contact with different amino acids. The
reaction among proline, hydroxyproline, and
the Isatin reagent yields a colored addition
product also known as the blue-colored
adduct. This test is performed as a useful
and relatively easy way to simply check for
the presence of proline since other tests
need further isolation in order to determine
the presence of proline in a solution while the
Isatin test does not require as many tedious
steps.
vii.) Folins McCarthy Sullivan’s Test
This test was used for the
determination of the presence of methionine.
The test has a high degree of specificity, as
it only yields a positive result with methionine
and a negative result with other amino acids.
The Folins McCarthy Sullivan's test also
introduced the process of acidification as it is
based on the reaction of nitroprusside and
the alkaline solution of methionine under the
process of acidification. Since both
tryptophan and histidine yield a red color
when reacted with nitroprusside, they added
acid to destroy any present tryptophan
through acid hydrolysis, and also
subsequently added glycine to eliminate the
presence of histidine. When Sodium
nitroprusside was added to an alkaline
methionine solution, the color red was
produced and the process of acidification
was completed.
viii.) Sakaguchi Test
The Sakaguchi Test is a qualitative
test performed to determine the presence of
arginine. This biochemical test works when
the guanidine group in Arginine reacts with
α-naphthol or 1-naphthol to produce a red-
colored product. The Sakaguchi reagent is
composed of 1-naphthol and sodium
hypobromite which act as an oxidizing agent
that initiates and facilitates the hydrogen
bonding of arginine grouped molecules. The
test’s reaction can be summarized below:
ix.) Histidine Test
The process of Bromination is
presented in this test wherein the Histidine
Test accounts for the introduction of Bromine
in any reaction or process. The Bromination
of histidine in an acidic solution is followed
sequentially by the neutralization of acid with
excess ammonia. Heating the solution
produces a blue-violet color.
x.) 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 nitric acid.
The nitrated tyrosine formed a complex with
mercury ions which produced a red-color
solution.
Page 7 of 9
B. QUALITATIVE TEST FOR PROTEINS
i.) Biuret Test
Proteins are polymers made from
amino acids. These amino acids are linked
through peptide bonds. The Biuret test is a
chemical test geared towards the
determination of these peptide bonds in each
analyte. In the experiment performed, two
analytes were given: egg albumin and gelatin
dispersion. The two analytes tested positive
for peptide bonds because they produced a
violet solution. This color change is in
accordance with the principle of the Biuret
Test which revolves around the Copper (II)
present in the biuret reagent that can bind
itself in the nitrogen atoms of the protein
peptides. The copper is then reduced to
Copper (I). The reaction of copper (II) and
Nitrogen then results in the displacement of
the peptide hydrogens. Then, four nitrogen
atoms donate lone pairs to form a coordinate
covalent bond with the cupric ion resulting in
the formation of a chelate complex that is
able to absorb light with wavelengths up to
540nm, thereby giving the solution its purple
color.
ii.) Xanthoproteic Test
This test is performed on two
analytes, egg albumin and gelatin
dispersion, to detect the presence of
aromatic amino acids such as phenylalanine,
tryptophan, and tyrosine. Both the egg
albumin and gelatin dispersion produced a
yellow color, indicating that they have
aromatic amino acids in their structures. The
yellow color change is a nitro derivative
caused by the nitration of the aromatic
groups of amino acids through heating with
concentrated Nitric acid.
iii.) Ninhydrin Test
This test is performed to detect the
presence of amines or ɑ-amino acids. A
deep blue color in the solution is obtained
with the presence of ammonia, primary,
secondary, and tertiary amines. However,
proline and asparagine are excluded from
this as they specially yield results of yellow
and brown, respectively. The mechanism of
the test involves the use of ninhydrin. The
amino group belonging to a free amino acid
undergoes a chemical reaction with
ninhydrin which acts as an oxidizing agent.
The reaction results in oxidative deamination
of the amino acid, freeing CO2, NH3, and an
aldehyde along with hydrindantin. The
ammonia then reacts with another molecule
of ninhydrin producing diketohydrin. This
complex explains the solution’s deep blue
color.
In the experiment, both the egg
albumin and gelatin dispersion solutions
yielded a deep blue indicating the presence
of alpha amino acids in their structures.
iv.) 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 nitric acid.
The nitrated tyrosine formed a complex with
mercury ions which produced a red-color
solution.
Out of the two specimens test, only
egg albumin produced a red-colored
solution, which indicated the presence of
tyrosine. On the other hand, the gelatin
dispersion did not yield the same red-colored
solution, which makes it negative for the
presence of tyrosine.
PART C. DETERMINATION OF THE UNKNOWN
To determine an unknown solution, a series
of tests were conducted to detect and confirm what
amino acids were present within the given unknown
solution. The tests were done through a lab
simulator.
For the conducted Ninhydrin Test, a test
used to detect the presence of alpha amino acids,
the solution yielded a deep blue color indicating that
the unknown solution had alpha amino acids
present. The reaction resulted in the oxidative
Page 8 of 9
deamination of the amino acid with the Ninhydrin as
the oxidizing agent, liberating CO2, NH3, and an
aldehyde along with hydrindantin. In the process,
ammonia reacts with another molecule of ninhydrin
and eventually produces diketohydrin, the complex
responsible for the deep blue color in the solution.
For the Xanthoproteic Test, the procedures
performed yielded an orange solution. This result
indicates the presence of aromatic amino acids such
as phenylalanine, tyrosine, and tryptophan. This
reaction was caused by the nitration of the aromatic
groups of amino acids through heating with
concentrated Nitric acid and in turn, produced an
orange nitro derivative.
With knowledge that an aromatic amino acid
is present in the solution, the Pauly's Diazo Test
was performed to test for the presence of tyrosine.
The test did not yield a positive result due to the
solution not changing to red with the Azo dye.
Through the process of elimination, only two
aromatic amino acids were left unchecked to
determine whether they were present in the
unknown solution. These aromatic amino acids were
phenylalanine and tryptophan.
To check for the presence of tryptophan, the
Hopkins Cole Test was performed. The proper
procedures were followed, and the test resulted in
the solution showing a purple ring which notes that it
is positive for the amino acid, tryptophan. This purple
ring was caused by the dehydration of tryptophan
through its reaction with glyoxylic acid in the
presence of concentrated sulphuric acid.
V. REFERENCES
What are proteins and what do they do?:
MedlinePlus Genetics. (2020). Medline Plus.
https://medlineplus.gov/genetics/understanding/ho
wgeneswork/protein/
Szalay, J. (2015). What is Protein? Livescience.
https://www.livescience.com/53044-
protein.html#:~:text=Chemically%2C%20protein%2
0is%20composed%20of,Institutes%20of%20Health
%20(NIH).
Bangash, A. (2020). Xanthoproteic test to find
aromatic amino acids-MBBS STUDY Stuff. Medical
Study Zone.
https://medicalstudyzone.com/xanthoproteic-test/.
A. (2020). Ninhydrin Test.
BYJUS.https://byjus.com/chemistry/ninhydrin-test.
A. (2013) Yellow Result from Ninhydrin Test? It’s
Avagadbro.
https://biochembro.wordpress.com/2013/02/18/yello
w-result-from-ninhydrin-test/.
Giri, D. (2020). Ninhydrin Test - Procedure, Uses,
Principle and Result. LaboratoryInfo.Com.
https://laboratoryinfo.com/ninhydrin-test/.
Basnet, A. (2020). Hopkins-Cole test (Adamkiewicz-
Hopkins’ test): Principle, Reaction, Reagents,
Procedure and Result Interpretation. Online
Biochemistry Notes.
http://biocheminfo.com/2020/04/05/hopkins-cole-
test-adamkiewicz-hopkins-test-principle-reaction-
reagents-procedure-and-result-interpretation/.
Basnet, A. (2020). Lead acetate test (Lead sulfide
test): Principle, Reaction, Reagents, Procedure and
Result Interpretation. Online Biochemistry Notes.
http://biocheminfo.com/2020/04/16/lead-acetate-
test-lead-sulfide-test-principle-reaction-reagents-
procedure-and-result-interpretation/.
Basnet, A. (2020). Pauly’s Test: Principle, Reaction,
Reagents, Procedure and Result Interpretation.
Online Biochemistry Notes.
http://biocheminfo.com/2020/04/17/paulys-test-
principle-reaction-reagents-procedure-and-result-
interpretation/.
Sapkota, A. (2020). Isatin Test-Definition,
OBjectives, Principle, Procedure, Result, Uses.
Microbe Notes. https://microbenotes.com/isatin-
test/.
Sapkota, A. (2020) Sakaguchi Test-Definition,
Objectives, Principle, Procedure, Result, Uses.
Microbe Notes.
https://microbenotes.com/sakaguchi-test/.
Sapkota, A. (2020) Millon’s Test-Definition,
Principle, Procedure, Result, Uses. Microbe Notes.
https://microbenotes.com/millons-test/.
A.(2020). Biuret Test. BYJUS. Biuret Test -
Checking for Peptide Bonds with Biuret Reagent
(byjus.com)
Page 9 of 9