Name: Jericho P.

Encarnacion CYS: BSES 2A-M Date Submitted: June 16, 2022

Experiment No. 17
Qualitative Analysis of Proteins

Introduction
The proteins are amino acids which are linked together through peptide
bonds. These proteins are categorize based on their function – transport
proteins, storage proteins, structural proteins, muscle contraction proteins, and
enzymes which catalyzes biological reactions. In addition, the sequence of amino
acids form proteins. However, the proteins that are biologically inactive are called
denatured proteins.
Objectives:
At the end of the experiment, the students should be able to:
1. Differentiate the amino acids from proteins based on their structure;
2. Identify different amino acids through Thin Layer Chromatography; and
3. Discuss how proteins and amino acids react in different solutions.
Materials and Apparatus
Test Compounds
Casein
Albumin
Gelatin
Milk
Egg white
Reagents
Dilute NaOH solution
0.01 M CuSO4
0.1% Ninhydrin solution
Nitric acid
Millon’s reagent
0.02% alpha-naphthol solution
 Bromine water
Hopkins-Cole reagent
Concentrated sulfuric acid
Lead (II) acetate crystals
Rubbing alcohol
Brine solution
Cold water
Hot water
Apparatus
Test tubes
Test tube rack
Dropper
Water bath
Glass rod
Containers
Spoon
Stopwatch
Procedure
A. Qualitative Tests for Proteins
As for Biuret test, a 1 mL 10% NaOH was added to 3 mL of each
protein suspension (albumin, casein, and gelatin) in three different test
tubes and mixed. After that, a drop of 0.01 M CuSO 4 was added to each
test tube and mixed well. The observations in terms of color were
recorded.
As for Ninhydrin test, a 5 drops 0.1% Ninhydrin solution were
added with the 2 mL of each protein suspension. Then, heated in a water
bath for 10 minutes. The observations were recorded.
As for Xanthoproteic test, a 1 mL concentrated HNO 3 to 3 mL of
each protein suspension. Afterwards, the test tubes were placed in a
water bath for 30 seconds and cooled in the test tube rack. Then, slowly
added with saturated NaOH drop by drop to each test tube until the
solutions became alkaline. The observations were recorded.
 As for Millon’s test, a 5 drops fresh Millon’s reagent were added to
the 3 mL of each protein suspensions. Then, carefully heated the mixtures
in water bath for 5 minutes. Meanwhile, the test tubes were cooled and
observed the change in colors.
As for Hopkins-Cole test, a 2 mL Hopkins-Cole reagent was added
to 3 mL of each protein suspension. Afterwards, when the test tubes were
inclined, the 1 mL concentrated sulfuric acid was added slowly through a
dropper. Then, the results were strictly observed and not stirred. The test
tubes were let stood for one to two minutes. The observations in terms of
color between two layers were recorded.
As for Sulfur reaction or Lead acetate test, a 5 mL 5% NaOH was
added with few crystals of Lead (II) acetate to the 3 mL of each protein
suspension. Then, the test tubes were heated in a water bath for five to
ten minutes and mixed occasionally. The observations in terms of color
change were recorded.
B. Denaturation of Proteins
Four glasses or any container were prepared and labeled as 1,2,3
and 4. Afterwards, 4 tbsp rubbing alcohol was placed in container 1, 4
tbsp brine solution was placed in container 2, 4 tbsp cold water was
placed in container 3, and 4 tbsp hot water was placed in container 4.
After that, 2 tbsp egg white was added to each container and set aside
for 15 minutes. Meanwhile, the containers were observed and recorded.
The procedure was repeated but the egg white was replaced with milk.
Data Analysis and Results Discussion
Table 1. Qualitative Test for Proteins
Test Compound
Test Procedures
Gelatin Casein Albumin
Gave an intense Gave a purple-blue Gave a light purple-blue
Biuret Test
purple-blue color color color
Intense blue color Intense blue color Intense blue color
Ninhydrin Test
formed formed formed
Xanthoproteic Pale yellow Dark yellow Yellow orange
Test precipitate formed precipitate formed precipitate formed
No red precipitate Pinkish red Reddish brown
Millon’s Test
formed precipitate formed precipitate formed
Reddish yellow ring Reddish Yellow ring
Hopkins-Cole Test No ring formed
formed formed
No black or gray No black or gray
Lea Acetate Test Black precipitate formed
precipitate formed precipitate formed
 The table above shows the qualitative tests done for identifying different
amino acids in given proteins. On Biuret test, the gelatin, casein, and albumin
gave off a purple-blue color in the test tube indicating that these samples have
peptide bonds which connect amino acids forming proteins. On Ninhydrin test,
the gelatin, casein, and albumin forms intense blue color complex when added
with Ninhydrin solution indicating the presence of alpha amino group of proteins
or free amino acids. On Xanthoproteic test, from the name itself “Xantho-” which
means yellow, this test has a positive result of yellow precipitate. This test
detects the presence of amino acids tyrosine and tryptophan. When proteins
containing these two amino acids react with concentrated nitric acid at high
temperature, the benzene ring in tyrosine and tryptophan became nitrated
causing a change in color. The gelatin, casein, and albumin are positive for this
test. On Millon’s test, casein and albumin forms a red precipitate indicating that
there are tyrosine present in these proteins, while gelatin do not form red
precipitate indicating that there are no tyrosine present. On Hopkins-Cole test, a
reddish-yellow or violet ring was formed when there are tyrosine and tryptophan
present in the proteins. In this test, only gelatin do not form any ring in between
two layers while casein and albumin forms reddish-yellow rings in between two
layers. On Lead (II) acetate test, the gelatin and casein do not form any black or
gray precipitate after treating with NaOH and Pb(Ac) 2 crystals while albumin
forms a black precipitate which indicates that among the three samples, only
albumin contains an amino acid cysteine.
Proteins are large biomolecules composed of one or more long chain of
amino acids linked together by peptide bonds. There are four aspects to protein’s
structure designated as: primary structure, secondary structure, tertiary structure,
and quaternary structure. Primary structure refers to the amino acids sequence in
proteins, secondary structure refers to the shape in which the long polypeptide
chain exists, tertiary structure refers to the overall shape of a simple protein
molecule, and quaternary structure is formed by several protein molecules. Some
tests are done to detect these amino acids in the protein samples.
In Biuret test, with the presence of alkali metals, the proteins react with
copper (II) ions forming a violet-colored complex called the Biuret. In
Xanthoproteic test, with the presence of concentrated nitric acid, there are certain
amino acids present in the proteins which undergo nitration. Some of these
amino acids are tyrosine and tryptophan forming yellow-colored xanthoproteic
acid. In Ninhydrin test, the proteins reacted with Ninhydrin reagent forms an
intense, blue-colored compound. In Millon’s test, the white precipitate of proteins
changes into brick red precipitate when heated indicating that there are certain
amino acids present such as tyrosine.
 Table 2. Denaturation of Proteins
Test Compounds Egg White Milk
The egg white solidifies White curds form and solid
Rubbing alcohol
and turn opaque white white precipitate forms
Brine solution No coagulation Still cloudy
Small amount of egg white
Forms yellow tinges in the
Cold water coagulates and becomes
milk
foggy
Cloudy and pure white in
Hot water Egg white coagulates
color

The table above shows the qualitative analysis of denaturation of proteins

in food such as egg white (albumin) and milk (milk protein). Upon testing, the
rubbing alcohol denatures protein which it breaks the bond that holds the part of
the protein in a folded shape where the egg white solidifies and turn opaque
white in color and the milk forms white curds or a powdered-like substance in the
container. The brine solution which composed of salt and water do not denature
the proteins. Therefore, there are no coagulation forms in the egg white and the
milk still the same. Cold water and hot water denature proteins such as albumin
and milk protein. As for egg white, the hot water coagulates the albumin first
before coagulation in cold water. As for milk, there are yellow tinges formed in
cold milk due to the separation of proteins from fats. These yellow tinges are the
fats situated in one place. In hot milk, the whey protein denatures and forms
whey protein- casein polymer which makes the milk cloudier and purer white in
color.
Conclusion
Based on the data and results, the following conclusions were drawn:
1. Proteins are polymers of amino acids that are linked together by peptide
bonds. These proteins are very large molecules with high molecular weight
that ranges from approximately 6500 to 200000 grams per mole. There are
several aspects to describe proteins based on their structure. The primary
structure refers to the amino acids sequence in proteins. The secondary
structure refers to the shape in which the long polypeptide chain exists. The
tertiary structure refers to the overall shape of a simple protein molecule. The
quaternary structure is formed by several protein molecules. However, the
proteins that are biologically inactive are called denatured proteins. Amino
acids are molecules which contains amino group and carboxyl group. The
amino acids found in proteins are the alpha-amino acids which means that
the amino group is attached to the alpha carbon.
2. Amino acids are the building blocks of peptides and proteins. The Thin Layer
Chromatography can be utilized to identify the different amino acids.
 Separation occurs as each component, being different in chemical and
physical composition, interacts with the stationary and mobile phases to a
different degree creating an individual spots on the plate. The solvent
mixtures were initially placed in TLC chamber and close the chamber. After
that, the paper was let inside the chamber and capillary reaction takes place.
Then dry the plates in hot air oven at 105 oC for 5 minutes. Here, the Ninhydrin
will react with the spotted amino acids and the resulting complex, which is
colored, makes them visible on the plate. After some time, mark the center of
the spots, then measure the distance of the spots from the origin (base line)
and calculate the Rf value with a formula of Rf value = Distance traveled by
the substance / Distance traveled by the solvent front from the origin.
3. Proteins and amino acids can be determined through different qualitative
tests. For Biuret test, in basic solution, molecules having two or more peptide
bonds will react with Cu2+ to generate a violet-pink complex. Because the
original Cu2+ solution is blue, the chemical being examined might be either an
amino acid or a dipeptide, or neither. For Xanthoproteic test, nitric reacts
chemically with the benzene rings on tyrosine and tryptophan. The aromatic
rings were nitrated in this reaction. If a sample contains tyrosine or
tryptophan, adding nitric acid to it and heating the mixture produces a yellow
solution. When a strong base is added to this yellow solution, it turns orange.
Most proteins will react positively in this test because they contain one or both
amino acids. For Ninhydrin test, the ninhydrin reagent will react with free
amino groups to produce a purple solution. A free amino group can be found
in almost all amino acids. With ninhydrin, several proteins also test positive.
For lead (II) acetate test, cysteine and methionine are sulfur-containing amino
acids. When one or both amino acids are present in a sample, the H 2S is
generated, which has a rotten egg odor. When a moistened piece of lead (II)
acetate paper is placed over a solution while H 2S is created, the H2S interacts
with the lead ion, generating a black or gray covering of lead (II) sulfide. The
presence of this black color indicates the presence of a sulfur-containing
amino acid. For denaturation, a protein can be denatured in a variety of ways.
Denaturation refers to the disruption of the secondary, tertiary, and
quaternary structures of a protein; it breaks the protein's natural folding,
rendering it inactive. When a protein is denatured, it frequently coagulates
and becomes a solid. Heat, organic solvents, agitation, acid or base, and
heavy metal ions are all denaturing agents.
Application
Proteins, together with carbs, fats, vitamins, and minerals, are essential
nutrients. If carbs or lipids are unavailable, they can be broken down and used as
a source of emergency energy. After intake, enzymes in the digestive tract break
protein down into smaller peptide chains, which are then broken down further into
individual amino acids. These tiny elements subsequently enter the bloodstream
 and are delivered to the cells from there. The amino acids are incorporated into
the cells, and proteins are formed.
References
Amritacreate (2016, February 5). Qualitative Analysis of Proteins - MeitY OLabs [Video].
YouTube.
https://www.youtube.com/watch?v=OsdhNtNNNds&feature=youtu.be
Channel, C. (2016, December 8). Identification of amino acid contained in proteins
[Video]. YouTube.
https://www.youtube.com/watch?v=40-WbMgU3cA&feature=youtu.be
Ramos, V. (2020, September 11). Qualitative Tests for Proteins [Video]. YouTube.
https://www.youtube.com/watch?v=6YPWipP-Qe8&feature=youtu.be
Vlab, A. (2011, January 14). Separation of Amino acids by TLC - Amrita University
[Video]. YouTube. https://www.youtube.com/watch?
v=tDaKxskUwA0S&feature=youtu.be
 APPENDICES
A. Preparation of Materials

CONTAINERS

Hot water Cold water

Brine
solution Rubbing Egg White
Milk alcohol

B. Denaturation of Egg White

 Egg white in Rubbing Alcohol Egg white in Brine Solution

Egg white in Cold Water Egg white in Hot Water

C. Denaturation of Milk Protein

Milk in Rubbing Alcohol Milk in Brine Solution
Milk in Cold Water Milk in Hot Water