Valiente, Claudine Joy D.

BSN1-B-17

QUALITATIVE TEST FOR PROTEINS

INTRODUCTION

Protein are biochemical molecules consisting of polypeptides joined by peptide

bonds between the amino and carboxyl groups of amino acid residues. Protein is a part of every
cell in the body. It helps the body to build and repair cells and tissues. Protein also plays an
important part of a healthy diet. Because it made up of chemical 'building blocks' called amino
acids. Our body uses amino acids to build and repair muscles and bones and to make hormones
and enzymes. They can also be used as an energy source. There 7 qualitative test for lipids. The
first one is Biuret test. This test is used to detect the presence of peptide bond. When treated
with copper sulphate solution in presence of alkali (NaOH or KOH), protein reacts with copper
(II) ions to form a violet colored complex called biuret. The second test is Ninhydrin test. This
is a test for amino acids and proteins with free –NH2 group. When such an –NH2 group reacts
with ninhydrin, an intense blue colored complex is formed. And then Xanthoproteic test It is an
identification test of protein and it gives a positive result with those proteins with amino acid
carrying aromatic group. When protein is treated with hot concentrated nitric acid, a yellow
colored substance is formed. The other test is Millon's test is an analytical reagent used to detect
the presence of soluble proteins. A reddish-brown coloration or precipitate indicates the presence
of tyrosine residue which occur in nearly all proteins. The Sakaguchi test is a chemical test used
for detecting the presence of arginine in proteins. The Hopkins-Cole test, also known as the
glyoxylic acid reaction, is a chemical test used for detecting the presence of tryptophan in
proteins. A protein solution is mixed with Hopkins Cole reagent, which consists of glyoxylic
acid. And the last test is Lead acetate test is generally regarded as selective for detection of
hydrogen sulfide .

MATERIALS

-Dropper, Test Tube, Test Tube Rack, Water bath

FOR THE PROTEIN SAMPLE: Albumin, Casein, and Gelatin
CHEMICALS: Sodium Hydroxide Solution, Molars of Copper sulfate, NinHydro Solution,
Nitric acid, Saturated Sodium Hydroxide, Fresh Millons Reagent, Sodium Hydroxide, Alpha
Naphthol, Bromine water, Hopkins cole reagent, Concentrated sulfuric acid.

METHODS

BIURET TEST- Add 1 ml. of 10% Sodium hydroxide solution into each of the protein sample and
mix. Then add 1 to 3 drops of 0.01 of molars of copper sulfate or until a definite color is produced.
NINHYDRIN TEST- Add 5 drops of 0.1% Nin Hydro Solution to each sample. And then heat it in
boiling water bath for 10 minutes.
XANTHOPROTEIC TEST- Slowly added 1 ml of concentrated nitric acid, to 3 ml of each of the
protein suspensions. And then place it in the water bath for about 30 seconds and place each test tube in a
rack to cool the solutions. Then add slowly, drop by drop, the saturated sodium hydroxide solution to
each test tube until the solutions are alkaline.
MILLON’S TEST- Add 5 drops of fresh Millons reagent to 3ml of each protein suspensions. Carefully
heat each mixture in boiling water bath for 5 minutes. And then cool the tubes and note the colors formed.
Caution: if too much reagent is used, the color may disappear in boiling.
SAKAGUCHI TEST- Add 1 ml of 10% NaOH and 1 ml of 0.02% alpha-naphthol solution to 3 ml of
each of the protein suspensions. After 3 minutes, add 2-4 ml of bromine water to each. A strong red color
can be stabilized by adding urea to destroy the excess hypobromite.
HOPKINS COLE TEST- Add 2 ml of Hopkins-Cole reagent to 3 ml of each of the 3 protein
suspensions. And then mix. Incline each test tube and with a dropper add slowly about 1 ml of
concentrated sulfuric acid down the side of the tube so that two layers will form. Do not stir.
Lastly, let stand for 1 to 2 minutes and note the color formed in between the layers.
LEAD ACETATE TEST- Add 5 ml of 5% Sodium Hydroxide and a few crystals of Pb(ac)2,
(Lead II acetate) to 3 ml of each protein suspensions. And then heat boiling water for 5-10
minutes with occasional mixing of the contents of the tube. describe the color change.

DATA:
QUALITATIVE TEST FOR PROTEINS

TEST SAMPLES RESULT

BIURET TEST ALBUMIN (+) Purple color/ Presence of
Proteins
CASEIN (+) Purple color/ Presence of
Proteins
GELATIN (+) Purple color/ Presence of
Proteins
NINHYDRIN TEST ALBUMIN (+) Purple color/ Presence of
Proteins
CASEIN (+) Purple color/ Presence pf
Proteins
GELATIN (+) Purple color/ Presence of
Proteins
XANTHOPROTEIC TEST ALBUMIN (+) Yellow color/ Presence of
Proteins
CASEIN (+) Yellow color/ Presence of
Proteins
GELATIN (+) Yellow color/ Presence of
Proteins
MILLON’S TEST ALBUMIN (+) Red Precipitate formed/
Presence of Protein
CASEIN (+) Red Precipitate formed/
Presence of Protein
GELATIN No color change/ No presence
of Protein
SAKAGUCHI TEST ALBUMIN (+) Red color/ Presence of

HOPKINS COLE TEST
LEAD ACETATE TEST
Protein
CASEIN (+) Red color/Presence of
Protein
GELATIN (+) Red color/Presence of
Protein
ALBUMIN (+) Tryptophan Presence/
Presence of Protein
CASEIN (+) Tryptophan Presence /
Presence of Proteins
GELATIN (-) Absence of Tryptophan /
No Presence of Proteins
ALBUMIN (+) Black Color presence/
Presence of Protein
CASEIN Little bit of Black Color
formed/ Presence of Proteins
GELATIN (-) Absence of Black Color/
No Presence of Proteins

ANALYSIS

1. BIURET TEST- In Biuret test it indicates that in Albumin, Casein and Gelatin Protein
samples it shows that there is a presence of protein in all the given suspensions as what the
results revealed. Because it has a purplish violet color formed in the test tubes.

2. NINHYDRIN TEST- In Ninhydrin test it stated that the protein suspensions such as albumin,
casein and gelatin have an intense blue or violet color formed in the test tubes. This implies that
there is a presence of protein in all the given suspensions as what the results revealed.

3. XANTHOPROTEIC TEST- In Xanthoproteic Test it indicates that all the results in the
Protein samples it shows that there is a Yellow formed in the test tubes. Moreover, albumin and
casein have a yellow orange color formed while gelatin have a light-yellow color formed.
However, it still implies that these protein suspensions have a presence of proteins as any kinds
of color that have a yellow is still positive result for this test.

4. MILLON’S TEST- In Millon’s Test it shows that the protein suspensions such as albumin
and casein have a white color precipitate formed which gradually turns into red while gelatin
have a red color formed. It implies that these protein suspensions have a presence of proteins as
red color is the positive result for this test.

5. SAKAGUCHI TEST- In Sakaguchi Test it indicates that the protein suspensions such as
albumin, casein and gelatin have a deep red color formed in the test tubes. This implies that there
is a presence of protein in all the given suspensions as what the results revealed.

6. HOPKINS COLE TEST- In Hopkins-Cole Test it shows in the protein samples such as
Albumin, Casein and gelatin that it has a brown colored ring at the junction of two layers while
gelatin have no brown colored ring at the junction of two layers as it is just clear as a glass.
However, to identify that there is a presence of proteins, there should be a violet-colored ring at
the junction of two layers in which there is none in the results of this test. Therefore, it implies
that these protein suspensions such as albumin, casein and gelatin does not have any presence of
proteins.
7. LEAD ACETATE TEST- In Lead Acetate Test It stated that albumin have a black color
formed while casein have a little bit of black color formed. It implies that if there is a black color
formed, then there is a presence of proteins. On the other hand, gelatin have no changes in color
as it does not form any black color in the test tube. It implies that there is no presence of proteins.

CONCLUSION

I conclude that proteins may be analyzed qualitatively using a variety of techniques. In Biuret
test there’s an appearance of purplish violet tint that indicates a positive Biuret test result. While
in the Ninhydrin test there’s a presence of a strong blue or violet hue that indicates a positive
result. The presence of yellow or yellow orange color is a positive result for the Xanthoproteic
test. The presence of red hue is required for Millon's and Sakaguchi tests. The existence of a
violet-colored ring at the junction of two layers is required for the Hopkins-Cole test. The
presence of black color is a positive result for the Lead Acetate test. As a result, the experiment's
hypothesis is accepted because if the color changes, then there is a presence of proteins.

REFERENCES:
https://microbenotes.com/xanthoproteic-test/?fbclid=IwAR0NygZBPa0ylM_PtSEQLyvUdVSJoqP-
quPAGqFiNbshDQFS1Txtn7_JWyM
https://microbiologynote.com/millons-test/?fbclid=IwAR2o1qRtkS-
xrUYgH44naHXnUxWMrcS0oS41f7sHkO2ln40XekFfpHx_2
https://sites.google.com/site/vernawang999/test-results?fbclid=IwAR2-zc-
cNDlAjn5PlJV2dnIVCkIXZFULcLOSzXnviSTZe9n4-IZpoMvkN3I