LABORATORY REPORT

MIC180
SEMESTER OCT 2021 – FEB 2022
LAB 2: Qualitative Tests for Amino Acids
NAME STUDENT ID
1 MUHAMMAD AIMAN AZAMUDDIN BIN ABDUL AZIZ 2022628476
2 ANIS NATASHA SHAHROM BINTI SHAHRIN 2022894238
3 ANISYA SAFFIA BINTI ZORHADI 2022477018
4 NURAMNA NAFESA BINTI RAZAK 2022840796
5 SORFINA MIRZA BINTI ZAFIZAL 2022841064
GROUP: AS1161E2
DATE OF SUBMISSION: 20 JANUARY 2023

MARKS

Objective, title and /4

Introduction

Experimental /2
procedure

Results: /4
data, figures, graphs,
tables, etc.

Discussion, /6
conclusion &
references

Post lab questions /4

 Total /20

TITLE: QUALITATIVE TESTS FOR AMINO ACIDS

INTRODUCTION:
Amino acids are building blocks for proteins. They share the same acid and amino
group, but the side chain differs. When amino acids are linked together to form an
amide, the product is called a peptide, and the newly formed bond is called a peptide
bond. If a few amino acids (up to 20) are linked, the molecule is called a polypeptide.
When amino acids (20-1000) are in the peptide chain, it is called a protein.

The α-amino acid can be detected using ninhydrin which forms a blue to blue-violet
colour as a positive indication. Other colours (yellow, orange, red) are negative. The test
is very sensitive and often used for colourimetric determination of amino acids solutions
or as a visualisation agent in the chromatography of amino acids. During the reaction,
the amino group will liberate ammonia; the ammonia will react with ninhydrin to form a
coloured complex.

The amino acid with thiol group (-SH) such as cysteine when heated in the presence of
alkali will produce sulphide ion. These ions can be detected by reaction with lead
cations- a black precipitate plumbum sulphide (PbS) is formed.

In Millon's Test, if the hydroxyphenyl side group is present, a red colour will be
observed. Tyrosine is the only amino acid which gives a positive test. However, any
molecule with phenolic –OH will react.

In Hopkins-Cole Test, heterocyclic side chain (indole chain) of tryptophan reacts to give
a purple to violet ring at the interface of two layers.
OBJECTIVE:
To conduct a series of qualitative tests for amino acids.

METHODS:
Test 1: Ninhydrin Test

a) 1 ml of glycine was pipetted into a test tube.

b) 5 drops of ninhydrin were added.
c) The mixture was heated in boiling water bath for 2 minutes.
d) The changes appeared were recorded.
e) The experiment was repeated by using stock solutions of tyrosine, tryptophan,
phenylalanine, and proline.

Test 3: Millon Test

a) 1 ml of glycine was pipetted into a test tube.

b) 5 drops of Millon reagent were added.
c) The test mixture was heated in a boiling water bath in 10 minutes.
d) The test mixture was cooled down to room temperature.
e) 5 drops of natrium nitric were added.
f) Any changes were recorded.
g) The experiment was repeated by using stock solutions of tyrosine and
phenylalanine.

Test 4: Hopkins-Cole Test

a) 1 ml of glycine were mixed with 1 ml of glacial acetic acid in a clean test tube.
b) The test tube was inclined and 1 ml of concentrated sulfuric were added but did
not mix.
c) Two layers should form.
d) The layers stand and the colour at the interface after 2-3 minutes were noted.
e) The experiment was repeated by using stock solutions of tyrosine and
tryptophan.
Test 5: Lead Sulphide Test

a) 1 ml of cysteine was pipetted into a test tube.

b) 1 ml of 40% NaOH were added.
c) The test tubes were swirled.
d) The test mixture was heated in a boiling water bath for 2 minutes.
e) The test tube was immersed in cold water to cool it down.
f) 1 ml of sodium plumbate were added.
g) Any changes were recorded.
h) The experiment was repeated by using stock solutions of cystine and methionine.
RESULTS:
Test 1: Ninhydrin Test
Sample Observation Conclusion
Glycine Blue Blue discoloration contains
protein.
Tyrosine No colour changes The sample does not
contain any of the amines,
amino acids or protein.
Tryptophan Brown Brown discoloration
contains an a-amino acid.
Phenylalanine Purple Purplish discoloration is
produced by the a-amino
acids.
Proline Yellow Yellow discoloration
because the test sample
contains proteinogenic
amino acids.

Test 3: Millon Test

Sample Observation Conclusion
Glycine No colour changes Negative test. No change
of colour because
hydroxyphenyl side group
is not present.
Tyrosine Brick red Positive test because a red
colour is observed.
Hydroxyphenyl side group
is present.
Phenylalanine No colour changes Negative test. No change
of colour because
hydroxyphenyl side group
is not present.
Test 4: Hopkins-Cole Test
Sample Observation Conclusion
Glycine No colour changes Negative test because no
appearance of a purple ring
between the two layers due
to absence of tryptophan
Tyrosine No colour changes Negative test because no
appearance of a purple ring
between the two layers due
to absence of tryptophan.
Tryptophan Colourless to violet Positive test because a
purple ring appears
between the two layers due
to presence of tryptophan.

Test 5: Lead Sulphide Test

Sample Observation Conclusion
Cysteine No colour changes Negative test because the
absence of sulphur-
containing amino acids.
Methionine No colour changes Negative test because
bond in methionine is not
release by the treatment
with NaOH.
DISCUSSION
In Ninhydrin test, blue colour in glycine is observed because it contains protein.
However, no colour changes occur in tyrosine. This is because the sample does not
contain any of the amines, amino acids or protein. As for tryptophan, the brown colour is
observed because the brown discoloration contains an a-amino acid. The purple colour
is observed in phenylalanine because the purplish discoloration is produced by the a-
amino acids. Lastly, the yellow colour is observed in proline because the test sample
contains proteinogenic amino acids.

In Millon test, no colour changes are observed in glycine and phenylalanine because
the hydroxyphenyl side group is not present. However, there are colour changes in
tyrosine which is brick red colour. This is because the hydroxyphenyl side group is
present.

In Hopkins-Cole test, no colour changes are observed in glycine and tyrosine. This is
shown as negative test because no appearance of a purple ring between the two layers
due to absence of tryptophan. Lastly, we detected the colour changes in tryptophan
which is colourless to violet. This shown as positive test because a purple ring appears
between the two layers due to presence of tryptophan.

In Lead Sulphide test, the observation is recorded as no colour change in cysteine

because the absence of sulphur-containing amino acids. As for methionine, there is no
colour changes occur because bond in methionine is not release by the treatment with
NaOH.
CONCLUSION
The qualitative test of amino acids is characterized by the colour changes, precipitation
and the ring formation based on which the amino acids can be detected and classified.
The colour changes are due to the change in the moiety or the structural configuration,
in which the functional group of amino acid react with the specific reagent to give
specific results.

POST LAB QUESTIONS

1) Choose a sample that showed an orange colour after it has been reached in
the Ninhydrin test.
Proline.
2) Ahmad had tested tyrosine, glycine, proline, with the Millon test. Only
tyrosine was observed to be positive in this reaction. Indicate the colour
observed in tyrosine.
Red.
3) A positive Hopkin’s Cole test is given by a purple violet ring that forms
between two layers of solutions. Name the specific compound present in
the positive sample that allows this happen.
Heterocyclic chain.
4) In this test, positive samples are indicated by the presence of black
precipitates. Select in which test this observation can be seen.
Presence of cysteine.
REFERENCES
• Tiwari, A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.

• Nigam S. C. and Omkar (2003). Experimental Animal Physiology and

Biochemistry. New Age International Pvt. Limited. New Delhi.

• D (2012). Biochemistry. Fourteenth Edition. Academic Publishers. Kolkata.

• Millon's reagent. R097. HiMedia Laboratories.

• Fearon, W R. “A Study of some Biochemical Tests. No. 2; The Adamkiewicz

Protein Reaction. The Mechanism of the Hopkins-Cole Test for Tryptophan. A
New Colour Test for Glyoxylic Acid,” The Biochemical journal 14,5 (1920): 548-
64.