TITLE : QUALITATIVE TEST FOR AMINO ACIDS.

Introduction :
Amino acids are building blocks for proteins. They share the same acid and amino
group but the side chain differ. 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 many amino
acids (20 1000 ) are in the peptide chain , it is called a protein.
The -amino acid can be detected using ninhydrin which forms a blue to blue-violet
colour as positive indication. Other colour ( yellow, orange, red ) are negative . The test is
very sensitive and often used for colorimetric determination of amino acid solutions or as a
visualization agent in chromatography of amino acids . During the reaction the amino group
will liberate ammonia , the ammonia will react with react with ninhydrin to form a coloured
complex.
Most protein give positive results in Xanthoprotein Test because of the presence of
phenyl group (-C6H5 ) . The phenyl group procedure coloured nitro compounds in reaction
with nitric acid.
The amino acid with thiol group (-SH ), such as cysteine when heated in the presence
of alkali will produced sulphide ions. These ions can be detected by reaction with lead cations
a black precipitate plumbum sulphide ( PbS ) is formed.
In Millons 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 the phenolic OH will react.
In Hopkins-Cole test, heterocyclic side chain ( indole group) of tryptophan reacts to
give a purple to violet ring at the interface of the two layers.
In Sakaguchi test , ammonia and ammonium ions give positive reactions in this test.

Objective :
To conduct a series of qualitative test for amino acids.

Procedure :

Ninhydrin Test
1.
2.
3.
4.
5.

1 ml of glycine was pipette in a test tube.

The 5 drops of ninhydrin was add.
The test mixture was heat in boiling water bath for 2 minutes.
Record any changes.
The experiment was repeated with stock solutions of tyrosine , tryptophan ,
phenylalanine and proline.

Xanthoproteic Test
1.
2.
3.
4.
5.
6.
7.

1 ml of glycine was pipette in a test tube.

1 ml of concentrated nitric acid was add.
The test tube was swirl and immerse the test tube in cold water to cool the down.
Any changes was record.
40 % of NaOH solution was add dropwise to change the pH solution to alkaline
Record the changes.
The experiment was repeated with stock solution if tyrosine , tryptophan , phenol and
phenylalanine.

Millon Test
1.
2.
3.
4.
5.
6.
7.

1 ml of glycine was add in the test tube.

5 drops of Millon reagent was add.
The test mixture was heat in the boiling water bath for 10 minutes.
The test tube was cool it down in room temperature.
5 drops of natrium nitric was add.
The changes was record in the table.
The stock solutions was repeated by using tryrosine and phenylalanine.

Hopkins-Cole-Test
1. 1 ml of glysine was mix with 1 ml of glacial acetic acid in a clean test tube.
2. The test tube was incline and slowly add 1 ml of concentration sulphuric acid but do
not mix.
3. The two layer was form.
4. The layers was stand and note the colour at the interface after 2-3 minutes.
5. The stock solutions was repeat using tryrosine and tryptophan.

Test for Presence of Cysteine

1.
2.
3.
4.
5.
6.
7.
8.

1 ml of cysteine was pipette in a test tube.

1 ml of 40% was add in NaOH.
The test tube was swirl.
The test mixture was heat in a boiling water bath for 2 minutes.
The test tube was immerse in cold water to cool the them down.
1 ml of natrium plumbat was add.
The changes was record in the table.
The stock solutions was repeated with tyrosine and methionine.

Sakagushi Test
1.
2.
3.
4.
5.
6.
7.

1 ml of arginine was add in a test tube.

1 ml of 40 % NaOH was add in test tube.
2 drops of napthol was add in test tube.
5 drops of bromine was add in the test tube.
The test tube was swirl.
Any changes that we can see was record in the table.
The procedure was repeated by using glycosiamine, methyl guanidine , creatine and
urea.

Result :
Ninhydrin Test

Sample
Glysine
Tyrosine
Tryptophan
Phenylalanine
Proline

Observation

Conclusion

Its colourless at the initial

and turn to blue at the final.
Its cloudy at the initial and
turn blue at the final.
Its brown colour at the initial
and blue at the final.
Its colourless at the initial
and turn to blue at the final.
Its cloudy at the initial and
final.

Presence of free amino group

Observation
Its colour colourless at the
initial and final.
Its colour colourless at the
initial and final.
Its colour colourless at the
initial and turn light orange
yellowish at the final.
Its colour colourless at the
initial and final.
Its colour colourless at the
initial and turn light orange at
the final.

Conclusion
Negative Phenyl Group

Observation
Its colourless at the initial
and final.
Its colourless at the initial
and turn to red at the final .
Its colourless at the initial
and final.

Conclusion
Absence of phenol group

Presence of free amino group

Presence of free amino group
Presence of free amino group
Absence of free amino group

Xanthoprotein Test
Sample
Glysine
Tyrosine
Tryptophan

Phenylalanine
Phenol

Negative Phenyl Group

Positive Phenyl Group

Negative Phenyl Group

Positive Phenyl Group

Millon Test
Sample
Glysine
Tyrosine
Tryptophan

Hopkins-Cole-Test

Presence of phenol group

Absence of phenol group

Sample
Glysine
Tyrosine
Tryptophan

Observation
Its colourless at the initial
and final
Its colourless at the initial
and final.
Its colourless at the initial
and form two layer with
yellow ring.

Conclusion
Negative Indole Group

Observation
Its colourless at the initial
and form black precipitate.
Its colourless at the initial
and final .
Its colourless at the initial
and final .

Conclusion
Positive result Thiol group

Observation
Its colourless at the initial
and turn to dark maroon at
final.
Its colourless at the initial
and turn to dark maroon at
final.
Its colourless at the initial
and turn black at final.
Its colourless at the initial
and turn to dark brown at
final.

Conclusion
Presence of Guanadine ion

Negative Indole Group

Positive Indole Group

Test for Presence of Cysteine

Sample
Cysteine
Tyrosine
Methionine

Negative result Thiol group

Negative result Thiol group

Sakaguchi Test
Sample
Arginine

Methyl guanidine

Creatine
Urea

Presence of Guanadine ion

Absence of Guanadine ion

Absence of Guanadine ion

Discussion :
Certain functional groups in amino acids and protein can react to produce
characteristically coloured product. The colour intensity of the product formed by a particular
group varies among proteins in proportion to the number of reacting functional , or free ,
groups present. In this experiment, various colour-producing reagent (dyes) will be used to
qualitatively detect the presence of certain functional groups in amino acids and protein.
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or
primary and secondary amines . When reacting with these free amines, a deep blue or purple
color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect
fingerprints , as the terminal amines of lysine residues in peptides and proteins sloughed off
in fingerprints react with ninhydrin . It is a white solid which is soluble in ethanol and
acetone at room temperature Ninhydrin can be considered as the hydrate of indane-1,2,3trione.
In Xanthoproteic test some amino contains aromatic groups that are derivatives of
benzene. These aromatic groups can undergo reactions that are characteristic of benzene and
its derivatives. One such reactions is the nitration of a benzene ring with nitric acid. The
amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo
nitration. The amino acid phenylalanine also contains a benzene ring but the ring is not
activated and therefore does not undergo readily nitration. The principle nitric acid gives
colour when heated with protein containing tyrosine ( yellow colour ) or tryptophan ( orange
colour ), the colour is due to nitration.
Millons test reagent gives positive results ( pink to dark red colour ) with proteins
containing the phenolic amino acid tyrosine . Some protein containing tyrosine will
initially from a white precipitate that turns red when heated , while others from a red solution
immediately.
Hopkins- cole test is the indole ring reacts with glyoxylic acid acid in the presence
of a strong acid to form a violet cyclic product. A positive indole will give observation
colourless at the initial and form two layer with yellow rings as a final results.
Although classified as a non-essential amino acid, in rare cases, cysteine may be
essential for infants, the elderly, and individuals with certain metabolic disease or who suffer
from malabsorption syndromes. Cysteine can usually be synthesized by the human body
under normal physiological conditions if a sufficient quantity of methionine is available.
Cysteine is catabolized in the gastrointestinal tract and blood plasma . In contrast, cystine
travels safely through the GI tract and blood plasma and is promptly reduced to the two
cysteine molecules upon cell entry.
The purpose for the Sakaguchi test used for the detection of a specific type of protein
with the amino acid containing the guanadine ion group. The sample arginine and methyl
guanidine was positive test and form final colour is dark maroon.

Conclusion :
Protein also make up the second largest portion of cells after water. They are larger
polymeric compounds that cells synthesize from various building blocks called amino acid.
Twenty different amino acids, which differ only in the structure of their side chains, are used
by human cells to build proteins. The side chain structure determine the class of the amino
acid , non-polar, neutral , basic or acidic. Human cells can synthesize most the amino acids
needed to build proteins. However, about nine amino acids called essential amino acids
cannot be synthesized by human cells and must be obtained from food.
For this experiment the objective is to conduct a series of qualitative test for amino
acids. I think we manage to do the experiment successfully and get a good result with help
from our lecture.

Refences :
1. Belitz , H.-D; Grosch , Werner; Schieberle , Peter (2009-02-27). Food Chemistry.
ISBN 9783540699330.
2. www.wikipedia.com.

3. Kamsani Ngalip, Nordin Ali, 01/06/2003. Biochemistry Lab Manual. UITM Negeri

Sembilan.