Colour Reaction

of Amino Acids
Prepared by:
Group#2
Quinto, Jasmine
Sta. Isabel, Rizzalyn
Yadao , Monique
Niazmand Saravani, Mostafa
Niazmand Saravani, Mojtaba
Seong, Jo-eun
Majidzadeh, Hossein

Proteins are complex, high

molecular weight biomolecules
composed of amino acids joined
together by peptide bonds. The
twenty amino acids normally
found in proteins differ in their
amino groups in the side chains.
These respond to specific
qualitative tests with certain
chemical compounds chemical
reagents and thus become the
basis of their detection whether
as free amino acids or in
combined form as in peptides or
proteins

Reactions of proteins may be

divided into three categories:
1.Reaction due to the presence
of specific chemical groups are
linkages in the protein molecule
2.Reaction due to the acidity or
basicity of the protein
3.Reactions due to the colloidal
nature of the proteins

Colour
Reactions of
Amino Acids
and Proteins

Biuret test
This is the test indicates the presence
of peptide linkages. The purplish to
violet colour is apparently due to the
cupric ions with the unshared electron
pairs of four nitrogen atoms. All
substance is proportional to the
number of peptide bonds give the test
and the intensity of the purple colour
produced id proportional to the
number of peptide bonds present.

Ninhydrin test
Amino acids reacts with ninhydrin
(tryketoninhydrindene hydrate) to
yield CO2, NH3 and aldehyde
containing one less carbon than the
amino acid. The reaction is also yields
a blue or purple colour useful for the
colorimetric determination of amino
acids.

Xanthoproteic test
This test is positive for proteins and
amino acids containing an aromatic
side chain (phenylalanine, tyrosine,
and tryptophan). The benzene ring
undergoes nitration with concentrated
HNO3 giving nitro derivatives which
are yellow in colour. Phenylalanine
does not response readily to this test
and requires H2SO4 as catalyst.

Millon-Nasse test
The phenolic group of tyrosine reacts
with Millon-Nasse reagent (HgSO4 in
H2SO4) forming an old rose or pink to
red complex upon heating. The
complex is probably the mercury salt
of the phenolic compound.

Bromine water test

The pyrrole ring of tryptophan
undergoes halogenation with Br2
water in the presence of amyl alcohol
giving a pink or lavender colour in the
alcohol layer.

Unoxidized sulfur
test
This is given by the sulfur containing
amino acids cysteine and cystine
(methionine is quite stable alkaline
lead acetate) whn boiled in strongly
alkaline solutions. The liberated sulfur
reacts with PbAc2 forming a brown to
black precipitate of lead sulfide.

Sakaguchi Reaction
The guanidinium group of arginine in
alkaline solution gives an orange or
red colour with alpha-naphthanol and
sodium hypobromite

Hopkins-Cole test
The indole ring of tryptophan reacts
with Hopkins-Cole reagent (glyoxylic
acid) and sulfuric acid to produce a
violet or ed purple color.

Objectives
1.To carry out the different colour
reaction testes for amino acids
and proteins;
2.Ro characterize amino acids
based on their reactions with
specific chemical reagents;
3.Ro identify the functional group
of the amino acid responsible
for the positive colour reaction.

Materials
1%solution of egg
albumin
0.1% solution of
tryptophan
Arginine
Cysteine
Tryrosine
Phenylalanine
10% formic acid
solution
0.1% ninhydrin
5% CuSO4

Saturated lead
acetate
10% NaOH
Millon-Nasse reagent
Bromine water
N-amyl alcohol
Alpha-naphtanol
solution
20%NaOH
Hopkins-Cole reagent

Apparatus
1.
2.
3.
4.
5.
6.

Hot plate/ stove

Alcohol lamp
600mL beaker
50mL beaker
Test tubes
Test tube rack

1. 10mL graduated
cylinder
2. Pipets
3. Droppers
4. Litmus paper
5. Wire guaze

Procedure

Preparation of Saliva
Solution
1.Collect 10mL of saliva using a
clean 50mL beaker
2.Dilute the saliva with an equal
volume of distilled water

Colour Reaction of
Amino Acids and
Proteins
The following tests will be performed on acid
soluble proteins of saliva solution and 1%
solution of egg albumin. Some or the tests
may require to be performed on specific
amino acids. Ain all of the following tests,
perform a blank test with distilled water
instead of the protein sample.

Biuret test
1. In 2 different test tubes, place1mL
each of the protein samples (saliva
solution and 1% solution of egg
albumin)
2. To each test tube add 1mL of
10%NaOH
3. Add 2 drops of CuSO4 solution.
Observe the result.
4. Run a blank test with distilled
water.

Ninhydrin test
1. In 2 different test tubes, place1mL
each of the protein samples (saliva
solution and 1% solution of egg
albumin)
2. Heat each of the protein solutions
with 0.25mL of 0.1%ninhydrin
3. Let cool and observe
4. Run a blank test with distilled
water.

Xanthoproteic test
1. In 2 different test tubes, place1mL
each of the protein samples (saliva
solution and 1% solution of egg
albumin)
2. To each protein solution, add 1mL
of concentrated HNO3.
3. Note if a heavy white precipitate is
formed. Heat carefully to boiling
and observe the colour of the
solution/precipitate.
4. Cool and make alkaline with 10%
NaOH. Note the colour change
5. Cool and make alkaline with 1%
solutions of tryptophan, tyrosine,
and phenylalanine in different test
tubes.

Millon-Nasse test
1. In 2 different test tubes, place1mL
each of the protein samples (saliva
solution and 1% solution of egg
albumin)
2. Add 2 to 3 drops of Millons reagent
to each of the protein solutions.
3. Boil in a boiling water bath for 5
minutes. Observe.
4. Perform an identical test with 0.1%
tryptophan solution.
5. Run a blank test with distilled
water.

Bromine water test

1. In 2 different test tubes, place1mL
each of the protein samples (saliva
solution and 1% solution of egg
albumin). And add 3 drops of
bromine water and 1mL of n-amyl
alcohol. Shake well
2. Observe the colour of the alcohol
layer.
3. Repeat the steps in with the 1%
tryptophan solution
4. Run a blank test with distilled
water.

Unoxidized sulfur test

1. Mix 1mL each protein samples in 3
different tubes with 1mL each of
10% NaOH and saturated lead
acetate, PbAc2.
2. Boil in a water bath for 3 minutes
and observe the result.
3. Perform an identical test with 0.1%
cystein and a bank test with
distilled water.

Sakaguchi Reaction
1. Place 1mL each of protein samples
in 3 different test tubes and add
2mL each of 20% NaOH and alpha
naphtanol solution.
2. Mix thoroughly and add 0.5mL of
freshly prepared bromine water.
Observe the result.
3. Perform an identical test with 0.1%
cystein and a bank test with
distilled water.

Hopkins-Cole test
1. Place 2mL each of sample solution
in 3 different test tubes and to each
tube add 1mL of Hopkins-Cole
reagent.
2. Incline the tube and carefully add
along the side 2mL at concentrated
H2SO4.
3. Observe the colour at the junction
of two liquids.
4. Perform an identical test with 0.1%
trytophan.

Test Solutions
Colour
Test

Saliva
solution

Tyr

Trp

Arg

CYs

Biuet
test

XXXX

XXXX

XXXX

XXXX

Ninhydri
n

XXXX

XXXX

XXXX

XXXX

XXXX

XXXX

XXXX

XXXX

XXXX

XXXX

Egg
Albumin

Gly

Xanthoproteic
Millon
test

XXXX

Bromine
water

XXXX

Unoxidiz
ed

XXXX

XXXX

Sakaguc
hi

XXXX

XXXX

Hopkinscole

XXXX

XXXX
XXXX
XXXX

XXXX