Experiment: 5

bcetives.o.guantify the amino acids present

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in the **********h**********lhseasn*
sample by Ninhydrine test
Ninhydrin Test Definition
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Ninhydrin test is a chemical test performed to detect the presence of ammonia,
primary/secondary amines, or amino acids. This test involves the addition of
ninhydrin reagent to the test sample that results in the formation of deep blue
Color, often termed as Ruhemann's purple, in the presence of an amino group.
Principle of Ninhydrin Test
Here ninhydrin acts as an oxidizing agent, and itself gets reduced. Ninhydrin roacts
with the amino group of the free amino acid in the test samplo and oxdizes
the compound, leading to delamination. In this reaction, two gassos get
released. These are ammonia (NH3) and carbon dioxide (CO2).
Reaction
O

OH H
R CH CoOH CO R C NH+ c
OH
NH2 C OH H

Ninydrin Reduced ninydrin

O
HO C.
+NH3 + CH -NC 3H,0
HO

Reduced ninhydrin Ninhydrin Violet-blue condensation product

Requirements
Reagents
Ninhydrin reagent: Dissolve 0.35g of ninhydrin in 100 ml ethanol (iso-propanol or 1:1
mixture of acetone/butanol may be used instead of ethanol).
Diluent solvent (for the quantitative test): Mix equal volumes of water and n-propanol.
Standard solution (1% protein solution)
Sample solution
Materials required
Test tubes
Test tube stand
Pipette
Water bath
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Procedure of Ninhydrin Test

in another
For qualitative analysis tube and 1 ml
of the test sample
solution in one test
protein
1. Take 1 ml of standard
dry test tube. the test tubes.
reagent to both allow cooling
to room

2. Add a few drops of ninhydrin 5 minutes and

then
the water bath for
in
3. Place the test tubes
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4.
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temperature.
Observe the formation of color
and note down

Ninhydrin Test
the result.

Interpretation of
Result and
Procedure, Result, Uses
Principle,
Test- Definition,
Ninhydrin

Positive
Negative Ninhydrin Test
Ninhydrin Test

Amino a c i d P r e s e n t

Amino acid Purple-colored

complex present
Absent Proline present

result and
a positive
the tube represents
complex in
of a
purple-colored
indicates the
lack of
The presence in the sample. result and
of amino acid negative
indicates the presence represents a
in the tube
of the complex
The absence samples.
acids in the sample. concentration of u n k n o w n
amino determine the
we c a n
From the graph, unknown samples.
acids in
Test ofamino for amino
Uses of Ninhydrin detect the presence
to monitor
the protection
is used to
Ninhydrin test
solid-phase peptide synthesis
also used in It is
This test is detect fingerprints.
used to
of proteins. commonly shed off in
acid analysis sensitive, it is and proteins
test is quite residues in peptides
As the ninhydrin amines of lysine
terminal
as the
possible ninhydrin.
react with in ammonia and
fingerprints with nitrogen
Test but also
Limitations of Ninhydrin with
a-amino groups

reacts not only reacts as the steric

Ninhydrin molecular weight proteins
other free amines effective to detect high
is not a-amino groups.
test the
The Ninhydrin from reaching
ninhydrin
hindrance limits the