Date:

Estimation of Amino Acids by Ninhydrin Method

Aim: To study the qualitative and quantitative estimation of Amino Acids.
Theory: There are 20 different amino acids present in our body. The amino acid has a
carboxyl group and an amino group with a variable side chain attached to a central carbon
atom.

Proteins are made up of amino acids. And hence, the study of amino acids gives us insights
into the structure and function of proteins.
Principle: α amino acids reacts with Ninhydrin involved in the development of color which
is explained by the following 5 steps:-
1. α amino acid + Ninhydrin Reduced Ninhydrin + α imino acid +H2O
This is an oxidative deamination reaction that elicit to hydrogen from the α
amino acid to produce an α imino acid. Also, the ninhydrin reduced and loses
an oxygen atom with the formation of water molecule.
2. α imino acid + H2O α keto acid + NH3
The rapid hydrolysis of NH group in the α imino acid will cause the formation
of an α keto acid with an ammonia molecule. This α keto acid further involved
in the decarboxylation reaction of the step.
3. α keto acid + NH3 aldehyde + CO2
The overall reaction for the above reactions is simply explained in reaction (4)
as follows:
4. α amino acid + 2Ninhydrin aldehyde + CO2 + final complex (BLUE) +
3H2O
 Ninhydrin which is originally yellow reacts with amino acid and turns deep purple. It is this
purple color that is detected in this method. Ninhydrin will react with a free α amino group.

Requirement:
Chemicals: Threonine (0.1mg\ml) i.e. 0.002 gm in 20 mL D.W.
Ninhydrin solution (0.2%) i.e. 0.05 gm in 25 mL acetone.
Ethanol (12.5mL D.W. + 12.5mL ethanol)
Glasswares: 7 test tubes, 250 mL beaker, conical flask, 2 1 mL pipettes, 5 mL pipette,
measuring cylinder.
Instruments: Colorimeter, weighing balance.
Miscellaneous: Cotton plug, distilled water.

Procedure:
1. Test tubes were prepared in accordance with the table.
2. Water bath was prepared. Test tubes were heated till color developed.
3. The colorimeter was set to zero with blank and absorbance was taken at 545 nm.
4. Graph of absorbance v\s concentration of A.A. was plotted and concentration of
unknown was estimated.
Observation Table:
Stock solution = 0.1 mg/mL
Test Amino acid D.W. Conc. Of Ninhydri D.W. 50% Absorbance
Tube solution (mL) amino n (mL) (mL) Ethanol(mL) at 540 nm
(Threonine) acid
(mL) solution
(0.1
mg/ml) Cotton
Blank 0.0 1.0 0.0 1 3 plug and 1
boiled
1 0.2 0.8 0.2 1 3 till 1
2 0.4 0.6 0.4 1 3 colour 1
3 0.6 0.4 0.6 1 3 develops. 1
4 0.8 0.2 0.8 1 3 1
5 1.0 0.0 1.0 1 3 Cool it. 1
6 U.K. - - 1 3 1

Result:

Conclusion:

GLP:
Switch on colorimeter one hour before usage.
Wipe the outside of cuvettes gently with tissue paper before inserting in the instrument.
Ninhydrin should be prepared and used with care as it is carcinogenic.

References:
http://vlab.amrita.edu/?sub=3&brch=63&sim=156&cnt=1
http://www.daviddarling.info/images/aminogroup.gif
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html
https://www.researchgate.net/post/what_is_the_principle_of_estimation_of_amino_acids