Biomedical Engineering Dept.

Bio Chemistry
Lab. Level 2 Evening
Semester 2

" Proteins Tests "

By
Mustafa Mohammed Hadi
(A-G3-) ‫مسائي‬

Supervised By

Assist.Lectu. Farah Hashim Hussein

Assist.prof.Dr. Sabah Saad Abdulsahib

2022 1443
Ninhydrin Test

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.

Objectives of Ninhydrin Test

- To detect the presence of amines and amino groups in the test solution.

- To quantify the amino acids present in the sample.

- To distinguish carbohydrates from amino acids.

Ninhydrin Test Principle

The amino group belonging to a free amino acid undergoes a chemical reaction
with ninhydrin, which behaves as an oxidizing agent. When exposed to ninhydrin,
the amino acid undergoes oxidative deamination, resulting in the liberation of
CO2, NH3, and an aldehyde along with hydrindantin (which is a reduced form of
ninhydrin).

Now, the ammonia goes on to react with another ninhydrin molecule to form
diketohydrin (which is also known as Ruhemann’s complex). This complex is
responsible for the deep blue colour. When the analyte contains Imino-acids like
proline, a yellow coloured complex is formed. When asparagine is used, the
colour of the resulting complex is brown.

2
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

Equipment

- Water bath
- Spectrophotometer

Ninhydrin Test Procedure

1- Take 1 ml of standard protein solution in one test tube and 1 ml of the test

sample in another dry test tube.

2- Add a few drops of ninhydrin reagent to both the test tubes.

3- Place the test tubes in the water bath for 5 minutes and then allow cooling to

room temperature.

4- Observe the formation of color and note down the result.

3
Results

1- The presence of a purple-colored complex in the tube represents a positive

result and indicates the presence of amino acid in the sample.

2- The absence of the complex in the tube represents a negative result and

indicates the lack of amino acids in the sample.

Uses of Ninhydrin Test

- Ninhydrin test is used to detect the presence of amino acids in unknown

samples.
- This test is also used in solid-phase peptide synthesis to monitor the protection
for amino acid analysis of proteins.
- As the ninhydrin test is quite sensitive, it is commonly used to detect
fingerprints. It is possible as the terminal amines of lysine residues in peptides
and proteins shed off in fingerprints react with ninhydrin.