RESULTS AND DISCUSSION

Amines, amino acids, and proteins are compounds that are very important in
peoples everyday lives. Amines are compounds that contain nitrogen in its structure
and are building blocks of amino acids. These amino acids are also important since they
compose the proteins that people need in their bodies to function properly. Different
types of amines are:
Primary amines (1 ): RNH2 (Only one R-group is attached to the Nitrogen)
Secondary amines (2 ): R2NH (It has 2 R-groups attached to the Nitrogen)
Tertiary amines (3 ): R3N (It has 3 R-groups attached to the Nitrogen)
4 tests were performed to identify an amine compound. These were the Solubility
of amines in aqueous acid test, Ninhydrin test, Biuret test, and Protein denaturation.
Solubility test of amines in aqueous acid test identifies whether the amine is soluble in a
polar solution. For this test, 3 samples were used: diethylamine, aniline, and N,Ndimethylaniline.

Diethylamine was tested first. This is characterized by a very pale yellow color
and is in its liquid state. This compound is a secondary amine. In the test, at the addition
of water, the test compound mixed completely and turned the solution into pale yellow,
confirming it is a polar compound. It was also confirmed that diethylamine is a base
because it turned the red litmus paper into blue. Because of this, the concentrated HCl
added reacted with this basic compound and it neutralized the final solution forming a
clear, colorless solution.

Aniline was tested next. This compound is described as an intense brown-orange

liquid and a primary amine. When H2O was added, it did not mix but instead settled at
the bottom of the test tube, confirming that it is denser than water and a nonpolar
compound. This is because of the bulky benzene ring attached to the nitrogen molecule.
It is neither an acid nor a base because it did not change the color of the red and blue
litmus paper. As a result, a beige yellow-colored solution was formed as smoke was
emitted at the addition of HCl.
Lastly, N,N-dimethylaniline, also a pale yellow liquid, was tested. At the addition
of water, it was found to be immiscible and less dense compared to H2O since the
compound settled at the top and formed its own yellow layer. It was also neutral when
tested with red and blue litmus paper. When concentrated HCl was added, it produced a
bright yellow color while smoke was also emitted.

Fig 1. The resulting solution of N,N-dimethylaniline

tested with HCl and water.

The second test performed was the Ninhydrin test. The Ninhydrin reagent used is
a yellowish liquid that was used to determine the presence of amines and free amino
acids and peptides. Alanine was the first compound tested and it is characterized as a
clear colorless liquid. After 30 minutes of color development, strong blue/violet color
appeared on the test spot making it a positive result. It means that an amine or a free
alpha amino acid is present in the compound.

Another compound tested was aniline. A distinct yellow color appeared on the
filter paper after color development signalling a negative result. Lastly, casein was
tested. A blue color developed on the filter paper making it a positive result, although it
was not as intense as the color produced from alanine. This means that the compound
may contain the functional group amine.

Fig 2. The filter papers that were just

dipped in the sample and reagent for
the Ninhydrin test.

Fig 3. The filter papers after 30 minutes of color

development.

The third test was the Biuret test. It made use of 10%NaOH which was a clear
solution and 2%CuSO4 which was a clear, light blue solution. These two mixtures make
up the Biuret reagent which tests for peptide bonds in protein. The presence of protein
in a compound can be found out through this test. A positive result would be the
appearance of blue, violet solution when the Cu2+ complexes with amide N atoms. The
forst test compound was alanine. At the addition of CuSO4, the solution turned into a
very light shade of blue. This is a positive result and therefore the compound may
contain a peptide bond.
The second compound was casein and after mixing, the solution turned into a
light shade of violet (pinkinsh violet) signalling the presence of protein in the compound.
Lastly, albumin was also tested and the final solution turned into a clear liquid with a

very faint pink color. This was considered to be positive and therefore the substance
may contain peptide bonds/protein.

The final test was protein denaturation. Protein denaturation changes the
conformation of protein structures without destroying the peptide bonds. It inactivates
the functionality of protein molecules. This was done through heat and strong acid. The
1% albumin was denatured using heat and a color change occurred. From yellowish
white, the color changed to a lighter shade than it had originally. This means that
albumin contains proteins that was denatured by heat, hence a positive result.
The 1% albumin was also denatured using a strong acid (concentrated HNO 3).
Heat was observed at the addition of the acid and a white insoluble layer also formed on
top of the solution. This means that protein was present in the compound because it
was denatured by the strong acid.

Fig 3. The denatured solution of 1% albumin

by the use of strong acid (HNO3)

CONCLUSION
There are different tests to confirm the presence of amines, amino acids and
proteins in different compounds. The first test is the solubility of amines in aqueous acid.
This test finds out whether the amine is highly basic and would react with the acid or it is
immiscible and forms a separate layer. It was found out that diethylamine is a highly
basic compound which forms a reversible reaction when a strong acid was added. It is
also miscible in water and therefore a polar compound.
Aniline was found to be neutral and is immiscible with water and HCl. N,Ndimethylaniline also formed a separate layer and it was classified as immiscible with the
aqueous acid.
The second test was the ninhydrin test. Both alanine and casein produced a dark
violet color on the test filter paper making a positive result. Aniline, on the other hand,
produced a yellow color and it was considered to be a negative result.
The third test was the Biuret test which made use of sodium hydroxide and
copper sulfate solution. All three test compounds: alanine, casein, and albumin returned
a positive result by producing a violet/ blue solution at the addition of copper sulfate.
Lastly, protein denaturation was also performed in two ways: by heat and by a strong
acid. Both ways denatured the 1% albumin by a difference in color or a formation of a
separate layer.
REFERENCES
Brilliant biology student: Biuret test for protein. Retrieved on Nov. 17, 2014 from:
http://web.njit.edu/~mitra/green_chemistry/EXP_3.htm

Green chemistry in teaching laboratory: Microwave induced reactions: Protein

denaturisation by heat. Retrieved on Nov. 17, 2014 from:
http://web.njit.edu/~mitra/green_chemistry/EXP_3.htm
Phenylamine as a primary amine. Retrieved on Nov. 17, 2014 from
http://www.chemguide.co.uk/organicprops/aniline/amine.html