RESULTS AND DISCUSSION Amines, amino amino acids, acids, and proteins proteins are compounds compounds that are are very important in people’s everyday lives. Amines are compounds that co ntain 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 ): R—NH2 (Only one R-group is attached to the Nitrogen) Secondary amines (2 ): R2—NH (It has 2 R-groups attached to the Nitrogen) Tertiary amines (3 ): R3—N (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 H 2O 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 H 2O 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 α 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%CuSO 4 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 Cu 2+ complexes with amide N atoms. The forst test compound was alanine. At the addition of CuSO 4, 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 (HNO 3)
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
