Amino Acids are the organic compounds which contain at least a carboxyl group and an amino group. In the field of chemistry and biology, we perform many tests to check the presence of these amino acids in different compounds. Paper Chromatograph is one of those most used science lab equipment which is used for the separation purposes and we can also do the separation of amino acids. Paper chromatography can also be used for many other purposes such as separation of colors from different chemicals including ink etc. One of the main purposes of this procedures is that we can identify and separate amino acids with the help of process of Paper Chromatography. Today, I am going to give you the complete procedure involving the separation and determination of amino acids by paper chromatography. First, let us have a look at the list of chemicals needed for the process.
Chemicals/Reagent and Apparatus for Determination of Amino Acids by Paper Chromatography:
The chemical agents used in this test will be 2% of Ammonia, propan-2-ol, Aluminium Foil, ninhydrin spray, separate test tubes containing 0.5M glycine, aspartic acid in 1.5% HCL, and leucine. So these were all the chemicals which we are going to need when we perform this test. The apparatus required for the test consist of Chromatograph paper, Capillary Tube, a Baker, and an oven. Since we know about all the chemicals and apparatus, read the following precautions which much be taken care of before performing the test.
Make sure that you take care of following precautions when you perform this test.
- Propane-2-ol is dangerous since it is highly flammable so make sure that you don’t light it.
- Ninhydrin which is dissolved in the ethanol is very dangerous for the skin since it forms a purple discoloration so keep away for it.
After taking care of these precautions, we can move forward to perform the experiment.
Procedure for Determination of Amino Acids by Paper Chromatography:
Here are all the steps that are taking part in the test. Make sure that you read them well and follow each and every step carefully.
- Take a beaker which can contain a volume of 500 cm3 and in it, mix 2% ammonia solution which must be 10 cm3 with propan-2-ol which must be 20 cm3. Make sure that the face of this beaker is being covered with aluminum foil and this solution is used as a solvent in the test.
- Now make the graph with the following specification on a chromatograph with must be 12×22 in length. Make a line of 1.5 cm from the bottom and away. On this line, with the distance of 2 cm, ten ‘X’ are marked.
- Now this paper must be given a cylindrical form which you will do by folding it and staple its ends. Make sure that they are stapled in a way that the two ends of meet never meet each other because then it helps in the faster movement of fluid.
- Then this cylindrically shaped chromatograph paper must be dried in an oven at the temperature of 100 degrees of more if want to it to be done quickly. 100 degrees is ideal, though.
- The solvent must be kept in that position for 1.5 hours at least since this is the ideal time. Shorter time period might not separate it very good.
- After some time, paper is removed from the oven let the solvent evaporate. When it is done, open the cylinder at the same place.
- Now remember that we mentioned that Ninhydrin solution. We will now spray it over the paper and let it dry for some time. Then again it is kept in the oven and this time between temperatures of 100 – 110 degrees.
- Now we can measure the distance traveled by each spot of the chemical by marking the ending point of each spot’s travel. You can determine the amino acids from the known spots and for the unknown spots, use the RF factor.
And your test is completed once you have followed all these steps.
So, this was the complete test for the determination of amino acids by paper Chromatography. Tell us by commenting your feedback below if you liked this article or not. You can also ask any question regarding the test. Keep visiting our website for more scientific tests.