Explain Procedure for Separation of Amino Acids by Paper Chromatography?
Now carry out the procedure following the steps given herewith:
1. Take a circular Whatman No 1 filter paper.
2. Take a Petri dish the diameter of which is slightly smaller than the diameter of the paper.
3. From the centre of the paper draw a circle of 2 cm diameter.
4. Mark the spots with a pencil for spotting on the circle.
5. With the help of a fine capillary tube spot the amino acid standards and unknown mixture on the marked spots. This process is called spotting. Allow the spot on the paper to dry completely.
6. Insert a cotton wick in the central hole of the filter paper.
7. Put some solvent in the Petri plate and place the paper on top of it ensuring that the wick dips into the solvent in the centre of the petridish. Cover the petridish.
8. Leave this arrangement for 3-3½ hours so that the solvent reaches nearly to the end of the circular paper.
9. Remove the lid and mark the solvent front with a lead pencil.
10. Dry the paper in air by clipping it on to a thread line.
11. For visualization of amino acid, spray ninhydrin over the paper and dry the paper again (for 10 minutes).
12. Areas where the amino acid has moved will get a yellow, pink or purple colour. Mark the areas with a pencil.
13. For identification of amino acids, estimate and measure the distance between the point of application of amino acid and the centre of the amino zone acid, as well as, the distance between the point of application and the solvent front.
14. Ratios of the two measurements give the Rf value of standard amino acid and that of an unknown amino acid to identify the amino acids.