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Separation of Amino Acids by Thin Layer Chromatography

Flash content link

Animation: https://vlab.amrita.edu/repo/BIOTECH/BIC/Sep_of_Aminoacid_TLC/index.swf

Simulation: https://vlab.amrita.edu/repo/BIOTECH/BIC/Sep_of_Aminoacid_TLC/Seperation_of_aminoacids_by_TLC_sumulation.swf


Materials Required:




  1. 2% solution of individual amino acids.
  2. Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by volume.
  3. Ninhydrin reagent.





  1. TLC plate.
  2. TLC chamber.
  3. Capillary tubes.
  4. Reagent spray bottle.
  5. Conical flasks.
  6. Beakers.




  1. Pour the solvent mixture in to the TLC chamber and close the chamber.
  2. The chamber should not be disturbed for about 30 minutes so that the atmosphere in the jar becomes saturated with the solvent.
  3. Cut the plate to the correct size and using a pencil (never ever use a pen) gently draw a straight line across the plate approximately 2 cm from the bottom.
  4. Using a capillary tube, a minute drop of amino acid is spotted on the line.
  5. Allow the spot to dry.
  6. Spot the second amino acid on the plate [enough space should be provided between the spots].
  7. Repeat the above step for spotting the unknown acid.
  8. Place the plate in the TLC chamber as evenly as possible and lean it against the side(immerse the plate such that the line is above the solvent). Allow capillary action to draw the solvent up the plate until it is approximately 1 cm from the end.
  9. Remove the plate and immediately draw a pencil line across the solvent top.
  10. Under a hood dry the plate with the aid of a blow dryer.
  11. Spray the dry plate with ninhydrin reagent.
  12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the faded spots of amino acids and make them visible as purple coloured spots.]
  13. After some time, mark the center of the spots, then measure the distance of the center of the spots from the origin and calculate the Rf values.


Rf value can be calculated using the formula:



The Rf values with butanol-acetic acid- water solvent are as follows: alanine 0.24, glutamic acid 0.25, glycine 0.2, leucine 0.58, valine 0.4, lysine 0.58, tyrosine 0.42.


Differences Encountered In a Real Laboratory:

In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab.


  1. Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch on the exhaust fan and make sure that all the reagents required for the experiment are available. If it is not available, prepare the reagents using the components shown in the reagent preparation.
  2. Care should be taken while handling reagents like Ninhydrin reagent. This reagent is a strong oxidizing agent and should not be inhaled or spilled on hands or other body parts.  Accidental spill of this reagent will cause severe itching sensation. Wash the spilled area with cold water and inform the lab assistant immediately.
  3. Hold the TLC plates by their side. Ensure that you do not touch the developing part of the TLC plate, because your finger prints will also get developed causing the result to be unclear.
  4. Make certain that the spots applied to the plate are above the surface of the eluting solvent.
  5. Before applying the second spot make sure that the previously applied spot is dried.
  6. Spot the components with proper space in between.
  7. Ensure that the chamber is saturated with the solvent vapour before you place the TLC plate in it.
  8. Give enough time for the solvent to advance up the plate.
  9. The top of the solvent must not advance up to or beyond the edge of the plates.






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