THIN LAYER CHROMATOGRAPHY

Aim:

To separate and identify the amino acids in mixture by thin layer chromatography

Introduction:

Thin layer chromatography is a simple, quick and inexpensive technique to identify the compound of interest in a mixture. The first reported use of a thin layer chromatography was in 1938 by two Russian scientists, N.A. Izmailov and M.S. Schreiber. This technique is highly useful in research laboratories to separate, identify and characterize the unknown compound. A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for support. The silica gel acts as the stationary phase and the solvent mixture acts as the mobile phase. In the ideal solvent system the compounds of interest are soluble to different degrees. Separation results from the partition equilibrium of the components in the mixture.

In the simplest form of the technique, a narrow zone or spot of the sample mixture to be separated is applied near one end of the TLC plate and allowed to dry. The strip or plate is then placed with this end dipping in to the solvent mixture, taking care that the sample spot/zone is not immersed in the solvent. As the solvent moves towards the other end of the strip, the test mixture separates into various components. This is called as the development of TLC plates. The separation depends on several factors; (a) solubility: the more soluble a compound is in a solvent, the faster it will move up the plate. (b) attractions between the compound and the silica, the more the compound interacts with silica, the lesser it moves, (c) size of the compound, the larger the compound the slower it moves up the plate. 

The plate is removed after an optimal development time and dried and the spots/zones are detected using a suitable location reagent. An important characteristic used in thin layer chromatography is Rf value.

Materials Required:

Reagents:

Individual amino acid solution (2% each)

Solvent mixture of normal butanol, acetic acid and water in the ratio 4:1:5 by volume.

Ninhydrin reagent.

Requirements:

TLC plate.

TLC chamber.

Capillary tubes.

Reagent spray bottle.

Conical flasks.

Beakers.

Procedure :

1. A uniform layer of silica gel was applied on clean glass plate/slide with the help of an

     applicator (spreader).  Thickness of layer 0.25mm was made.

2. TLC plates were activated by heating in an oven at 100-150 C for about half an hour.

3. Poured the solvent mixture in to the TLC chamber and closed the chamber.

4. The chamber was not disturbed for about 30 minutes so that the atmosphere in the jar became    

     saturated with the solvent.

5. By pencil gently drawn a straight line across the plate approximately 2 cm from the bottom.

6. Using a capillary tube, a minute drop of amino acid was spotted on the line.   Allowed the spot

    to dry.

7. Spotted the second amino acid on the plate [enough space provided between the spots].

8. Repeated the above step for spotting the unknown acid.

9. The plate was placed in the TLC chamber (immersed the plate such that the line is above the

     Solvent).

10. Allowed capillary action to draw the solvent up the plate until it is approximately 1 cm from   

      the end.

11. Removed the plate and immediately drawn a pencil line across the solvent top and allowed  

      the plate to dry

12. Sprayed the dry plate with ninhydrin reagent.

14. Dried the plates in hot air oven at 105°C for 5 min. [Ninhydrin  react with the faded spots

      of amino acids and made them visible as purple coloured spots.]

15. Then marked the center of the spots on the plates, measured the distance of the center of the

      spots from the origin and calculate the Rf values.

Result:

The given amino acids were identified as ______   based on Rf value and results were tabulated.

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

Ref:  vlab.amrita.edu,. (2011). Separation of Amino Acids by Thin Layer Chromatography.