Tlc

Thin Layer Chromatography

1. Advance techniques in biotechnology Ms. Sadhana Gupta

2. Practical protocol on thin layer chromatography

3. AIM : To Separate the given sample of amino acids by thin layer chromatography . REQUIREMENTS – Glass chamber , silica gel G ,pipettes or graduated cylinders , beakers , amino acid solution ,distilled water, ,stirring rod , tissue paper ,calcium carbonate, glass rod, tlc plate , , butanol, acetic acid, spray b ottles ,ninhydrin PRINCIPLE - Thin layer chromatography (TLC) depends on the separation principle. The separation relies on the relative affinity of compounds towards both the phases. The compounds in the mobile phase move over the surface of the stationary phase. The movement occurs in such a way that the compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. Therefore, the separation of the mixture  is attained. On completion of the separation process, the individual components from the mixture appear as spots at respective levels on the plates. Their character and nature are identified by suitable detection techniques

4. An overview of TLC

5. PROCEDURE - First step in tlc is sterilization of tlc plate in hot air oven. 2)Make a slurry of silica gel and calcium carbonate. 3)Coat the tlc plate with silica gel slurry and allow it to dry at room temperature for 10 minute 4)Activate the TLC plate in hot air oven for 1 hr. 5)Apply sample spots with the help of capillary tube. 6)Pour the mobile phase(solvent-butanol:aceticacid:water-12:3:5) into the TLC chamber and to maintain equal humidity, place a moistened filter paper in the mobile phase . 7)Place the tlc plate in glass chamber containing solvent.

6. 8)Allow the sample to run on gel plate. 9)As the solvent run ¾ th remove the plate and allow it to dry for few minutes. 10)With the help of a sprayer bottle, spray color producing reagent on gel plates. 11)Keep the plates in hot air oven for 5-10 minutes 12)Once the spots are developed, take out the plates and dry them. The sample spots can be observed under a UV light chamber. 13) Now calculate the RF value of samples. Calculation - Rf value = Distance travelled by solute Distance travelled by solvent

7. Steps of TLC

8. Rf VALUE OF SOME AMINO ACIDS

9. RESULT - The Rf value of samples were calculated successfully PRECAUTIONS- Care should be taken while handling reagents like Ninhydrin reagent. 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. Make certain that the spots applied to the plate are above the surface of the eluting solvent      

10. Before applying the second spot make sure that the previously applied spot is dried. Spot the components with proper space in between. Ensure that the chamber is saturated with the solvent vapour before you place the TLC plate in it. Give enough time for the solvent to advance up the plate. The top of the solvent must not advance up to or beyond the edge of the plates.