Thin layer chromatography

Thin layer chromatography

Aim

To perform thin layer chromatography on the given amino acids and to estimate their Rf values

Principle

Thin layer chromatography (TLC) is a chromatographic technique used to separate mixtures. Chromatography is the separation of two or more compounds or ions by the distribution between two phases, one which is moving and the other which is stationary.  These two phases can be solid-liquid, liquid-liquid or gas-liquid.

Thin-layer chromatography or TLC, is a solid-liquid form of chromatography where the stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or combination of solvents.  TLC is a quick, inexpensive technique that can be used to determine the number of components in a mixture, verify a substance’s identity, monitor the progress of a reaction, etc.

The stationary phase is a polar absorbent, usually finely ground alumina or silica particles.  This absorbent is coated on a glass slide or plastic sheet creating a thin layer of the particular stationary phase.  a single solvent or combination of solvents can be used as the mobile phase.  

TLC can also provide a chromatographic measurement known as an Rf value.  It is the distance travelled by the sample or analyte divided by distance travelled by the solvent front in chromatography. The Rf value is the “retardation factor” or the “ratio-to-front” value expressed as a decimal fraction.

Rf   = Distance travelled by the solute

Distance travelled by the solvent

Materials required

1. Thin layer of silica gel

2. Separation chamber

3. Solvent system –n-butanol: acetic acid: water (4:1:5)

4. Spraying agent: 1 gm of ninhydrin in 100 ml of acetone

5. Standard amino acid solution: 10 mg/ml glycine and tryptophan were prepared

6. Oven at 110^o C

Procedure

1. Clean and dry glass slides were taken and an aqueous slurry of silica gel G was poured on the surface of the slide and evenly spread over it.  The plates were allowed to dry.  The plates were allowed to dry.  They were then activated by heating at 110^oC for one hour in oven and allowed to cool.

2. The solvent was poured into the chromatographic chamber (glass beaker) and kept closed and undisturbed so that the chamber gets saturated with vapors of the solvent.

3. A line was drawn 1 cm from one end of the slide and the amino acids were spotted on this line at individual equidistant spots with separate capillary tubes.  Then the plates were kept inside the chromatographic chamber in such a way that the spots of amino acids were above the solvent system.  

4. The chamber was then closed and kept undisturbed for one – two hours.  Then the plate was taken out and solvent front was marked.  After the plate was dried, ninhydrin was sprayed and purple spots appeared in positions of amino acids.  It was then kept in oven at 110^o C for 5 minutes to fix the spots.  

5. The Rf value was calculated.

Observation

Aminoacid	Distance travelled by solute (cm)	Distance travelled by solvent (cm)	Rf Value
Glycine	-	-	-
Tryptophan	-	-	-

Result

Thin layer chromatography was performed on the given amino acids and the Rf value for glycine was found to be ……… and tryptophan was found to be ………….