Paper chromatography

Paper chromatography

Aim

To perform paper chromatography on the given aminoacid and to determine its Rf value.

Principle

Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.  They all have a stationary phase (a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates and gets separated.  In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.

Paper chromatography is specially used for separation of mixture having polar and non polar compounds, for separation of amino acids, to determine organic compounds, biochemicals in urine etc., for determination of hormones, drugs, for evaluation of inorganic compounds like salts and complexes, etc.

Based on the way the development of chromatogram on the paper is done there are 5 types of chromatography.  

1. Ascending chromatography: As the name indicates, the chromatogram ascends. Here the development of paper occurs due the solvent movement in upward direction on the paper.  The solvent reservoir will be at the bottom of beaker.

2. Descending chromatography: Here the development of paper occurs due to solvent travel downwards on the paper.  The solvent reservoir will be at the top. The movement of solvent is assisted by gravity besides capillary action.

3. Ascending- descending mode: Here solvent first travels upwards and then down wards on the paper.

4. Radial mode: Here the solvent travels from center towards periphery of circular chromatography paper. The entire system is kept in a covered petridish for development of chromatogram.  

5. Two dimensional chromatography: Here the chromatogram development occurs in two directions at right angles. In this mode the samples are spotted to one corner of rectangular paper and allowed for first development. Then the paper is again immersed in mobile phase at right angle to previous development for second chromatogram.

The distance travelled by the solute relative to the solvent is called the Rf value. For each compound it can be worked out using the formula

Rf   = Distance travelled by the solute

Distance travelled by the solvent

Materials required

1. Separation chamber

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

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

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

Procedure

1. Strips of whatmann No.1 filter paper was taken (7X2 cm).  A line was drawn 1 cm away from one end of the paper and amino acid solution was spotted separately using capillary tube on to this line.  

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. Then the paper containing aminoacids were then kept inside the chromatographic chamber in such a way that the spots of amino acids were above the solvent system.  

4. The chamber is then closed and kept undisturbed for one – two hours.  Then the paper was taken out and solvent front was marked.  

5. After the paper was dried at 62-72^oC, ninhydrin was sprayed and kept at 105^oC for 5 minutes to visualize the spots.  Purple spots appeared in positions of amino acids.  

6. The Rf value was calculated.

Observation

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

Result

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