PAPER CHROMATOGRAPHY

Aim:

To separate and identify the amino acids in mixture by Paper chromatography

Introduction:

Chromatography is a physicochemical method for separation of complex mixtures and was discovered in 1903 by Russian-Italian botanist M. S. Tswett. It is the most powerful technique to separate and identify the substances in to the individual from the mixture based on their physicochemical properties. The compounds are separated on the basis of their partition coefficient between two immiscible phases.  The stationary phase is may be a solid or liquid, mobile phase is may be a solid, liquid or gas.

In paper chromatography the solutes applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. Components of the mixture are carried along with the solvent up the paper to varying degrees, depending on the compound's preference to be adsorbed onto the paper versus being carried along with the solvent. The paper is composed of cellulose to which polar water molecules are adsorbed, while the solvent is less polar, usually consisting of a mixture of water and an organic liquid. The paper is called the stationary phase while the solvent is referred to as the mobile phase. Performing a chromatographic experiment is basically a three-step process: 1) application of the sample, 2) "developing" the chromatogram by allowing the mobile phase to move up the paper, and 3) calculating Rf values and making conclusions. In order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, we can calculate an "Rf value" for each separated component in the developed chromatogram. An Rf value is a number that is defined as:

Materials Required:

Amino acid solution (2% each)

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

Ninhydrin reagent.

Chromatography chamber

Micro syringe or capillary tube

Ninhydrin sprayer/ Atomiser

Procedure:

1. Whatman filter paper was cut into a rectangular piece of desired measurement.

2. A line of origin was marked approximately 2 cm from the bottom.

3. The given amino acid samples were placed as spots on the line of origin.

4. The paper was placed in a tank/chamber containing the solvent n butanol, acetic acid and  

     water.

5. The setup was left undisturbed for capillary action to draw the solvent up the paper until it is   

    approximately 1 cm from   the end.

5. After this process removed the paper and immediately drawn a pencil line across the solvent   

    top and allowed  the paper to dry.

6. Sprayed the locating reagent ninhydrin and dried the paper in oven at 105°C for 5 min.

7. Spots were located and the Rf value was determined using the formula.

Result:

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