Structural Biochemistry/Chromatography/Paper

Basic Concept of Paper Chromatography

Though this is a different kind of chromatography, it still separates mixtures of substances into the individual components, molecules, even atoms. The size and concentration of the component is determinant of the component's rate. The stationary phase, which is either a solid or a liquid supported by a solid, is absorbed in a uniform manner in paper chromatography. On the contrary, the mobile phase, being gas or liquid, serves as the solvent. Compounds can travel as far as the solvent does when the paper is dipped in a container filled with solvent. These compounds travel at different rates and separated into distinctly colored dots on the paper. The solvent that is used can be either nonpolar or polar. These properties affect the solubility of the compounds and components in the particular mixture. Polar components will be attracted to the water molecules attached to the cellulose (paper) and not attracted to a nonpolar solvent. The chromatogram will not contain the polar components, given that it doesn't climb up the paper with the nonpolar solvent. These components spend more time in the stationary phase rather than the mobile phase therefore the rate of moving up the paper is slow. If it were the opposite and nonpolar components were in a polar solvent, then the same thing will occur. The mobile phase can be various organic solvents or mixture. The compound can be stained with iodine in order to visualize where they have traveled easily. The stationary phase can be called a paper chromatogram. Usually, one will split the paper into individual lanes so that multiple trials can be done with one paper. Also it will allow the experimenter to compare the differences or similarities present in each lane depending on how far the compound has traveled.

The paper is placed in a container with a shallow layer of a suitable solvent or mixture of solvents in it. Sometimes the paper is just coiled into a loose cylinder and fastened with paper clips top and bottom. Then the cylinder stands in the bottom of the container. The container is covered to make sure that the atmosphere in the beaker is saturated with solvent vapor. Saturating the atmosphere in the beaker with vapor stops the solvent from evaporating as it rises up the paper. As the solvent slowly travels up the paper, the different components of the ink mixtures travel at different rates and the mixtures are separated into different colored spots.

The distance travelled relative to the solvent is called the Rf value. Its formula is: Rf = distance traveled by compound (a.k.a. the solute) / distance traveled by solvent. Thus, the higher the Rf value, the further the compound has traveled up the paper. The main benefit of the Rf value is that we can now compare values similar values and conclude that they are indeed the same compound

General Scheme

References

http://www.chemguide.co.uk/analysis/chromatography/paper.html