Paper Chromatography: Overview

It is widely used for the separation and identification of compounds of biochemical interest.

It works because of differences in the polarity and solubility of amino acids.

They are often used for the initial screening of urine samples for drug metabolites because they are inexpensive and quick.

The differential partitioning of the samples (solutes) between a polar stationary phase and a nonpolar mobile phase.

By capillary action.

The organic components of the solvent have a lower affinity for the paper and so they move up faster. They form the mobile phase. The water in the solvent is strongly absorbed to the polar -OH groups of the paper and so is the stationary phase.

Solutes are applied near the bottom of the paper. The solvent moves up the paper and over the solutes. The individual solutes will be partitioned between the stationary and mobile phases according to their relative solubility in each phase. The more soluble the solute in the nonpolar, organic, mobile phase, the farther it will move.

The distance the solute travels is relative to the distance the solvent moves is called the Rf value.

Rf= distance traveled by the solute from the origin/distance traveled by the solvent from the origin.

The unknown is identified using the known samples. More specifically, identification of the unknowns is accomplished by a comparison of their Rf values and colors with those of the known reference amino acids.

Amino acids are polar due to their carboxyl and amino groups attached to the alpha-carbon. They can be seperated based on the polarity of their R-groups. Some of these R-groups are more soluble in the mobile phase because of their nonpolar R-groups, while others are insoluble due to charged or highly polar R-groups.

Amino acids are visualized by spraying the completely chromatogram with ninhydrin reagent.

Ninhydrin reacts with primary amino acids to give a purple color and with proline, a secondary amino acid, to give a yellow color.

Non-polar molecules will travel farther than polar molecules.

(1) Forgot to spray the paper with ninhydrin. (2) Forgot to dry the chromatography paper (3) Did not spot the amino acids well enough.