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Paper chromatography is a procedure used to separate substances in a mixture. In a biology lab, this is usually a solution of liquid plant pigments :
these contain different kinds of chlorophylls and other colored photosynthetic pigments.

* A small, concentrated sample of a mixture is placed on the chromatography paper – above the line of a solvent mixture.

* Paper is then placed in contact with a solvent solution, at its bottom.

* Solvent is drawn up through the paper due to capillary action.

* As solvent hits the spot, it dissolves many of the pigments.

* Some pigments have a greater attraction for the chromatography paper, so they move a lesser distance.

* Some have a lesser attraction, so they move a greater distance.

Experimental setup

Example of a result


Another example, showing how the different pigments each moved a different distance.

The sample will separate into consistent patterns – as long as the solvent, paper, and time it runs, stay the same.

(Time matters because the longer the amount of time that the experiment runs, the further the pigments will move.)

Different solvents will change the separation pattern of the mixture.

The retardation factor, Rf value, of a pigment is a statistic used to identify how a pigment behaves.

Each pigment has it’s own specific Rf (for the same solvent, same time)

Calculation of Rf

Retardation factor (Rf) is defined as the ratio of the distance traveled by the center of a spot, to the distance traveled by the solvent front


Calculating Rf chromatography

Movement depends on the polarity of the molecules

from Bio C2005/F2401x 2010 Lec.5  Lawrence Chasin, September 221, 2010

How can we separate a solution of amino acids? Paper chromatography does this based on the differential solubility of the different amino acids in organic (non-polar) solvents.

This solubility is determined by the nature of the amino acid’s side group.

The amino acid mixture is spotted onto a filter paper; one edge of the paper is immersed in a mixture of aqueous and non-aqueous solvents.

The liquid will be drawn up the paper by capillary action.

As it rises, the water in the liquid mixture is bound by the paper (cellulose, with its many OH groups), forming a stationary water layer, or stationary phase.

The organic solvent (e.g., propanol) moves up without as much interaction with the solid cellulose; it is considered the mobile phase.

The amino acids will be constantly equilibrating between being in the mobile organic phase or the stationary water phase.

The more polar the side chain, the more time the amino acid will spend in the stationary phase.

The more hydrophobic the side chain, the more time it spend in the mobile organic phase.

By using a series of different solvents, all 20 amino acids can be separated in this way. It works for many other organic molecules as well.

The distance that an organic molecule moves in a particular chromatographic system is called the Rf, which stands for mobility Relative to the Front:

that is, the distance the organic molecule in question migrated divided by the distance that the front of liquid has risen on the paper at that time.

Rf’s in a particular solvent and at a given temperature are reproducible and are published for many organic compounds, including all the amino acids



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