Rf values depend on temperature, quality of the paper used and the composition of the solvent used.
For these reasons, it is usual to run samples of known substances at the same time as you run the sample of the unknown mixture.
Animated Tutorial : Ascending Paper Chromatography Technique#
Ascending Paper Chromatography Technique
Application of the Sample
A starting point is marked on the paper using pencil. (Do not use a pen containing an ink that will be soluble in the mobile solvent phase)
A spotter, such as a thin capillary tube, is used to place a very small spot of the solution mixture on the paper.
The spot is dried.
The paper is placed in a closed vessel containing a small amount of the mobile phase solvent so that one end of the paper is under the surface of the liquid and so that the starting point marked on the paper is above the solvent.
The mobile phase solvent will rise slowly up the paper by capillary action.
The components of the mixture to be separated will also rise up the paper, but at a slower rate than the mobile phase.
Components with low solubility in the stationary phase and high solubility in the mobile solvent phase move up the paper quickly.
Components with high solubility in the stationary phase and low solubility in the mobile solvent phase move slowly up the paper.
When the solvent front (the level of the mobile solvent phase) has almost reached the top of the paper, the position of the solvent front is marked and the paper set aside to dry.
If the components are different colours, you can see the position of each component on the paper.
If the components are colourless, there are a number of techniques used to detect each component:
reactions with colour producing reagents, eg, spraying colourless amino acid spots with ninhydrin produces visible purple spots
placing the sample under ultra violet light (if the sample fluoresces or absorbs ultra violet light)
Calculating the Retardation Factor, Rf
A sample containing a mixture of amino acids was separated using the ascending paper chromatography technique described above.
The results of the experiment are shown below.
Calculate the retardation factor, for each of the amino acids in the mixture.
Measure the distances travelled by the solvent and each component
Calculate the retardation factor, Rf, for each component.
Describing Relative Solubility The further up the paper the sample spot moves, the more soluble it is in the mobile solvent phase and the less soluble it is the stationary phase.
more soluble in mobile solvent phase
least soluble in stationary phase
least soluble in mobile solvent phase
most soluble in stationary phase
*The paper can be impregnated with different solvents, eg, silicone oil, for the stationary phase.
#Other paper chromatography techniques include descending (mobile phase descends down the paper under gravity) and radial horizontal (mobile phase is continuously added to the centre of a horizontal, circular piece of paper).