Chromatography Techniques

Paper chromatography works only if the substances being investigated can dissolve in the chosen solvent. Water is one possible solvent, but other solvents may be needed because not every substance is soluble in water.

The two phases

Every chromatographic method has two phases. In paper chromatography, the paper is the stationary phase, which stays in place. The solvent is the mobile phase, which moves through the paper.

As the solvent rises through the paper, each component is continually distributed between the two phases. It may be carried along while it is in the mobile phase, but its movement is held back by its attraction to the stationary phase. A component that is more soluble in the solvent and less strongly attracted to the paper generally spends more time moving with the solvent, so it travels further. A component that is less soluble or more strongly attracted to the paper travels less far. These differences cause the components of a mixture to separate into spots.

Carrying out paper chromatography

Draw a straight origin line in pencil near the bottom of a strip of chromatography paper. Mark the sample positions on this line, then use a separate capillary tube for each sample to add a small, concentrated spot. Separate tubes prevent one sample contaminating another.

Put a shallow layer of the chosen solvent into a beaker. Suspend the paper vertically so that its lower edge touches the solvent but the origin line remains above the solvent level. If the samples are submerged, they may dissolve into the solvent at the bottom instead of travelling up the paper.

Allow the solvent to rise through the paper. Remove the paper before the solvent reaches the top and immediately mark the solvent front in pencil, because the solvent may evaporate. Let the chromatogram dry. Pencil is used for both lines because graphite is insoluble and will not travel with the solvent; pen ink might dissolve and form misleading spots.

Reading a chromatogram

A mixture can produce several spots because its components may travel different distances. If an unknown sample has a spot at the same height as a known reference run alongside it, this suggests that both contain the same substance. Comparing $R_f$ values makes this comparison more precise.

The comparison is valid only under the same conditions. Different compounds have different $R_f$ values in different solvents, so an unknown should be compared with reference substances using the same solvent and stationary phase. Changing the solvent can alter both the separation and the $R_f$ values.

A pure compound produces a single spot in all solvents. An impure substance or mixture may produce more than one spot, although two components can sometimes remain together in one solvent. Therefore, one spot from one chromatogram does not prove purity conclusively; trying another solvent may reveal additional components.

Calculating an Rf value

The retention factor is calculated using

Measure both distances from the origin line. For a substance, measure to the centre of its spot; for the solvent, measure to the marked solvent front. Use the same distance unit for both measurements. The units cancel, so $R_f$ has no unit.

For example, if the centre of a spot is $3.2\ \text{cm}$ above the origin and the solvent front is $8.0\ \text{cm}$ above it,

This means that the substance travelled $0.40$ of the distance travelled by the solvent. Because a spot should not move beyond the solvent front, an $R_f$ value normally lies from 0 to 1. Give calculated values to an appropriate number of significant figures; here, both measurements have two significant figures, so $0.40$ is appropriate.