Chemistry 2.9 - 2.10 - Reading chromatograms

How paper chromatography separates

Black ink can look like one substance, yet a drop may contain water and several dissolved dyes. Paper chromatography separates those soluble dyes without making new substances. The finished pattern of separated spots is a chromatogram.

Two phases are involved:

• The mobile phase is the solvent that moves through the paper. Water is a suitable mobile phase for many washable inks.
• The paper contains the stationary phase, which stays fixed while the solvent moves.

At the visible, macroscopic level, the solvent rises through the paper, a spot of mixed ink spreads into separate spots, and different dyes finish at different heights. At the particle level, dye particles that dissolve in the moving solvent are carried upwards, while attractions to the stationary phase hold them back. Each dye has its own balance between these two effects.

A dye that is more soluble in the mobile phase and less strongly attracted to the stationary phase spends more time moving with the solvent, so it travels farther. A dye that is less soluble in the solvent or more strongly attracted to the stationary phase travels more slowly and finishes lower down. The separation is therefore caused by different movement rates, not by dyes reacting or simply by one dye being "heavier" than another.

The solvent must be able to dissolve the substances being separated. If a dye is insoluble in water, it may remain on the start line; strong retention can also keep a soluble dye near the baseline. If no useful separation occurs, a different suitable solvent would be needed to investigate it.

Calculate retention factors

Read each sample vertically above its labelled start position. In a successful run where the sample dissolves, one spot is consistent with a pure substance, while two or more separated spots show a mixture. One spot is not absolute proof of purity because components can overlap. A spot that stays on the start line does not provide useful separation evidence: the solvent may be unsuitable or the substance strongly retained.

An unknown may contain a known dye when their spots line up at the same height on the same chromatogram. This supports the match but cannot prove it uniquely. When chromatograms are run separately, compare $R_f$ values only if the solvent, paper and other conditions are the same. A matching colour alone is not enough, and even a matching $R_f$ is supporting evidence rather than unique proof of identity.

Measure both distances from the pencil start line. Use the centre of a broad spot rather than its leading edge. $R_f$ has no unit because the same distance unit cancels in the ratio. Its value lies from 0 to 1: a stationary spot has $R_f = 0$, while no spot can travel beyond the solvent front.