Understanding chromatograms

A chromatogram is what you are left with after a paper chromatography run has finished and dried. To interpret it you need to find three things:

• the baseline — the pencil line where the spots started;
• the solvent front — the highest line the solvent reached before you took the paper out;
• the centre of each spot — the point you measure to.

Components that are more soluble in the solvent (and less attracted to the paper) travel further up the paper. So a spot near the top moved a long way; a spot near the baseline barely moved.

Counting components. The number of separate spots from a single mixture tells you the minimum number of substances in that mixture. One spot = a pure substance; two or more spots = a mixture.

Top tip: always measure from the baseline, never from the bottom edge of the paper. The bottom edge sat in the solvent and is not where the spots started.

The retardation factor (Rf) turns a chromatogram into a number you can compare.

$R_f = \dfrac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}}$

Both distances are measured from the baseline:

• distance moved by the spot = baseline → centre of the spot;
• distance moved by the solvent = baseline → solvent front.

Because the spot can never travel further than the solvent, Rf is always between 0 and 1, and because it is one distance divided by another distance, it has no units.

Worked numbers. If a spot's centre is 3.6 cm above the baseline and the solvent front is 4.8 cm above the baseline:

$R_f = \dfrac{3.6}{4.8} = 0.75$

A second example — spot 2.1 cm, solvent front 6.0 cm:

$R_f = \dfrac{2.1}{6.0} = 0.35$

Keep the answer tidy. Give Rf to 2 decimal places unless the question says otherwise, and never put a unit (no cm, no %) after it.

A given substance always travels the same fraction of the way up the paper in the same solvent at the same temperature, so it always has the same Rf. That makes Rf a kind of fingerprint you can use to identify an unknown.

Two ways to identify a spot:

1. Run references alongside. Put the unknown mixture and known reference substances on the same baseline. A component of the unknown that lines up level with a reference spot (same height = same Rf) is that substance.
2. Compare with a data table. Calculate the unknown's Rf and match it to a published Rf value — but only valid if the same solvent and temperature were used as in the table.

Worked identification. A dye in a food colouring travels 4.5 cm while the solvent travels 6.0 cm, so its Rf = 4.5 ÷ 6.0 = 0.75. A reference table (same solvent) lists: Sunset Yellow 0.55, Tartrazine 0.75, Carmine 0.40. The dye matches Tartrazine (Rf 0.75).

Why the conditions must match. Change the solvent and a substance's Rf changes, because its balance between dissolving in the solvent and sticking to the paper changes. So an Rf value only identifies a substance when the solvent and temperature are stated and identical.

Coloured substances (inks, food dyes, plant pigments) show up by themselves. But many important substances — most famously amino acids from a protein — are colourless, so the chromatogram looks blank.

To see them you develop the chromatogram:

• spray with a locating agent such as ninhydrin, then warm gently — amino acids appear as purple / pink spots; or
• view the dried paper under ultraviolet (UV) light, under which some substances fluoresce (glow).

Only after the spots are visible can you mark their centres and measure the distances to work out Rf.

Exam phrasing. If asked "the spots are colourless — how can you see them?", the mark is for named locating agent ninhydrin (or UV light), not just "use a chemical to colour them in".