The paper stays still — it is the stationary phase. The solvent (ethanol here) soaks upward through the paper — it is the mobile phase. As the solvent sweeps past the ink spot it dissolves the dyes and carries them along. A dye that is very soluble in the solvent spends most of its time riding with the solvent, so it is carried a long way up. A dye that is less soluble, and clings more strongly to the paper, keeps being left behind and only travels a short way. Because each dye strikes its own balance, the mixture spreads out into separate spots — that is the separation.
Graphite is insoluble in the solvent, so a pencil line stays exactly where you drew it for the whole run. A pen line is itself made of ink — the ink would dissolve and travel up the paper along with your sample, smearing colour everywhere and making the chromatogram useless.
The solvent must reach the spot by creeping up the paper, not by touching it directly. If the spot began below the ethanol surface, the dyes would simply dissolve straight off the paper into the beaker, and there would be nothing left to separate. That is why the solvent is poured only about 1 cm deep and the start line sits safely above it.
Ethanol evaporates easily. The watch-glass lid keeps the air inside the beaker saturated with solvent vapour, so the solvent is not constantly evaporating off the strip as it climbs. Without the lid the run is slower, uneven, and the beaker slowly empties itself into the room.
The dried strip with its pattern of spots is called a chromatogram. Our "purple" ink turned out to be a mixture of a blue dye and a red dye. A pure substance gives a single spot; a mixture gives two or more. You can also identify an unknown by running it side by side with known substances — spots at the same height (same Rf) are very likely the same substance.
Rf = distance moved by the substance ÷ distance moved by the solvent
Measure both distances from the start line — the substance to the centre of its spot, the
solvent to the front you marked. Rf is always between 0 and 1 (a spot can never
overtake the solvent carrying it), and it is only fixed for a given
solvent at a given temperature — change either and the Rf changes.
That is exactly what this experiment demonstrates: the two strips ran for different times, so every distance on strip 2 is smaller — but divide each dye's distance by its own solvent front and the ratios come out identical. Mark the solvent front in pencil the moment the strip comes out: once the strip dries, the front is invisible and Rf can no longer be calculated.
Many substances (sugars, amino acids, drugs) separate perfectly well but cannot be seen. The chromatogram is then sprayed with a locating agent — a chemical that reacts with the spots to make them coloured — or viewed under ultraviolet (UV) light, which makes some substances glow.
| Term | Meaning |
|---|---|
| chromatogram | The finished paper with its pattern of separated spots. |
| solvent front | The furthest level the solvent reaches — mark it before the paper dries. |
| Rf value | Distance moved by substance ÷ distance moved by solvent (same solvent, same temperature). |
| locating agent | A chemical sprayed on to reveal colourless spots. |
| baseline / start line | The pencil line the sample starts on — pencil because graphite is insoluble. |