2.9–2.11Chromatography
Chromatography can be used to separate mixtures, and can give information to help identify substances.
Chromatography involves a stationary phase and a mobile phase. Separation depends on the distribution of substances between the phases. In paper chromatography the paper is the stationary phase and the solvent is the mobile phase.
You should be able to explain how paper chromatography separates mixtures. The explanation: the solvent moves up the paper, carrying the substances with it. A substance that is more attracted to the solvent than to the paper spends more time in the mobile phase and travels further; one that is more attracted to the paper spends more time in the stationary phase and hardly moves. Because different substances have different distributions between the two phases, they end up at different heights.
The ratio of the distance moved by a compound — the centre of the spot from the origin — to the distance moved by the solvent can be expressed as its Rf value:
Rf = distance moved by substance ÷ distance moved by solvent
Different compounds have different Rf values in different solvents, which can be used to help identify the compounds.
The compounds in a mixture may separate into different spots depending on the solvent, but a pure compound will produce a single spot in all solvents. That last sentence is how chromatography is used to suggest whether a substance is pure — and why "one spot in this solvent" is weaker evidence than "one spot in every solvent tried".
Worked example
A spot's centre is 3.6 cm from the pencil line. The solvent front is 8.0 cm from the pencil line. Calculate the Rf value.
- Both distances are measured from the same pencil baseline.
- Rf = distance moved by substance ÷ distance moved by solvent = 3.6 ÷ 8.0.
- = 0.45.
- Rf has no units, because it is a ratio of two lengths, and it is always less than 1.
Answer: 0.45 — quoted to two significant figures, matching the precision of the measurements.
Required practical 6
Investigate how paper chromatography can be used to separate and tell the difference between coloured substances
- Draw a baseline on the chromatography paper in pencil, about 1 cm from the bottom. Pencil is used because ink would dissolve and run up with the samples.
- Put a small, concentrated spot of each sample on the baseline, letting each dry before adding more.
- Pour solvent into the container to a depth below the baseline, then stand the paper in it. If the solvent covers the baseline the samples dissolve into it instead of travelling up the paper.
- Put a lid on the container so the solvent does not evaporate, and leave it until the solvent front is near the top.
- Remove the paper, immediately mark the solvent front in pencil, and let it dry.
- Measure from the baseline to the solvent front, and to the centre of each spot, and calculate each Rf value.
You should be able to interpret chromatograms and determine Rf values from chromatograms, and provide answers to an appropriate number of significant figures. You should also be able to suggest how chromatographic methods can be used for distinguishing pure substances from impure substances.
Common mistake
Measure to the centre of the spot, not its top or bottom edge, and take both distances from the same pencil baseline. Measuring the solvent front from the bottom of the paper instead makes every Rf value too small.
- Chromatography
- a technique that separates mixtures using a stationary phase and a mobile phase, separation depending on the distribution of substances between the two
- Rf value
- the distance moved by a substance divided by the distance moved by the solvent, used to identify substances in chromatography
Before you practise
- The solvent is the mobile phase and the paper is the stationary phase; a substance moves further the more soluble it is and the less it is attracted to the paper.
- Start the pencil line above the solvent level, or the spots dissolve away into the tank.
- Use pencil, not pen — ink would separate too.
- A pure substance gives one spot; a mixture gives more than one.
- Rₑ = distance moved by the spot ÷ distance moved by the solvent, measured from the pencil line to the centre of the spot.
- Rₑ has no units and is always less than 1.
- Identify a substance by running a known sample beside the unknown in the same solvent — the Rₑ only holds for that solvent.