What is chromatography?
Chromatography is a way to separate the parts of a mixture. The word means "colour writing", because the first experiments separated coloured plant pigments.
Every kind of chromatography has two parts:
- Stationary phase: the part that does not move. Example: paper, a thin layer of silica on a plate, or powder packed in a tube.
- Mobile phase: the part that moves. Example: a liquid solvent (like water or ethanol) or a gas (like helium).
Each substance in the mixture is pulled two ways. The mobile phase carries it forward. The stationary phase holds it back. A substance that sticks more to the stationary phase (or dissolves less in the mobile phase) moves more slowly. So different substances end up in different places. That is the whole idea.
Two ways of sticking: adsorption (the substance clings to the surface of a solid, as in TLC and column) and partition (the substance shares itself between two liquids, as in paper chromatography, where water is held in the paper fibres).
Paper chromatography and the Rf value
- Draw a base line in pencil (ink would run) about 2 cm from the bottom.
- Put a small, concentrated spot of the mixture on the line.
- Dip the paper in solvent. The solvent level must be below the base line, or the spot dissolves into the pool.
- Cover the tank so the solvent does not evaporate. Let the solvent climb.
- Take the paper out before the solvent reaches the top. Mark the solvent front at once.
The Rf value
Rf = distance moved by the spot Γ· distance moved by the solvent front, both measured from the base line (to the centre of the spot).
Rf has no unit and is always between 0 and 1. For one substance, with the same solvent, paper and temperature, Rf is always the same. So we can identify an unknown by matching its Rf with a known substance run on the same paper.
A mixture gives several spots. A pure substance gives one spot. A substance that does not dissolve in the solvent stays on the base line (Rf = 0).
Two-way chromatography
If two spots overlap, turn the paper 90Β° and run it again in a second solvent. The spots now spread out in two directions.
Thin-layer chromatography (TLC)
In TLC the stationary phase is a thin layer of silica gel (SiOβ) or alumina on a glass, plastic or aluminium plate. The mobile phase is an organic solvent.
- TLC is faster than paper and gives sharper spots, so it separates better.
- Silica is polar. Polar substances stick to it more and have low Rf. Non-polar ones move further.
- A more polar solvent pulls everything further up, so all Rf values go up.
Seeing colourless spots
Many organic compounds have no colour. We use a locating agent:
- UV light: plates with a fluorescent dye glow, and the spots show as dark or glowing patches.
- Iodine vapour: spots turn brown.
- Ninhydrin spray: amino acids turn purple.
Checking purity and following a reaction
Run the product beside a pure sample. Same single spot at the same Rf = pure. Extra spots = impurities or left-over starting material.
Column chromatography
A glass tube is packed with silica gel or alumina (stationary phase). The mixture is added at the top. Solvent, called the eluent, is poured in and flows down.
The parts of the mixture move down as separate coloured bands. The part that sticks least moves fastest and comes out (elutes) first. We collect each band in a separate flask, then evaporate the solvent.
Column chromatography is used to collect larger amounts of pure substance, not just to look at them. Chemists often run a quick TLC first to choose a good solvent for the column.
Gas chromatography (GC) and retention time
In GC the mobile phase is an unreactive carrier gas (helium or nitrogen). The stationary phase is a thin liquid coating (or a solid) inside a very long, thin, coiled column kept in an oven. The sample must turn into a gas.
Each substance takes its own time to pass through. The retention time is the time from injecting the sample to the moment the substance reaches the detector. It depends on:
- how much it sticks to (or dissolves in) the stationary phase,
- its boiling point (more volatile = comes out sooner),
- the column temperature and gas flow rate.
The detector draws a chromatogram: one peak for each substance. The position of a peak (retention time) tells what it is, when compared with known substances under the same conditions. The area under a peak tells how much is present. GC is often joined to a mass spectrometer (GC-MS) to identify each peak with certainty.
HPLC
High-performance liquid chromatography pushes a liquid mobile phase through a tightly packed column at high pressure. It also gives retention times and peaks, and works for substances that would break down if heated.
Try it at home
Felt-pen chromatography: pencil base line, ink dot, 1 cm of water in a glass, wait 10 minutes, mark the front, measure and work out Rf for each colour. Do it with two black pens from different brands: do they contain the same dyes? Then predict: if you use salt water or vinegar instead, will the Rf values change? Check in the free-play step of the 3D.
Key formulas and definitions
- Rf = distance moved by spot Γ· distance moved by solvent front
- 0 β€ Rf β€ 1 (no unit)
- Distance of spot = Rf Γ distance of solvent front
- Retention time = time from injection to the peak at the detector
- Amount of a substance β area under its peak (GC/HPLC)
- Sticks more to stationary phase β lower Rf, later elution, longer retention time
Worked examples
1. The solvent front moved 8.0 cm. A blue spot moved 2.0 cm. Find its Rf.
Line 1: Rf = spot distance Γ· solvent distance. Line 2: Rf = 2.0 Γ· 8.0. Line 3: Rf = 0.25.
2. A dye has Rf = 0.60. The solvent front moved 9.5 cm. How far did the dye move?
Line 1: spot distance = Rf Γ solvent distance. Line 2: = 0.60 Γ 9.5. Line 3: = 5.7 cm.
3. An unknown spot moved 3.3 cm when the solvent moved 6.0 cm. Known Rf values in this solvent: caffeine 0.30, aspirin 0.55, paracetamol 0.75. Which is it?
Line 1: Rf = 3.3 Γ· 6.0 = 0.55. Line 2: Compare with the list: aspirin has Rf 0.55. Line 3: The unknown is most likely aspirin (confirm by running it beside pure aspirin on the same plate).
4. On a silica TLC plate, compound A is very polar and compound B is non-polar. Which has the bigger Rf?
Line 1: Silica is polar, so polar substances stick to it more. Line 2: A sticks more, so it moves less. Line 3: B has the bigger Rf.
5. A GC chromatogram shows peaks at 2.1 min (ethanol) and 5.0 min (an unknown). Which leaves the column first, and why?
Line 1: Retention time = time to reach the detector. Line 2: Ethanol, 2.1 min, arrives first. Line 3: It interacts less with the stationary phase and/or is more volatile.
6. In a GC trace, peak X has area 300 units and peak Y has area 100 units. The detector responds equally to both. What percentage of the mixture is X?
Line 1: Total area = 300 + 100 = 400. Line 2: % of X = 300 Γ· 400 Γ 100. Line 3: = 75%.
Common mistakes
- Measuring from the bottom edge of the paper instead of from the base line. Both distances start at the base line.
- Drawing the base line in ink. The ink dissolves and runs, giving extra spots. Use pencil.
- Putting the solvent above the base line. The spot washes into the solvent instead of climbing.
- Thinking that the substance which sticks most comes out of a column first. It is the opposite: least stuck comes out first.