Every kind of chromatography works the same way: a mobile phase carries the mixture over a stationary phase, and each component moves at its own speed depending on the balance between how much it dissolves in the mobile phase and how strongly it is held by the stationary phase. Components that favour the mobile phase move faster and separate out.
Two phases, one balance
Separation always depends on the balance between solubility in the moving phase and retention by the stationary phase. A component that is more soluble in the mobile phase (or less strongly held by the stationary phase) travels further/faster. AQA covers three types:
| Type | Stationary phase | Mobile phase | Measured by |
|---|---|---|---|
| Thin-layer (TLC) | solid coating on a plate | solvent moving up the plate | Rf value |
| Column (CC) | solid packed in a column | solvent moving down the column | time / volume to elute |
| Gas (GC) | solid, or a solid coated with a liquid | an unreactive carrier gas | retention time |
🧪 Exam-style questions
In general terms, what determines the distance a spot travels in TLC?
Source: AQA A-Level Chemistry past papers.
Thin-layer chromatography & Rf values
In TLC, a plate is coated with a solid stationary phase; a spot of the mixture is placed on a pencil start line and the plate stands in a shallow layer of solvent (the mobile phase), which rises up the plate carrying the components different distances. Each component’s position is fixed by its own balance between dissolving in the solvent and sticking to the plate.
Rf = distance moved by the spot ÷ distance moved by the solvent front
Rf is always less than 1 (the spot can’t out-run the solvent) and, measured under the same conditions, is characteristic of a substance — so you identify components by comparing their Rf to known standards.
- Draw the start line in pencil (ink would run) and keep the solvent level below it.
- Put a lid on the beaker so the atmosphere is saturated with solvent vapour (stops the plate drying out and the solvent evaporating).
- Colourless spots (like amino acids) are made visible with a locating / developing agent — ninhydrin or UV light.
- If two components have the same Rf in one solvent, run two-way (2-D) TLC with a second solvent to separate them.
🧪 Exam-style questions
On a TLC plate, an amino acid spot moves 3.4 cm while the solvent front moves 10.0 cm from the start line. Calculate its Rf value, and state how a colourless amino acid is made visible.
Show answer
Rf = 3.4 ÷ 10.0 = 0.34. 1 mark
Spray with ninhydrin (or view under UV light). 1 mark
A protein is hydrolysed to a mixture of amino acids, spotted onto a TLC plate, and run in solvent 1; the plate is then dried, turned through 90° and run in solvent 2. Suggest why two different solvents are used.
Show answer
Some amino acids have the same Rf (do not separate) in the first solvent; a second solvent separates those that overlapped. 1 mark
Source: AQA A-Level Chemistry past papers.
Gas chromatography & GC-MS
In gas chromatography the mobile phase is an unreactive carrier gas and the stationary phase is a solid (or a solid coated with a liquid) packed in a long column held at high temperature. The mixture is vaporised and swept through; each component takes a characteristic retention time to pass through and reach the detector.
A component that is more soluble in the mobile phase (or less strongly retained by the stationary phase) passes through faster — a shorter retention time. One held more strongly by the stationary phase takes longer. You identify components by comparing retention times with standards run under the same conditions, and the size of each peak shows how much of that component is present.
GC-MS
Feeding the separated components straight into a mass spectrometer gives GC-MS: the GC separates the mixture and the MS identifies each component from its mass spectrum. Its limitation: a mass spectrometer cannot distinguish isomers, because isomers have the same molecular formula and therefore the same Mr / m/z.
🧪 Exam-style questions
A sample of cyclohexene is contaminated with cyclohexanol and separated by column chromatography (silica stationary phase, hexane mobile phase). Explain why cyclohexene has a shorter retention time than cyclohexanol.
Show answer
Cyclohexene is less polar than cyclohexanol. 1 mark
So cyclohexene has a greater affinity for the (non-polar) mobile phase / hexane (while cyclohexanol is held more strongly by the polar silica, e.g. by hydrogen bonding) — so cyclohexene moves through faster. 1 mark
A mixture of two isomeric dipeptides is analysed by GC-MS. Explain why the two can be separated by gas chromatography, but why mass spectrometry does not let you tell them apart.
Show answer
GC: the two have different retention times… 1 mark
…because they have a different balance between solubility in the mobile phase and retention by the stationary phase (different affinity for the phases). 1 mark
MS: they give the same m/z values… 1 mark
…because the isomers have the same molecular formula / Mr. 1 mark
Source: AQA A-Level Chemistry past papers.
Reading a chromatogram
Whatever the technique, the exam asks the same two things: separate (explain the balance of phases) and identify (compare Rf or retention time with standards). Keep the language precise — it is always the balance between solubility in the mobile phase and retention by the stationary phase.
- Measuring Rf from the wrong place — always from the start line, to the centre of the spot and to the solvent front.
- Explaining separation with only half the idea — you need both solubility in the mobile phase and retention by the stationary phase.
- Claiming MS can identify isomers — it can’t (same Mr); that is GC’s job.
- Ink start lines, no lid, or solvent above the start line — all ruin the chromatogram.
Examiner reports note that phase-balance answers lose marks for naming only one phase — state both the affinity for the mobile phase and the retention by the stationary phase for the mark.
- Principle: separation by the balance between solubility in the mobile phase and retention by the stationary phase.
- TLC: stationary = solid on a plate, mobile = solvent rising. Rf = distance moved by spot ÷ distance moved by solvent front (always < 1). Locate colourless spots with ninhydrin or UV.
- GC: mobile = carrier gas, stationary = solid or liquid-coated solid; components identified by retention time. More soluble in / less retained by the column → shorter retention time.
- GC-MS: GC separates, then the mass spectrometer identifies each component. Limitation: it cannot distinguish isomers (same Mr).
- Identifying: compare Rf values or retention times with those of known standards run under the same conditions.