AQA A-Level Chemistry: Chromatography (7405)

This guide teaches section 3.3.16 Chromatography of the AQA AS and A-level Chemistry specification (7404/7405), version 1.2, July 2026, for AS and A-level exams from June 2016 onwards. The whole section is marked “A-level only”, so it is not AS content. It sits in the organic chemistry section, which the specification lists under A-level Paper 2, and Paper 3 can assess any content and any practical skill. It includes Required practical 12, separation of species by thin-layer chromatography.

For quick recall, use the revision notes. To test yourself, use the practice questions. The course hub is AQA A-Level Chemistry, the printable checklist lists every specification point, and the free 10-minute diagnostics show where your gaps are.

What this unit covers

Spec point (3.3.16)	What you must be able to do	Status
Purpose	Explain that chromatography separates and identifies the components of a mixture	A-level only
Thin-layer chromatography (TLC)	Describe it: a plate coated with a solid, with a solvent moving up the plate	A-level only
Column chromatography (CC)	Describe it: a column packed with a solid, with a solvent moving down the column	A-level only
Gas chromatography (GC)	Describe it: a column packed with a solid, or a solid coated by a liquid, with a gas passed through under pressure at high temperature	A-level only
How separation happens	Explain separation as a balance between solubility in the moving phase and retention by the stationary phase	A-level only
Rf values and retention times	Calculate Rf values from a chromatogram; compare Rf values and retention times with standards to identify substances	A-level only
GC with mass spectrometry	Explain how mass spectrometry analyses the components separated by GC	A-level only
Required practical 12	Separate species by thin-layer chromatography (AT a, i and k)	A-level only

The specification suggests using TLC to identify analgesics, or to identify transition metal ions in a solution.

How chromatography separates a mixture

Every type of chromatography has two phases. The stationary phase stays in place: the solid on a TLC plate, the solid packed in a column, or the solid (or liquid-coated solid) in a GC column. The moving phase flows past it: a liquid solvent in TLC and CC, and a gas in GC. (Many books call it the mobile phase.)

Each component is shared between the two phases. In the specification’s words, separation depends on the balance between solubility in the moving phase and retention by the stationary phase.

• A component that is very soluble in the moving phase and weakly held by the stationary phase spends most of its time moving. It travels far (TLC), leaves the column first (CC) or has a short retention time (GC).
• A component that is strongly held by the stationary phase travels a short distance or comes out later.

Two components separate only if their balances differ. If they are the same, you see one spot or one peak.

Worked example 1: predicting the order of separation

A mixture is run on a TLC plate whose stationary phase is polar. The solvent is much less polar. Compound J can form hydrogen bonds; compound K cannot and is only weakly polar. Which has the larger Rf value?

1. The polar stationary phase interacts more strongly with J, because J can hydrogen bond.
2. So J is retained more strongly and spends less time in the moving phase.
3. K is held only weakly and dissolves well in the less polar solvent.
4. K moves further up the plate, so K has the larger Rf value.

The answer depends on both phases. With a much more polar solvent, J would dissolve better in the moving phase and its Rf value would rise. Always explain using both phases.

Thin-layer chromatography (TLC)

In TLC, a plate is coated with a thin layer of solid, the stationary phase. Small spots of each sample go on a pencil line near the bottom (the baseline), and the plate stands in a shallow layer of solvent in a covered container. The solvent rises up the plate. Near the top, you remove the plate and mark the solvent front at once.

Coloured substances, such as many transition metal complexes, show as visible spots. Colourless ones must be located: the specification names ultraviolet light and developing agents such as ninhydrin for amino acids (section 3.3.13.2). The apparatus list (AT i) allows thin-layer or paper chromatography; the Rf method is the same for both.

Calculating Rf values

Rf = distance moved by the spot from the baseline
     ---------------------------------------------
     distance moved by the solvent front from the baseline

• Measure both distances from the baseline, and measure to the centre of the spot.
• Rf has no units and lies between 0 and 1.
• An Rf value only applies to one stationary phase, one solvent and one set of conditions.

Worked example 2: calculating Rf values

The solvent front is 7.20 cm above the baseline. Two spots have centres 1.80 cm and 4.68 cm above it.

Spot 1: Rf = 1.80 / 7.20 = 0.25
Spot 2: Rf = 4.68 / 7.20 = 0.65

The Rf values are 0.25 and 0.65. The second component is less strongly retained (or more soluble in the solvent) than the first.

Worked example 3: identifying substances with standards

A crushed analgesic tablet is dissolved and spotted on a TLC plate. Beside it, on the same baseline, are three pure standards, A, B and C. The solvent front ends 6.40 cm above the baseline.

Spot	Distance from baseline / cm	Rf
Standard A	1.92	0.30
Standard B	3.52	0.55
Standard C	4.80	0.75
Tablet, spot 1	1.92	0.30
Tablet, spot 2	4.80	0.75

1. Divide each distance by 6.40 cm (shown in the table).
2. Tablet spot 1 matches A; tablet spot 2 matches C; nothing matches B.
3. Conclusion: the tablet contains A and C, and no B was detected.

Standards go on the same plate because Rf values change with the solvent, the stationary phase and the conditions; side by side, sample and standard get identical treatment. A match is good evidence, not proof: two substances can share an Rf value in one solvent. Repeating in a different solvent is a further test.

Column chromatography (CC)

A vertical column is packed with a solid, the stationary phase. The mixture is added at the top and solvent is run down through the column. Weakly retained components that are very soluble in the solvent move down fastest and leave the bottom first; strongly retained ones follow later. Because the components leave one after another, you can collect them in separate containers (fractions) and obtain each one separately.

For J and K from worked example 1, on a column of the same polar solid with the same solvent, K leaves first. The reasoning is the same as for TLC; only the direction of solvent flow differs.

Gas chromatography (GC)

The column is packed with a solid, or with a solid coated by a liquid. A gas, the moving phase, is passed through under pressure at high temperature. The sample is injected, vaporises and is carried through. A detector at the end records each component as it emerges, giving a peak.

The retention time is the time from injection until a component reaches the detector. Strongly retained components have long retention times. Retention times depend on the column, its temperature and the gas flow, so you compare them with standards run under the same conditions. Raising the column temperature makes components spend more time in the gas, so retention times get shorter.

Worked example 4: identifying components by retention time

Standards run on the same column, temperature and gas flow give retention times of 3.2 min (D), 4.5 min (E) and 7.8 min (F). A sample gives peaks at 3.2 min and 7.8 min only.

The peaks match D and F, so the sample contains D and F; no E is detected. Two substances can share a retention time on one column, so a match is strong evidence, not certainty. That is one reason GC is combined with mass spectrometry.

Mass spectrometry of the components separated by GC

In GC-MS, the gas leaving the column passes into a mass spectrometer. GC has already separated the mixture, so the components arrive one at a time and each gives its own mass spectrum. The molecular ion gives the relative molecular mass, and a high-resolution m/z value fixes the molecular formula (section 3.3.6.2).

How a mass spectrometer works is in the atomic structure guide; precise-mass calculations are taught in the organic analysis guide.

Worked example 5: GC-MS

One GC peak gives a molecular ion at m/z = 100.0635. Which of C₆H₁₂O, C₅H₈O₂, C₄H₈N₂O and C₅H₁₂N₂ is it? Use H = 1.0078, C = 12.0000, N = 14.0031 and O = 15.9949.

C6H12O  : 6(12.0000) + 12(1.0078) + 15.9949             = 100.0885
C5H8O2  : 5(12.0000) + 8(1.0078) + 2(15.9949)           = 100.0522
C4H8N2O : 4(12.0000) + 8(1.0078) + 2(14.0031) + 15.9949 = 100.0635
C5H12N2 : 5(12.0000) + 12(1.0078) + 2(14.0031)          = 100.0998

Only C₄H₈N₂O matches. GC separated this component; mass spectrometry identified its formula.

Required practical 12: separation of species by TLC

The specification lists AT a (recording measurements), AT i (thin-layer or paper chromatography) and AT k (safe handling of chemicals). A typical method:

1. Wear gloves and handle the plate by its edges.
2. Draw a pencil baseline about 1 cm from the bottom. Pencil does not dissolve in the solvent; ink would dissolve and separate.
3. With a fine capillary tube, put small, concentrated spots of each sample and standard on the baseline, spaced apart. Let them dry.
4. Add solvent to a depth below the baseline, so the samples do not dissolve into the solvent pool.
5. Stand the plate in the container and add a lid, which keeps the air saturated with solvent vapour so solvent does not evaporate from the plate.
6. Near the top, remove the plate and mark the solvent front in pencil straight away.
7. Dry the plate, then locate colourless spots (for example under ultraviolet light).
8. Measure from the baseline to each spot centre and to the solvent front. Calculate Rf values and compare with the standards.

Safety (AT k): check the hazards of the solvent and any developing agent first. Organic solvents are often flammable, so keep them away from flames, and use a fume cupboard if vapours are harmful. Do not look directly at an ultraviolet lamp.

Common errors

• Measuring from the bottom edge of the plate, or to the top of a spot, instead of baseline to spot centre.
• Writing Rf with units, or getting a value above 1 by dividing the wrong way round.
• Comparing an Rf value with one from a different solvent or plate.
• Explaining separation with one phase only, or by “how heavy” a substance is.
• Stating that GC uses a liquid moving phase.
• Saying a single matching Rf value or retention time “proves” identity.

Where next

Condense this with the revision notes, then try the practice questions. The transition metal ion tests in reactions of ions in aqueous solution pair with the suggestion of using TLC on transition metal ions.

Official syllabus

AQA AS and A-level Chemistry specification (7404/7405), version 1.2, July 2026, for AS and A-level exams June 2016 onwards (AQA). Section 3.3.16 Chromatography (A-level only), including Required practical 12.