OxfordAQA A-Level Chemistry: Chromatography (9620)

This guide covers 3.3.16 Chromatography (subsection 3.3.16.1) in the OxfordAQA International AS and A-level Chemistry (9620) specification, Version 5.3 (International AS and A-level exams from May/June 2020 onwards). Chromatography belongs to the International A2 content, so everything on this page is International A-level only. In the scheme of assessment it falls within the organic content of Unit 4, and Unit 5, the practical and synoptic unit, covers all content. This section has no required practical.

Afterwards, the revision notes condense the topic and the practice questions check your understanding. The course hub gathers every topic, the printable checklist sets out each specification point, and a quick diagnostic tells you which topics to start with.

What this unit covers

Section	You should be able to	Status
3.3.16.1 Purpose	Explain that chromatography can separate and identify the components in a mixture	International A-level only
3.3.16.1 TLC	Describe thin-layer chromatography: a solid coating on a plate, and a solvent that rises up the plate	International A-level only
3.3.16.1 CC	Describe column chromatography: solid packed into a column, with solvent flowing down through it	International A-level only
3.3.16.1 GC	Describe gas chromatography: a column of packed solid (which may carry a liquid coating), with a gas forced through under pressure at high temperature	International A-level only
3.3.16.1 Separation	Explain separation as a balance between a component’s solubility in the moving phase and its retention by the stationary phase	International A-level only
3.3.16.1 Rf values	Calculate Rf values from a chromatogram	International A-level only
3.3.16.1 Identification	Compare retention times and Rf values with standards to identify different substances	International A-level only
3.3.16.1 GC-MS	Explain the use of mass spectrometry to analyse the components separated by GC	International A-level only

Two other sections feed into this one: 3.3.13.2 (amino acids separated by TLC, located with ninhydrin or ultraviolet light, identified by Rf values; see amino acids and proteins) and 3.3.6.2 (molecular formulae from precise masses).

The two phases and why components separate

Every chromatography method has a stationary phase, which stays put, and a moving phase, which flows over or through it.

Method	Stationary phase	Moving phase
Thin-layer (TLC)	A solid coated on a plate	A solvent, moving up the plate
Column (CC)	A solid packed in a column	A solvent, moving down the column
Gas (GC)	A packed solid in a column, which may carry a liquid coating	A gas, under pressure at high temperature

As the moving phase passes, each component keeps moving between the two phases. The key idea in the specification: separation depends on the balance between a component’s solubility in the moving phase and its retention by the stationary phase.

Strong retention holds a molecule back; high solubility in the moving phase carries it forward. A component whose balance favours the moving phase travels further up a TLC plate, comes out of a column sooner, or reaches a GC detector sooner. Two components separate only when their balances are different. Change either phase and you change the balance, which is why the same pair of substances can separate in one solvent and not in another.

On a polar stationary phase, the forces from shapes, polarity and intermolecular forces decide retention: a molecule that can hydrogen bond is held more firmly than a non-polar one of similar size.

Worked example 1: one spot, then two

A mixture of two colourless compounds, X and Z, is run on a TLC plate in solvent 1. After locating the spots, there is a single spot 3.2 cm above the baseline; the solvent front is 8.0 cm above it. A second plate, run in solvent 2, shows two spots at 1.2 cm and 3.9 cm, with the front at 7.5 cm.

Solvent 1:  Rf = 3.2 / 8.0 = 0.40   (X and Z together)
Solvent 2:  Rf = 1.2 / 7.5 = 0.16
            Rf = 3.9 / 7.5 = 0.52

In solvent 1 the two compounds have the same balance between the phases, so they travel together. Solvent 2 shifts their balances by different amounts, so they separate. The lesson: a single spot in one solvent does not prove a sample is pure.

Thin-layer chromatography (TLC)

A TLC plate carries a thin, even layer of solid. To run it:

1. Draw a pencil baseline near the bottom edge (ink would dissolve and run).
2. Spot every sample and standard on the baseline, keeping the spots small, concentrated and well apart.
3. Stand the plate in a covered container, with the solvent below the baseline so the spots are not washed into it.
4. When the solvent is close to the top, lift the plate out and draw a pencil line at the solvent front before it evaporates.
5. Make colourless spots visible. For amino acids, section 3.3.13.2 names ninhydrin as a developing agent; ultraviolet light is the other option it gives.

Calculating Rf values

       distance travelled by the spot (baseline to spot centre)
Rf  =  --------------------------------------------------------
       distance travelled by the solvent (baseline to front)

Rf is a pure number from 0 to 1. A value only holds for the stationary phase, solvent and conditions it was measured with.

Worked example 2: three spots in millimetres

A chromatogram has its solvent front 72 mm above the baseline. Spot centres are 13 mm, 40 mm and 61 mm above the baseline.

Spot 1: Rf = 13 / 72 = 0.18
Spot 2: Rf = 40 / 72 = 0.56
Spot 3: Rf = 61 / 72 = 0.85

Spot 1 is the most strongly retained. Use the same unit for both distances.

Worked example 3: naming an unlabelled amino acid

An unlabelled bottle holds one of four amino acids. A solution of it is spotted on one plate beside standards of all four. Amino acids are colourless, so after the run the plate is sprayed with ninhydrin (section 3.3.13.2). The solvent front is 9.6 cm above the baseline. The distances are illustrative values for this one plate.

Spot	Distance / cm	Rf
Serine standard	2.1	0.22
Glycine standard	2.6	0.27
Alanine standard	3.6	0.38
Valine standard	5.5	0.57
Unknown	2.6	0.27

1. Divide each distance by 9.6 cm to get the Rf values in the table.
2. The unknown’s Rf, 0.27, matches glycine and no other standard.
3. Serine is only 0.05 lower, so the match is less secure than it looks. Rerun the unknown and all four standards in a different solvent. If the unknown still lines up with glycine, the identification is much stronger.

Column chromatography (CC)

In column chromatography, a vertical column is packed with a solid. The mixture goes on at the top and solvent runs down through the solid. Components with a balance that favours the solvent move down faster and leave the bottom first. The liquid coming out is collected in a series of tubes (fractions), so the components end up in different containers. So CC can give you each component on its own, in larger amounts than a TLC plate holds.

Worked example 4: checking the fractions

A mixture of benzaldehyde and benzoic acid is run through a column packed with a polar solid. Eight fractions are collected and each one is checked by TLC alongside pure standards of both compounds.

Fractions	TLC result
1 to 2	No spot
3 to 5	One spot, level with the benzaldehyde standard
6	Two spots, level with both standards
7 to 8	One spot, level with the benzoic acid standard

1. Benzaldehyde comes off first. Its –CHO group is polar, but benzoic acid’s –COOH group can form hydrogen bonds with the polar solid, so the acid is retained more strongly.
2. Combine fractions 3 to 5 for benzaldehyde, and 7 to 8 for benzoic acid.
3. Keep fraction 6 out of both, because it contains the two compounds together.

Gas chromatography (GC)

In GC, the column holds a packed solid, which may carry a thin liquid coating. A small sample is injected and vaporised, and a gas carries it through the column under pressure at high temperature. A detector at the far end records a peak as each component emerges.

A component’s retention time is how long it takes to reach the detector, timed from the moment the sample is injected. A component retained strongly by the stationary phase has a long retention time. Retention times change with the column, the temperature and the gas flow rate, so you identify a component by comparing its retention time with a standard run under the same conditions.

Worked example 5: matching peaks to standards

A solvent blend is checked by GC. Standards run on the same column, at the same temperature and gas flow, give these retention times: ethyl ethanoate 2.6 min, propyl ethanoate 4.1 min and butyl ethanoate 6.3 min. The blend gives peaks at 2.6 min, 5.3 min and 6.3 min.

1. The 2.6 min peak matches ethyl ethanoate.
2. The 6.3 min peak matches butyl ethanoate.
3. No peak appears at 4.1 min, so propyl ethanoate is not detected.
4. The 5.3 min peak matches none of the standards. Something else is present, and GC alone cannot say what it is.

A matching retention time is strong evidence, not proof: different substances may come through one column at the same time.

Mass spectrometry of the separated components (GC-MS)

The specification includes using mass spectrometry to analyse the components separated by GC. The GC column’s outlet feeds a mass spectrometer, which the separated components enter one after another, each giving a separate spectrum. Its molecular ion peak gives Mr, and a precise m/z value pins down the molecular formula. How a time of flight instrument works is covered in atomic structure, and the precise-mass method is taught in organic analysis.

Worked example 6: identifying the unknown peak

The 5.3 min component from worked example 5 is analysed by GC-MS. Its molecular ion is at m/z = 98.0729. The candidates are C₆H₁₀O, C₅H₆O₂, C₅H₁₀N₂ and C₄H₆N₂O. Precise masses: C = 12.0000, H = 1.0078, N = 14.0031, O = 15.9949.

C6H10O  : 6(12.0000) + 10(1.0078) + 15.9949             = 98.0729
C5H6O2  : 5(12.0000) + 6(1.0078) + 2(15.9949)           = 98.0366
C5H10N2 : 5(12.0000) + 10(1.0078) + 2(14.0031)          = 98.0842
C4H6N2O : 4(12.0000) + 6(1.0078) + 2(14.0031) + 15.9949 = 98.0479

All four have a whole-number Mr of 98, but only C₆H₁₀O matches the measured value.

Common errors

• Measuring from the plate’s bottom edge, or to the leading edge of a spot. Both distances start at the baseline; the spot distance ends at its centre.
• Dividing the solvent-front distance by the spot distance, which gives a value above 1.
• Explaining separation with one phase only. Name both phases and say how the component interacts with each.
• Calling the GC moving phase a liquid. It is a gas; any liquid is coated on the solid in the stationary phase.
• Treating one spot, or one matching value, as proof of purity or identity.
• Saying the mass spectrometer separates the mixture. GC separates it; mass spectrometry analyses each component.

Where next

Next, read the revision notes and try the practice questions. For more precise-mass work, see the organic analysis practice.

Official syllabus

OxfordAQA International AS and A-level Chemistry (9620) specification, Version 5.3, for International AS and A-level exams May/June 2020 onwards (OxfordAQA). Section 3.3.16 Chromatography (International A2), subsection 3.3.16.1.