OxfordAQA A-Level Chemistry: Chromatography (9620) – Revision Notes

These notes condense 3.3.16 Chromatography (subsection 3.3.16.1) from the OxfordAQA International AS and A-level Chemistry (9620) specification, Version 5.3, for International AS and A-level exams from May/June 2020 onwards. As International A2 content, the whole section is International A-level only. It is part of the organic content assessed in Unit 4, and Unit 5 can test any content.

Full explanations and six worked examples are in the chromatography study guide. When you are ready to test yourself, use the practice questions. For the rest of the course, use the course hub and printable checklist; a free 10-minute diagnostic points to your weakest topics.

What the specification asks for

3.3.16.1 outcome	In one line
Purpose	Chromatography separates and identifies the components in a mixture
Types	TLC, column chromatography (CC) and gas chromatography (GC)
How it works	Balance between a component’s solubility in the moving phase and its retention by the stationary phase
Identification	Retention times and Rf values, compared with standards
GC-MS	Mass spectrometry analyses the components that GC has separated
Skill	Calculate Rf values from a chromatogram
Skill	Compare retention times and Rf values with standards

Linked outcome from 3.3.13.2: amino acids can be separated by TLC, located with developing agents such as ninhydrin or with ultraviolet light, and identified by their Rf values (see the amino acids and proteins notes).

Key terms

Term	Meaning
Stationary phase	The part that stays fixed: the solid on the plate or in the column, or the solid (which may be coated by a liquid) in a GC column
Moving phase	The solvent (TLC, CC) or gas (GC) that flows past the stationary phase
Chromatogram	The developed plate, or the detector trace, that records the separation
Baseline	Pencil line on a TLC plate where samples are spotted
Solvent front	The furthest point the solvent reached before the plate was removed
Rf value	How far the spot centre moved divided by how far the solvent moved, each measured from the baseline
Retention time	Time from injecting a sample into a GC column to a component reaching the detector
Standard	A pure sample of a known substance, run alongside the unknown
Fraction	A portion of liquid collected from the bottom of a column

One idea explains every method

Each component moves between the two phases. How fast it travels is set by the balance between its solubility in the moving phase and its retention by the stationary phase.

Balance favours…	TLC	CC	GC
Moving phase	Large Rf	Leaves the column early	Short retention time
Stationary phase	Small Rf	Leaves the column late	Long retention time

Two substances with the same balance do not separate. Change the solvent, the solid or the conditions, and the balance changes.

On a polar stationary phase, retention rises with polarity: a molecule that forms hydrogen bonds is held more strongly than one with only weak dipoles, which is held more strongly than a non-polar one of similar size.

The three methods side by side

	TLC	CC	GC
Moving phase	A solvent	A solvent	A gas
Stationary phase	Thin solid layer on a plate	Solid packed into a column	Packed solid, may be liquid-coated
Flow	Up the plate	Down the column	Through the column, under pressure, at high temperature
Main result	Spots and Rf values	Separate fractions of each component	Peaks and retention times
Typical use	Quick identification and checking purity	Obtaining each component on its own	Identifying volatile components; feeds GC-MS

Rf values

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

• A pure number between 0 and 1.
• Measure both distances from the baseline, to the centre of the spot.
• Only compare with standards on the same plate, in the same solvent.

Worked reminder. Solvent front 7.5 cm, spot centre 3.3 cm: Rf = 3.3 / 7.5 = 0.44.

Method in steps: identifying by Rf

1. Spot the sample and the standards side by side on one baseline.
2. Run the plate, then mark the solvent front straight away.
3. Locate colourless spots (ultraviolet light, or ninhydrin for amino acids).
4. Calculate the Rf of every spot.
5. Match each sample spot to a standard with the same Rf.
6. A sample spot with no match is a substance not among the standards.
7. To strengthen a match, rerun in a different solvent.

TLC technique in brief

Step	Why
Pencil baseline	Graphite does not dissolve; ink would run with the solvent
Solvent level below the baseline	The spots must be carried up, not dissolved into the solvent
Small, concentrated spots	Large spots spread out and overlap
Lid on the container	Keeps the air full of solvent vapour so the plate does not dry as the solvent rises
Mark the front immediately	The solvent evaporates and the front can no longer be seen

Column chromatography

• Mixture added at the top; solvent run down through the packed solid.
• The component with the balance most in favour of the solvent leaves first.
• Collect the liquid in a series of fractions; check each fraction by TLC against standards.
• Combine fractions that contain only one component; leave out fractions that contain two.

Gas chromatography and GC-MS

• Sample injected and vaporised; a gas carries it through the column under pressure at high temperature.
• A detector records a peak for each component as it emerges.
• Identify a peak by matching its retention time to a standard run on the same column, at the same temperature and gas flow.
• A peak with no matching standard is an unknown: send it to the mass spectrometer.
• GC-MS: the column’s output passes straight into a mass spectrometer. Components arrive one at a time, so each gives its own spectrum. The molecular ion gives Mr, and a precise m/z value fixes the formula; the method is in the organic analysis notes (section 3.3.6.2).

Method in steps: precise m/z to formula

1. Write down every candidate formula (all have the same integer Mr).
2. Total the precise masses of all the atoms in each candidate.
3. Pick the total that equals the measured m/z.
4. Show every sum, not just the one that matches.

Must-know distinctions

Don’t confuse	Difference
TLC flow vs CC flow	Up the plate vs down the column
Moving phase in GC	A gas, never a liquid; a liquid can only be part of the stationary phase
High Rf vs long retention time	High Rf means weak retention; a long retention time means strong retention
Separating vs identifying	GC separates; mass spectrometry analyses each separated component
One spot vs pure	One spot in one solvent does not prove purity
Matching vs proving	A matching Rf or retention time is evidence, not proof

Quick self-test

1. A spot centre is 3.8 cm above the baseline and the solvent front is 5.0 cm above it. Calculate the Rf value.
2. In a solvent where the front rises 8.5 cm, a compound has Rf = 0.62. How far above the baseline is its spot?
3. The solvent front is 50 mm and a spot is 41 mm from the baseline. A student divides 50 by 41 and reports Rf = 1.22. Find the error and give the correct value.
4. State the moving phase in gas chromatography.
5. In which method does the solvent move down through the stationary phase?
6. A GC component has a long retention time. What does this tell you about its balance between the phases?
7. Why is ninhydrin sprayed on a plate used to separate amino acids?
8. A tripeptide is hydrolysed and the TLC plate shows only two spots. Give two possible reasons.
9. The molecular ion of a component leaving a GC column is at m/z = 96.0573. Is it C₆H₈O, C₅H₄O₂ or C₅H₈N₂? (C = 12.0000, H = 1.0078, N = 14.0031, O = 15.9949)
10. Why must standards for TLC be run on the same plate as the sample?
11. A column is checked fraction by fraction. One fraction shows both components on TLC. What should you do with it?

Answers

1. Rf = 3.8 / 5.0 = 0.76.
2. 0.62 × 8.5 = 5.27, so about 5.3 cm (2 s.f., like the data).
3. The ratio is upside down; an Rf value cannot exceed 1. Rf = 41 / 50 = 0.82.
4. A gas.
5. Column chromatography.
6. It is retained strongly by the stationary phase compared with its solubility in the moving phase.
7. Amino acids are colourless; ninhydrin is a developing agent that makes their spots visible so Rf values can be measured.
8. One amino acid appears twice in the tripeptide, or two different amino acids have the same Rf in this solvent.
9. C₆H₈O = 6(12.0000) + 8(1.0078) + 15.9949 = 96.0573; C₅H₄O₂ = 96.0210; C₅H₈N₂ = 96.0686. It is C₆H₈O.
10. Rf values depend on the solvent, the stationary phase and the conditions, so only values from the same run can be compared.
11. Keep it separate from the pure fractions of each component (it could be run through the column again).

Where marks are usually lost

• Taking measurements from the bottom of the plate or to the leading edge of a spot; both distances start at the baseline, and the spot distance ends at its centre.
• Writing the Rf ratio upside down, giving a value above 1.
• Adding units such as cm to an Rf value.
• Explaining separation with one phase only, or with “mass” or “size” alone, instead of the balance between both phases.
• Getting the flow direction wrong: TLC solvent rises, column solvent falls.
• Describing the GC moving phase as a liquid.
• Defining retention time without its starting point, the moment of injection.
• Comparing an Rf or retention time with a value from a different plate, solvent or column.
• Claiming one spot proves purity, or a single match proves identity.
• Choosing a formula from a precise m/z without showing the sums for all the candidates.

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.