1. How Chromatography Separates a Mixture
Chromatography is used to separate and identify the components in a mixture.
Every type of chromatography has two phases:
- the mobile phase (moving phase), which carries the components along;
- the stationary phase, which does not move and holds the components back.
The Key Idea
Separation depends on the balance between solubility in the moving phase and retention by the stationary phase. A substance that is more soluble in the mobile phase, or less strongly held by the stationary phase, moves further or faster.
| Type | Stationary phase | Mobile phase |
|---|---|---|
| Thin-layer (TLC) | A plate coated with a solid | A solvent moves up the plate |
| Column (CC) | A column packed with a solid | A solvent moves down the column |
| Gas (GC) | A column packed with a solid, or with a solid coated by a liquid | A gas passed through the column under pressure at high temperature |
2. Thin-Layer Chromatography (TLC)
Method
- Draw a pencil start line near the bottom of the plate. Pencil does not dissolve in the solvent.
- Spot the sample and the standards on the start line.
- Put the plate in a beaker with solvent. The solvent must be below the start line, so the spots do not dissolve into the solvent.
- Put a lid on the beaker. This keeps the atmosphere saturated with solvent vapour.
- Remove the plate when the solvent is near the top. Mark the solvent front straight away.
Locating colourless spots
- Ultraviolet light: the plate has a fluorescent coating, so the spots appear dark.
- A developing agent: for example, ninhydrin turns amino acid spots purple.
Calculating Rf
Rf value
Rf = distance moved by the spot ÷ distance moved by the solvent front
Measure both distances from the start line.
Measure both distances from the start line.
Worked Example: Rf Value
A spot moves 3.6 cm. The solvent front moves 8.0 cm. Calculate the Rf value.
Rf = 3.6 ÷ 8.0 = 0.45
Rf has no units. It is always less than 1.
Identifying substances and testing purity
- Identify a substance by comparing its Rf with a standard run on the same plate in the same solvent. In the diagram, the mixture M contains A and B.
- A pure substance gives one spot. This is one way to test the purity of a product in RP10 (prepare a pure organic solid and test its purity).
3. Column Chromatography
- A column is packed with a solid, for example silica or alumina.
- The solvent (the eluent) runs down through the column.
- The components come out at different times. They are collected as separate fractions.
- It is used to purify products on a larger scale than TLC.
4. Gas Chromatography (GC)
- An inert carrier gas (for example helium or nitrogen) carries the vaporised sample through a heated column.
- The column holds a solid, or a solid coated with a liquid.
- Each component leaves the column at a different time.
Retention time
The time from injection of the sample to detection of a component. Compare it with standards run under the same conditions: the same column, temperature and flow rate.
The area under each peak shows the relative amount of each component.
Examiner Tip
A component that is more strongly retained by the stationary phase takes longer to leave the column. It has a longer retention time.
5. GC-MS
- The components leave the GC column one at a time. They pass straight into a mass spectrometer.
- The mass spectrum of each component identifies it. Use the molecular ion peak and the fragmentation pattern, for example by comparing the spectrum with a spectral database.
- Uses: forensics, drug testing and environmental analysis.
In short: GC separates the mixture, and MS identifies each component.
6. Separating Amino Acids by TLC
- A protein is hydrolysed into its amino acids. The amino acids are separated by TLC.
- They are colourless. Locate them with ninhydrin (purple spots) or UV light.
- Identify each amino acid by its Rf value, compared with standards run in the same solvent.
See Amino Acids, Proteins and Cisplatin for protein hydrolysis.
Go deeper
These UK AQA pages cover the same ideas in more detail. They may include content that is not in 9620.
7. Practice Questions
Question 1 (1 mark)
A spot moves 4.2 cm and the solvent front moves 7.0 cm. Calculate Rf.
Show Answer & Mark Scheme
Answer: 4.2 ÷ 7.0 = 0.60 (1)
Question 2 (2 marks)
Explain why the start line is drawn in pencil and must be above the solvent level.
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Answer:
- Pencil (graphite) does not dissolve or run (1).
- Otherwise the spots would dissolve into the solvent in the beaker (1).
Question 3 (3 marks)
Explain how amino acids can be identified after TLC.
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Answer:
- Spray with ninhydrin or use UV light to show the spots (1).
- Measure the Rf value of each spot (1).
- Compare with the Rf values of known amino acids (standards) in the same solvent (1).
Question 4 (2 marks)
Explain why two compounds have different retention times in GC.
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Answer:
- They have a different balance between solubility in the moving gas phase and retention by the stationary phase (1).
- The more strongly retained compound takes longer to leave the column (1).
Question 5 (2 marks)
Why is GC linked to mass spectrometry?
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Answer:
- GC separates the mixture (1).
- MS identifies each separated component from its mass spectrum (1).