Topic 15 · page 27 of 28
Column chromatography
High-performance liquid chromatography and gas chromatography pass a mixture through a column. Components leave at different retention times, and the detector, often a mass spectrometer, identifies and measures each.
Read first: Chromatography and Rf values
Chromatography in a column 15.23
Paper and thin-layer chromatography work on a flat surface. Column chromatography packs the stationary phase into a tube (the column) and pushes the mobile phase through it. The sample is injected at one end and the separated components come out of the other, where a detector records them.
Two kinds are needed here:
| Gas chromatography (GC) | High-performance liquid chromatography (HPLC) | |
|---|---|---|
| mobile phase | an unreactive carrier gas, such as helium or nitrogen | a liquid solvent, pumped at high pressure |
| stationary phase | a liquid held on a solid support inside a coiled column in an oven | a solid (finely divided silica) packed in the column |
| sample must be | volatile: vaporised by the heated injector | dissolved in a solvent, so volatility is not needed |
In both, the components separate because each is attracted to the stationary phase to a different extent and is carried by the mobile phase to a different extent.
Retention time 15.23
A component that is strongly attracted to the stationary phase takes longer to leave the column, so it has a longer retention time. A component that is more soluble in the mobile phase, or less strongly held, leaves sooner.
The retention time under fixed conditions is a property of a compound, so it can be compared with the time for a known compound to identify it. The area under a peak is proportional to the amount of that component, once the detector has been calibrated.
Worked exampleComposition from peak areas
Illustrative peak areas are A = 0.132, B = 0.280 and C = 0.180 (arbitrary units). Find the percentage of each component, assuming the detector responds equally to all three.
- Total area = 0.132 + 0.280 + 0.180 = 0.592
- A: 0.132 ÷ 0.592 × 100 = 22.3%
- B: 0.280 ÷ 0.592 × 100 = 47.3%
- C: 0.180 ÷ 0.592 × 100 = 30.4%
Using the mass spectrometer as the detector 15.23
A column separates substances, but the retention time alone does not prove what a substance is. The outflow can be fed straight into a mass spectrometer, which records the mass spectrum of each component as it comes off the column. The molecular ion peak gives the relative molecular mass and the fragments help to identify the compound. This combination is called GC–MS (or LC–MS for the liquid version).
Applications include:
- forensics: identifying drugs, explosives or accelerants (such as petrol) found at a crime scene or a fire;
- drug testing in sport: finding banned substances and their breakdown products in a urine or blood sample;
- food and environmental analysis: detecting pesticides, flavour compounds and pollutants.
Check yourself
- State the mobile phase and the stationary phase in gas chromatography.
- What is meant by retention time? Why do two compounds have different retention times?
- Explain why a gas chromatogram of a mixture shows peaks of different areas.
- Explain why a mass spectrometer is often attached to a gas chromatograph.
- Give two applications of these techniques.
Answers cover these until you have tried the questions
- The mobile phase is an unreactive carrier gas (such as helium or nitrogen). The stationary phase is a liquid held on a solid support inside the column.
- Retention time is the time from injection to the compound reaching the detector. The compounds differ in how strongly they are attracted to the stationary phase and how soluble they are in the mobile phase, so they travel through the column at different speeds.
- The area of each peak is proportional to the amount of that component in the mixture, so components present in larger amounts give larger peaks.
- The column separates the components, but retention time does not prove what each is. The mass spectrometer records the mass spectrum of each as it leaves, from which its identity can be found.
- Any two: forensic analysis of drugs or accelerants; drug testing in sport; analysing pesticides or pollutants in food and water.