How does chromatography separate and identify the components of a mixture?
Explain the principles of chromatography, including gas chromatography (GC) and high-performance liquid chromatography (HPLC), and interpret chromatograms.
How GC and HPLC separate mixtures by differential partitioning between a mobile and stationary phase, and how retention time and peak area are used to identify and quantify components.
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What this dot point is asking
You must explain the partition principle behind chromatography, distinguish GC from HPLC, and read a chromatogram to identify and quantify components.
The core principle
Every form of chromatography has two phases:
- a mobile phase that carries the sample through the system, and
- a stationary phase that the sample interacts with as it passes.
The result is that each component travels at its own speed and emerges separately. The time from injection to a component leaving the column is its retention time.
Gas chromatography (GC)
In GC the mobile phase is an inert carrier gas (such as helium or nitrogen) and the stationary phase is a high-boiling liquid or polymer coated on the inside of a long, thin column held in a temperature-controlled oven.
- The sample is vaporised on injection, so GC suits volatile substances that do not decompose on heating.
- Components separate by a combination of their volatility and their attraction to the stationary phase.
- A detector at the end produces a peak for each component.
High-performance liquid chromatography (HPLC)
In HPLC the mobile phase is a liquid solvent pumped at high pressure through a column packed with a fine solid stationary phase.
- Because nothing is vaporised, HPLC suits substances that are non-volatile or heat-sensitive (large biological molecules, pigments, pharmaceuticals).
- Separation depends on the relative attraction of each component to the liquid mobile phase versus the solid stationary phase.
Reading a chromatogram
A chromatogram plots detector signal against time. Each peak is a component.
Why this matters for environmental monitoring
GC and HPLC let analysts separate complex environmental mixtures - traces of pesticides, hydrocarbons, or pollutants in water and air - and both identify and measure each component, even at very low levels.
Exam-style practice questions
Practice questions written in the style of SACE Board exam questions on this dot point, with worked answer explainers. The year tag is the paper they imitate, not the source.
2022 SACE Stage 23 marksA sample of one soft drink was analysed using HPLC. A polar mobile phase and a non-polar stationary phase were used. The retention times were saccharin 0.93 min, caffeine 2.37 min, benzoic acid 4.78 min. Use the data in the table to identify and explain which of the three additives is the most polar.Show worked answer →
The most polar additive is saccharin (shortest retention time, 0.93 min).
The stationary phase is non-polar and the mobile phase is polar.
A polar substance has a stronger attraction to the polar mobile phase and a weaker attraction to the non-polar stationary phase, so it spends more time moving with the mobile phase.
It is therefore carried through the column fastest and elutes with the shortest retention time. Saccharin elutes first, so it is the most polar. One mark for the identity, two for the reasoning linking polarity to the phases and retention time.
2024 SACE Stage 23 marksHPLC is used to separate and identify vitamins in dietary supplements. Vitamin C is more polar than vitamin B2. Using the principles of chromatography, explain how vitamin B2 was separated from vitamin C in the column.Show worked answer →
Separation depends on the different attractions of the two vitamins to the stationary and mobile phases.
As the sample moves through the column, each component partitions between the mobile phase and the stationary phase according to its polarity.
Vitamin C is more polar, so (with a non-polar stationary phase and polar mobile phase) it is attracted more strongly to the mobile phase, moves faster, and has a shorter retention time, eluting first.
Vitamin B2 is less polar, interacts more with the stationary phase, is retained longer, and elutes later. The difference in retention times separates the two. One mark each for partitioning, the faster more-polar component, and the slower less-polar component.
2023 SACE Stage 23 marksThin layer chromatography (TLC) is used to detect performance-enhancing drugs. The stationary phase was more polar than the mobile phase. RF values were acetazolamide 0.40 and stanozolol 0.95. Explain whether stanozolol is more polar or less polar than acetazolamide.Show worked answer →
Stanozolol is less polar than acetazolamide.
The stationary phase is more polar than the mobile phase.
A higher RF (0.95) means stanozolol travelled further up the plate, so it spent more time in the less-polar mobile phase and was attracted less strongly to the polar stationary phase.
Acetazolamide has a lower RF (0.40), so it is more strongly attracted to the polar stationary phase, indicating it is more polar. Therefore stanozolol, with the higher RF, is the less polar substance.