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In 1903, the Russian-Italian botanist Mikhail Tsvet poured an extract of green leaves into the top of a glass column packed with powdered chalk and washed it through with solvent. As the liquid flowed down, the pigments spread into separate coloured bands: green chlorophylls and yellow-orange carotenoids. He called the technique chromatography, from the Greek for “colour writing”.
Today, chromatography is one of the most widely used techniques in science. It tests athletes for banned drugs, checks the purity of medicines, measures pollutants in water, identifies ink in forged documents and analyses the flavours in food. Most of these uses have nothing to do with colour, but the core idea is exactly Tsvet’s.
The core idea
Every form of chromatography has two phases:
- A stationary phase that stays in place: paper, a coating on a plate, a packed column, or a liquid film on the inside of a tube.
- A mobile phase that moves through or over the stationary phase: a liquid solvent, or a gas.
A mixture is introduced at one end. As the mobile phase moves, it carries the components of the mixture with it. But each component is also attracted to the stationary phase, to a different degree.
Components that interact more strongly with the stationary phase move more slowly. Components that prefer the mobile phase move faster. Over the length of the paper, plate or column, the components spread apart and separate.
A useful analogy: imagine a group of people walking along a street lined with shops. Everyone walks at the same speed, but some people stop to look in every window while others hardly stop at all. By the end of the street, they’ve spread out according to how interested they are in the shops.
Two ways substances “stick”
The attraction to the stationary phase happens in one of two main ways (and often a combination):
Partition. The stationary phase is a liquid (such as water held in paper fibres, or a liquid coating inside a GC column). Each component dissolves partly in the stationary liquid and partly in the mobile phase, distributing itself between them according to its relative solubility. This is the main mechanism in paper chromatography and gas–liquid chromatography.
Adsorption. The stationary phase is a solid (such as silica or alumina). Components stick to its surface by intermolecular forces, including hydrogen bonds and dipole–dipole attractions, and are then displaced by the moving solvent. This is the main mechanism in thin-layer and column chromatography on silica.
Either way, the principle is the same: different substances spend different fractions of their time in the stationary phase, so they travel at different speeds.
What decides how far a substance travels
- Polarity. On a polar stationary phase like silica or paper, polar substances are held more strongly and travel less far. Non-polar substances travel further.
- Solubility in the mobile phase. More soluble substances travel further.
- Size and shape (in some techniques, such as size-exclusion chromatography).
- Volatility (in gas chromatography, more volatile substances travel faster).
- Charge (in ion-exchange chromatography).
Changing the mobile phase changes the result. A more polar solvent competes more effectively for the polar stationary phase and pushes polar substances further. That’s why chemists often test several solvents to find the one that gives the best separation.
Measuring how far substances travel
In planar techniques (paper and TLC), the key measure is the Rf value:
Rf = distance moved by the substance ÷ distance moved by the solvent front
In column techniques (gas chromatography and HPLC), substances come out of the end of the column one after another, and the key measure is the retention time: how long each takes to pass through.
Both Rf and retention time are characteristic of a substance under fixed conditions, so they can be used to identify it by comparison with known standards.
The main types of chromatography
| Technique | Stationary phase | Mobile phase | Main uses |
|---|---|---|---|
| Paper chromatography | water in cellulose paper | liquid solvent | teaching, separating dyes and pigments |
| Thin-layer chromatography (TLC) | thin layer of silica or alumina on a plate | liquid solvent | quick purity checks, monitoring reactions |
| Column chromatography | silica or alumina packed in a column | liquid solvent | purifying larger amounts of compounds |
| Gas chromatography (GC) | liquid or polymer coating inside a long thin column | inert gas (helium, nitrogen) | volatile substances: fuels, perfumes, drugs in blood |
| High-performance liquid chromatography (HPLC) | very fine particles in a steel column | liquid pumped at high pressure | medicines, food additives, biological molecules |
| Ion-exchange chromatography | charged resin | aqueous buffer | proteins, water softening, separating ions |
| Size-exclusion chromatography | porous gel beads | liquid | separating molecules by size, e.g. proteins, polymers |
Each has its own detailed guide: paper chromatography, thin-layer chromatography, gas chromatography, HPLC and column chromatography.
Analytical vs preparative
Chromatography serves two different purposes:
- Analytical chromatography identifies what’s in a mixture and how much of each component there is. Tiny samples are enough. Paper, TLC, GC and analytical HPLC are mainly analytical.
- Preparative chromatography separates and collects useful amounts of pure substances. Column chromatography and preparative HPLC are used by chemists to purify products after a reaction.
Chromatography combined with other techniques
Chromatography separates; it doesn’t always identify. So it’s often coupled with a detector that identifies each separated component:
- GC–MS (gas chromatography–mass spectrometry) separates a mixture, then the mass spectrometer identifies each component by its mass and fragmentation pattern. It’s a gold-standard method in forensic toxicology, drug testing and environmental analysis.
- LC–MS does the same for liquid chromatography, handling larger and less volatile molecules such as proteins and many drugs.
Where chromatography is used
- Medicine and sport: testing blood and urine for drugs, including anti-doping tests at major sporting events.
- Pharmaceuticals: checking the purity and content of every batch of medicine.
- Food and drink: detecting additives, contaminants, pesticides and flavour compounds.
- Environment: measuring pollutants such as pesticides, PFAS and hydrocarbons in water and soil.
- Forensics: analysing inks, fibres, explosives residues and fire accelerants.
- Biology: purifying proteins and separating DNA fragments.
A short history of a big idea
After Tsvet’s pioneering work, chromatography was largely ignored for decades. It revived in the 1930s and 1940s, when chemists began using it to separate vitamins and other natural products. In 1952, Archer Martin and Richard Synge won the Nobel Prize in Chemistry for developing partition chromatography, and Martin, with Anthony James, went on to invent gas–liquid chromatography the same year. HPLC developed rapidly from the late 1960s, and today chromatography underpins much of modern chemical analysis.
Key takeaways
- Chromatography separates mixtures using a stationary phase and a mobile phase.
- Components that interact more strongly with the stationary phase move more slowly, so the mixture spreads out.
- Separation works by partition (dissolving between phases) or adsorption (sticking to a surface).
- Planar methods use Rf values; column methods use retention times.
- Chromatography is often combined with mass spectrometry to both separate and identify components.
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