Chromatography basics

Why it matters

Chromatography is how labs separate and measure the parts of a mixture: drugs in a tablet, pesticides in water, amino acids in a protein digest, drugs of abuse in urine. Chemists, biochemists and medical laboratory staff all use it, and many technician jobs are built around running and maintaining HPLC or GC systems. This lesson explains the principles so you can follow what an instrument is doing and read a chromatogram.

The core idea

Every chromatography method has two phases:

• A stationary phaseThe part of a chromatography system that stays put, such as a coated plate, packed column or coated capillary. that stays put: a coated plate, a packed column or a coated capillary.
• A mobile phaseThe solvent or gas that moves through the stationary phase and carries the sample. that moves through it: a solvent (liquid chromatography) or a gas (gas chromatography).

Each compound in the sample divides its time between the two phases. Compounds that interact more strongly with the stationary phase move more slowly and come out later. That difference in speed separates them.

Thin-layer chromatography (TLC)

TLC is the simplest form. The stationary phase is a thin layer of silica on a plate. You spot the sample near the bottom, stand the plate in a shallow pool of solvent in a closed chamber, and let the solvent climb by capillary action. When the solvent front is near the top, you mark it, dry the plate, and see the spots (often under UV light or with a stain).

The retention factorIn TLC, the distance a spot travels divided by the distance the solvent front travels (Rf). (Rf) describes how far a spot moved:

Rf = distance travelled by the spot / distance travelled by the solvent front

Worked example. The solvent front moved 8.0 cm. Spot A moved 2.0 cm (Rf 0.25) and spot B moved 6.0 cm (Rf 0.75). On normal silica, which is polar, the more polar compound sticks more and has the lower Rf, so A is more polar than B. TLC is quick and cheap: it is used to check whether a reaction is finished or whether a sample is pure. It is mostly qualitative.

High-performance liquid chromatography (HPLC)

HPLC pumps liquid mobile phase at high pressure through a column packed with very small particles. The parts of a system are:

1. Solvent reservoirs and pump: deliver a steady flow, either at a fixed composition (isocratic) or a changing one (gradient).
2. Autosampler: injects a precise volume of each sample.
3. Column: the stationary phase. The most common is reversed-phase C18: a non-polar coating, with a polar mobile phase such as water and acetonitrile or methanol. Non-polar compounds are retained longer.
4. Detector: often UV-visible absorbance, sometimes fluorescence or a mass spectrometer (LC-MS).
5. Data system: draws the chromatogram.

Gas chromatography (GC)

GC uses an inert carrier gas (helium, hydrogen or nitrogen) as the mobile phase and a long, thin coated capillary column in a temperature-controlled oven. The sample must be volatile (or made volatile) and stable when heated. Compounds separate by boiling point and by interaction with the coating. Common detectors are the flame ionisation detector (FID) for organic compounds and the mass spectrometer (GC-MS), which also identifies compounds by their fragmentation pattern. GC suits solvents, fuels, fragrances, blood alcohol and many environmental contaminants.

Reading a chromatogram

A chromatogram plots detector signal against time. Each peak is a compound (or several compounds that did not separate).

• Retention timeThe time at which a compound's peak appears, used with a standard to help identify it.: when the peak appears. Under fixed conditions it helps identify the compound, by comparison with a standard.
• Peak area (or height): proportional to the amount. With a calibration curve from standards, area gives concentration.
• ResolutionHow well two neighbouring peaks are separated; overlapping peaks give unreliable areas.: how well two neighbouring peaks are separated. Overlapping peaks give unreliable areas.
• Peak shape: tailing or split peaks often point to column or method problems.

Regulated labs run a system suitability testA standard injection that must meet limits for retention time, resolution, peak shape and repeatability before samples are run. before samples: a standard injection that must meet limits for retention time, resolution, peak shape and repeatability. If it fails, you do not run samples.

Choosing a method

Common mistakes

• Treating retention time alone as proof of identity. Two compounds can share a retention time; MS or a second method confirms it.
• Running samples after a failed system suitability test.
• Letting the solvent pool rise above the TLC spots (the sample dissolves into the pool).
• Integrating peaks inconsistently, which changes the reported amount.

Quick recap

• Separation happens because compounds split their time differently between a stationary and a mobile phase.
• TLC gives Rf; HPLC uses liquid under pressure, often on reversed-phase C18; GC uses a carrier gas for volatile compounds.
• Retention time helps identify, peak area quantifies, and resolution tells you whether you can trust the areas.
• System suitability must pass before samples run.

Hands-on chromatography is learned in your teaching lab and on the job, under supervision.

Try it

This lesson is quiz-only. Open the Do tab to check your understanding.