Chromatography: TLC & Column (AQA A Level Chemistry): Video

Hi, I'm Eleanor with 3 years of experience teaching Chemistry, and this video is about the principles of chromatography, thin-layer chromatography and column chromatography.

All chromatography techniques work on the same principle, so TLC and column chromatography differ only in how that principle is set up, and the Rf value is how the result is read off either one.

Chromatography is a technique that enables the separation of mixtures. Every technique uses the same principle: the components of a mixture dissolve in a fluid, the mobile phase, and flow through another material, the stationary phase, at varying rates. How fast each component moves depends on the balance between how soluble it is in the mobile phase and how strongly the stationary phase retains it. That balance is what separates the mixture, and it is the same balance whichever technique you use.

We'll start with those principles, then thin-layer chromatography, then column chromatography, and finish with how you read the results.

Chromatography separates mixtures, and the techniques on this course are thin-layer chromatography, column chromatography, and gas chromatography, which is sometimes called gas-liquid chromatography. The latter will be covered in another video. In all of them the mobile phase carries the sample and the stationary phase retains it. The rate of separation depends on how the components interact with the stationary phase, which is their retention, and how soluble they are in the mobile phase. Each technique pairs those two phases differently. In TLC and column chromatography the mobile phase is a liquid solvent and the stationary phase is solid silica or alumina, while in gas chromatography the mobile phase is an inert carrier gas.

Thin-layer chromatography analyses small samples by separating them. The stationary phase is a thin metal sheet coated in alumina or silica, and the solute molecules adsorb onto its surface. The mobile phase flows over it: a polar or non-polar liquid solvent that carries the components. Water and alcohol are polar solvents, alkanes are non-polar.

To run it, you draw a baseline in pencil at the bottom of the plate, spot a pure reference compound on the left and the sample on the right, and stand the plate in a beaker of solvent with the spots above the solvent level. The solvent travels up the plate, dissolving the compounds as it goes, and where it stops is marked as the solvent front. A component more soluble in the solvent travels further, so the separation you see is that balance between solubility and affinity for the stationary phase.

In column chromatography the column is a long vertical glass tube, or a burette in the laboratory. An inert solid, usually powdered silica gel or alumina, is the stationary phase packed into the column, and a liquid solvent is the mobile phase. The sample is dissolved in solvent and added carefully at the top with a pipette, without disturbing the surface, and more solvent, the eluent, is added on top. The sample flows through under gravity, and fresh solvent is added so the column does not dry out.

The same balance applies to both here: the component with the greatest attraction to the stationary phase takes the longest time to flow through the column.

Each component in a mixture has a unique retention factor, the Rf value, calculated by dividing the distance the compound moves by the distance moved by the solvent. Rf is dimensionless and always between 0 and 1, and these values can be used alongside other analytical data to deduce the composition of mixtures.

On a TLC plate, the reference compounds spotted next to the sample allow the components of the mixture to be identified. If the components are coloured their spots are easy to see. If they are not, the spots are located using UV light, ninhydrin or iodine vapour, and drawn around in pencil.

In column chromatography, coloured components can be identified by Rf value and colourless ones by fluorescence under UV light. In gas-liquid chromatography the term used is retention time instead: the time from the moment of injection to when a component exits the chromatography tube.

The baseline on a TLC plate must be drawn in pencil. Any other medium would interact with the sample components and the solvents used in the analysis.

Thin-layer chromatography runs a solvent up a coated plate. Column chromatography runs a solvent down a packed tube under gravity. In both, the Rf value reads the result off, comparing the distance the component travelled with the distance the solvent travelled. And in both, what actually separates the mixture is the same balance: solubility in the mobile phase against retention by the stationary phase.

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