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Thin-layer chromatography is the organic chemist’s pocket tool. It takes a few minutes, uses a tiny amount of sample, costs very little, and answers the questions chemists ask most often: Is my product pure? Has my reaction finished? Which solvent should I use to purify this? It’s paper chromatography’s faster, sharper, more versatile cousin.
How TLC differs from paper chromatography
| Paper chromatography | Thin-layer chromatography | |
|---|---|---|
| Stationary phase | water held in cellulose fibres | thin layer of silica gel (SiO₂) or alumina (Al₂O₃) on glass, aluminium or plastic |
| Separation mechanism | mainly partition | mainly adsorption |
| Speed | 30–60 minutes or more | often 5–15 minutes |
| Spot sharpness | broader spots | sharper spots, better separation |
| Solvents | mostly water or alcohols | wide range of organic solvents |
| Visualising colourless spots | ninhydrin, iodine | UV (with fluorescent plates), stains, iodine |
The principle is the same: components of a mixture distribute between the stationary phase and a moving solvent, and travel different distances. See chromatography.
Why silica separates by polarity
Silica gel’s surface is covered in Si–OH groups, which make it very polar. Polar molecules, especially those that can hydrogen bond (alcohols, carboxylic acids, amines), stick to the surface strongly and move slowly. Non-polar molecules (hydrocarbons, halogenoalkanes) barely stick and travel with the solvent.
So on a silica TLC plate:
- Non-polar compounds → high Rf (travel far)
- Polar compounds → low Rf (stay near the baseline)
Equipment
- TLC plates (silica gel, often with a fluorescent indicator, labelled F₂₅₄)
- Pencil and ruler
- Fine glass capillary tubes for spotting
- TLC developing chamber, or a beaker with a watch glass or lid
- Solvent (the “eluent”), e.g. mixtures of hexane and ethyl ethanoate
- UV lamp (254 nm) and/or iodine chamber or chemical stain
- Tweezers
- Eye protection; UV-protective glasses if using a UV lamp
Method
- Handle the plate by the edges only. Fingerprints contain oils that show up as spots.
- Draw a baseline in pencil about 1 cm from the bottom. Press lightly so you don’t scrape off the silica.
- Dissolve samples in a small amount of volatile solvent (such as ethyl ethanoate or dichloromethane).
- Spot each sample on the baseline with a capillary tube, keeping spots under about 2 mm. Allow each spot to dry.
- Prepare the chamber: add eluent to a depth of about 0.5 cm, below the baseline. Put a piece of filter paper against the inside wall and cover the chamber for a few minutes so the air becomes saturated with solvent vapour.
- Place the plate in the chamber with tweezers, baseline at the bottom, and replace the lid.
- Let the solvent rise until it’s about 1 cm from the top. Don’t disturb the chamber.
- Remove the plate, immediately mark the solvent front in pencil, and let it dry.
- Visualise the spots (see below) and circle them lightly in pencil.
- Calculate Rf values: distance from baseline to spot centre ÷ distance from baseline to solvent front. See calculating Rf values.
Seeing colourless spots
Most organic compounds are colourless, so they need to be made visible.
UV light (non-destructive). Plates labelled F₂₅₄ contain a fluorescent material that glows green under 254 nm UV light. Compounds that absorb UV at that wavelength, especially aromatic compounds and those with conjugated double bonds, block the glow and appear as dark spots on a green background. Circle them in pencil while the lamp is on. Never look directly at the UV lamp; wear UV-protective glasses.
Iodine vapour (semi-destructive). Place the dried plate in a closed jar containing a few iodine crystals. Iodine vapour dissolves in or reacts with many organic compounds, staining them yellow-brown. The spots fade, so mark them quickly.
Chemical stains (destructive). Dip or spray the plate with a reagent and heat gently:
- Potassium permanganate: compounds that can be oxidised (alkenes, alcohols, aldehydes) show as yellow-brown spots on a purple background.
- Ninhydrin: amino acids and amines show as purple or pink spots.
- Vanillin or anisaldehyde stains: many compounds give different colours, which helps distinguish them.
Stains are used in a fume cupboard with appropriate precautions.
Choosing the solvent
The eluent controls how far compounds move. For silica plates, a more polar eluent competes more strongly for the silica surface and pushes all compounds further up the plate.
A common approach is to mix a non-polar solvent (hexane) with a polar one (ethyl ethanoate) and adjust the ratio:
| Eluent (hexane : ethyl ethanoate) | Polarity | Effect on Rf values |
|---|---|---|
| 9 : 1 | low | compounds stay low; good for non-polar mixtures |
| 4 : 1 | moderate | often a good starting point |
| 1 : 1 | high | polar compounds move up |
| pure ethyl ethanoate | higher still | even polar compounds move near the front |
Aim for Rf values between about 0.2 and 0.6 for the compounds you care about, with clear gaps between spots.
What TLC is used for
1. Checking purity
A pure compound gives one spot. Extra spots reveal impurities. Running the plate in two different eluents increases confidence, since two compounds might coincidentally share an Rf in one solvent.
2. Identifying compounds
Spot the unknown next to authentic samples of likely candidates on the same plate. A useful trick is the co-spot: in the middle lane, spot both the unknown and the reference on top of each other. If they’re the same compound, the co-spot runs as a single spot; if not, it separates into two.
3. Following a reaction
Spot the starting material, the reaction mixture, and a co-spot of both, on the same plate, at intervals during a reaction:
- At the start, the reaction mixture shows only the starting material’s spot.
- As the reaction proceeds, a new product spot appears and the starting material spot fades.
- When the starting material spot has gone, the reaction is complete.
This lets chemists decide when to stop a reaction without guessing.
4. Choosing conditions for column chromatography
The eluent that gives good separation on TLC is the starting point for purifying a larger amount by column chromatography.
Common problems
| Problem | Likely cause | Fix |
|---|---|---|
| Streaks instead of spots | sample overloaded, or very polar/acidic compound | dilute the sample; add a little acid or base to the eluent |
| All spots at the baseline | eluent not polar enough | increase the polar component |
| All spots near the front | eluent too polar | increase the non-polar component |
| Crooked solvent front | plate touching the chamber wall or filter paper; uneven silica | place the plate carefully |
| Spots disappear | compound evaporated, or iodine stain faded | mark spots quickly |
| Extra spots on every lane | contaminated eluent or fingerprints | use clean solvent; handle plates by the edges |
Safety
- Wear eye protection.
- Organic solvents are flammable and often harmful: use them in a fume cupboard, away from flames.
- Don’t look directly at UV lamps; wear UV-protective glasses.
- Handle iodine and stains in a fume cupboard.
- Don’t breathe silica dust from scraped plates.
Key takeaways
- TLC uses a thin layer of silica or alumina as the stationary phase and an organic solvent as the mobile phase.
- On silica, non-polar compounds travel further (higher Rf); polar compounds stay lower.
- Colourless spots are made visible with UV light, iodine vapour or chemical stains.
- Adjust eluent polarity to get Rf values around 0.2–0.6.
- TLC checks purity, identifies compounds by co-spotting, follows reactions and guides column chromatography.
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