In thin-layer chromatography (TLC), the stationary phase is a thin layer of silica (SiO2) or alumina (Al2O3) spread on a glass, metal or plastic plate. The mobile phase is a liquid solvent. Components separate by adsorption: the more strongly a component is adsorbed onto the stationary phase, the more slowly it moves.
1. Draw a pencil baseline about 1 cm from the bottom of the plate.
2. Spot the mixture, and known reference compounds, on the line using a capillary tube.
3. Stand the plate in a beaker containing a little solvent. The solvent level must be below the baseline, or the spots would dissolve into the solvent.
4. Cover the beaker with a lid so the air inside is saturated with solvent vapour, which stops the solvent evaporating from the plate.
5. When the solvent has nearly reached the top, remove the plate and immediately mark the solvent front.
6. Locate colourless spots: many plates contain a fluorescent dye, so spots show up as dark patches under a UV lamp. Iodine vapour or a chemical developing agent can also be used.Each spot is identified by its Rf value:Rf = distance moved by spot ÷ distance moved by solvent front Worked example: a spot moves 2.4 cm and the solvent front moves 6.0 cm. Rf = 2.4 ÷ 6.0 = 0.40.TLC is quick and needs only tiny samples, so it is used to follow the progress of a reaction and to check the purity of a product: a pure compound gives one spot.
1. Draw a pencil baseline about 1 cm from the bottom of the plate.
2. Spot the mixture, and known reference compounds, on the line using a capillary tube.
3. Stand the plate in a beaker containing a little solvent. The solvent level must be below the baseline, or the spots would dissolve into the solvent.
4. Cover the beaker with a lid so the air inside is saturated with solvent vapour, which stops the solvent evaporating from the plate.
5. When the solvent has nearly reached the top, remove the plate and immediately mark the solvent front.
6. Locate colourless spots: many plates contain a fluorescent dye, so spots show up as dark patches under a UV lamp. Iodine vapour or a chemical developing agent can also be used.Each spot is identified by its Rf value: