(s) (i) the principles and uses of paper and thin layer chromatography to…

The idea

Chromatography separates molecules by differential partitioning between a mobile phase and a stationary phase. In paper chromatography the stationary phase is water held in cellulose fibres; in thin layer chromatography (TLC) it is a thin layer of silica gel or cellulose on a glass or plastic plate. The solvent (mobile phase) moves up by capillary action, carrying dissolved molecules. Molecules that interact more strongly with the stationary phase travel more slowly, so each compound moves a characteristic distance. The result is expressed as an Rf value: Rf = distance travelled by the spot ÷ distance travelled by the solvent front. Rf is a ratio with no units and is always less than 1. Because Rf depends on the solvent and conditions, comparisons must use the same solvent and temperature.

What earns the marks

• Paper chromatography uses water held in cellulose as the stationary phase; TLC uses a thin layer of silica gel or cellulose on a plate as the stationary phase.
• In both methods the mobile phase is a solvent that moves through the stationary phase, carrying the dissolved molecules with it.
• Separation depends on differential partitioning: molecules with greater affinity for the stationary phase move more slowly than those with greater affinity for the mobile phase.
• Rf = distance travelled by the spot ÷ distance travelled by the solvent front; it is a unitless ratio less than 1.
• Rf values are only comparable when the same solvent, temperature and conditions are used, so known standards are run alongside samples.
• Uses include separating and identifying amino acids, sugars and photosynthetic pigments in biological mixtures.

Common mistakes and how to put them right

• Thinking the solvent front is the top of the paper or plate.

  Put it right: The solvent front is the furthest point the solvent itself has reached, and it must be marked before it dries.

• Measuring Rf from the origin to the top of the paper rather than to the centre of the spot.

  Put it right: The measurement is from the origin line to the centre of the spot.

• Assuming Rf values are fixed constants for a substance.

  Put it right: They depend on solvent, temperature and stationary phase, so conditions must be standardised for comparison.

• Confusing which phase is stationary.

  Put it right: In paper chromatography the cellulose holds water as the stationary phase, while the moving solvent is the mobile phase.

Exam tips

• Always state both phases explicitly: name the stationary phase and the mobile phase for the method described.
• When calculating Rf, show the two measured distances and divide spot distance by solvent-front distance, keeping the answer below 1.
• Link separation to relative affinity for the two phases rather than saying molecules are simply 'pulled up' at different speeds.
• For identification questions, mention comparing the sample Rf with Rf values of known standards run under the same conditions.