RP12

• Chromatography


  An analytical technique used to separate and identify components of a mixture
• Thin-layer chromatography (TLC)


  • Typically used in A-level chemistry to identify amino acids after the hydrolysis of a peptide
• Preparation of the TLC Plate

  1. Draw a pencil line 1.5cm from the bottom

  2. Use a capillary tube to apply a small drop of sample, reference (b) and reference (y) to the pencil line

  3. Allow the spots to completely dry
• Developing the TLC Plate

  1. Add no more than 1cm depth of solvent to the developing tank

  2. Place the TLC plate into the developing tank

  3. Allow the solvent to rise up the plate

  4. Mark the position of the solvent front with a pencil

  5. Allow the plate to dry in a fume cupboard
• RF value


  The ratio of the distance travelled by the spot to the distance travelled by the solvent
• Analysing results when components are colourless

  • Use UV light to temporarily reveal the spots and circle in pencil to calculate RF values
  • Spray with a locating agent like ninhydrin to stain the molecules and make the spots visible
• Analysing results when amino acids have similar RF values

  • Use two-dimensional TLC to give a greater degree of separation
  • Spot the sample in the corner of a square TLC plate and run in one solvent, then turn 90 degrees and run in a different solvent
• Amino acids have similar structures, so it is not unusual for them to have similar RF values in a given solvent
• Wearing plastic gloves to hold the TLC plate is essential to prevent contamination
• Adding the developing solvent to a depth of not more than 1cm is essential, as a deeper solvent could dissolve the mixture from the plate
• Allowing the solvent to rise to the top of the plate is not essential, as the RF value will be the same regardless of how far the solvent travels
• Allowing the plate to dry in a fume cupboard is essential, as the solvent is toxic and should not be inhaled
• Locating the positions of the amino acids on the TLC plate and determining their RF values


  1. Spray with a developing agent or use UV light

  2. Measure the distances from the initial pencil line to the spots (x)

  3. Measure the distance from the initial pencil line to the solvent front line (y)

  4. Calculate: RF value = x / y
• Why amino acids have different RF values

  They have different polarities, so they have different retention times on the stationary phase or different solubilities in the developing solvent