12 - THIN-LAYER CHROMATOGRAPHY

• PREPARATION OF TLC PLATE?

  1. Wear gloves
  2. Draw pencil line on TLC plate, 1.5 cm from bottom
  3. Use capillary tube to apply small drop of solution and references onto pencil line
  4. Allow to completely dry
  5. Can repeat to increase concentration
• DEVELOPING THE TLC PLATE?

  1. Add no more than 1 cm solvent to developing tank
  2. Place TLC plate into developing tank
  3. Allow solvent to rise up plate
  4. Remove plate, mark position of solvent front with pencil
  5. Allow to dry in a fume cupboard
• ANALYSING WHEN COMPONENTS ARE COLOURLESS?

  e.g. amino acids

  1. UV light used to temporarily reveal spots, circle in pencil before calculating Rf
  2. Or spray locating agent (e.g. ninhydrin) to stain molecule so it is easily seen
• ANALYSING WHEN AMINO ACIDS HAVE SIMILAR Rf?

  similar structures so sometimes similar Rf in given solvent

  • use two-dimensional TLC
  • sample of amino acids spotted in corner of square TLC plate
  • run as usual so compounds separate
  • turn 90 degrees and run in different solvent
  • Rf per solvent is calculated and compare to data values
• ANALYSING RESULTS?

  • calculate Rf
  • compare to data values
• WHY IS IT ESSENTIAL TO WEAR GLOVES?

  prevent contamination of TLC by hands
• WHY IS IT ESSENTIAL TO ADD A SOLVENT OF DEPTH NO MORE THAN 1 cm?

  may dissolve mixture from plate if too deep
• WHY IS IT ESSENTIAL TO DRY IN FUME CUPBOARD?

  solvent is toxic and should be not inhaled
• WHY DO AMINO ACIDS HAVE DIFFERENT Rf VALUES?

  • different polarities
  • different solubility in mobile phase
  • different affinity in stationary phase so different retention times