Chromatography

• Chromatography


  An analytical technique that separates components in a mixture between a mobile phase and a stationary phase
• Mobile phase


  • Liquid
  • Gas
• Stationary phase


  • Solid (as in thin-layer chromatography, TLC)
  • Liquid or solid on a solid support (as in gas chromatography, GC)
• Solid stationary phase


  • Separates by adsorption
• Liquid stationary phase


  • Separates by relative solubility
• If the stationary phase was polar and the moving phase was non-polar e.g. hexane

  Non-polar compounds would pass through the column more quickly than polar compounds as they would have a greater solubility in the non-polar moving phase
• Separation by column chromatography

  Depends on the balance between solubility in the moving phase and retention in the stationary phase
• Gas-liquid chromatography GC


  The mobile phase is an inert gas such as nitrogen, helium, argon, the stationary phase is a liquid on an inert solid
• Types of chromatography


  • Thin-layer chromatography (TLC)
  • Column chromatography (CC)
  • Gas chromatography (GC)
• Retention times


  Used to identify different substances
• Rf values


  Used to identify different substances
• Thin-layer chromatography method


  1. Draw pencil line 1 cm above bottom of TLC plate

  2. Mark spots for each sample

  3. Use capillary tube to add tiny drop of each solution

  4. Add solvent to chamber, level below pencil line

  5. Place TLC plate into chamber

  6. When solvent reaches 1 cm from top, remove plate and mark solvent level

  7. Allow plate to dry in fume cupboard

  8. Place plate under UV lamp

  9. Calculate Rf values
• Two directional chromatography


  Separates a complex mixture by using two different solvents in sequence
• HPLC


  High performance liquid chromatography, a type of column chromatography commonly used in industry, with a solid silica stationary phase and a liquid mobile phase
• Column chromatography (CC)


  1. Glass tube filled with stationary phase (usually silica or alumina)

  2. Filter or plug used to retain solid

  3. Solvent added to cover powder

  4. Mixture to be analysed dissolved in minimum solvent and added to column

  5. Solvent or solvent mixture run through column

  6. Time for each component to reach end of column recorded (retention time)
• Gas-liquid chromatography


  • Mobile phase is an inert gas, stationary phase is a high boiling point liquid absorbed onto a solid
  • Retention time can be used to identify substances
  • Number of peaks indicates number of components, area under peaks indicates abundance
• Factors that can change retention times in gas-liquid chromatography

  GC column temperature, column length, flow rate
• It is important to use an inert carrier gas such as helium or nitrogen in gas-liquid chromatography