Chromatography

• Chromatography can be used to separate and identify the components in a mixture
• Types of chromatography

  • Thin layer
  • Column
  • Gas
  • High performance liquid
• Thin layer chromatography (TLC)

  • The stationary phase is the plate which has SiO2 on it
  • The mobile phase is the solvent
  • Its use to separate drugs and analyse antibiotic
• Column chromatography

  • The stationary phase is the solid matrix (powder in column)
  • The mobile phase is the solvent
  • Its used to purify substances e.g. separating out a catalyst
• Gas chromatography

  • The stationary phase is the long coiled tube packed with powder
  • The mobile phase is inert gas mainly N2, He and H2
  • Can be used for blood testing
  • Can be used with a mass spectrometer to find a substance e.g. drugs in athletes
• Substances separated according to relative affinity (attraction) to stationary and mobile phase.

  • If stronger affinity for mobile phase, then the substances move quickly up the plate
  • If stronger affinity for stationary phase, then they move slower
• Ways to make spots more visible in chromatography:

  • YOU can use UV light (ultraviolet)
  • Ninhydrin
• Method : thin layer chromatography

  1. Wearing gloves, draw a pencil line just over 1cm above bottom of a TLC plate and mark spots for each sample equally spaced along line
  2. Use a capillary tube add a tiny drop of each solution to a different spot, allow it to dry and add another drop repeat two more times
  3. Add solvent to a large beaker with a lid and then place the TLC plate into the chamber just touching the solvent and make sure the lid is on tight
• Method : TLC chromatography pt2

  1. Wait for the solvent to reach near the top of the plate and then remove it and mark with a pencil the solvent line
  2. Place the plate in fume cupboard to dry
  3. Then place plate under UV lamp in order to see the spots. Don't allow your hand to be under the lamp. Draw around them lightly in pencil .
  4. Calculate the Rf value for the observed spots
• Why do we wear gloves when holding the TLC plate

  To avoid any contamination from hands to plate
• Why do you add the solvent to a depth not more than 1cm?

  So it won't dissolve the mixture from the plate
• Why do we allow the plate to dry in a fume cupboard?

  Because the solvent is toxic
• How do you work out Rf values

  Distance moved by spot / Solvent front
• Chromatography column

  A) Mobile phase

  B) Sample

  C) Stationary phase

  
• What determines the distance travelled by a spot in TLC

  Solubility in the mobile phase and the retention by the stationary phase
• You used ninhydrin to see amino acid spots on the TLC plate due to the spots being colourless