practical 12

• describe a method to separate amino acids using chromatography

  1. wear gloves and draw a pencil baseline at the bottom of the plate
  2. use a capillary tube to add a dot of each sample on the baseline
  3. allow to air dry
  4. add solvent to a beaker and place the plate in ensuring the solvent sits below the baseline
  5. cover the beaker to prevent the solvent from evaporating
  6. leave until the solvent reaches just below the top and mark the solvent front in pencil
  7. allow to dry in a fume cupboard
• describe how you would identify unknown amino acids from chromatography

  1. observe plate under UV light
  2. mark dots on plate with pencil (where amino acids had travelled)
  3. calculate rf values
  4. compare with the database and identify unknown amino acids
• why should gloves be worn during TLC?

  to prevent contamination of the chromatography plate
• why does the solvent need to sit below the baseline?

  to ensure it doesn't mix with the samples as they would dissolve and not move up the plate
• why does the beaker need to be covered and air tight?

  to prevent the solvent from evaporating
• how do you calculate rf values?

  distance travelled by the sample / distance travelled by the solvent front
• why does the paper need to be dried in a fume cupboard?

  toxic solvent will evaporate
• why does the solvent need to rise to near the top of the plate?

  so the solvent front can be marked in pencil and rf values can be calculated
• why does the solvent need to be below the baseline?

  so the solvent does not dissolve the solute (spots)so solute moves up the plate effectively
• why should tiny drops of each solution be used?

  so they don't merge
• why should the baseline be drawn in pencil?

  pencil is insoluble in the solvent so will not affect the results
• why do TLC plates need to be observed under UV?

  spots of organic solutes are colourless so this makes them visible
• what needs to be sprayed onto TLC plates to make amino acids visible?

  ninhydrin
• what do rf values tell you about the affinity of the solute?

  low rf value - moves slowly as has a stronger affinity for the stationary phase

  high rf value - moves quickly as has a stronger affinity for the mobile phase
• what is the mobile phase in chromatography?

  substance that flows over the stationary phase

  TLC - solvent

  Column - solvent

  Gas - inert gas
• what is the stationary phase in chromatography?

  substance that is fixed in place (solid or solid coated in liquid)

  TLC - powder on glass plate

  Column - powder

  Gas - powder
• what solvents are used in chromatography?

  non-polar: alkanes eg. hexane

  polar: water or alcohols
• what power is used as the stationary phase in chromatography?

  silicon oxide (SiO2) or aluminium oxide (Al2O3)
• why can substances (such as amino acids) be separated by TLC?

  they have different affinities to the mobile and stationary phases