3.16 Chromatography

• • Chromatography can be used to separate and identify the components in a mixture.
  • Types of chromatography include:
  • • thin-layer chromatography (TLC) – a plate is coated with a solid and a solvent moves up the plate
  • • column chromatography (CC) – a column is packed with a solid and a solvent moves down the column
  • • gas chromatography (GC) – a column is packed with a solid or with a solid coated by a liquid, and a gas is passed through the column under pressure at high temperature.
• • Separation depends on the balance between solubility in the moving phase and retention by the stationary phase.
  • Retention times and Rf values are used to identify different substances.
• Amino acids can be located on a chromatogram using developing agents such as ninhydrin or ultraviolet light and identified by their Rf values.
• State why each amino acid has a different Rf value.

  • each amino acid has different (relative) affinity/attraction to/solubility in stationary and mobile phases
  • allow reference to different solubility in solvent OR different affinity for stationary phase
• Suggest a suitable reagent for the hydrolysis of a protein

  • CONCENTRATED Hydrochloric acid
• State in general terms what determines the distance travelled by a spot in TLC. [1]

  • (Balance between) solubility in moving phase and retention by stationary phase
• To obtain the chromatogram, the TLC plate was held by the edges and placed in the solvent in the beaker in the fume cupboard. The lid was then replaced on the beaker. Give one other practical requirement when placing the plate in the beaker.

  • Solvent depth must be below start line (ignore safety)
• TLC practical procedure

  Wear plastic gloves

  • Essential – to prevent contamination from the hands to the plate
• Add developing solvent to a depth of not more than 1 cm3

  • Essential – if the solvent is too deep it will dissolve the mixture from the plate
• In gas-liquid chromatography GC

  • the mobile phase is a inert gas such as nitrogen, helium, argon.
  • The stationary phase is a liquid on an inert solid.
• If the stationary phase was polar and the moving phase was non- polar e.g. hexane.

  • Then 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. (Think about intermolecular forces)
• RP12

  • Lid– to prevent evaporation of toxic solvent
  • tiny drop – too big a drop will cause different spots to merge
  • dry in a fume cupboard as the solvent is toxic
  • UV lamp used if the spots are colourless and not visible
• Two directional chromatography

  • To separate a mixture that has components of different solubility in solvents, do chromatography with two different solvents.
  • A spot of the mixture on a TLC plate is first separated with one solvent.
  • Then the TLC plate is rotated 90 degrees and the plate is placed in a second solvent for a second separation to take place
• This mixture was separated by COLUMN chromatography. Outline briefly why chromatography is able to separate a mixture of compounds. Practical details are not required.

  • M1 phase or eluent or solvent (or named solvent) is moving or mobile 1
  • M2 stationary phase or solid or alumina/silica/resin 1
  • M3 separation depends on balance between solubility or affinity (of compounds) in each phase
  • OR different adsorption or retention
  • OR (amino acids have) different Rf values
  • OR (amino acids) travel at different speeds or take different times
• The dipeptide in part (d) is hydrolysed in acid conditions and the mixture produced is analysed by column chromatography. The column is packed with a resin which acts as a polar stationary phase. Suggest why lysine leaves the column after alanine.

  • M1 In acid lysine has double positive or more positive charge 1
  • M2 (Lysine ion) has greater affinity / greater attraction / adheres better / sticks better to polar / stationary phase

  
• Explain why J and K (cys-asp dipeptide) can be separated by gas chromatography

  • Different retention times / dipeptides appear at different times.
  • Different balance between solubility in the moving phase / gas carrier and retention by the stationary phase / column OR different relative affinity for mobile and stationary phases.
• Explain why J and K using electrospray ionisation, does not enable you to identify them

  • Same m/z values. 1
  • Both dipeptides / J and K have same molecular formula / Mr.