chromatography and analysis

• 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
• TLC Chromatography (thin-layer chromatography)

  1. Take chromatography paper and draw a pencil line 1.5cm from bottom

  2. With a capillary tube put a small drop of amino acid on pencil line

  3. Roll up paper and stand it in a large beaker

  4. The solvent in the beaker should be below the pencil line

  5. Allow to stand for 20 mins and mark final solvent level

  6. Spray paper with ninhydrin and put in oven
• Rf value


  Distance moved by amino acid / distance moved by the solvent
• Some substances won't separate because similar compounds have similar Rf values. So some spots may contain more than one compound
• Thin-layer chromatography


  1. Draw a pencil line 1 cm above the bottom of a TLC plate and mark spots for each sample, equally spaced along line

  2. Use a capillary tube to add a tiny drop of each solution to a different spot and allow the plate to air dry

  3. Add solvent to a chamber or large beaker with a lid so that is no more than 1cm in depth

  4. Place the TLC plate into the chamber, making sure that the level of the solvent is below the pencil line. Replace the lid to get a tight seal

  5. When the level of the solvent reaches about 1 cm from the top of the plate, remove the plate and mark the solvent level with a pencil. Allow the plate to dry in the fume cupboard

  6. Place the plate under a UV lamp in order to see the spots. Draw around them lightly in pencil

  7. Calculate the Rf values of the observed spots
• Gas-Liquid Chromatography


  The mobile phase is a gas such as helium and the stationary phase is a high boiling point liquid absorbed onto a solid
• Retention time


  The time taken for a particular compound to travel from the injection of the sample to where it leaves the column to the detector
• Some compounds have similar retention times so will not be distinguished
• Basic gas-liquid chromatography


  Will tell us how many components there are in the mixture by the number of peaks and the abundance of each substance by the area under each peak
• It is also possible for gas-liquid chromatography machine to be connected to a mass spectrometer, IR or NMR machine, enabling all the components to be identified in a mixture
• GC-MS is used in analysis, in forensics, environmental analysis, airport security and space probes
• Calibration


  Known amounts of a pure component can be passed through the GC machine. The calibration curve will give the retention time of the component and the area under the curve (the peak integration value) will be a measure of the pure concentration. This can then be compared with the retention times and integration values of the components in the mixture to work out the amounts and proportions of the components in a mixture
• Testing for functional groups

  • Alkene: Bromine water (orange colour decolourises)
  • Carbonyl: 2,4-DNP (orange precipitate formed)
  • Aldehyde: Tollens' Reagent (silver mirror formed)
  • Carboxylic acid: Carbonate ions CO3^2- (effervescence of CO2 evolved)
  • 1^o 2^o alcohol and aldehyde: Sodium dichromate and sulphuric acid (orange to green colour change)
  • Haloalkane: Warm with aqueous silver nitrate in ethanol (slow formation of white precipitate)
  • Phenols: React with sodium and sodium hydroxide, won't react with carbonate ions CO3^2- (fizzing with sodium but no reaction with sodium carbonate)