Chromatography

Last updated Jun 26, 2023 Edit Source

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• Chromatography is a group of techniques used to separate mixtures of compounds
  • Chroma - Colour
  • Graph - To Draw
  • It is a separation technique, but for the course we go into more depth
• A mobile phase moves through or across a stationary phase
  • Stationary Phase
    • This phase does not move
    • Compounds in the mixture are attracted to it (absorbed) and slowed down
    • Either a solid or a liquid
    • e.g. special chromatography paper for paper chromatography
  • Mobile Phase
    • This phase moves
    • The more soluble compound in the mixture are carried faster as the mobile phase moves
    • Either a liquid or a gas
    • e.g. the solvent in paper chromatography

• The mobile phase (solvent) is drawn up the stationary phase by capillary action
• Capillary action occurs due to the stationary phase being polar
  • The sample substances are attracted to the stationary phase and so they cling onto the stationary phase allowing them to be dragged upwards
• Adhesive Forces are between molecules of different substances
  • Adhesion enables solvent molecules to bond to stationary phase
• Cohesive Forces are between molecules of the same substance
  • Cohesion enables solvent molecules to bond to each other and move each other through stationary phase
• Diagram:
• Video:

• # Paper Chromatography

  

  • Test tube is filled slightly with solvent → mobile phase
    • For the paper chromatography to work, the solvent must be able to dissolve the mixture
    • Water is normally a good solvent
    • The solvent must be polar
  • Line of origin is drawn on the chromatography paper → stationary phase
    • When the stationary phase is placed in the test tube, the line of origin must be above the solvent
    • The line of origin must be drawn with an insoluble mixture
      • e.g. pencil is a good option
        • pen must never be used since it will dissolve
    • The stationary phase must be polar as the different parts of the mixture need to have different levels of attraction to it
  • Dot of mixture is placed on line of origin
    • It must not be submerged in the solvent
  • The mobile phase is absorbed up the paper and will drag the mixture along the paper
    • This will happen over a slow period of time
  • The dye will separate into different colours at different points as it is drawn up the paper by capillary action
    • The further a colour is dragged, the less attraction it has to the paper (less polar)
      • More distance along paper → Less polar

• # Thin Layer Chromatography (TLC)

  • TLC uses same principals as paper chromatography
  • Capillary action still draws the solvent up the matrix
    • However while the molecules in paper chromatography are separated based on mass, in TLC, separation often depends upon solubility or charge
  • Silica Gel → Stationary Phase
    • The silica gel is on top of a glass plate
    • Silica gel is very polar
  • Solvent → Mobile Phase
  • Method
    • A dry sample is placed in the silica gel matrix
      • A lid should be used on top of the beaker to prevent the solvent from evaporating
        • This also applies to paper chromatography
    • As the solvent front moves up the gel, it dissolves the sample and carries it up the matrix with it
    • Some of the particles in the sample stick more strongly to the silica gel than others, so they lag behind the solvent
      • The less polar the substance, the further it will travel due to less attraction between it and the silica gel
    • Eventually the different substances in the sample separate out with similar molecules travelling a similar distance
    • These substances are not actually visible by normal means, you must use a UV lamp or some other special technique to illuminate the different substances
  • Advantages of TLC:
    • Silica gel is reusable
    • Glass plate is rigid unlike chromatography paper
      • Easier to use

• # Gas Chromatography (GC)

  • For gas chromatography, the mobile phase is an inert gas
    • Inert means non-reactive
      • This is very important as you don’t want the mobile phase to be reacting with your samples
  • The stationary phase is usually a long, thin tube of silica gel
  • Diagram
  • Method:
    • The sample is injected into the machine
      • Sample is vaporised
    • Sample is pushed by the inert gas through a long tube
      • This tube is usually coiled and can be very long, exemplifying the results
    • A detector at the end of the tube records the intensity of the amount of substances (including the solvent which is usually the first substance to make it to the end) that make it there at a given time
      • This is then graphed and displayed on a monitor
  • Substances are separated because they take different times to travel through the tube with some clinging to the stationary phase more and some not
    • The more a substance is attracted to the stationary phase, the longer it takes to make it through the tube

• # High Performance Liquid Chromatography (HPLC)

  • HPLC is a development of column chromatography where the mobile phase is pumped through the column at high pressure
    • It is better and faster than standard column chromatography
  • The mobile phase is passed thru (using pressure) the stationary phase
  • Diagram: 400

• Retention Factor ($R_f$)
  • How far the solutes have moved relative to the solvent’s movement
    • The further a substance travels, the greater the retention factor
  • $R_f$ = Solute distance/Solvent distance