Chromatography

• Chromatography


  Chroma = Colour; Graphein = Writing/Express
• Mikhail Tswett


  Russian botanist who invented chromatography in 1901
• Substances used by Mikhail Tswett


  • Petroleum ether
  • Chlorophyll
  • Calcium carbonate
  • Glass
• Mikhail Tswett's chromatography experiment


  Separated plant pigments using a column of calcium carbonate and petroleum ether
• Chromatography


  Separation and purification technique
• Basic principle of chromatography


  • Components in a mixture have different tendencies to: Adsorb onto a surface, Dissolve in a solvent
• Adsorption


  The adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface
• Requirements for chromatographic methods


  • A stationary phase (Static part- Solid or Liquid), A mobile phase (Moving part- Liquid or Gas)
• Paper chromatography experimental setup


  1. Draw a pencil line across the width of the paper strip, about one centimetre from the bottom end (base line)

  2. Place a concentrated solution of the mixture on the middle section of the pencil line using a capillary tube

  3. Hang the strip in a beaker/glass containing solvent. Note: The solvent level should be below the base line (should not touch the base line)
• Retention factor (Rf) value


  How much a substance is held back by the paper
• Calculating Rf values


  Rf = Distance travelled by substance / Distance travelled by solvent
• Separation of components in paper chromatography

  • Components of mixture separates because of its difference in: Adsorption on to the stationary phase and solubility in the mobile phase
• Separation of components in paper chromatography


  1. Solvent rises up by capillary action, taking some of the coloured substances with it

  2. Different substances have different solubilities so will travel at different rates, causing the substances to spread apart

  3. Substances with higher solubility will travel further than the others
• Retention Factor (Rf)


  Characteristic property of a given compound in a given solvent on a particular stationary phase
• Locating agent for colourless mixtures (amino acids)


  Ninhydrin binds to the chemicals in the spot, producing coloured spot or glowing under UV light
• Identifying amino acids present in a mixture


  • 1,2,3, 4 & 5 : Known amino acids and M : Unknown mixture of amino acids
• Locating agents for colourless mixtures


  Ninhydrin - used to locate colourless mixtures of amino acids, Resorcinol - used to locate colourless mixture of glucose
• Baseline drawn in ink is a mistake as the ink will separate and interfere with the chromatogram
• Baseline below solvent level is a mistake as the spots will dissolve into the solvent and not move up
• Baseline must be drawn in pencil as graphite/pencil is not soluble
• Baseline must be above the solvent, otherwise spots will dissolve into the solvent
• Key points in paper chromatography

  • Number of spots determined number of substances present in the mixture
  • Spots in the same position means it's the same substance
  • Substances which are MOST SOLUBLE travels the furthest
  • Spots that remain on the baseline indicates it is not soluble, therefore need to change the solvent
  • Water soluble substances use water as solvent, other substances use ethanol/alcohol as solvent
  • Colourless spots are sprayed with locating agent to identify position and make it visible
  • To determine number of substances in a sample of mixture: count the NUMBER OF SPOTS on the chromatogram
  • To identify substance in a sample of mixture: calculate Rf value and compare to Rf value chart