practical biochemistry 3

• chromatography

  • stationary phase - this is either the chromatography paper or TLC plate - paper is made of cellulose, TLC is often a sheet of plastic coated with a layer of silica gel or aluminium hydroxide (all have free -OH groups pointing outwards)
  • mobile phase - solvent for biological molecules ; we can use water or ethanol for non polar

  -> mobile phase flows through and across the stationary phase, carry biological molecules
• chromatography 2

  to identify the the pigments you can use the relative distance travelled by calculation the Rf value

  • distance from pencil line to centre of spot of pigment
  • distance from pencil line to solvent front
• chromatography with colourless molecules there are solutions :

  1. ultraviolet light - TLC plates have a chemical which fluoresces under UV light (under plate most of it will glow except the pigment spot)
  2. ninhydrin - to see amino acids allow to dry and spray with ninhydrin so it binds to amino acids
  3. iodine - allow plate to dry and place in an enclosed container with a few iodine crystals - iodine forms a gas which binds to the molecules in each spot
• TLC (thin layer chromatography) is commonly used to monitor the progress of reactions because it works relatively quicker than other methods