chromotography

• Chromatography separates dissolved substances by their solubility
• Paper chromatography -

  • The mixture we want to separate is placed on paper.

  • This is then stood in a solvent (liquid).

  • As the solvent moves along the paper, it dissolves the ink.

  • It carries the different substances in the mixture at different speeds, and they become separated.

  • The more soluble the substance the further it will move up the chromatography paper
• TLC is Thin Layer Chromatography - Thin Layer Chromatography can be used to separate biological molecules and compounds. It can be used to separate and then identify amino acids in a solution

  
• Mobile phase - Organic solvent, the substance in chromatography that carries the soluble components of the mixture
• Stationary phase - thin layer of silica gel on sheet of glass or metal, the substance in chromatography that holds back the components that are attracted to it.
• The rate at which the different amino acids in the solvent move through the silica gel depends on the interactions they have with the silica in the stationary phase, and their solubility in the mobile phase. Different amino acids have different R groups, so they all have different affinities for the solvent
• 1. Wearing gloves draw a pencil line 2cm from the bottom, only touch the plate by the edges.
  2. mark Four equally spaced points along the pencil line
  3. spot the amino acid solution onto the first pencil mark using a capillary tube. let it dry and repeat then label with a pencil
  4. repeat for the next 3 amino acids
  5. place the plate in the solvent jar and close the lid. The solvent was no more than 1cm deep
  6. leave the plate in the solvent until about 2cm from the top. draw a line across the solvent front and leave to dry
  7. In a fume cupboard spray the plate with ninhydrin spray
• Amino acids are colourless, so to make them visible a developing agent is used, such as ninhydrin or ultraviolet light
• The Rf value stands for the retention value. This is the distance travelled by the pigment divided by the distance travelled by the solvent.

  Rf = Distance moved by Component / Distance moved by solvent