Chromatography

• what is thin layer chromatography?

  a technique used to separate the individual components of a mixture

  • can be used to separate and identify a mixture of amino acids in solution
• what is the stationary phase?

  • the layer of absorbent material
  • silica gel or aluminium oxide coated on a rigid surface - glass, plastic, aluminium
  • doesn’t move
• what is the mobile phase?

  • picks up the amino acids, moves through stationary phase and amino acids are separated
  • moving substance
  • the water or ethanol moving
• what factors affect how quickly the molecules travel along the paper or TLC plate?

  • interactions (hydrogen bonds) they have with the silica in the stationary phase
  • solubility in the mobile phase
  • temperature
  • attraction to the paper (adhesion)
• how do you calculate Rf values?

  distance moved by substance / distance moved by solvent
• describe how UV light is a method of observing the results if molecules being separated are colourless

  • TLC plates have a chemical that fluoresce under UV light - any molecules on the plate will block the UV light, so these areas won’t glow under UV light
• describe how Nihydrin is a method of observing the results if the molecules being separated are colourless

  • This is a chemical that stains amino acids
  • one the plate is dry, spray is with nihydrin and the amino acids will appear as brown or purple spots
• describe how iodine is a method of observing the results if the molecules being separated are colourless

  • once the plate is dry, place it in a container with a few iodine crystals
  • the iodine forms a gas, which binds to the molecules on the plate
• what does Rf stand for?

  retardation factor
• what is Rf?

  • every pigment will have a specific Rf value for a certain solvent
  • used to identify pigments by comparing to known values
  • will always be a number between 0 and 1
• why is a pencil used to draw lines of the TLC plate?

  pencil will remain stationary (won’t move with the mobile phase) and isn’t soluble (pen is soluble)
• why must gloves be worn when handling TLC plates?

  you may contaminate the paper with biological materials present on your hands (e.g. lipids)
• why is a mixture of solvents usually used as the mobile phase when separating a mixture of amino acids?

  • there are some polar amino acids that aren’t soluble in water
  • true to cater to different solubility of amino acids
• why is the solvent added to no more than 1cm depth?

  so that the solvent doesn’t touch the pencil line - substance would dissolve in the solvent and not move up the paper with the solvent
• why is the Rf values might be different if a different solvent is used?

  substances may have different solubility in different solvents
• why is the Rf values different when filter paper is used instead of a TLC plate?

  different levels of attraction and absorbance
• why is it important that the sides of the TLC plate do not touch the sides of the container?

  • solvents on the sides of the container may cause contamination
  • adhesion to sides of container may affect mobile phase