Principles of chromatography

• what is chromatgraphy?

  relative affinity of molecule for stationary and mobile phase
• what happens if something a has a greater affinity for mobile phase?

  moves further up paper
• what is the stationary phase of SEC?

  beads in a colum
• what is the mobile phase of SEC?

  buffer running through column
• what physical properties of proteins aid purification?

  charge (ion exchange), hydrophobicity (hydrophobic interaction), biorecognition (affinity, eg ligand specificity), size (size exclusion)
• what does SEC mean?

  size exclusion chromatography (also called gel filtration chromatgraphy)
• Ion exchange types:

  • anion exchange
  • cation exchange
• cation exchange chromatography:

  • The negative charge, on the resin bead is counterbalanced by positively charged protein.
  • These counter ions are loosely attached to the matrix and can change places with ions (proteins) of similar charge in solution
• Ion exchange basis:

  • protein with a similar charge (cation exchange = more negative, anion exchange = more positive) will travel further through the matrix based on the functional groups attached to the amino acids
• Hydrophobic interaction chromatograph:

  • separate protein based on the extent of hydrophobic amino acids at the molecule surface
  • Resins use hydrophobic side chain in the immobile phase of the column
  • proteins are applied at high ionic strength (salt) - salt pulls away water from protein hydrophobic patches associate with bead
  • least hydrophobic protein elute first
  • decrease salt so water molecules are able to mask hydrophobic protein patches
  • most hydrophobic protein elute last
• what increases the solvation energy of protein?

  the interaction between the protein and a water molecule
• how is a ligand linked to the column?

  using spacer arms (14 long hydrocarbon chain)
• what is the function of a spacer arm?

  space ligand away from bead

  Allows protein of interest to more easily to bind to ligand
• What makes up beads in SEC?

  polymeric sugar molecules
• Why do smaller molecules take longer to come through SEC?

  because they fit into the pores on the bead meaning the path through is longer
• what is the void volume in SEC?

  Substances that do not pass through the pores of the bead
• what is an elution profile?

  Shows the molecular weight of substances passed through the column