Separation of molecule by ion exchange chromatography

Separation of molecule by ion exchange chromatography

  • Proteins are complex ampholytes i.e. they have both positive and negative charges and can be separated from a mixture of compounds on the basis of net positive or negative charge that carry.
  • Isoelectric point of a protein (pI) is the pH at which its net charge is zero (i.e., number of positive and negative charges are equal).
  • Therefore, proteins will have either a net negative charge or net positive charge depending on the pH of solution and thus, it is possible to separatrdy use either an anion exchanger or a cation exchanger. 

 Principle

  • A solution containing protein is applied to the ion exchanger.
  • Protein bind to the ion exchanger depending upon net charge at that particular pH and on    the ionic strength of the mobile phase.
  • Bound protein is then eluted out from the stationary phase by increasing the concentration of counter ions or by changing pH, which alters the charge on the protein.
  • Weakly charged proteins is displaced from the stationary phase with lower concentration of counter ion than highly charged protein.
  • This results in separation of protein based upon its net charge.  
  • Extent of separation and purification (e.g. Enzyme) can be determined by computing its specific activity.
  • Specific activity of the enzyme: It is the ratio of enzyme activity to mass of protein in the sample, usually expressed as units of activity per milligram of protein U/mg.
  • As the enzyme is purified, other proteins in the mixture are eliminated while most of the enzyme activity is retained.
  •  By determining the specific activity before and after purification, one can determine the purification and yield of the enzyme.
    • Last modified: Monday, 19 March 2012, 6:37 AM