Hydrophobic interaction chromatography [HIC] Theory and Principle


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Hydrophobic Interaction chromatography,utilizes the reversible interaction of protein and the hydrophobic ligand for the separation of protein mixtures.
Proteins, separates out based on, the increasing order of hydrophobicity.
Proteins containing, hydrophobic and hydrophilic regions are applied to a HYDROPHOBIC INTERACTION CHROMATOGRAPHY column, in a high-salt buffer.

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Hydrophobic interaction chromatography [HIC] Theory and Principle

  1. 1. Hydrophobic Interaction Chromatography [HIC] Theory & Principle Bio-Resource www.technologyinscience.blogspot.com
  2. 2. Hydrophobic Interaction Chromatography www.technologyinscience.blogspot.com
  3. 3. Hydrophobic Interaction Chromatography - Introduction • HIC is a powerful technique for separation and purification of bio molecules. • This technique was initially termed as “Salting Out”. • HIC is widely used for Protein purification, isolating protein complexes and studying protein folding and unfolding. www.technologyinscience.blogspot.com
  4. 4. Hydrophobic Interaction Chromatography • Sample molecules(Proteins) containing hydrophobic and hydrophilic regions (amino acids) are applied to an HIC column in a high-salt buffer. • The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. • The more hydrophobic the molecule, the less salt is needed to promote binding. • Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. • Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution buffer. www.technologyinscience.blogspot.com
  5. 5. Hydrophobic Interaction Chromatography - Theory • Salting Out and Hydrophobic Interaction. • Thermodynamic Theory. • Surface Tension, van der Waals Forces. www.technologyinscience.blogspot.com
  6. 6. Salting Out and HIC • Precipitation by Salting out and HIC are based on same phenomena. • In HIC, hydrophobic interaction happens at much lower salt concentration than that happens in the precipitation (Salting out). www.technologyinscience.blogspot.com
  7. 7. hofmeister series salt www.technologyinscience.blogspot.com
  8. 8. Salting in – Salting out www.technologyinscience.blogspot.com
  9. 9. HIC – Salting out www.technologyinscience.blogspot.com
  10. 10. Thermodynamic Theory - HIC • Thermodynamic theory of HIC directly relates to Gibb’s Free Energy equation: ΔG = ΔH – TΔS ΔG – Gibbs Free Energy change ΔH – Change in enthalpy or heat T –Temperature in Kelvin ΔS – change is disorder or randomness www.technologyinscience.blogspot.com
  11. 11. HIC Thermodynamics • When a Hydrophobe immersed in water, it will be surrounded by water molecules. An ordered shell of water molecule will be formed around the hydrophobe. • When salt is added, the water molecules forms hydrogen bonding with the salt causing the disruption of water shell. • When protein is introduced into such system, to minimize entropy hydrophobic regions of the proteins are forced to merge to the hydrophobe of the HIC media. www.technologyinscience.blogspot.com
  12. 12. HIC Hydrophobe - Protein Interaction Mechanism Hydrophobe water shell around hydrophobe Disordered water shell after addition of salt To minimize entropy protein is forced to merge towards the hydrophobe www.technologyinscience.blogspot.com
  13. 13. Surface Tension, van der Waals Forces • A third theory suggests that van der Waals forces are responsible for the hydrophobic interaction in HIC. • This is supported by the fact that these forces should be increased as the order of water increases in the presence of salt. www.technologyinscience.blogspot.com
  14. 14. HIC Media / Matrix In HIC, media is described based on • Ligand • Ligand Density • Available Capacity: It is the actual amount of protein that can bind to the media. If flow rate is included in the defined conditions then it is called as Dynamic Binding Capacity. www.technologyinscience.blogspot.com
  15. 15. Dynamic Binding Capacity Dynamic Binding Capacity is based on these factors: • Salt Concentration • Flow Rate • Temperature • pH to a lesser extent www.technologyinscience.blogspot.com
  16. 16. HIC Media – Dynamic Bindin Capacity www.technologyinscience.blogspot.com
  17. 17. HIC Matrix - Characteristics Characteristics of good HIC Matrix • High Binding Capacity - Larger Area will results in high capacity binding • Physical Stability • Chemical Stability • Matrix should be Inert www.technologyinscience.blogspot.com
  18. 18. Salts Used in HIC Most commonly used salts in HIC are • Ammonium Sulfate, • Sodium Sulfate, • Sodium Chloride, • Potassium Chloride, and • Ammonium acetate. www.technologyinscience.blogspot.com
  19. 19. HIC Ligands • Ligands Used in Hydrophobic Interaction Chromatography meida has Alkyl or Aryl Groups. • Alkyl - Butyl, Octyl, Ether, Isopropyl - Eg: Butyl S • Aryl - Phenyl - Eg: Phenyl 650 • Alkyl shows pure hydrophobic character while Aryl shows mixed behaviour. • Ligand attachment to the matrix in HIC is done through glycidyl ether. www.technologyinscience.blogspot.com
  20. 20. Binding and Elution in HIC Binding is done at high salt concentration • 1 – 2 M Ammonium Sulfate or • 3M Nacl Elution is performed by reducing the salt concentration • Elution of proteins from the HIC will be in the increasing order of hydrophobicity. www.technologyinscience.blogspot.com
  21. 21. References • Protein Purification Technical Resources, GE Amersham • Protein Purification Technical Resources, Biorad • Protein Purification Technical Resources, TOSOH Biosciences • Hydrophobic Interaction Chromatography:Fundamentals and Applications in Biomedical Engineering, Andrea Mahn • Hydrophobic Interaction Chromatography, Encyclopedia of Bioprocess Technology • Calibration and Optimization of Hydrophobic Interaction Chromatography , Alex Olsson www.technologyinscience.blogspot.com
  22. 22. Bio-Resource http://www.technologyinscience.blogspot.com/2013/07/things- to-look-for-in-hydrophobic.html www.technologyinscience.blogspot.com