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Thursday, July 18, 2013

Things to look for in a Hydrophobic Interaction Chroamtography


Things to look for in a Hydrophobic Interaction chroamtography are
  • Purity
  • Speed
  • Resolution
Sample viscosity can interfere in resolution so sample should be diluted prior to loading if the sample is viscous.

In HIC media is described based on
  1. Ligand
  2. Ligand Density
  3. 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.
HIC matrix dynamic binding capacity
Dynamic Binding Capacities of Different Matrices

Dynamic Binding Capacity is based on these factors:
  • Salt Concentration
  • Flow Rate
  • Temparature
  • pH to a lesser extent

Most commonly used salts in HIC are

Ammonium Sulfate, Sodium Sulfate, Sodium Chloride, Potassium Chloride, and Ammonium acetate.

Characteristics of good HIC Matrix

  • High Binding Capacity - Larger Area will results in high capacity binding
  • Physical Stability
  • Chemical Stability
  • Matrix should be Inert

Check the stability of protein at different salt concentrations.

Proteins shouldn't precipitate at high salt concentration, if protein precipitates there will no or very low yield.

Elution from the HIC column is in the increasing order of hydrophobicity.

Ligands Used Hydrophobic Interaction Chromatography meida has Alkyl or Aryl Groups.
Phenyl 650


  • Alkyl - Butyl, Octyl, Ether, Isopropyl - Eg: Butyl S
  • Aryl - Phenyl - Eg: Phenyl 650 - 
Alkyl shows pure hydrophobic character while Aryl shows mixed behaviour.

Binding Condition: 1M - 2M Ammonium Sulfate or 3M NaCl most frequently used.


For tightly bound proteins use harsh conditions like washing with 0.5M - 1.0M NaOH, 70% Ethanol or 30% IPA followed by water washes or salt free buffer washes.

References

Encyclopedia of Bioprocess Technology: FERMENTATION,BIOCATALYSIS, & BIOSEPARATION

Technical Resources on Chromatographic Media From GE, TOSOH, Shimadzu