Hydrophobic interaction chromatography (HIC) is an effective method for purification and separation of proteins (especially enzymes) based on differences in their surface hydrophobicity. Since this method does not use organic solvents like reversed phase chromatography, there is only a little loss in enzyme activity and the tercially structure of proteins (denaturation). COMSOSIL 5HIC is a packed column for hydrophobic interaction chromatography on silica gel base.

Characteristics

• No need for organic solvents.(Protein is not denatured.)
  
• Allows high pressure and high flow rate because of silica gel base.
  
• No problem with swelling like with polymer gel bases.
  
• Economic solution.

Separation of protein standards

Column: COSMOSIL 5HIC 4.6mmI.D.×50mm Mobile phase A: 20 mmol/l Phosphate buffer +100 mmol/l Na2SO4 + 1.5mol/l (NH4)2SO4(pH6) B: 20 mmol/l Phosphate buffer +100 mmol/l Na2SO4(pH6.7) A 100 %->B 100 % 10min linear gradient Flow rate 1.0 ml/min Temperature 30 C Detection UV 220 nm Sample 1. Myoglobin 0.5 mg/ml   2. B-Lactoglobulin 1.0 mg/ml   3. Hemoglobin 2.5 mg/ml   4. BSA 1.0 mg/ml Injection vol. Mixture 2.0 µl

In hydrophobic interaction chromatography, at first the high (NH4)2SO4 concentration buffer (1~2 mol/l) is used, to facilitate adsorbing of proteins to the stationary phase. Next, lower salt concentration applied gradually to remove the proteins of lower hydrophobicity from the stationary phase.

Separation of Crude B-Glucosidase

Column: COSMOSIL 5HIC 4.6mmI.D.×50mm Mobile phase A: 20 mmol/l Phosphate buffer +100 mmol/l Na2SO4 + 2.0 mol/l (NH4)2SO4(pH6) B: 20 mmol/l Phosphate buffer +100 mmol/l Na2SO4(pH6.7) A 100 %->B 100 % 10 min linear gradient Flow rate 1.0 ml/min Temperature 30 C Detection UV 220 nm Sample 10.0 mg/ml Injection vol. 1.5 µl