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.
Application
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 |
MATERIAL CHARACTERISTICS | |
COSMOSIL 5HIC | |
Silica gel | high purity porous spherical silica |
Average particle size | 5 µm |
Average pore size | approx. 300Å |
Specific surface area | approx. 150m²/g |
Cosmosil 5HIC Column
| Item | Catalog | Price ($U.S.) | Buy |
|---|---|---|---|
| 5HIC – Analytical Column, 4.6 x 50mm | 08882-00 | $412.00 | Add to cart |
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