Creative Enzymes builds efficient purification methods on the foundation of comprehensive understanding and practical experiences of enzyme separation. We provide a group of isolation and purification methods to satisfy the post-expression needs in further applications. Given particular situations of each sample, Creative Enzymes chooses and sometimes develops the most suitable purify method in each case. Hydrophobic interaction chromatography (HIC) represent one of the most widely used methods in enzyme purification. The expertise of Creative Enzymes on high-quality HIC will support your research demands.

Hydrophobic interactions have a great importance in the biological systems. They are the dominant force in protein folding and structure stabilization, besides, it plays important roles in other biological processes like antibody-antigen reactions and enzyme-substrate recognition. HIC takes advantage of the hydrophobicity of proteins promoting its separation on the basis of hydrophobic interactions between immobilized hydrophobic ligands and non-polar regions on the surface of proteins. HIC is now an established and powerful separation technique in laboratory-scale, as well as in industrial-scale purification of enzymes. In HIC purification process, high-salt buffer is used to expose the hydrophobic region in enzyme molecules, subsequently, the hydrophobic region is adsorbed by the base matrix. The more hydrophobic group in molecule, the less salt is needed to promote binding.

HIC and reverse-phase chromatography (RPC) are closely related LC techniques. In practice, however, they are different. Adsorbents for RPC are more highly substituted with hydrophobic ligands than HIC adsorbents. Protein binding to RPC adsorbents is usually very strong, which requires the use of non-polar solvents for their elution. RPC has found extensive applications in analytical and preparative separations of mainly peptides and low molecular weight proteins that are stable in aqueous-organic solvents. Compared with RPC, the polarity of the complete system of HIC is increased by decreased ligand density on the stationary phase and by adding salt to the mobile phase.

To obtain success in a chromatographic process for a specific application, main parameters need to be considered when selecting HIC media and optimizing separation processes:

• Ligand type and degree of substitution
• Type of base matrix
• Type and concentration of salt
• pH
• Temperature
• Additives

Creative Enzymes has applied HIC in purification of a great variety of enzymes and accumulated extensive experiences. We carefully examine the characteristic of target enzymes and purification environment to select proper stationary and fluid mobile phases. HIC is considered to be an efficient separation technique, therefore it can combine with other approaches, ignoring the order. Creative Enzymes provides combined purification processes as part of isolation and purification services based on particular demands. Our services are guaranteed with first-in-class quality. Please contact us for technical consultation and service quotations.

Figure 1: The schematic diagram showing hydrophobic interactions between: (A) proteins in an aqueous solution, and (B) between proteins and a hydrophobic ligand on an HIC adsorbent.

Figure 2: The schematic showing a gradient elution of a protein mixture using HIC. In the diagram, the salt concentration is linearly decreased (from high salt to low salt), which results in elution of both impurities and the target enzyme.