FAQs on Purification with Hydrophobic Interaction Chromatography

FAQs on Purification with Hydrophobic Interaction Chromatography

How do I determine which HIC resin will work best for my application?

The binding mechanism of target molecules to HIC media is a function of the molecule’s hydrophobicity and varies for each molecule. If you are not familiar with how your target interacts with any of our TOYOPEARL HIC resins each should be scouted to determine which offers the best selectivity and recovery for your target and its impurity profile. In general, the more hydrophobic the target molecule the less hydrophobic the solid support. Conversely, the less hydrophobic the molecule the more hydrophobic the solid support.  These tests can be easily performed using our SkillPak columns. Available in both 1 mL and 5 mL size columns, the SkillPak HIC column libraries are an economical choice as it costs less than individual resin samples and requires minimal feedstock to generate data.

Will higher concentrations of transition metals in my buffer form complexes with the functional groups on the resin and tie up binding sites? 

The answer to this question is no. The available HIC functional groups are designed to be inert except for the ability to form hydrophobic interactions with the proteins. There are some instances, however, where very slight interaction with the buffer components and the resin solid support could occur. More importantly, the question is whether the transition metals interact with the proteins in the feedstock. This will be dependent on the proteins themselves and not the HIC resin.

I have run a gradient from high salt to no salt in my buffer and I still have protein that hasn’t eluted. How can I elute it? 

If it is the target molecule of interest, low concentrations of ethanol or even acetonitrile can be used when the protein is still sticking to the column after washing with elution buffer. A chaotrope such as urea or guanadine HCl can also be used in low concentrations to denature proteins and cause them to elute from the column. Otherwise caustic can be used to strip the column to get rid of unwanted contaminants. A less hydrophobic HIC phase may also be found which would not require the use of post gradient modifiers to the system.

Does it matter what pH I operate at when running HIC?  

It is recommended to operate within a pH range that is ideal for protein stability. pH can influence how effectively the target molecule will bind and elute from a column but this is case specific and may not show the same trend going from one pH to another for different molecules. some of our customers use pH changes to elute thier protein. Once again the importance of maintaining the proteins stability and activity is paramount.