I am attempting to separate Hepatitis B surface antigen (HBsAg, 25Kd) using 1000A reverse phase Column. However, I am having difficulty in acheiving adequate separation. I would like to ask if anybody could suggest other suitable columns for this separation.

Depends on what you're trying to separate it *from*, what the purpose of the separation is (purification? assay? do you have to maintain biological activity?), and whether you're trying to run an exising method from the literature or starting from scratch.

Possibilities include:
- ion exchange
- HIC
- affinity
- size exclusion
- different reverse phase material

Can you give us more details on what you're trying to accomplish and what the specific problem is?

I am trying to separate the HBsAg from homogenised plant extract. The purpose of the assay is to develop a quanitation assay for the antigen. There is no need to maintain biological acticity. The pI of the antigen is 4.6. I am using an TFA/ Acetonitrile/ Propna-2-ol mobile phase system to elute this extremely hydrophobic antigen.

I have been trying to run an exisiting method from the literature but found difficulty in trying to reproduce the data.The main problem with the reverse phase separation is the difficulty in obtain reproducible and adequate separation.

Would the separation of this extremely hydrophobic membrane protein be better suited to ion exchange chromatography?

Sorry to keep asking so many questions, but there is a (allegedly!) a method to my madness. Do you have lots of interfering peaks in your extract, or are there only a few, some of which happen to elute near your analyte.

If the answer is "lots of peaks", then a wholesale change of separation type is likely to simply exchange one set of interferences for another. In that case, I'd be tempted to see what I could do to maximize plate counts (i.e., get the peaks as narrow as possible), then try to make relatively small changes in selectivity to move your analyte and the interferences away from each other (if possible!). Changing pH or adding a different organic modifier might be possible choices here.

If you have a relatively small number of peaks which just happen to elute close to one another, then a change in mechanism might be more useful. I'd be tempted to look at HIC (hydrophobic interaction chromatography, also sometimes called "salting out" chromatography with a decreasing ammonium phosphate salt gradient. The separation is based on hydrophobicity (like reversed phase), but since you're separating the native form of the proteins (rather than the denatured form as in reversed-phase), the selectivity is often different.

Any suggestions on specific columns/conditions out there?

-- Tom Jupille / LC Resources