I'm having some trouble with an HPLC-HIC column we routinely use as a final enzyme purification step. It's a Sigma Phenyl-Sepharose we've used without problems in the past, but due to decreasing resolution of our protein, I washed it with 6M urea to remove strongly bound protein, and re-equilibrated 3 hrs with buffer (25 mM Na-OAc, pH6). Now every injection of protein, or BSA for that matter, in 0.5-1 M ammonium sulfate, results in a large front peak (most likely denatured protein) and no subsequent peaks, - any reason why this would be the case, and potential solutions ?

Uh, I hate to say this, but it sounds like a dead column. It might be more cost-effective to just get a new one rather than spend a lot of time trying to resurrect the old one.

You are, of course, right - trying to get a loaner now from Supelco. But what is the deal with washing HIC columns with 6M urea ? From my experience, I wouldn't try it again.

This is only hand-waving, but my understanding is that things like urea are "chaotropes" that work in part by decreasing the surface tension of the water. The salts used in HIC are "anti-chaotropes" which increase the surface tension. Possibly the urea decreased the surface tension enough to "wet" the column packing differently ??

Maybe someone elst can contribute an explanation??

On a similar note, I'll have to fork out $500 for a new column, but first I want to try an old LC-HINT column we have lying about (Supelco, 5 um). Has anyone used these before, and if so, what type of mobile phase is used ??