Chromatography Forum

I am currently using a diphenyl column, which provides great resolution of a complex protein sample (typical ACN/TFA gradient).
If you run a water blank on the next sample, the same proteins come off (smaller amounts).
If I wash the column with 5-10 volumes ACN/IPA (reverse flow), no great effect. Same proteins come off.
Any suggestions, or experience people have had with these sticky proteins and how others have managed to wash them off a hydrophobic column at runs end.

Thanks,

Sticky proteins

You have to have some water (say 30%) in your washing eluent, as well as some acid or a base - if the column is compatible with the latter.
Use IPA rather than ACN or if you insist on ACN, then the IPA should be at least twice as much as the ACN.

Best Regards

Since you are seeing it, on a blank following a sample injection, then again after numerous washes, I would look somewhere other than the column. It is possible that you have a carryover effect occurring. It may be that you need a more aggressive rinse cycle after an injection, or even that you need a more aggressive rinse solution.

A quick way to check would be to make a sample injection then make a series of blank injections and observe the trend if any that the protein peaks exhibit in the blank injections. If they are trending downward then it is likely carryover from the autosampler.

How big are your proteins, and what's the pore size of your packing?

Thanks for all your suggestions.

The protein range is from 35-70kD. The particle size ids 2.7um with a 300A pore size.
I eliminated the column today and injected a water blank, running the normal method.
As thought, UV absorbing material was detected right away, having no column to attach to.
Thus proteinaceous material is binding somewhere (injector?) in the system.

Any suggestions now?

Thanks again

Yes. Use my suggestion above only for a syringe/injector wash instead of eluent for column wash.

Best Regards