Loss of sensitivity SEC-column

[dingemap]

Some time ago i bought a TSKgelG5000PWXL column. We use this column for the examination of the moleculair size of PRP-TT (a glycoprotein used as vaccin). A contamination in this product is sucrose (concentration about 8%).
After a while the respons of the PRP-TT is decreasing (at the same concentration and inj. vol.). I have clean the column but it stays the same. Other components (like dextran blue and sodiumazid) don't have this loss of respons.

Do anyone have a solution for this problem ?

[tom jupille]

I would guess that your column has picked up contaminants or developed active sites to which your protein is adsorbing, hence the decreasing recovery. This is actually a common phenomenon with silica based packings, and a standard "fix" is to make a few large injections of an inexpensive protein (we used to use BSA) to tie up the active sites. This treatment has to be repeated every now and then.

The problem is less common with the polymer-based materials. The risk with applying the fix I just described on a polymer column is that you may make the problem worse (your protein may interact with residual BSA adsorbed to the packing). I would try cleaning the column again and see if that helps. If not, try the BSA trick (you have nothing to lose).

[HW Mueller]

Dingemap, are you really loosing material on the column or did your peaks get broader with a resulting deterioration of the integration? If this was the case you may have the same problem that I have with a TSKgel Super SW 3000 (I posted on this, talked extensively to the manufacturer: no solution yet). After using TFA (or higher perfluoros) the peaks broadened badly, partially irreversible. I don´t know whether acidity or other factors caused this. Having been partially recoverable indicates that stripping via pH can not be the sole reason for the observation. It could be instructive to hear more about your conditions.
(The partial improvement after the use of TFA came about by "keeping the protein happy and solvated" with phosphate buffered saline [PBS] as mobile phase as well as protein solvent, and apparently by washing something off the column. Incidentally, keeping the protin well solvated can be an alternative to Tom´s "loading" with a protein.)