Chromatography Forum

I am trying to run a 10mL protein G column on the AKTA explorer. The load was not binding to the column correctly and I realized the pH seemed really high (~10). I ran PBS (checked the pH of the bottle and it was ~7) over the column and it took 50mL (5CV) to get to pH 7.

Trying to troubleshoot, I am now running PBS through the column bypass position to try and figure out where the high pH is coming from. Like I said, I checked the pH of the PBS buffer and it is correct at 7. It is running through A11. The only other A1 buffers are ethanol and a low pH washing buffer. Still, the buffer that is coming out of A pump is at pH 9.

The system is really old and the only thing I can think is that there is something in the lines that's raising the pH. I really have no good logical eplanation of how this pH 7 buffer could suddenly be pH 10.

Are you using sodium hydroxide solutions in any cleaning steps between runs or after a few days?

Try purge the system and remove any residual buffers, perhaps some form of residual basic salts have precipitated, thus left in the system, which are dissolved in your new solvents

Try purge the system and remove any residual buffers, perhaps some form of residual basic salts have precipitated, thus left in the system, which are dissolved in your new solvents

Use hot water for this purpose (60°C in the bottle will be OK). Good luck!