Air issues

[floguigui]

Hello,
I'm doing SEC analysis of proteins, using a TSK-GEL SuperSW3000 column from TOSOH and a guard Super SW guard Column from TOSOH too. Mobile phase is sodium phosphate 0.05M and sodium perchlorate 0.4M. My solvent bottle went empty and I guess some air came in the column. After washing with water (millipore quality) and putting a new bottle of solvent, my pressure is about 100 psi higher than before.
Do you think this will have an impact on the analysis? If yes, in which way?

Thanks a lot!

FG

[Gerhard Kratz]

If you pump air on the column the packing can be destroyed. Please disconnect your detector, flush the column in reversed mode and hope that it is still working. If it was only a small bubble it should not affect the packing. Add maximum Acetonitril to your mobile phase and flush your column by reversed flow. Please rins your detector as well, best with a syringe, but be carefull, it is not recommended to do boddy building. Good luck.

[Markus Laeubli, Metrohm]

As eluent is on the column again, first thing I would do is runing a standard chromatogram and check performance.
Reversing the column is quite often not recommended by the manufacturers. Therefore check the column documentation prior of reversing the column.

[floguigui]

hello,
I took off the column, wash the system with MeOH 5% then water. Then I put the column, wash it with 1) milipore Water (1hr, 0.35 mL/min)
2) mobile phase (1hr, 0.35 mL/min)

Then I inject a standard Gel filtration, perforances are good (nearly as good as before). This is a good point. But my monitored pressure inside the system is still higher...This should mean the packing is somehow affected, isn't it? Or something is the system (pump, injector, pipes, detector...)?

Thanks for the quick answers!
FG

[HW Mueller]

During the time you spend here you could have long since found out where the restriction is located.
my guess is that you "caked" out some remaining protein on a frit while running dry. In any case, why are you worried if it works?

[floguigui]

Hello,
Its more curiosity which drives me to ask these questions I'm quite new in the business and I was just wondering if anyone can give me advices (which is definitely the case).
Concerning my analysis, only the vera first samples injected with solvent (after purging) give strange signal, the rest seems fine!
Have a nice week end!
FG