Chromatography Forum

Hi All,

I’m having some trouble with a steadily dropping peak area over the course of a run. Assay details are as follows:

Equipment:
Waters e2695 separations module
Waters W2475 fluorescence detector
Mono Q 5/50 (IEC) or Superdex 200 10/300 (SEC)
PTFE sample tubes

Reagents:
Filtered Mobile Phase containing Salts, Tris Base and 10% Polysorbate-80 at PH7

Run Time:
40 or 30mins depending on the assay. Total run length, 30+hrs

Sample:
Protein (blood clotting factor)

I run two methods, a gradient IEC method and an isocratic SEC method. In both assays I see a drop in peak area throughout the run. To this end we have bracket injections throughout the run and drift injections at the end.

I have introduced a new reference standard* and have not managed to get beyond the first or second bracket injection before Peak Area becomes too low (system suit. agreement <97%). If I could figure out what aspect of the assays causes the drop in Peak Area I’m sure I could take steps to negate the effect. Any Ideas?

Note: My current best guess is maybe the PS-80 is oxidizing or there may be some other change in the Mobile Phase. I have tried using it fresh and leaving it 48hrs but not often enough to notice a pattern.

* The new Ref. standard has a lower protein concentration. This means that when reconstituted in 500µl, the sample is more dilute. We alter the on-column protein conc. by adjusting the injection volume accordingly.

Well I think I figured it out. We were using PTFE sample vials with a resealable septum in the cap. Subsequently, every time an injection was made, a vacuum was building up in the vial meaning the next injection was harder to make.

The reason this had suddenly become such an issue is that the protein concentration of my reference has decreased by around 40% so I must inject 40% more each time. from now on I will use split septa caps or split the sample between two or three vials.

this topic can now be closed.