Chromatography Forum

Hi All,

I'm having an issue with a gradient method. Setup as follows:

Kit:
-Waters e2695 Separations Module
-GE Mono-Q 5/50 GL Column
-Waters W2475 Fluorescence detector

Reagents:
-Mobile Phase A: Tris Base, CaCl2, PS-80
-Mobile Phase B: Tris Base, CaCl2, 1M NaCl, PS-80

Sample:
-Blood clotting factor (protein)
-Injection time 40mins

The assay starts at 75%A:25%B and ends at 30%A:70%B the goes to 100%B at the end of the injection for clean-up. It is this last step that it gets stuck at.

Usually I see 350~450psi but if I set the system to 1ml/min immediately after stalling it will hover between -14psi and 14psi. I then set the system to Line A, pressure goes up, back to Line B and we're back to normal.

It doesn't seem to coincide with a particular injection. It can occur after 3 injections or 43. very frustrating.

Any help would be much appreciated!!!

Several things that come to mind.

First one, the 2695, doesn't really like to pump at back pressures of <500 psi, add some restriction (a short run of 5 thou tubing) between the the injector and the column, try and get the pressure around 1000 psi (the actual number is not important).

Might this is a degasser issue (look for liquid in the degasser exhaust line) . The bottle sinkers might be partially plugged, that could also cause this.

AA

Cheers AA, that's some good advice. I'd never thought of a degasser issue. I'll check it out!

I wasn't aware that the system struggled bellow 500psi but I had already added a little 5 thou tubing on the off chance it might help. I'm currently running at around 450psi so maybe I'll add a little more. I'm nervous of pushing it too hard though as it is a glass column. The leaflet that came with it states the recommended max overpressure is 580psi so I'll aim for that.

Adding tubing prior to the column will not impact the pressure the column sees.

Good point, I've added it between the column and detector this time however as I wasn't seeing any benefit from having it before. If it stops again tonight I'll go back to putting it before the column and ramp the pressure up as suggested.

I've got 550psi (which will increase to around 580psi by the end of each injection) and I've ensured the degasser is fully primed and the waste tubes aren't obstructed and aren't sitting in liquid.

I have a run underway but it's 30hrs so I won't know for sure until Thursday. I'll keep you posted.

Note: a little more background that might be useful: I have been running the method for around 4 years and usually see my current problem with new columns out of the box and occasionally very old columns. The reason I have posted this time is that at the moment it is occurring every assay, with multiple columns of varying age and on two different systems. The only change has been a new reference standard which is about 40% weaker than the previous reference and as such my injection volumes for standards and samples have increased accordingly.

Well it stopped last night at the start of an injection. I really can't see any pattern. I'll put a load of tubing prior to the column and give it one last try while my phase is still in date then it'll have to wait till after Xmas haha.

The engineer is coming out today for a different machine so I'll mention it to him. I was trying to kid myself that I wouldn't need vendor help and that it would start working eventually as it always has in the past. I'll keep you all posted.

OK, so the engineer was here for an hour. I showed him the instrument method, some chromatograms and he had the system open and he was pretty stumped.

Current theory is that air is getting past the degasser somehow (maybe when the sample is being taken). There's a bubble somewhere in the system, probably in the column, that is only showing itself occasionally and only when it switches to 100% line B after around 32mins.

I'm not sure how best to test this theory or what to do about it. It' has been suggested that I run 100% Acetonitrile through it backwards but it will have to wait till the new year.

If anybody has any other suggestions let me know

The only other issue that I could think may be the problem is a faulty GPV. You could try running C/D instead of A/B that might i.d. the problem. Are you absolutely sure that the bottle sinkers are not the issue? Take them off and try your run.

OK, so with New Year out of the way I am back at it.

I did as you advised AA and switched the bottle sinkers and had my first two runs that didn't cut out on me. Thanks for all your Help!

I am curious as to why dirty bottle sinkers would cause the system to cut out in the way I described. Would it be air or salt opposites that would interfere with the run?

I'll come back if there are any more developments but for now it seems to have worked

Edit: oh, also, sorry I didn't try the bottle sinker thing earlier. I was pretty burned out by the time I posted on here haha.

Clogged sinkers create a vacuum, which is not something you want with LC pumps. They're designed to push really hard but don't pull very well. Glad it was something simple -well done!