Baseline problems -Acquity (Concave and Convex)

[AA]

Make your weak wash 1:1 methanol/water.

[AdrianF]

The problem could be the 3ul injection - why so small? try filling the 10ul loop completely and see what happens.

Are you using different autosamplers?

[Rob Burgess]

Hi Adrian,

The method has been validated with 10µL. We are in an overload posistion to gets sens for imps. If we injected 10µL we would lose Rs for peaks on the tail of the mainj peak - there is a very fine line for this method unfortunately.

I will get back with further results / conclusions when I have time next week. I think I need to follow up on the needle wash idea to be honest and then get back to this forum.

[AdrianF]

If the method has been validated at 10ul I think you should stick with it. if necessary dilute your sample. I am all for varying a method but even to me that is a 'bridge to far'!

[Rob Burgess]

Sorry my mistake, its should have read the method has not been validated for 10µL.

The injection volume of the method is 3µL and this has been fully validated to ensure Rs is assured when running the method.

A colleague of mine has run another method with AcN gradient - I'll let you all know the outcome in due course.

[Rob Burgess]

With regards to just the smile curvature of the 1st chromatogram (10 mm flow cell) it appears that the light guided flow cell is definitely at fault here. It is even explained in the Waters publication regarding use of their Median Baseline Filter.

I have done some further runs with the high sensitivty flow cell and the gradient profile is as expected - i.e. steadiliy rising rather than just convex. I'm not sure what has solved it, but a user before me was running AcN grads where before I was running just MeOH. Also I've changed the needle wash solvents to:

Weak needle wash - 50% water 25: AcN 25 % MeOH
Strong needle wash - 50% MeOH and 50% AcN

The sensitivity check of my method is now visible!

I will check the analytical flow cell tomorrow (and perhaps run some AcN grads down it) to see if I can get rid of the concave baseline affect with this cell as well. I'll keep everyone posted how I get on....

[Rob Burgess]

With regards to just the smile curvature of the 1st chromatogram (10 mm flow cell) it appears that the light guided flow cell is definitely at fault here. It is even explained in the Waters publication regarding use of their Median Baseline Filter.

I have done some further runs with the high sensitivty flow cell and the gradient profile is as expected - i.e. steadiliy rising rather than just convex. I'm not sure what has solved it, but a user before me was running AcN grads where before I was running just MeOH. Also I've changed the needle wash solvents to:

Weak needle wash - 50% water 25: AcN 25 % MeOH
Strong needle wash - 50% MeOH and 50% AcN

The sensitivity check of my method is now visible!

I will check the analytical flow cell tomorrow (and perhaps run some AcN grads down it) to see if I can get rid of the concave baseline affect with this cell as well. I'll keep everyone posted how I get on....

[Rob Burgess]

It was definitely a flow cell problem. A waters engineer came in and replaced the 10 mm analytical flow cell and "voila" case solved - expected gradient baseline rise from my method - something to watch out for methinks!

[danko]

Hi Rob,

What was wrong with the flow cell that was replaced?
Also, what caused the wavy baseline?

Thanks for sharing.

Best Regards