Baseline problems -Acquity (Concave and Convex)

[Rob Burgess]

Hi All,

I seem to be having a problem with the baseline output of our new Acquity system. Instead of a steadily rising baseline from a gradient as per our Agilent 1100 systems (Image 1), I am getting either a concave (Image 2 - std. 10mm flow cell) or convex (Image 3 - 25mm high sensitivity flow cell). I'm guessing it is an optic effect of some sort. However it isn't just a problem affecting the "look" of the chromatogram it appears to be cutting the sensitvity of detection for my method.

Any ideas anyone?

Image 1:

Image 2:

Image 3:

[GoldLeader]

Rob,

See the post below - "Humidity Affects Baseline." This is precisely what we see when this happens. Can you correlate yours to changes in humidity/lab temperature?

Jeff

[Uwe Neue]

The differences in the drifts between the three detectors are refractive index effects. I can't tell you anything about the sensitivity, since I do not see the same peaks in the three chromatograms.

[danko]

Hi Rob,

My first impression is that the column is not equilibrated at the time of injection – in the case of the 2 Acquity generated chromatograms. I’m not sure what happens after the 45 min. runtime, you show data of, but it seams to me that there is no equilibration time programmed following the gradient.
Maybe the equilibration is not shown? Or do you just perform a new injection after 45 min?
Have you tried to reproduce these injections and if so, do the baselines overlay perfectly?

Best Regards

[Rob Burgess]

Please note that images 2 & 3 are ith the same detector, all that is different is the flow cell was changed.

The chromatograms generated are preceeded by three equilibrating blank injections and the gradient profile is reproducible.

I have spoken to a Waters tech guy about this, and he says its something to do with the Total internal reflectance of the cel itself. It could be contamination of the cell, but I feel this is unlikely as it is brand new and we haven't run much down it!

I'm not sure about the humidity argument - our lab temperature/humidity is fairly constant and the Agilent is located in the same lab, so why isn't that affected?

[danko]

Rob, would you please post the gradient table/s?

Best Regards

[Rob Burgess]

MP A: Water + 0.1% formic acid
MP B: MeoH + 0.1% formic acid

Gradient: 15 to 60% B in 50 mins

[Rob Burgess]

Sorry that should be 15 to 60% B in 40 mins

[danko]

Thanks,

It is quite obvious to me that the 5 min equilibration time left, is inadequate.
Try adding 5 minutes more equilibration (10 min initial conditions after the gradient in all) and see if it works better.

Best Regards

[Rob Burgess]

Not sure I agree with that - it looks fine and consistent with the Agilent system and the gradient profile is consistent for the Acquity.

[danko]

I think we need some details.

Is the gradient table for the Agilent system the same as the one for the Acquity?
What about the flow rate?
Do you use the same column?
Dimensions?
Does the chromatogram represent the total runtime in all cases?

Best Regards

[Rob Burgess]

Gradient table exactly the same for both systems.
Flow aret is 0.8 mL/min - for both systems
Same column was used for both systems Waters X-Bridge C18, (150 x 4.6) 3.5µm
Chromatograms represent total run time (including 5 mins re-equilibration).

I think the focus needs to be on the flow cell design to answer this - I'm very curious to ask if anyone else has seen anything like this...

[AA]

The baseline effect is due to refractive index changes, it is the nature of these light guided flow cells to do this. This, however, is not likely the cause of the reduced signal. The injection technique is quite different than your 1100. The mode of injection that you choose on the Acquity system and the nature of the weak and strong washes will impact how much mass of your compoun gets on column. So, what is the injection volume, what is the sample diluted in, and what is the composition of the weak and strong, and what injection mode did you use?

[Rob Burgess]

Hi AA,

I think you are right, I need to investigate the injection part of to discover why I'm getting a loss in sensitvity as well.

So here goes:

Compound (& impurities) dissolved in 50/50 v/v MeOH/0.1M HCL

Injection volume is 3µL

Weak wash solvent is MP A i.e. Water +0.1% formic
Strong wash solvent is MP B i.e. MeOH + 0.1% formic

Injection mode is partial loop needle overfill (currently fitted with a 10µL loop)

[danko]

Rob,
I’m far from convinced that your problem is the flow cell.
I’d guess that this wavy baseline is caused either by some pollution (but it wouldn’t be reproducible) or pumping/mixing problems. Have you monitored the pressure?
I also agree with AA that strong wash or sample solvent could cause a similar problem, but in your case the baseline is wavy even before an injection is performed (see especially chromatogram No 2, where the injection is performed in the beginning of a valley). The baseline at that time should be flat. In order to investigate the influence of the injection, you could just inject “nothingâ€