never get a stable baseline

[jcg200]

Dear Forum:

Please help me with the baseline problem. I set up Waters 2695 Alliance with a 2998 PDA detector using the following parameter:

Column: Whatman Partsil 10-SCX
Column T: 25 celcius degree
Wavelength: 210 nm
Mobile phase: 25% 25 mM KH2PO4 adjusted to ph=4.5; 75% acentonitrile

After I flushed the system (with column and detctor flow cell) with water and 100% methanol, I still couldn't get a stable baseline.
Here is what the baseline looks like:
https://docs.google.com/file/d/0Bw8z-jp ... sp=sharing

Please help trouble shooting the baseline.

Thanks so much!

[mattmullaney]

Hi jcg200,

Is the degasser working well and did you do anything to degas the eluent after mixing the ACN and aqueous salt eluents together? Also, did you allow the final mixed eluent to warm to room temperature before setting the HPLC up to run?

If you have isopropyl alcohol, you could flush the cell with it to help remove air bubbles...or a 50:50 mix of MeOH/deionized water--these might be better for "pushing" bubbles out of the detector cell.

Good Luck!

[jcg200]

The degaser is on all the time with a pressure reading less than 1 psi. The mobile phase was filtered through 0.45um filter proior to use. The mixture of ACN and buffer is cold indeed during mixing. But the baseline still fluctuates after the container sits there overnight.

The system is always flushed with 50% MOH in water during its setup. I also suspect the detector flow cell may be contaminated, but I am not sure how to clean it.

Hi jcg200,

Is the degasser working well and did you do anything to degas the eluent after mixing the ACN and aqueous salt eluents together? Also, did you allow the final mixed eluent to warm to room temperature before setting the HPLC up to run?

If you have isopropyl alcohol, you could flush the cell with it to help remove air bubbles...or a 50:50 mix of MeOH/deionized water--these might be better for "pushing" bubbles out of the detector cell.

Good Luck!

[mattmullaney]

Understood and Thank You.

Okay...as I recall, Waters recommends that the degasser be turned off when eluent isn't actively flowing (don't know if you do this or not), but otherwise all seems to be well.

A Good Procedure to remove bubbles is in place, check.

In the past, I have used hot water (60 deg Celsius) with a drop of detergent, 30% v/v phosphoric acid in water or 6N nitric acid to clean 2487 detector cells. I don't see any recommendations in the 2998 manual for cleaning solutions, but any of these are safe for use with the Alliance Separations Module. A caveat with using acids as cleaning agents--the LC must be flushed out with deionized water after cleaning to neutrality, this can take overnight. How much acid to use? 30-50 mL should work quite well, applied to the cell at a low flow rate, say 0.25 mL/min. It's not too bad to open the detector, remove the flow cell, and look to see if the windows of the TaperCell are occluded, too.

Another possibility could be a dying lamp, in general Waters allows for 2000 operating hours or one year from purchase as expiry criteria for their lamps. Empower will likely have a note for the lamp usage for your detector.

See what you think.

[HPLCaddict]

I'm not very familiar with ion-exchange chromatography, but you're trying to equillibrate an ion-exchange column with a buffer that is no buffer at this pH. Could this be part of the problem? And is it usual to have that much organic modifier in your eluent in ion-exchange?

[Gerhard Kratz]

Baseline Looks like Pulsation of the pump, at 210nm not surprising. Baseline fluctuation, I guess your flow rate is 1ml/min can come from temperature differences. My recommendation is to purge the pump, without a column connected, with Isopropanol. You can check if the puls damper is ok. Please check out the following publication:Journal of Chromatography, 210 (1981) 241-254
Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHROM. 13,686
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF NUCLEOBASES,
NUCLEOSIDES AND NUCLEOTIDES
II. MOBILE PHASE COMPOSITION FOR THE SEPARATION OF CHARGED
SOLUTES BY ION-EXCHANGE CHROMATOGRAPHY
Maybe a Change of the buffer is needed. Good luck

[LCbob]

Hi

At 210nm for DAD, it doesn't look too bad to me. You have noise of approx. 0.1mAu and drift of approx. 1.8mAu. I cannot find the typical OQ specs for this detector ( which would be at 254nm ), but my guess is it would pass drift and fail noise slightly.

Try water @1ml/min and a wavelength of 254nm and see how the baseline behaves.

[aceto_81]

Also, do you see a pattern in the fluctuations?
eg every hour the baseline rises for 10 minutes, and then falls slowly back for 50 minutes, ...
Or does it really seems random?

HTH

Ace

[jcg200]

Thank you all for comments.

To dig into this baseline fluctuation, I made two zero micro liter injections of 100% MeOH. One with a flow rate of 0, and the other with a flow rate of 1.5 ml/min.
Please see the links for the chromatograms.

https://docs.google.com/file/d/0Bw8z-jp ... sp=sharing
https://docs.google.com/file/d/0Bw8z-jp ... sp=sharing

[aceto_81]

For what I can see you need to search for some influence in cycles of about 25 minutes.
eg a airco which starts to cool every 25 minutes, ....

Ace

[AA]

I fixed one like this years ago by wrapping some insulation around the piece of tubing going from the outlet of the column to the inlet of the detector. Cant say if that will work here, but it is an easy cheap thing to try.

[mattmullaney]

aceto_81 and AA make a good point and good observation. It's a lot less likely that it's a dirty cell with the baseline fluctuations being periodic in nature. Lamp usage...well hopefully that would be easy to check in Empower, and I get the feeling that isn't the trouble here.

I'm kind of curious myself, I know that when I've done specificity studies as part of a team, sometimes ion exchange was used as a comparative separation method to reversed-phase separation. Is this eluent composition fairly typical of that for ion exchange work? I'm a reversed-phase/normal phase kind of guy, myself.

[tom jupille]

On those last two figures -- which is which? both of them say 1.5 mL/min. Assuming that they are 0 and 1.5 in that order, the problem shows up only under flowing conditions. Which makes it look suspiciously like the degasser cycling on and off. Are you *sure* it's working OK? Maybe bypass the degasser temporarily to check/

[jcg200]

Many thanks for the comments.

I ran the system over the weekend to see how the baseline changes in a 240 minutes period. Please click the links for the chromatograms.

https://docs.google.com/file/d/0Bw8z-jp ... sp=sharing
https://docs.google.com/file/d/0Bw8z-jp ... sp=sharing

It seems the pumps plays a role in it, considering the 20-min cycles when the flow is on. Does it mean the pumps need service or repair?

There appears also some cycles when the flow is off. I am not sure where it comes from.

Does these cycles affect analysis?

[mattmullaney]

Hi jcg200,

It seems, based on the chromatograms, that this baseline Could Affect your Results...that answer is easy...particularly if the peaks you obtain are small. If this is an assay, maybe there isn't such a problem.

The oscillations are more or less regular, so the degasser remains a possible error source, and in fact since you had the flow off for one figure, the degasser seems to be a good potential source of the trouble. It is probably worthwhile to take Tom Jupille's suggestion and run the eluent through the detector while bypassing the degasser.

If the degasser seems to function well over time--as you mentioned earlier--could it be that something like an ion-pairing agent was used with this Alliance system? Degassers do have large surface areas, and contaminants, once in the system, may be hard to remove...though this idea is difficult to reconcile with the regularity of the baseline oscillation. Seems very worthwhile to check the baseline behavior without a degasser in place. Another easy thing to do...does the baseline behave in a similar way with, say, 100% MeOH or 3:1 ACN/Water?