Chromatography Forum

I agree with HPLCaddict in that you would have a longer retention time with methanol as well as a higher pressure as the gradient progresses, versus a cetonitrile. I would make fresh mobile phase to quickly rule out this possibility.

I just remembered something that gave us really strange problems similar to this a few months back. The degasser on our Agilent system was not working properly and we got strange results with pressure and erratic retention times as well. If you can swap in a different degasser see if that will make things look better.

I never thought a degasser would cause such problems until it happened to us.

My guess is that you have a gradient proportioning problem. Jiri Urban has a nice set of viscosity/composition curves for ACN/water systems at http://www.chromatographer.com/mobile-phase-viscosity/ Depending on your temperature, the back pressure should peak at somewhere around 30% ACN. A partially plugged column frit would scale the entire verticasl axis of the pressure curve up, but should not change the shape.

If this were my problem, I would run a "stairstep" test (http://www.lcresources.com/resources/TSWiz/hs450.htm) to verify that the gradient proportioning system is functioning correctly.

I agree with HPLCaddict in that you would have a longer retention time with methanol as well as a higher pressure as the gradient progresses, versus a cetonitrile. I would make fresh mobile phase to quickly rule out this possibility.

I will do that ASAP.

\Erik

I just remembered something that gave us really strange problems similar to this a few months back. The degasser on our Agilent system was not working properly and we got strange results with pressure and erratic retention times as well. If you can swap in a different degasser see if that will make things look better.

I never thought a degasser would cause such problems until it happened to us.

I've dealt with a faulty degasser as well, however I don't think this is the issue here, as I get similar pressure profiles with a another (but similar) column on our 2 systems.



However the retention times are not similar.

\Erik

My guess is that you have a gradient proportioning problem. Jiri Urban has a nice set of viscosity/composition curves for ACN/water systems at http://www.chromatographer.com/mobile-phase-viscosity/ Depending on your temperature, the back pressure should peak at somewhere around 30% ACN. A partially plugged column frit would scale the entire verticasl axis of the pressure curve up, but should not change the shape.

If this were my problem, I would run a "stairstep" test (http://www.lcresources.com/resources/TSWiz/hs450.htm) to verify that the gradient proportioning system is functioning correctly.

I will do the proportioning test, however I'm still confused that various combinations of channels (e.g. BC, CB, CD) gives rise to identical retention times. I would have expected swapping from BC to CB (e.g. swapping C for B as ACN channel) would have resulted in an earlier elution as channel C (supposedly delivering less solvent than anticipated) now would deliver water.

\Erik

Ok so I've performed the proportioning test on both systems. I used ACN on the main channel and ACN with 0.1% acetone on the other channel. The absorption drops appeared following opening, clicking a random spot in the gradient table and closing the "setup pump" menu in the software during the run - kind of weird never seen that before.

(The overlay has been shifted 2% for clarity)


It seems to me, that channel D of the suspected system delivers what it's supposed to.

\Erik

I've dealt with a faulty degasser as well, however I don't think this is the issue here, as I get similar pressure profiles with a another (but similar) column on our 2 systems.



However the retention times are not similar.

This is the original method with the "old" solvents just with another C18 column?

You doesn't change anything except the column?

Are this the first injections on this coloumn?

Beside the problem with the pressure:

http://www.sepscience.com/Techniques/LC ... PLC-Method

I run on my HPLCs every morning a blank injection with my main method. (Just halved all times.)

Without this the first sample of a day often has no reproducible retention times on my systems.

I'm running with H2O/ACN on a C18 like you, one with TFA and one with formic acid. Just the composition of the solvents are a little bit different. ( H2O/ACN 77/23 -> 15 min -> 23/77 -> 5min ->23/77)

This is the old solvent just a different column, same method on 2 different systems.

I usually include a blank run prior to the run in mention, using the same method.

\Erik

This is the old solvent just a different column, same method on 2 different systems.

I usually include a blank run prior to the run in mention, using the same method.

\Erik

Could the "old" solvent be contaminated with Methanol? If so you could be getting the increased pressure from the Methanol.

I don't think it's the solvent, since the run with the other column, gave rise to similar pressure on the 2 individual systems.

The weird pressure profile seems to be related to the specific column. However there is an issue with the pump as well, but

I'm confused since the proportioning test gave similar results on the 2 systems, but I get longer retention times on the system in question, regardless of the column used.

I'll get back once I know more

Thanks for the inputs

\Erik

Hi again

So I did the proportioning test on the A and B channels of the pump. It turned out that when running on channel B, I get no signal at all, indicating that something is wrong with the pump. The proportioning valve unit have to be replaced I guess.

I have replaced the pump with a binary one from our old 1100 system, and now I get the same retention time (+ ½min) as on the 1260 system. The pressure profile still looks a bit odd, but I have purchased a new column and I'll try that one out today.

Thanks again
Erik

Before replacing the proportioning valve, try two more things:
1.) Clean the solvent filters in an ultrasonic bath.
2.) Flush the system with hot water (60 °C) for some minutes. Use 25 % of each channel at minimum 5 ml/min with the purge valve opened. If you experience pressure > 7-8 bar during this procedure, replace the PTFE frit.

Good luck
Jörg

Before replacing the proportioning valve, try two more things:
1.) Clean the solvent filters in an ultrasonic bath.
2.) Flush the system with hot water (60 °C) for some minutes. Use 25 % of each channel at minimum 5 ml/min with the purge valve opened. If you experience pressure > 7-8 bar during this procedure, replace the PTFE frit.

Good luck
Jörg

Thanks Jörg, I'll give that a try.

\Erik

Before replacing the proportioning valve, try two more things:
1.) Clean the solvent filters in an ultrasonic bath.
2.) Flush the system with hot water (60 °C) for some minutes. Use 25 % of each channel at minimum 5 ml/min with the purge valve opened. If you experience pressure > 7-8 bar during this procedure, replace the PTFE frit.

Good luck
Jörg

If you are using the glass/ceramic Agilent frits you can clean them by soaking in 50% nitric acid, this removes any accumulated algae very well.

Also to check if the proportioning valve is having problems I have taken 4 100ml graduated cylinders, placed each line in one cylinder then fill to the 100ml mark. Run with purge valve open at 25% each and 4ml/min and after 30 minutes check volumes. If working correctly they should all be at 70ml. If not you know what channel is bad.