Agilent HPLC Quaternary. Buffer/ACN mixing problem?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I am running a gradient of B (ACN) in A (50 mm phosphate buffer + 0.025% Et3N), overall from 2% B to 60% B in 14 min, with 4 min equilibration, XBridge C18 4.6x100 mm, 2.5 um column, 1 mL/min. 40C. Max pressure 200 bars. I am getting terrible reproducibility of retention times and pressure curves. Retention times shift 1- 3 minutes. No problems with binary pump (Agilent) What are your thoughts?
> 50 mmol buffer is usually buggy with at least agilent 1260 quaternary and gradient. We use ~ ≤ 44 mmol to get no pressure issues. Time shift is strange for me.
Best regards,
Dmitriy A. Perlow
Lengthen the post run equilibration time a few minutes before the subsequent injection and see if that helps.
Another approach is to tweak your MP prep so that you can start at higher than 2% for the A line. Mixing is usually an issue w/ high pressure mixing systems at the extremes.
Thanks,
DR
Image
How much does the pressure curve vary? Is it a smooth line fluctuating about 1bar or is it a fuzzy line fluctuating 5-10bar?
The past is there to guide us into the future, not to dwell in.
I wish I could post HPLC trace and pressure trace (can I?), but there is an obvious correlation between the pressure, ACN%, and retention time shift. Which tells that there is more ACN in the beginning sometimes, both pressure and retention shift confirms. I am trying now to lower buffer concentration. Again, this is a low pressure mixing. I think that the proportional pump is affected by the high buffer concentration. I wonder if Agilent makes them from different materials (?).
The valves can go bad or clog over time. You can test the accuracy of the valves by placing the mobile phase tubes into 100ml graduated cylinders all filled to 100ml with water, set the gradient to equal parts of each and a flow of 1ml/min and let it flow to the bypass valve for an hour or two, then look and see if equal volumes were pulled from each cylinder. You can do all 4 channels of the quat pump and know if one is malfunctioning.

If it just happens to be a plug, use a 10ml syringe with an adapter and attach it to the end of the line that goes into the bottom of the pump, put the solvent lines in a beaker(after removing the filters on the ends) and set the flow to something low and the valve to 100% on A then after you start the pump, that valve will open and you can push water backwards through the valves to clean them out. Switch to B, wait for the click then push another 10ml through that one, so on and so forth until you have flushed all 4. You might want to bypass the degasser so you don't push any contaminate from the valve back into the degasser. I did this last week to one of mine and on channel B it was hard to push solvent through but suddenly it popped lose and began to flow really easily, the gradient worked great after that.

If it continues to give uneven flow rates when you do the graduated cylinder check again, then the valve solenoids are not working properly and the mixer needs to be replaced, simple just remove the lines, the two screws and the wires unplug like a network cable. I don't think that part is super expensive.

Also if the degasser is not functioning correctly, it will give high pressure swings because micro air bubbles will form as the valve solenoids switch and that causes pressure problems. If it is getting well degassed solvents, the pressure swing should be about 1bar when running isocratic.
The past is there to guide us into the future, not to dwell in.
Recommend you skip the sample for now and verify correct operation of the pump and HPLC in general with stds and verification tests first (verify pump, grad valve, filters are all fine and no leaks).

These articles may help you solve the problem:

"Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)"; https://hplctips.blogspot.com/2014/01/d ... -hplc.html

"HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/h ... hange.html
Also quaternary pump manual recommends no to use < 5 % from any channel.
Best regards,
Dmitriy A. Perlow
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