Drifting retention times

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

7 posts Page 1 of 1
I am looking for ideas as to why my retention times are decreasing. We are running Prep or semi-prep methods and performing multiple injections of the same sample; we have encountered this problem before but this is a particularly severe example.

Column 10x250mm Atlantis T3 (C18)
Mobile phase A=water, B=Acetonitrile, C=2% Formic acid in water
Flow rate 4.6 mls/min
Initial 45%A, 50%B, 5%C
30mins 55%A, 60%B, 5%C
31mins 0%A, 95%B, 5%C
32mins 0%A, 95%B, 5%C
33mins 45%A, 50%B, 5%C
cycle time 45 mins.
Injection volume 4 mls

My peaks of interest elute at 31 and 32 minutes as a partially resolved pair. For the first 15 or so injections these retention times were fairly constant, then there was a period where the retention decreased by 0.5 to 1 minute per run, then it would stabilise again, then drift again, etc, etc. By the end of 54 injections the retention times were approx 14 minutes and the separation very poor.

Does anyone have any ideas on whats causing this and/or what we can do to try and get the retention back?

Many thanks
Chris, what is nature of your compounds? Do you know structures? If your compounds are eluting at 31 and 32 minutes with partial resolution may be you need to revisit your method. If they are ionic compounds you might want to try mixed-mode separation and add ion-exchange mode to improve resolution.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
These pair of molecules elute at 31 and 32 minutes respectively at 95% ACN. In order to elute them earlier and achieve baseline separation you may want to add 5% MeOH in increcrements up to 50%. It is very rare to have molecules that have similar solubilities in 3 solvents!
HPLC chemist wrote:
These pair of molecules elute at 31 and 32 minutes respectively at 95% ACN.

Actually, that's the composition at the head of the column, not what the column itself is actually "seeing".

At first I was going to suggest a longer time (column volumes) of 95% ACN to wash off uneluted crap off the column before the next injection.

But upon second reading, in addition to the above, I think I'd reverse-gradient down to 45%A, 50%B, and 5%C and KEEP IT AT THAT COMPOSITION 10 minutes to reequilibrate the column prior to the next injection. That can be done as part of the run or as a post-run command.

If that helps, you can experiment with decreasing that equilibration time, but note that "too long" is way better than "too short".
If you send me a sample I can screen it for you on several mixed-mode columns free of charge. I need to know structures. We can sign NDA/CDA if needed.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
Take Vlad up on his offer. Maybe Vlad can provide some more-modern smaller particle size and/or narrower columns so you can decrease flow rate/save a ton of solvent and likely speed up the analysis time.
Thanks for your replies. We are a small company that specialises in generating metabolites by biotransforming compounds using a range of microorganisms. We generally do not know what the parent structures are and each project is a "one off" so we cannot spend lots of time optimising each separation. In this particular case the reaction produced 5 different "+16" peaks, assumed to to be 5 different hyroxylated metabolites of the parent. The pair mentioned are two of these.

As HPLC Chemist noted the 31 and 32 minute retention times are within the "usable" portion of the gradient, the baseline disturbance (RI effect?) at the end of the gradient starts at 34 minutes. I might try replacing some of the acetonitrile with methanol and see if that changes the selectivity, but I am skeptical. I have not found that a fruitful approach in the past and with two very similar structures...?

The sample was from an initial fractionation LC separation and doesn't contain alot of late eluting material, I did try adding a longer re-equilibration time in a couple of the runs without any effect, and I dont think this would explain the progressive and erratic nature of the problem.

Update - it seems to be a problem with the proportioning valve, still under investigation but running a 30% isocratic system with acetonitrile in line B gave very different retention to putting acetonitrile in line A and running 30% of A. This is very irritating as it's a new system - Waters 2545 pump.

Thanks again - Chris
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