UPLC Instant Retention Time Shift

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

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Hi everyone

We are currently seeing some strange things happening on our Waters Alliance H-Class systems. We are new to the use of these systems and not very familiar with all the problems we may occur although we are finding out your margins for small “errors” are reduced. Our retention time is shifting without warning. We have been using this method for nearly 12 months and have not encountered this problem. Occasionally we had seen the peak shift out but only at certain times i.e.

The system had been left for a number of days
Mobile phase was aging
If mobile phase is prepared as a mixture of 80:20, pH 3.0 phosphate: Acetonitrile opposed to being used as a gradient with inline mixing

Following a good system purge the peak always shifts back to the typical retention time of 0.6 minute


A number of analyses performed on the system thought out the morning, retention time of the peak of interest is 0.6 minute, total run time of 1.0 minute. 3 hours later additional analysis performed and no peaks are observed. Upon investigating found that the peak had shifted from 0.6 minute to 1.1 minute. Nothing had changed and the system had not been touched between analyses.

Method details:

Gradient analysis – pH 3.0 phosphate buffer: Acetonitrile, mixing inline 80:20 at 0.6 mL/min
Wavelength at 280 nm, injection of 1.5 micro litres
Column is Acquity UPLC® BEH C18, 1.7 m, 2.1 x 50 mm at ambient temperature

Mobile phases are degassed and filtered through 0.2 filters, all reagents are of appropriate quality, samples are prepared in compatible reagents and good lab practice is employed.

Can anyone give any advice as to what might be causing this sudden shift? What can we do to eliminate this occurring or minimize the odds of this happening? :?

Any suggestions are appreciated and I thank you all for you time in advance. If you need any additional information please ask and I will provide.
Hi Jana,

Please, do you happen to actively sparge your eluent as the sample queue is running? If so, possibly you're losing ACN--greater retention as eluent loses eluting strength. Possibly, is the eluent bottle in an area that gets warmed up over time? Same thing can happen, differential evaporation of solvent...ACN out, weaker eluent.

If the separation is at ambient conditions--please, do you mean that the column lies within a column enclosure set at ambient temperature, or is the column open to the laboratory environment?
In the latter case, how constant is the temperature in your lab? Particularly for polar analytes, variations in temperature can make a large difference in retention time, generally at cooler temps, peaks will take a bit longer to elute at a constant eluent flow.

Also, how long do you normally use this eluent? ACN can be lost to evaporation over time even at constant lab temperature...the pH value of the eluent mixture could also change due to absorption of carbon dioxide...causing peaks to suffer change in retention time.

One more thought...the peak area, does it change in value when its "normal" elution moves to a later time...if the peak area increases, then the pump or the flow has changed...flow has become less in value, maybe due to air in the pump, a leak, or dirty check valves. Though also...changes in the mobile phase (losing % organic), stationary phase (crud or something else) or a decrease in the temperature where the column resides could do this...as hinted at above.

Here, a t0 check can help in troubleshooting...in my small opinion not enough organizations/people do this. No one's fault...and everyone's fault...

...except now I re-read your description, and None of What I Said really would result in a Sudden Retention Change...ugh!

Please, see what you think. I'll do the same on my end.

Hi Matt

Thank you for your input. Here are some answers to your thoughts/questions:

The temperature in our laboratory is fairly stable at approx. 21 degrees C and I don't believe we are losing any ACN. Our laboratory, location of instruments has not changed and previously we have run this method very successfully for near on 12 months.

By stating that the column is at ambient temperature I am saying it is enclosed in the column compartment but the oven is off (not set). As previously stated I don't believe that the temperature should be causing the issue. However I did fail to mention that both the columns are new (the old columns we were using started to fail and produce splitting peaks). Is UPLC column tecnhology reliable? Are there still bugs being ironed out by UPLC column manufacturers which cause differences between column batches?

All the solutions were freshly prepared when we first encountered the mysterious shift in retention. All the solutions have been confirmed as correct preparation etc. To me it is unlikely to be responsible for the sudden shift as after the system lines are purged the retention time returns to normal for a time period.

A new development is that we purge all the lines etc. and being injecting and it is only if we leave the system flowing with no injections running for a period of time (anything from 45 mintues to 3 hours) that we see a dramatic shift in the next analysis.

The peak area stays typical regardless of retention time! The peaks also look beautiful, symmetrical and typical regardless of how late they elute. :scratch:

I am thinking pump/mixing something like that would be the cause although why all of a sudden and on two independant instruments?! :?

Has anyone experiance problems with mixing 100% aqueous and 100% organic on the Waters H-Class systems or for UPLC? It is only now we are experiancing difficulties. :(
You normally mix your eluents on-line? So there's no possibility of retention time shifts because of ACN evaporation. Did you do robustness studies of the method, i.e. retention behaviour after pH/eluent composition/temperature changes?
I'm not familiar with the Aquity but it should be definitely better than what you're experiencing. Maybe it's a sticky check valve caused by pure Acetonitrile?

Generally I'm a big fan of premixed eluents as it may keep you from a lot of troubles (bacterial growth in the buffer, pump problems with pure acetonitrile, less noise in the baseline, ...). I'd try to premix and run the analysis with the mobile phase attached to all channels. Do you see differences when using different channels to pump the mobile phase?
Hi Jana and HPLCaddict,

I've not experienced any mixing problems with the Acquity-H in terms of mixing 100% buffer and 100% organic...but something to check here may be what you use for Seal Wash (Waters calls this Wash in their Aquity-H manual) and Purge solvents...Seal Wash is to be mostly buffer/additive-free water and a small proportion of organic, Purge is to be mostly organic (but not neat organic) and a bit of buffer/additive-free water...Not a Good Idea to use the Same Solution for Both Wash and Purge.

Temp not a problem, good...my thought on these U(H)PLC type columns is that they are at least as reliable if not more so than particles of greater size...BEH and Phenomenex Gemini (part polymer part silica, though Gemini isn't < 2 um...) are both nice materials, certainly any of Agilent's, Restek's...whomever's...in my experience, not a whole lot of difference. Got a Lot more use out of Bonus RP in 1.7 um size than 5 um for DNPH-carbonyl separations, well over 2500 injections as compared to around 1000-1250 for the 5 um material, but this is anecdotal.

I misunderstood before, you are dial-a-mixing rather than pre-mixing...you're not losing ACN, good. I am in HPLCaddict's camp on pre-mixing, though in the "Ideal World" there should be No Difference. His suggestion in switching the aqueous and organic lines to see what happens is an excellent idea.

Chemical and Physics causes are removed...must be rapid changes in the proportioning of aqueous and organic--that is, the pumping and/or proportioning are at fault, agreed overall. I've had the same experience as HPLCaddict, Lots of Trouble over the years with sticking check valves and ACN, less problems with some vendors than others...or perhaps a micro-leak (can happen also) on the organic line, though don't know if that would pop up suddenly. You could try running MeOH or THF on your ACN line, or sonicating the ACN line check valves in MeOH...see if you can get consistent retention times once again. (Do this to get rid of polymer build-up from the ACN, like acrylonitrile, for example).

Leaving things running also would seem to lend credence to sticky organic line check valves...they may not have time to "seize up" if they're always moving, or have had time to move a while before use...until they decide to get stuck again, of course. Would also "sort of" explain why you'd have the same thing happen on two systems simultaneously, though this might be coincidence, but then, if the same ACN is used on both...

One thing is strange to me...peak moves to later retention time but doesn't increase in area? Interesting...seems that the longer a peak would reside in the UV detector, the greater the peak area should become...in an isocratic separation...wow, not even a little increase in area between RT @ ca. 0.6 min and @ 1.1 min? Interesting...

Please, see what you think, and my thanks to You Both!
The system had been left for a number of days
Mobile phase was aging
If mobile phase is prepared as a mixture of 80:20, pH 3.0 phosphate: Acetonitrile opposed to being used as a gradient with inline mixing

How do each of these elements effect your RT?
Hello everyone,

I am also having the same problem with UPLC H-class. After running about 13 to 14 injections (Approx. 6-7 hours), abnormal Retention time shifting observed.

Please help me to solve this.

Thanks in advance
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