Decrease in peak area after pause

[brinker]

Twice now within a couple of weeks, we've had a situation where, in the middle of a large overnight batch, the autosampler had an error when injecting and paused the batch. As a result, the MS (an Orbitrap QExactive HF) and LC (UltiMate 3000) kept running at initial conditions for several hours, until the analyst arrived the next morning and reset things. I think we've figured out what's causing the injection error, that's not my question. Both times, after the batch was restarted, the analyte peaks had decreased substantially (about half) from what they were before the error. My question is, why? Fortunately we had an internal standard that compensated, so that the adjusted standard curves from before and after the error were pretty comparable, but I'm curious as to why this happened. We wouldn't have gotten that much drift if the samples had kept injecting continously. Any suggestions?

[Chromavore]

My wild guess for this would be some form of ion suppression from a build of mobile phase additives, if for example you are using a buffered mobile phase A and unbuffered mobile phase B. Try a thorough flush before reinitiating the sequences. This is assuming you mean the mass spec response has decreased and not the UV/LC-detection response.

alternatively you may have sample solution stability issues and your samples require injection within a certain time from preparation?

you may also also be seeing some kind of phase collapse/column fouling from being held at initial conditions for long periods resulting in peaks that are broader/shorter and are losing some area/TIC to the baseline, a column/system flush should also help with this.

any more info on what you are running/columns/initial conditions etc might reveal more to more experienced users but those would be my starting points.

good luck,
Chromavore

[brinker]

Thanks for these suggestions. Yes, I do mean the mass spec response. The samples are pretty stable, we've checked them over a period of several months, so I don't think it's that. They're kept at 4C in the autosampler. Also, there are no buffers (or acids) in the solvents, just water, methanol, and acetonitrile. They're UHPLC grade solvents and Milli-Q water. I suppose it could be something in one of the solvents, but it would seem unlikely? I can go back and check whether the later peaks are broader.

The initial solvent is 50% water, 25% acetonitrile, 25% methanol. The column is a Thermo Betasil phenyl hexyl column.

Thanks for any suggestions!

[Multidimensional]

After checking the instrument's LOG FILE for specific errors (to determine what caused the Injector to stop the analysis), check these items:
(1) Be sure to include separate, column wash methods to remove any built up material from fouling the column over time. Fouling often leads to reduced area counts and/or over-pressure limits. Adding separate flush methods which use solvent(s) which are stronger than the analysis mobile phase helps to reduce fouling over time and improve reproducibility.

(2) "Leave the vial cap slightly loose so it does not make a full seal. *This prevents a vacuum forming inside the vial, resulting in injection volumes which may be lower than the selected volume. "Loose caps" can greatly improve accuracy and reproducibility when larger OR multiple volumes are injected from the same vial. Additionally, if the total sample vial volume is very small (i.e. ~ 200 ul), utilize a vial insert of the correct dimensions and type for improved accuracy. When using vial inserts, check that the needle height is correct for the vial insert used. Do not use the entire sample volume! Never use more than 90% of the vial volume or air may be aspirated resulting in invalid data collection." *Please refer to the free article at this link for more information ["HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?"; https://hplctips.blogspot.com/2023/07/h ... uld-i.html].