I'm running a Waters UPLC-TQD system. I've been experiencing challenges with repeatability of peak areas across analytes , columns and methods.
I'm currently trouble shooting the issue. I'm running repeat injections of a Caffeine standard. The concentration is 100 ppb, injection volume is 10 ul. Areas start off being repeatable, then drop and change . Minimum value = 5455, maximum = 14419, median 13081. Signal-to-noise ratio is also all over with a minimum = 40870, maximum = 585158 and median =161406.
I've checked that there aren't pressure drops / spikes on the UPLC, no leaks that I can detect (pressure test passed, but I can't test up to the pre-column heater as I don't have a blank to blank off at this point). I've repeatedly primed and purged the injector, the purge solvent is mobile phase.
On the MS side I've done a resolution tune (passed) and have calibrated.
Any comments / suggestions as to how I can improve repeatability? %RSD = 25
*I hope that you are not using the MS data for quantification as that can result in wide variability (due to non-optimized settings and the fact that most MS data is only used for qualitative ID, not quant). If you can not get a std such as caffeine to work perfectly, then there is no point in running samples.
General peak area or retention time variation problems that I see as a consultant are often caused by: methods that do not follow good chromatography fundamentals such as: use proper K primes; the column is not properly washed with a stronger solvent than used in the mobile phase after each analysis (leading to fouling); inadequate equilibration; integration settings are not appropriate; detection settings are not appropriate; sample preparation issues and/or lack of hardware maintenance. Troubleshooting must start with the basics (Instrument OQ and PV) and is something that can not be done via the web (only in-person, on-site, reviewing the specific equipment used and methods chosen). If possible, hire a local professional to review the methods and systems used. A second set of "eyes" on the problem can be very helpful to quickly find the cause.
Here is a link to a general article that addresses these types of common problems. Perhaps it will help ?
- "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."; https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
iquid mass spectrometry enables the analysis of complex mixtures of small particles and is widely applied in a variety of research fields. Rapid developments in the devices used to extract information are determined by the advantages of this technology through a distinct set of retention time and mass-to-charge ratio. Which acts as a relative measure of the abundance of the compound that produces the feature, God willing, provided you with some information.
Reproducibility can be checked by making replicate measurements and computing standard deviation and relative standard deviations for different sets of values of retention time for a given measurement so as to assure precision.
For any one who actually reads and understand my question, this is the mostly likely answer.
Although this is a relatively new system, prior to my arrival there was an instance of high level contamination with a pesticide, requiring a source clean. The behaviour that we are currently seeing indicates that the contamination went beyond the source and that we are seeing RF discharge happening. The symptoms that are most indicative of this are flat baselines and angular peaks as a result of mass shifts. It is time to strip and clean.
Hi Nial.
For me your problem may come from your injector.
Do you use an autosampler ?
If yes then first replace the seals you can (syringe seal first if there is one).
then try again your repeatablity test.
hope it's useful.
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