Chromatography Forum

My pre-run, blank, and samples using different solvents (water, methanol, and acetonitrile) show the same peaks. I've tried flushing the column to remove the peaks, however when I'm trying the pre-run again, the same peaks appear. What could be the possible reason for this? Thank you.

Most likely cause would be mobile phase. What mobile phase are you using? Also if you are using mass spec, can you not get an analysis of that peak?

Does it matter? It can be really difficult to get rid of all background peaks completely, especially in a very sensitive instrument. If you are looking for particular targets, or differences between samples, quite often background masses are an irritation that can be ignored rather than a disaster that must be solved. They can come from mobile phase, or from lingering contaminants (for example if you've run samples with significant levels of DMSO you may see DMSO and its cluster ions for a long time).

If you're looking for families of known targets, it may be better to work with extracted ion chromatograms and avoid background masses; if you're looking at broad-scale profiling, you can filter out background masses/background peaks.

Most likely cause would be mobile phase. What mobile phase are you using? Also if you are using mass spec, can you not get an analysis of that peak?

i have tried two binary eluent system, water-methanol and water-acetonitrile. still same result. the pre-run, blank, even the qc pooled sample gives the same peaks.

Does it matter? It can be really difficult to get rid of all background peaks completely, especially in a very sensitive instrument. If you are looking for particular targets, or differences between samples, quite often background masses are an irritation that can be ignored rather than a disaster that must be solved. They can come from mobile phase, or from lingering contaminants (for example if you've run samples with significant levels of DMSO you may see DMSO and its cluster ions for a long time).

If you're looking for families of known targets, it may be better to work with extracted ion chromatograms and avoid background masses; if you're looking at broad-scale profiling, you can filter out background masses/background peaks.

i still dont have known targets since i am still focusing on untargeted metabolomics. i tried flushing the column to remove the contaminants, but after trying the pre-run, still same peaks appear. even my blank, qc, and samples have the same peaks.

I agree with lmh that these peaks do not necessarily matter, even in untargeted approaches where one may filter them out either during acquisition (e.g. exclusion list for DDA) or data processing. With the advent of ultra-sensitive instruments in full scan mode, there is no way to get perfectly flat TICs.

However, could you share which m/z you observe and in which mode (pos or neg)? Maybe one of us has seen these peaks and can provide further recommendation.

Not enough information to answer, but one of the very first things that comes to mind is carryover contamination. Have you properly serviced the auto-injector? Has the system been properly cleaned and serviced? Injectors wear out quickly and a worn or scratched valve rotor or seal will continue to create extra peaks when a blank, sample or anything is run.

"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/c ... on-in.html