As shown in Figure 1, when analyzing samples, the signal intensity is stable when the ACN concentration is below 20%. However, as the ACN concentration gradually exceeds 20%, the MS1 signal drops sharply. Figure 2 displays the low-concentration ACN test. If the ACN concentration is kept at a maximum of 20% for sample elution, the MS1 signal remains relatively normal, only showing a significant drop in MS1 intensity when the ACN concentration is raised to 76% for column washing. However, if the ACN concentration is increased to 64% during the sample elution process (Figure 3), the MS1 signal intensity sharply decreases much earlier.
I would like to ask those with mass spectrometry operation experience, what could be the potential reasons for this problem? We have checked the column and the tubing in between and found no leakage. The mass spectrometer has also been inspected by an engineer and no issues were found.
Hi! I assume that You use nanoLC for separation. The first thing that comes to my mind is to check Your nanospray, and do it not only in starting conditions (4% ACN) but also in higher concentrations of organic phase (I usually used 30% for benchmarking). It often happens, that nanospray gets way worse with higher ACN concentrations, especially if You use stainless steel emmiters. If You can observe any instability of the spray cone (e.g. bubbling) after going higher with ACN, then You have a source of Your problem ;).
Good luck!
Thank you for your reply. Yes, I am using NanoLC with a stainless steel emitter. I have checked the spray performance of the emitter at 60% ACN and observed no bubble formation. Therefore, issues related to sample atomization can be ruled out as the cause.@ Pawel Pasikowski
Ok, so another thing I would check would be the performance of the ACN pump. BTW, I assume You use the same amount of mobile phase additive (I guess 0,1% formic acid?) to both water and ACN phase, am I right?
Another thing, this might be sample related. I guess You could get similar results by cleaning Your sample on C18 column/tip and eluting with 20-30% ACN...
And last but not least, this could have something to do with incomplete sample loading. I would try to prolong the time of sample loading to a trap column a bit and then check if You get similar results. Actually, this probably should be the first thing to try.
Good luck!
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