Thank you so much for this!!! I was wondering if you happen to have a scientific source for this equation? You saved this grad students week. Thanks again!!!
Thanks a lot for the clear explanation of dwell time, Deeply from my heart I appreciate your work. Too many Lcmsms experts tried to explain but they couldn't, the reason that they were not fully understood dwell time concept. One last favor, could you please explain MRM detection window?
I guess it depends on your vendor and the software - so I am only guessing what you mean by this term. My guess is that it refers to a time controlled experiment where you enter a RT window for each MRM and then that MRM experiment is only collected during the specified time window. Other MRMs may also be occurring during that time, controlled by their own RT acquisition window.
Hi, could I have another one question please? I performed the separation of ciprofloxacin and its metabolites on two different columns - Kinetex F5 and CSH UPLC fluorophenyl column. Better separation was achieved on the Kinetex column. On the CSH UPLC fluorophenyl column desethylenciprofloxacin elutes for almost 3 minutes. There is a great tailing here. Do you think this is due to the fact that both columns are prepared with a different technology? Because both columns contain pentafluorophenyl and yet the separation is different.
Thank you for your kind words. Unfortunately this was a one of, that I recorded for something else when I was a consultant and just decided to post it on youtube.com
Thank you so much this video is made me understand easily. Moreover, I would like to know if I want to check data point of each compound after finished run from 20 mixed compounds. How to calculate data point of that by hand? The point is my dwell is not the same number and individual peaks are show different width.
You can certainly step thru the spectra to see how many data points you have. And if you are doing good gradient chromatography the peak widths should be about the same unless you are doing something wrong - the exception would be peaks at the beginning or the end of the gradient. Those can be a little wider, and that's ok, as you are looking to find the # of data points for the narrowest peaks - extra points are fine
very helpful video, just wondering if you need to take into account the internal standard in the calculation or should it be removed to calculate the dwell time? IS normally monitors one transition, how do we manipulate the equation to take care of this fact?
Hello. I would like to ask you for advice. Does the mobile phase have a major effect on the optimization of the mass spectrometer? When optimizing the mass spectrometer, I chose a mixture of acetonitrile and water as the mobile phase. However, when optimizing the HPLC method, I chose a mixture of buffer and methanol as the mobile phase. I would like to know if the change of the mobile phase has an effect on the mass spectrometry (finding of precursor and product ions, voltage of ion optics and collision energy)
The mobile phase can affect the precursor, for example enhancing M+H over M+Na or creating M+NH4 instead of M+H. If the precusor does not change, it will have zero effect on any of the product ions, the ion optics, or the CE
Thank you so much!!!!! I had been taught this differently by two people and needed to confirm!! What happens if you set 20 msec/point (dwell time)? Will the system add more points (>20)? I guess the higher the dwell time the better right?
You set the dwell time so that you have the appropriate number of points across the peak. As long as you are above the manufacturer's minimum dwell time, a longer dwell time does not matter. The intensity of the signal is the intensity of the signal at that point in time, and measuring it for longer will not increase that intensity.
smoothing on the data processing side (after acquisition) is always possible in any vendor's software but it's always more desirable to smooth DURING acquisition by setting to correct dwell time - if you have too few points post acquisition smoothing can make the peak visually look better, but your data will be inconsistent
Depends on whether you are doing qualitative work or quantitative. If you are on a quad you want to maximize your full scan time because it directly affects your sensitivity. For qual, you want a minimum of the scans across your LC peak but a better number would be 5-8 so you can tells it's a peak. If your peak was 6 secs wide, then you'd probably want a rate of one spectrum per second to get 6 data points, but no slower than 2 sec per scan acquiring 3 spectra. Any more than 6 and you are giving up sensitivity that you don't have to give up. For quantitative analysis you'd want at least twelve scans across the peak, because that's how many is needed to accurately and precisely measure the area under the peak. Probably shoot for 15 secs to make sure you get at least 12. Any more than that and you are throwing away sensitivity. So for our 6 sec wide peak above, you'd have to be scanning a 1 spectrum every 0.5 sec, or 2 spectra per second
Thank you for the response! I am just doing a little mass detective work using Full scan (Q1) to determine what form the compound of interest is in M+H, M+NH4, M+Na etc.
@@Gmen595 You can't really without an accurate mass system that you can trust - but what you can look for is mass differences - M+H, from that +17 is NH4, +22 is M+Na, + 38 is M+K - so if you see multiple ions look for those patterns to deduce what is what
It actually has nothing to do with the sensitivity as we are talking here about triple quads which are parked at selected masses and are not in a scanning mode. Too many points become a practical issue in that you start to see the irregularity of the spray itself, and integrator algorithms, which look for slope changes, may only integrate a portion of the chromatographic peak
