Newbie in HPLC-MS: does this look alright?

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

16 posts Page 1 of 2
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Hi All,

So, I got a brand-new Shimadzu HPLC-MS system (with a single quad), and I find the noise really high... Apparently, it's normal but is it really?!

I am using Formic 0.1% Water/ACN (fresh bottles) and run it at 1mL/min on an Ascentis Express 5x2.1 x2.7.

I am scanning from 100 to 600 m/z.

Thanks for any insights you might have!

K
One source of such high-frequency noise could be overly high sampling rate (short dwell time). Meaning the detector has to do measurements too frequently. This means that there's not enough time to gather a lot of signal. But the random noise stays more or less the same - so you end up with lower signal-to-noise ratio.

You have 5646 scans, which seems quite high. It means that the measurement is taken ever ~50 milliseconds. I think I typically see injections with less than 1000 scans per 5 min.
Software Engineer at elsci.io (my contact: stanislav.bashkyrtsev@elsci.io)
Thanks a lot for your help!

I think it's getting a little bit better! I still wonder if this is what one should expect or not :)

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If this is the tic for scan 100-600 m/z then you sum alot of noise. How does it look if you extract the xic for your compound? Why scan mode and not sim?
Thanks a lot!

My SIMs are fine, flat line and nice peaks - but we do organic syntheses and the idea is to run mixture of unknown compounds to try to find out what they are.

I have a GC-MS too, where thinks are a bit easier to see (but not everything likes being heated up at 280C in the injector!)

I was just wondering if such a signal/noise ratio is "normal" in TIC in HPLC-MS.
The noise depends on several parameters in HPLC-MS (e.g. mobile phase type, solvent brand, column, whether a maintenance was just performed on the HPLC, source parameter values etc.), but it is notoriously high on single and triple quadrupoles in full scan mode. By increasing the scan time you have flattened the trace and decreased the noise but, as we can see in the chromatograms you posted, the s/n ratio for your peak at 1.7 min hasn't changed. I am not saying it was a bad idea to increase the scan time, it was actually very appropriate in your case (1000 Da/s is the fastest rate modern quads can efficiently deal with, and lower rates would always be preferable) but it had no effect on s/n ratios. To improve signal-to-noise ratios, and assuming that your HPLC and mobile phases are reasonably clean (meaning there is nothing abnormal with your instrument - you can ask your Shimadzu contact about it, they must have hundreds of examples in-house) you can display the BPI chromatogram instead of the TIC, which should improve things a bit. Another option would be to check which ions are the highest (usually small ions between 50-150 Da) and, if possible, start the scan at a higher mass. Otherwise increase the amounts injected, if possible.
KevinBe wrote:
Thanks a lot!

My SIMs are fine, flat line and nice peaks - but we do organic syntheses and the idea is to run mixture of unknown compounds to try to find out what they are.

I have a GC-MS too, where thinks are a bit easier to see (but not everything likes being heated up at 280C in the injector!)

I was just wondering if such a signal/noise ratio is "normal" in TIC in HPLC-MS.


Then I understand. Looks like you run es+ and es- but I could read table wrong. I dont know your ms spec but seems like you would get too low dwell times running in es+ and es- with polarity switch. I would trying a narrower scan range and run either es+ or es- to compare . I dont get good results on our system running such a large mass range and getting 15 points across uhplc peak, you sum alot of spectra with bad s/n when you have poor ion statistics.

I might add that I find that acetonitrile can give a lot of background in scan mode. Methanol gives far less background. Dont know what grade you use but getting UHPLC MS grade is likely worth the cost. I find that different brands and batches of MS grade also differ a lot in background.
Thanks!
So I tried your recommendations, but it didn't lead to any major improvements.

1. Standard scan from 100 to 600 m/z in +/- mode.

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2. +/- mode scanning from 100 to 400 m/z

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3. + mode only scanning from 100 to 400 m/z
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Yes, did not change much I am afraid. Was expecting that you would get a more stable baseline with less noise in it, like you hade in the first screen. Don't have any good advice I am afraid more than trying to figure out where the background signal comes from (solvents vials etc). I also get a lot of ions in MS scan mode so not sure how much background one can get rid of. Clean solvents and high grade consumables is a start.
Thanks a lot though!

My base line is mostly due to an m/z of 143. No clue what is it. It has always been there since the new HPLC has arrived. The solvents are brand new, so is the column.
Waters has a great document listing common background ESI+ and ESI- ions (can be found with a Google search). The closest thing I found for m/z 143 is ESI+ m/z 144 which is tripropylamine (TPA)
"Have you tried explaining it to the rubber duck?"
When our new uhplc system was installed the waters enginer had to clean it with both acid wash formic acid, basic wash NH4 and lots of flushing with ipa to get rid of contaminants from production. If it is a new system it might have some contaminants left from production. New systems are not clean.
per_oxid wrote:
When our new uhplc system was installed the waters enginer had to clean it with both acid wash formic acid, basic wash NH4 and lots of flushing with ipa to get rid of contaminants from production. If it is a new system it might have some contaminants left from production. New systems are not clean.


On Agilent engineer I know uses a 25% mixture of Cyclohexane, Acetonitrile, Isopropyl and I cant remember if the fourth was methanol or something else. He would run that slowly overnight without a column in place to flush out the system.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
On Agilent engineer I know uses a 25% mixture of Cyclohexane, Acetonitrile, Isopropyl and I cant remember if the fourth was methanol or something else. He would run that slowly overnight without a column in place to flush out the system.


The Agilent flushing mix is
50% IPA
25% ACN
15% Cyclohexane
10% DCM
I will guess that your organic chemists are creating libraries by reacting one mixture of compounds (Ma) with another mixture of compounds (Mb).

Suppose that the smallest molecule that can be lost is H2O from a condensation reaction of one of Ma with one of Mb.
Your lowest m/z to start data acquisition is then

[Ma lowest + Mb lowest - H2O + H]+ (but, also drop an additional m/z 18 in case the product molecule can lose an H2O)

Suppose that some compounds can actually do addition, with no loss of a small neutral.
Your highest m/z to end acquisition is then
[Ma highest + Mb highest + H]+ (allow some more m/z to see possible Na+ and K+ adducts).

If you know what reactants your chemists are using, you can set up your scan range quite easily.

BTW, if you still have to run the wide scan-range, there was a paper in Analytical Chemistry in about 1997-2000 that showed mathematically that the best S/N ratio summing across a chromatographic peak is obtained by ONLY taking the scans that are at least 37% of the peak maximum height.

If you sum baseline-to-baseline across the whole peak you get too much noise and too little signal.

Regards,
JMB
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