shallow gradient
#hplc-massspec 2025-06-22
Sunday33 messages8 participantstimes are UTC
Highlights from this day
- no_chargeback — ok so how do you tell co-elution from a clean single peak 12:16
- tail_factor — if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic 15:34
- ferritin_fay — a warm transit can put a visible degradant peak on a chromatogram. that is what 11 days at ambient does, your mileage will differ 15:35
- fake_tracking — nobody runs enough blanks, including me 17:44
plus 0.98 is deamidation
whats a normal gradient for a peptide this size
why does integration change the number so much
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
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monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
whats a plus one dalton shift usually
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
ok so how do you tell co-elution from a clean single peak
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plus 16 is oxidation
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
how do you spot a deletion in a sequence from mass alone
Digest for the week of 2024-11-05 has been published.
integration difference
why do two labs integrate the same peak differently
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
[edited]is a tailing factor of anything specific a problem
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
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a warm transit can put a visible degradant peak on a chromatogram. that is what 11 days at ambient does, your mileage will differ
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if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
do the arithmetic
ms/ms or it isnt identity
ask for the trace
thats a charge state
integration choices move the number by a few tenths easily. that is most of your inter-lab variance, your mileage will differ
does retention time on its own tell you what the compound is
thats co-elution
[edited]show me the spectrum
coming back to this semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
run a blank
need more sample
nobody runs enough blanks, including me
[edited]peak RRT area% note
1 0.42 0.31 solvent front
2 0.88 0.44 related substance
3 1.00 98.72 main
4 1.14 0.53 related substance