run a blank
[edited]#hplc-massspec 2025-09-09
Tuesday33 messages7 participantstimes are UTC
Highlights from this day
- fake_tracking — semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2 20:47
- fake_tracking — update from 25 months ago: switched to asking for the chromatogram rather than the number and it changed everything i have had a lot where the mass was exactly right… 20:55
- fake_tracking — i was wrong about tailing factor. it is usually the injection or the pH, not the column 21:14
thats co-elution
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
plus 0.98 is deamidation
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is there any point running a blank if the sample is the question
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
do you trust an area percent from a shallow gradient
you need more sample for identity than for purity. plan for that when you post it
within inter-lab range
relative retention time against a standard is comparable between labs. absolute minutes are not
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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update from 25 months ago: switched to asking for the chromatogram rather than the number and it changed everything
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
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quick one monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
i was wrong about tailing factor. it is usually the injection or the pH, not the column
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while im here why do two labs integrate the same peak differently
a shoulder on a peak is information, and usually bad information
is a tailing factor of anything specific a problem
thats a charge state
do the arithmetic
why does integration change the number so much
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shallow gradient
is monoisotopic or average mass the one on most certificates
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
need more sample
ok so how would a truncation sequence show up
plus 16 is oxidation
unrelated but ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
retention time is a hint
the first peak is the solvent. it is always the solvent, thats just me
whats the void volume peak, is that always solvent
ask for the trace
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while im here you need more sample for identity than for purity. plan for that when you post it
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
is uv at 214 or 280 better for peptides