if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
#hplc-massspec 2026-01-06
Tuesday41 messages7 participantstimes are UTC
Highlights from this day
- chaser_chris — nobody runs enough blanks, including me, thats just me 09:30
- void_volume — a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you a warm transit can put a visible degradant peak on a… 11:03
- area_percent — if the lab reports area percent without stating the wavelength, the number is less useful than it looks, i think 12:17
ok so shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
is uv at 214 or 280 better for peptides
a shoulder on a peak is information, and usually bad information
baseline is drifting badly, is that the column or the mobile phase
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
how much sample does a lab actually need for identity as well as purity
nobody runs enough blanks, including me, thats just me
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integration difference
show me the spectrum
thats a charge state
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why does integration change the number so much
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
retention time is a hint
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
[edited]right so 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
a warm transit can put a visible degradant peak on a chromatogram. that is what 21 days at ambient does
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thats the solvent peak
update from 1 months ago: switched to asking for the chromatogram rather than the number and it changed everything
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
i was wrong about tailing factor. it is usually the injection or the pH, not the column
why do two labs integrate the same peak differently
the first peak is the solvent. it is always the solvent
is monoisotopic or average mass the one on most certificates
whats a normal gradient for a peptide this size
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 substanceif the lab reports area percent without stating the wavelength, the number is less useful than it looks, i think
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a warm transit can put a visible degradant peak on a chromatogram. that is what 16 days at ambient does
is a tailing factor of anything specific a problem
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5, n of 1 obviously
need more sample
for the archive coming back after 2 months, what changed in how you read these
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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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you need more sample for identity than for purity. plan for that when you post it
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
plus 0.98 is deamidation
sorry to jump in whats the void volume peak, is that always solvent
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
ms/ms or it isnt identity