update from 24 months ago: switched to asking for the chromatogram rather than the number and it changed everything
#hplc-massspec 2025-09-23
Tuesday37 messages11 participantstimes are UTC
Highlights from this day
- ferrous_ash — shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly 17:38
- deamidation — right so if the lab reports area percent without stating the wavelength, the number is less useful than it looks, ill dig out the number 18:31
- VialBot — Channel stats, last 30 days: 22 messages from 35 members. 19:06
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
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a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
thats co-elution
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
coming back after 24 months, what changed in how you read these
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
you need more sample for identity than for purity. plan for that when you post it
ask for the trace
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
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the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
plus 0.98 is deamidation
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
right so if the lab reports area percent without stating the wavelength, the number is less useful than it looks, ill dig out the number
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 substanceshow me the spectrum
has anyone ever had a result where the mass was right and the purity was wrong
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
i was wrong about tailing factor. it is usually the injection or the pH, not the column
Channel stats, last 30 days: 22 messages from 35 members.
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ok so i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
sorry to jump in semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
why does integration change the number so much
nobody runs enough blanks, including me, happy to be corrected
thats the solvent peak
why do two labs integrate the same peak differently
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
a shoulder on a peak is information, and usually bad information
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
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unrelated but does ms/ms actually prove identity or just make it very likely
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plus 16 is oxidation
integration difference
ok so monoisotopic and average diverge more the bigger the molecule. say which one you are quoting, anyway
need more sample
is uv at 214 or 280 better for peptides
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
plus sixteen, is that always oxidation