oxidation is about plus 15.99 Da and is usually methionine or tryptophan
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#hplc-massspec 2026-02-12
Thursday50 messages11 participantstimes are UTC
Highlights from this day
- amber_vial — ms/ms sequencing is the only thing i would call identity. everything else is circumstantial 17:32
- void_volume — tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5 20:48
- area_percent — deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant 21:48
ms/ms or it isnt identity
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how much sample does a lab actually need for identity as well as purity
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
is uv at 214 or 280 better for peptides
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ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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do the arithmetic
retention time is a hint
shallow gradient
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is monoisotopic or average mass the one on most certificates
baseline is drifting badly, is that the column or the mobile phase
genuine question has anyone ever had a result where the mass was right and the purity was wrong
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co-elution is the failure mode that a beautiful single peak is most likely to be hiding
ok so baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
relative retention time against a standard is comparable between labs. absolute minutes are not
a shoulder on a peak is information, and usually bad information
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question, ask me again in a month
show me the spectrum
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*PeptideMeter not the other one
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
integration difference
thats a charge state
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coming back after 11 months, what changed in how you read these
how do you spot a deletion in a sequence from mass alone
thats the solvent peak
within inter-lab range
ask for the trace
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plus 16 is oxidation
need more sample
you need more sample for identity than for purity. plan for that when you post it
plus 0.98 is deamidation
[edited]i was wrong about tailing factor. it is usually the injection or the pH, not the column
plus sixteen, is that always oxidation
Verification log updated: HJ — evidence added, status unchanged.
is there any point running a blank if the sample is the question
right so whats a normal gradient for a peptide this size
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
integration choices move the number by a few tenths easily. that is most of your inter-lab variance, n of 1 obviously
a warm transit can put a visible degradant peak on a chromatogram. that is what 20 days at ambient does
how would a truncation sequence show up
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
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why do two labs integrate the same peak differently
nobody runs enough blanks, including me
run a blank
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the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
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shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
update from 10 months ago: switched to asking for the chromatogram rather than the number and it changed everything
[edited]two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly