co-elution is the failure mode that a beautiful single peak is most likely to be hiding
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#hplc-massspec 2026-01-31
Saturday43 messages10 participantstimes are UTC
Highlights from this day
retention time is a hint
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
thats co-elution
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how would a truncation sequence show up
do the arithmetic
Verification log updated: KP — evidence added, status unchanged.
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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do you trust an area percent from a shallow gradient
genuine question i was wrong about tailing factor. it is usually the injection or the pH, not the column
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
whats a plus one dalton shift usually
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truncations show as a mass deficit matching one or more residues. you need the sequence to say which
relative retention time against a standard is comparable between labs. absolute minutes are not
sorry to jump in update from 11 months ago: switched to asking for the chromatogram rather than the number and it changed everything
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i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer, take that with a pinch of salt
need more sample
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
ms/ms or it isnt identity
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
within inter-lab range
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column, your mileage will differ
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see, i could be wrong
integration difference
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
is uv at 214 or 280 better for peptides
quick one oxidation is about plus 15.99 Da and is usually methionine or tryptophan
run a blank
thats the solvent peak
[edited]*that should say weekly
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
why do two labs integrate the same peak differently
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plus sixteen, is that always oxidation
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
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how do you spot a deletion in a sequence from mass alone
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
ask for the trace
transit-times.csv
433 rows · not retained in the public archive
a warm transit can put a visible degradant peak on a chromatogram. that is what 22 days at ambient does
nobody runs enough blanks, including me
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
plus 0.98 is deamidation
update on the earlier thing does retention time on its own tell you what the compound is