while im here i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
#hplc-massspec 2025-11-04
Tuesday49 messages12 participantstimes are UTC
Highlights from this day
- lyophile_liv — while im here i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer two labs a point or two apart on the same lot is… 16:15
- VialBot — Digest for the week of 2025-05-22 has been published. 16:43
- peak_split — if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result 17:18
- karl_fischer — nobody runs enough blanks, including me, i think 17:21
- VialBot — Testing queue: 5 submissions open, 87 awaiting dispatch. 18:53
sorry to jump in shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
integration difference
a shoulder on a peak is information, and usually bad information
run a blank
coming back to this integration choices move the number by a few tenths easily. that is most of your inter-lab variance
thats a charge state
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Digest for the week of 2025-05-22 has been published.
while im here a warm transit can put a visible degradant peak on a chromatogram. that is what 26 days at ambient does
ask for the trace
plus 16 is oxidation
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
whats a plus one dalton shift usually
[edited]retention time is a hint
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
[edited]if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
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thats co-elution
nobody runs enough blanks, including me, i think
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ms/ms or it isnt identity
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semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
plus 0.98 is deamidation
i was wrong about tailing factor. it is usually the injection or the pH, not the column
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
show me the spectrum
do the arithmetic
update from 10 months ago: switched to asking for the chromatogram rather than the number and it changed everything
[edited]slightly off topic but you need more sample for identity than for purity. plan for that when you post it
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
Testing queue: 5 submissions open, 87 awaiting dispatch.
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
why do two labs integrate the same peak differently
[edited]the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
thats a charge state
thats the solvent peak
shallow gradient
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant, n of 1 obviously
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
food_noise_offco-elution is the failure mode that a beautiful single peak is most likely to be hiding
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
i have a number at about a third of what i expected, is that a charge state
need more sample, take that with a pinch of salt
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
sorry to jump in does ms/ms actually prove identity or just make it very likely
update from 21 months ago: switched to asking for the chromatogram rather than the number and it changed everything
coming back after 6 months, what changed in how you read these
unrelated but monoisotopic and average diverge more the bigger the molecule. say which one you are quoting, still working it out
has anyone ever had a result where the mass was right and the purity was wrong
slightly off topic but is monoisotopic or average mass the one on most certificates