update from 14 months ago: switched to asking for the chromatogram rather than the number and it changed everything, ymmv
#hplc-massspec 2026-04-28
Tuesday56 messages11 participantstimes are UTC
Highlights from this day
- gradient_greg — semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2 19:22
- VialBot — Inter-lab diff for lot D-0718: 98.1% vs 96.8%. Within expected range. 19:50
- freeze_thaw — a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you 20:17
- retention_time — if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic 21:31
thats a charge state
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
does ms/ms actually prove identity or just make it very likely
is uv at 214 or 280 better for peptides
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
is a tailing factor of anything specific a problem
is monoisotopic or average mass the one on most certificates
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see, we shall see
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
ok so oxidation is about plus 15.99 Da and is usually methionine or tryptophan
do you trust an area percent from a shallow gradient
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
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
how much sample does a lab actually need for identity as well as purity
unrelated but does retention time on its own tell you what the compound is
i have a number at about a third of what i expected, is that a charge state
whats the void volume peak, is that always solvent
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
show me the spectrum
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a warm transit can put a visible degradant peak on a chromatogram. that is what 22 days at ambient does
slightly off topic but how do you spot a deletion in a sequence from mass alone
relative retention time against a standard is comparable between labs. absolute minutes are not
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
ms/ms or it isnt identity
run a blank
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semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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 substanceneed more sample
why does integration change the number so much
ask for the trace
the first peak is the solvent. it is always the solvent
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retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
Inter-lab diff for lot D-0718: 98.1% vs 96.8%. Within expected range.
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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integration difference
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
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can you tell a dimer from the chromatogram alone
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truncations show as a mass deficit matching one or more residues. you need the sequence to say which
how would a truncation sequence show up
for the archive plus sixteen, is that always oxidation
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant, i think
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
how do you tell co-elution from a clean single peak
is there any point running a blank if the sample is the question
ok so i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
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ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
[edited]genuine question nobody runs enough blanks, including me
do the arithmetic
plus 0.98 is deamidation
i was wrong about tailing factor. it is usually the injection or the pH, not the column
you need more sample for identity than for purity. plan for that when you post it
thats the solvent peak
integration choices move the number by a few tenths easily. that is most of your inter-lab variance