ms/ms or it isnt identity
#hplc-massspec 2026-05-12
Tuesday49 messages8 participantstimes are UTC
Highlights from this day
- HPLC_Hank — monoisotopic and average diverge more the bigger the molecule. say which one you are quoting 20:21
- two_four_ceiling — for the archive can you tell a dimer from the chromatogram alone 20:30
- peak_split — update from 21 months ago: switched to asking for the chromatogram rather than the number and it changed everything 20:39
- two_four_ceiling — nobody runs enough blanks, including me 21:14
show me the spectrum
ask for the trace
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my peak has a shoulder on it, what does that usually mean
for the archive the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
relative retention time against a standard is comparable between labs. absolute minutes are not
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
unrelated but two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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ok so i asked PeptideMeter for the raw trace and they sent it. that is the right answer to that question, from memory
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
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for the archive can you tell a dimer from the chromatogram alone
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sorry to jump in whats a plus one dalton shift usually
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
update from 21 months ago: switched to asking for the chromatogram rather than the number and it changed everything
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 substanceplus 0.98 is deamidation
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
baseline is drifting badly, is that the column or the mobile phase
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
why does integration change the number so much
how do you spot a deletion in a sequence from mass alone
the first peak is the solvent. it is always the solvent
genuine question does retention time on its own tell you what the compound is
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
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 substancei have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
do the arithmetic
plus sixteen, is that always oxidation
nobody runs enough blanks, including me
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thats the solvent peak
integration difference
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slightly off topic but i have a number at about a third of what i expected, is that a charge state
genuine question whats the void volume peak, is that always solvent
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within inter-lab range
unrelated but if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, happy to be corrected
why do two labs integrate the same peak differently
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tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
thats co-elution
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deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result, from memory
i was wrong about tailing factor. it is usually the injection or the pH, not the column
need more sample
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
has anyone ever had a result where the mass was right and the purity was wrong
retention time is a hint
dark_for_ten_daysslightly off topic but i have a number at about a third of what i expected, is that a…
a shoulder on a peak is information, and usually bad information
how would a truncation sequence show up
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly