plus 16 is oxidation
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#hplc-massspec 2026-01-25
Sunday54 messages11 participantstimes are UTC
Highlights from this day
- vienna_vial — deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant 10:56
- fake_tracking — how do you spot a deletion in a sequence from mass alone 12:41
- hydrate_hana — i was wrong about tailing factor. it is usually the injection or the pH, not the column 12:49
- fake_tracking — a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you 14:39
nobody runs enough blanks, including me
show me the spectrum
is a tailing factor of anything specific a problem
how much sample does a lab actually need for identity as well as purity
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
do the arithmetic
whats a normal gradient for a peptide this size
why does integration change the number so much
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
shallow gradient
follow up can you tell a dimer from the chromatogram alone
is uv at 214 or 280 better for peptides
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
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you need more sample for identity than for purity. plan for that when you post it
whats the void volume peak, is that always solvent
retention time is a hint
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
right so relative retention time against a standard is comparable between labs. absolute minutes are not
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
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i asked VendorInvestigate for the raw trace and they sent it. that is the right answer to that question, i think
does ms/ms actually prove identity or just make it very likely
coming back after 4 months, what changed in how you read these
On this day 5 years ago this channel logged 117 messages.
how do you tell co-elution from a clean single peak
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column, from memory
plus 0.98 is deamidation
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do you trust an area percent from a shallow gradient
how do you spot a deletion in a sequence from mass alone
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retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
i was wrong about tailing factor. it is usually the injection or the pH, not the column
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the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
coming back to this i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
thats the solvent peak
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
baseline is drifting badly, is that the column or the mobile phase
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ms/ms or it isnt identity
need more sample
genuine question my peak has a shoulder on it, what does that usually mean
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
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two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
update from 8 months ago: switched to asking for the chromatogram rather than the number and it changed everything, still working it out
the first peak is the solvent. it is always the solvent
i have a number at about a third of what i expected, is that a charge state
is there any point running a blank if the sample is the question
integration difference
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does retention time on its own tell you what the compound is
*PeptideMeter not the other one
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
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ask for the trace
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214nm sees the amide backbone so it sees everything. 280 only sees aromatics
genuine question is monoisotopic or average mass the one on most certificates
update from 3 months ago: switched to asking for the chromatogram rather than the number and it changed everything
thats co-elution
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