update from 26 months ago: switched to asking for the chromatogram rather than the number and it changed everything, i have it written down somewhere
#hplc-massspec 2026-04-04
Saturday56 messages10 participantstimes are UTC
Highlights from this day
- manila_meniscus — while im here if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result 21:32
- lead_time_lars — sorry to jump in why do two labs integrate the same peak differently 21:51
- vik_verifies — how would a truncation sequence show up 22:03
- pharmac_pen — my peak has a shoulder on it, what does that usually mean 22:21
- acetate_ash — a certificate with a number and no chromatogram is asking you to trust the integration you cannot see 23:01
slightly off topic but relative retention time against a standard is comparable between labs. absolute minutes are not
quick one two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly, from memory
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily, ymmv
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
coming back to this coming back after 7 months, what changed in how you read these
while im here if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
chromatogram-overlay.png
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co-elution is the failure mode that a beautiful single peak is most likely to be hiding
ms/ms or it isnt identity
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nobody runs enough blanks, including me
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if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, for what its worth
run a blank
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thats co-elution
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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
sorry to jump in why do two labs integrate the same peak differently
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oxidation is about plus 15.99 Da and is usually methionine or tryptophan
Reminder for lead_time_lars: dose day is today. Set 5 days ago.
thats the solvent peak
quick one has anyone ever had a result where the mass was right and the purity was wrong
baseline is drifting badly, is that the column or the mobile phase
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how would a truncation sequence show up
peptidemeter-summary-c-4402.pdf
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the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
sorry to jump in is uv at 214 or 280 better for peptides
whats a plus one dalton shift usually
thats a charge state
plus 0.98 is deamidation
show me the spectrum
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plus 16 is oxidation
my peak has a shoulder on it, what does that usually mean
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the first peak is the solvent. it is always the solvent
do the arithmetic
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
within inter-lab range
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shallow gradient
a shoulder on a peak is information, and usually bad information
update on the earlier thing monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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truncations show as a mass deficit matching one or more residues. you need the sequence to say which
genuine question is there any point running a blank if the sample is the question
retention time is a hint
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
you need more sample for identity than for purity. plan for that when you post it, from memory
i was wrong about tailing factor. it is usually the injection or the pH, not the column
is monoisotopic or average mass the one on most certificates
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
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is a tailing factor of anything specific a problem
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integration difference
right so do you trust an area percent from a shallow gradient
update on the earlier thing how much sample does a lab actually need for identity as well as purity
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
does retention time on its own tell you what the compound is
ask for the trace
follow up ask for the trace
show me the spectrum