if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
#hplc-massspec 2026-02-25
Wednesday56 messages12 participantstimes are UTC
Highlights from this day
- fasting_insulin — is monoisotopic or average mass the one on most certificates 04:54
- VialBot — New independent result logged — ERP, lot A-2418, purity 98.1% (Janoshik). 04:55
- luerlok — ms/ms sequencing is the only thing i would call identity. everything else is circumstantial 06:41
- luerlok — does retention time on its own tell you what the compound is 09:20
the first peak is the solvent. it is always the solvent
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
show me the spectrum
is monoisotopic or average mass the one on most certificates
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New independent result logged — ERP, lot A-2418, purity 98.1% (Janoshik).
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do you trust an area percent from a shallow gradient
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run a blank
coming back after 17 months, what changed in how you read these
i have a number at about a third of what i expected, is that a charge state
quick one oxidation is about plus 15.99 Da and is usually methionine or tryptophan
is there any point running a blank if the sample is the question
you need more sample for identity than for purity. plan for that when you post it, ask me again in a month
how would a truncation sequence show up
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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ms/ms or it isnt identity
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right so semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2, for what its worth
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
plus 16 is oxidation
slightly off topic but shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily, ask me again in a month
a warm transit can put a visible degradant peak on a chromatogram. that is what 6 days at ambient does
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
nobody runs enough blanks, including me
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
i was wrong about tailing factor. it is usually the injection or the pH, not the column
need more sample
update from 6 months ago: switched to asking for the chromatogram rather than the number and it changed everything
thats a charge state
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unrelated but if the lab reports area percent without stating the wavelength, the number is less useful than it looks
right so the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly, your mileage will differ
does retention time on its own tell you what the compound is
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 substance214nm sees the amide backbone so it sees everything. 280 only sees aromatics
is uv at 214 or 280 better for peptides
relative retention time against a standard is comparable between labs. absolute minutes are not
ask for the trace
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
integration difference
whats a normal gradient for a peptide this size
why do two labs integrate the same peak differently
co-elution is the failure mode that a beautiful single peak is most likely to be hiding, i have it written down somewhere
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
retention time is a hint, for what its worth
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
thats co-elution
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
has anyone ever had a result where the mass was right and the purity was wrong
retention time is a hint
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
do the arithmetic
a shoulder on a peak is information, and usually bad information
while im here is a tailing factor of anything specific a problem
for the archive how do you tell co-elution from a clean single peak