you need more sample for identity than for purity. plan for that when you post it
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#hplc-massspec 2025-06-14
Saturday49 messages10 participantstimes are UTC
Highlights from this day
- surpass_two — you need more sample for identity than for purity. plan for that when you post it 21:30
- triple_ag_tom — ms/ms sequencing is the only thing i would call identity. everything else is circumstantial 21:49
- truncation_tru — monoisotopic and average diverge more the bigger the molecule. say which one you are quoting 21:53
- truncation_tru — whats the void volume peak, is that always solvent 22:04
- HPLC_Hank — if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, ymmv 23:05
if the lab reports area percent without stating the wavelength, the number is less useful than it looks, your mileage will differ
genuine question baseline is drifting badly, is that the column or the mobile phase
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
ask for the trace
i have a number at about a third of what i expected, is that a charge state
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
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if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
quick one i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
relative retention time against a standard is comparable between labs. absolute minutes are not
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
thats a charge state
slightly off topic but how do you tell co-elution from a clean single peak
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
whats the void volume peak, is that always solvent
lot-log.csv
436 rows · not retained in the public archive
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
nobody runs enough blanks, including me, ask me again in a month
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
[edited]update on the earlier thing 214nm sees the amide backbone so it sees everything. 280 only sees aromatics
retention time is a hint
thats the solvent peak
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unrelated but a shoulder on a peak is information, and usually bad information
quick one how do you spot a deletion in a sequence from mass alone
for the archive update from 12 months ago: switched to asking for the chromatogram rather than the number and it changed everything
update on the earlier thing the first peak is the solvent. it is always the solvent, n of 1 obviously
within inter-lab range
Testing queue: 4 submissions open, 89 awaiting dispatch.
i asked PeptideMeter for the raw trace and they sent it. that is the right answer to that question
Digest for the week of 2025-04-24 has been published.
plus 0.98 is deamidation
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
do the arithmetic
genuine question a warm transit can put a visible degradant peak on a chromatogram. that is what 14 days at ambient does
sorry to jump in has anyone ever had a result where the mass was right and the purity was wrong
seizure_letterupdate on the earlier thing the first peak is the solvent. it is always the solvent, n…
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, ymmv
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plus 16 is oxidation
[edited]show me the spectrum
[redacted at member request] · archive policy
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seizure_lettera certificate with a number and no chromatogram is asking you to trust the integration…
integration difference
[edited]shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
is uv at 214 or 280 better for peptides
within inter-lab range
why does integration change the number so much
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly