i was wrong about tailing factor. it is usually the injection or the pH, not the column
#hplc-massspec 2026-05-10
Sunday54 messages11 participantstimes are UTC
Highlights from this day
- rowan_reads — a certificate with a number and no chromatogram is asking you to trust the integration you cannot see 16:50
- forty_five_kg — the first peak is the solvent. it is always the solvent 19:01
- auckland_aliquot — semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2 20:01
- two_labs_two_answers — how do you tell co-elution from a clean single peak 20:19
nobody runs enough blanks, including me
[edited]truncations show as a mass deficit matching one or more residues. you need the sequence to say which, ill dig out the number
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
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214nm sees the amide backbone so it sees everything. 280 only sees aromatics, i could be wrong
does ms/ms actually prove identity or just make it very likely
thats the solvent peak
is monoisotopic or average mass the one on most certificates
ms/ms or it isnt identity
right so how would a truncation sequence show up
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
retention time is a hint
plus 16 is oxidation
update on the earlier thing the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
need more sample
slightly off topic but plus sixteen, is that always oxidation
*that should say weekly
shallow gradient
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
show me the spectrum
a warm transit can put a visible degradant peak on a chromatogram. that is what 15 days at ambient does
quick one integration choices move the number by a few tenths easily. that is most of your inter-lab variance
coming back after 24 months, what changed in how you read these
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whats a plus one dalton shift usually
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update from 10 months ago: switched to asking for the chromatogram rather than the number and it changed everything
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily, not advice obviously
the first peak is the solvent. it is always the solvent
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if the lab reports area percent without stating the wavelength, the number is less useful than it looks, thats just me
ok so tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
relative retention time against a standard is comparable between labs. absolute minutes are not
has anyone ever had a result where the mass was right and the purity was wrong
thats co-elution
oxidation is about plus 15.99 Da and is usually methionine or tryptophan, i have it written down somewhere
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer, i could be wrong
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
plus 0.98 is deamidation
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
i have a number at about a third of what i expected, is that a charge state
*that should say weekly
why do two labs integrate the same peak differently
within inter-lab range
how much sample does a lab actually need for identity as well as purity
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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do the arithmetic
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how do you spot a deletion in a sequence from mass alone
M = 4113.6
[M+2H]2+ = (4113.6 + 2.02) / 2 = 2057.8
[M+3H]3+ = (4113.6 + 3.02) / 3 = 1372.2
observed 1372.3 -> consistentsorry to jump in does retention time on its own tell you what the compound is
Batch lookup SG-1177: 5 independent reports on file, earliest 2025-08-13.
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
a shoulder on a peak is information, and usually bad information
how do you tell co-elution from a clean single peak
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whats a normal gradient for a peptide this size
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ask for the trace
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