plus 16 is oxidation
#hplc-massspec 2026-03-02
Monday51 messages10 participantstimes are UTC
Highlights from this day
- two_four_ceiling — retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily 19:19
- birch_bloods — semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2, thats one data point integration choices move the number by a few tenths easily.… 20:50
- gradient_greg — can you tell a dimer from the chromatogram alone 21:25
- peak_split — two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly 22:31
whats a normal gradient for a peptide this size
why do two labs integrate the same peak differently
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result, not advice obviously
plus sixteen, is that always oxidation
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
ask for the trace
has anyone ever had a result where the mass was right and the purity was wrong
[edited]how would a truncation sequence show up
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
medutest-report-kp-0925.pdf
3 pages · 846 KB · not retained in the public archive
shallow gradient
does ms/ms actually prove identity or just make it very likely
is a tailing factor of anything specific a problem
run a blank
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
a warm transit can put a visible degradant peak on a chromatogram. that is what 24 days at ambient does
integration difference
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
[edited]i have a number at about a third of what i expected, is that a charge state
Reminder for gradient_greg: dose day is today. Set 6 days ago.
do the arithmetic
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
within inter-lab range
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
coa-kp-0925.pdf
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why does integration change the number so much
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while im here 214nm sees the amide backbone so it sees everything. 280 only sees aromatics
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2, thats one data point
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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while im here how do you tell co-elution from a clean single peak
baseline is drifting badly, is that the column or the mobile phase
medutest-report-e-2205.pdf
3 pages · 536 KB · not retained in the public archive
while im here whats a plus one dalton shift usually
whats the void volume peak, is that always solvent
you need more sample for identity than for purity. plan for that when you post it, someone check my working
slightly off topic but a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
show me the spectrum
is monoisotopic or average mass the one on most certificates
ms/ms or it isnt identity
retention time is a hint
thats a charge state
can you tell a dimer from the chromatogram alone
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monoisotopic and average diverge more the bigger the molecule. say which one you are quoting, not advice obviously
the first peak is the solvent. it is always the solvent
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
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plus 0.98 is deamidation