a warm transit can put a visible degradant peak on a chromatogram. that is what 15 days at ambient does
#hplc-massspec 2026-01-23
Friday49 messages12 participantstimes are UTC
Highlights from this day
- apob_over_ldl — coming back after 16 months, what changed in how you read these 17:59
- never_for_sale — if the lab reports area percent without stating the wavelength, the number is less useful than it looks 18:46
- warsaw_vial — semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2 19:53
- assay_not_purity — the honest position is that hobby-level interpretation of a chromatogram is worth something but not much 20:00
- fern_freezes — while im here whats the void volume peak, is that always solvent 21:19
update on the earlier thing i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
coming back after 16 months, what changed in how you read these
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oxidation is about plus 15.99 Da and is usually methionine or tryptophan
does ms/ms actually prove identity or just make it very likely
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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
update on the earlier thing 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
is monoisotopic or average mass the one on most certificates
why do two labs integrate the same peak differently
ask for the trace
plus 0.98 is deamidation
coa-a-2418.pdf
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214nm sees the amide backbone so it sees everything. 280 only sees aromatics
[edited]if the lab reports area percent without stating the wavelength, the number is less useful than it looks
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do the arithmetic
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
can you tell a dimer from the chromatogram alone
is uv at 214 or 280 better for peptides
nobody runs enough blanks, including me
update on the earlier thing i was wrong about tailing factor. it is usually the injection or the pH, not the column
plus sixteen, is that always oxidation
do you trust an area percent from a shallow gradient
ms/ms or it isnt identity
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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whats a plus one dalton shift usually
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly, for what its worth
ok so integration choices move the number by a few tenths easily. that is most of your inter-lab variance
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
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how much sample does a lab actually need for identity as well as purity
whats a normal gradient for a peptide this size
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
you need more sample for identity than for purity. plan for that when you post it
how do you tell co-elution from a clean single peak
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
thats a charge state
shallow gradient
is a tailing factor of anything specific a problem
thats co-elution
coming back to this how would a truncation sequence show up
while im here whats the void volume peak, is that always solvent
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need more sample
truncations show as a mass deficit matching one or more residues. you need the sequence to say which, still working it out
run a blank
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result, n of 1 obviously
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, ill dig out the number