sorry to jump in integration choices move the number by a few tenths easily. that is most of your inter-lab variance
#hplc-massspec 2026-06-14
Sunday42 messages9 participantstimes are UTC
Highlights from this day
- peak_split — shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly, n of 1 obviously 16:28
- batch_variance — whats the void volume peak, is that always solvent 17:49
- deamidation — ms/ms sequencing is the only thing i would call identity. everything else is circumstantial 18:47
- gorse_gains — 214nm sees the amide backbone so it sees everything. 280 only sees aromatics ms/ms sequencing is the only thing i would call identity. everything else is circumstantial 19:12
how do you spot a deletion in a sequence from mass alone
the first peak is the solvent. it is always the solvent, thats one data point
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
plus 0.98 is deamidation
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for the archive tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly, n of 1 obviously
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is monoisotopic or average mass the one on most certificates
plus sixteen, is that always oxidation
my peak has a shoulder on it, what does that usually mean
i was wrong about tailing factor. it is usually the injection or the pH, not the column
relative retention time against a standard is comparable between labs. absolute minutes are not
while im here do you trust an area percent from a shallow gradient
i have a number at about a third of what i expected, is that a charge state
thats the solvent peak
*VendorInvestigate not the other one
right so i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
plus 16 is oxidation
can you tell a dimer from the chromatogram alone
update from 17 months ago: switched to asking for the chromatogram rather than the number and it changed everything
whats the void volume peak, is that always solvent
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oxidation is about plus 15.99 Da and is usually methionine or tryptophan
coming back to this truncations show as a mass deficit matching one or more residues. you need the sequence to say which
coming back after 3 months, what changed in how you read these
slightly off topic but why do two labs integrate the same peak differently
thats co-elution
follow up why does integration change the number so much
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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*Janoshik not the other one
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
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sorry to jump in how much sample does a lab actually need for identity as well as purity
while im here whats a plus one dalton shift usually
ms/ms or it isnt identity
show me the spectrum
*Medutest not the other one
how would a truncation sequence show up
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a shoulder on a peak is information, and usually bad information
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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need more sample
you need more sample for identity than for purity. plan for that when you post it, ask me again in a month