integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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#hplc-massspec 2025-06-12
Thursday32 messages7 participantstimes are UTC
Highlights from this day
- is_this_forever — integration choices move the number by a few tenths easily. that is most of your inter-lab variance 15:53
- nhs_pathway_nell — follow up co-elution is the failure mode that a beautiful single peak is most likely to be hiding, ask me again in a month 18:49
- nhs_pathway_nell — while im here a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you 19:09
- monoisotopic — truncations show as a mass deficit matching one or more residues. you need the sequence to say which, n of 1 obviously 19:30
thats co-elution
has anyone ever had a result where the mass was right and the purity was wrong
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
coming back after 9 months, what changed in how you read these
do the arithmetic
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is there any point running a blank if the sample is the question
retention time is a hint
is a tailing factor of anything specific a problem
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
the first peak is the solvent. it is always the solvent
ask for the trace
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
plus sixteen, is that always oxidation
need more sample
baseline is drifting badly, is that the column or the mobile phase
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
plus 16 is oxidation
[edited]do you trust an area percent from a shallow gradient
thats the solvent peak
how do you spot a deletion in a sequence from mass alone
relative retention time against a standard is comparable between labs. absolute minutes are not
plus 0.98 is deamidation
follow up co-elution is the failure mode that a beautiful single peak is most likely to be hiding, ask me again in a month
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214nm sees the amide backbone so it sees everything. 280 only sees aromatics
while im here a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
why does integration change the number so much
truncations show as a mass deficit matching one or more residues. you need the sequence to say which, n of 1 obviously
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show me the spectrum
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 -> consistent🤝5💀3
my peak has a shoulder on it, what does that usually mean