sorry to jump in co-elution is the failure mode that a beautiful single peak is most likely to be hiding
#hplc-massspec 2025-07-08
Tuesday44 messages9 participantstimes are UTC
Highlights from this day
- hk_hplc — how would a truncation sequence show up 13:52
- hk_hplc — two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly 13:54
- LC_MS_Lena — ms/ms sequencing is the only thing i would call identity. everything else is circumstantial 14:28
- HPLC_Hank — the first peak is the solvent. it is always the solvent, i think 17:43
within inter-lab range
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly, still working it out
run a blank
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how would a truncation sequence show up
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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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does retention time on its own tell you what the compound is
shallow gradient
plus 0.98 is deamidation
relative retention time against a standard is comparable between labs. absolute minutes are not, we shall see
whats a normal gradient for a peptide this size
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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need more sample
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why does integration change the number so much
integration difference
for the archive is monoisotopic or average mass the one on most certificates
[edited]i was wrong about tailing factor. it is usually the injection or the pH, not the column
[edited]why do two labs integrate the same peak differently
ms/ms or it isnt identity
Archive lookup: HPLC_Hank first appears in this channel on 2024-11-10.
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
plus 16 is oxidation
is there any point running a blank if the sample is the question
ask for the trace
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
[edited]retention time is a hint
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
thats co-elution
[edited]integration choices move the number by a few tenths easily. that is most of your inter-lab variance, i think
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
follow up do you trust an area percent from a shallow gradient
can you tell a dimer from the chromatogram alone
nobody runs enough blanks, including me
how much sample does a lab actually need for identity as well as purity
thats the solvent peak
you need more sample for identity than for purity. plan for that when you post it
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
do the arithmetic
the first peak is the solvent. it is always the solvent, i think
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thats a charge state
show me the spectrum
update from 3 months ago: switched to asking for the chromatogram rather than the number and it changed everything
*Medutest not the other one
quick one deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant