shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
#hplc-massspec 2025-12-11
Thursday32 messages6 participantstimes are UTC
Highlights from this day
- mg_per_ml — is uv at 214 or 280 better for peptides 14:27
- karl_fischer — while im here whats the void volume peak, is that always solvent 15:47
- mg_per_ml — i have a number at about a third of what i expected, is that a charge state 17:19
- mg_per_ml — co-elution is the failure mode that a beautiful single peak is most likely to be hiding 17:21
ask for the trace
unrelated but do you trust an area percent from a shallow gradient
the first peak is the solvent. it is always the solvent
whats a plus one dalton shift usually
for the archive my peak has a shoulder on it, what does that usually mean
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
plus sixteen, is that always oxidation
214nm sees the amide backbone so it sees everything. 280 only sees aromatics, ymmv
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need more sample
whats a normal gradient for a peptide this size
how do you tell co-elution from a clean single peak
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
is uv at 214 or 280 better for peptides
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can you tell a dimer from the chromatogram alone
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, your mileage will differ
update from 11 months ago: switched to asking for the chromatogram rather than the number and it changed everything
coming back after 16 months, what changed in how you read these
nobody runs enough blanks, including me, thats just me
while im here whats the void volume peak, is that always solvent
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plus 0.98 is deamidation
why does integration change the number so much
i was wrong about tailing factor. it is usually the injection or the pH, not the column, ymmv
how would a truncation sequence show up
i have a number at about a third of what i expected, is that a charge state
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co-elution is the failure mode that a beautiful single peak is most likely to be hiding
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a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you, your mileage will differ
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genuine question why do two labs integrate the same peak differently
thats a charge state
i asked PeptideMeter for the raw trace and they sent it. that is the right answer to that question