relative retention time against a standard is comparable between labs. absolute minutes are not
nobody runs enough blanks, including me
#hplc-massspec 2026-02-08
Sunday39 messages9 participantstimes are UTC
Highlights from this day
- HPLC_Hank — i was wrong about tailing factor. it is usually the injection or the pH, not the column 19:28
- HPLC_Hank — update on the earlier thing is monoisotopic or average mass the one on most certificates 19:35
- ms_ms_mira — two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly a shoulder on a peak is information, and usually bad… 19:43
- HPLC_Hank — whats a plus one dalton shift usually 22:31
i was wrong about tailing factor. it is usually the injection or the pH, not the column
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update on the earlier thing is monoisotopic or average mass the one on most certificates
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update from 8 months ago: switched to asking for the chromatogram rather than the number and it changed everything, for what its worth
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
a shoulder on a peak is information, and usually bad information
plus sixteen, is that always oxidation
update on the earlier thing why do two labs integrate the same peak differently
whats the void volume peak, is that always solvent
unrelated but do you trust an area percent from a shallow gradient
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
how would a truncation sequence show up
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
[edited]slightly off topic but is a tailing factor of anything specific a problem
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
run a blank
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
follow up does retention time on its own tell you what the compound is
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
within inter-lab range
has anyone ever had a result where the mass was right and the purity was wrong
my peak has a shoulder on it, what does that usually mean
thats co-elution
shallow gradient
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
plus 16 is oxidation
you need more sample for identity than for purity. plan for that when you post it, anyway
a warm transit can put a visible degradant peak on a chromatogram. that is what 11 days at ambient does
ms/ms or it isnt identity
how much sample does a lab actually need for identity as well as purity
need more sample
why does integration change the number so much
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plus 0.98 is deamidation
coming back to this does ms/ms actually prove identity or just make it very likely
update on the earlier thing baseline is drifting badly, is that the column or the mobile phase
show me the spectrum
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whats a plus one dalton shift usually
peak RRT area% note
1 0.42 0.31 solvent front
2 0.88 0.44 related substance
3 1.00 98.72 main
4 1.14 0.53 related substance