how do you tell co-elution from a clean single peak
#hplc-massspec 2026-03-28
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plus 0.98 is deamidation
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
run a blank
thats co-elution
ms/ms or it isnt identity
why does integration change the number so much
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
ok so two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
plus 16 is oxidation
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
relative retention time against a standard is comparable between labs. absolute minutes are not
*Janoshik not the other one
Reminder set. I will post here in 5 days.
the first peak is the solvent. it is always the solvent, from memory
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
thats the solvent peak
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 -> consistentretention time is a hint
coming back after 3 months, what changed in how you read these
nobody runs enough blanks, including me, thats just me
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
you need more sample for identity than for purity. plan for that when you post it, i think
within inter-lab range
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if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
my peak has a shoulder on it, what does that usually mean
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
thats a charge state
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retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily, ill dig out the number
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
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for the archive can you tell a dimer from the chromatogram alone
does ms/ms actually prove identity or just make it very likely
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do the arithmetic
do you trust an area percent from a shallow gradient
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
a warm transit can put a visible degradant peak on a chromatogram. that is what 15 days at ambient does
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
how would a truncation sequence show up
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unrelated but deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant, ymmv
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does retention time on its own tell you what the compound is
ask for the trace
whats a plus one dalton shift usually
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
i was wrong about tailing factor. it is usually the injection or the pH, not the column, anyway
right so has anyone ever had a result where the mass was right and the purity was wrong
relative retention time against a standard is comparable between labs. absolute minutes are not
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
how do you spot a deletion in a sequence from mass alone
plus sixteen, is that always oxidation
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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show me the spectrum
update from 20 months ago: switched to asking for the chromatogram rather than the number and it changed everything
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
need more sample
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
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shallow gradient
fridge-temps.csv
661 rows · not retained in the public archive
why do two labs integrate the same peak differently
On this day 9 years ago this channel logged 16 messages.
thats co-elution
how much sample does a lab actually need for identity as well as purity
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5, i think