relative retention time against a standard is comparable between labs. absolute minutes are not
#hplc-massspec 2026-03-18
Wednesday45 messages10 participantstimes are UTC
Highlights from this day
run a blank
does ms/ms actually prove identity or just make it very likely
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
whats the void volume peak, is that always solvent
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
shallow gradient
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
thats the solvent peak
[edited]truncations show as a mass deficit matching one or more residues. you need the sequence to say which
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tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
i was wrong about tailing factor. it is usually the injection or the pH, not the column
do the arithmetic
nobody runs enough blanks, including me, thats just me
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got a mass spec printout with my order and the biggest peak is at 1372 which is nowhere near 4113 so i assume its fake
do not assume that. 1372 is almost exactly what you would expect
how
electrospray does not usually give you the bare molecular weight. it gives you the molecule carrying protons, and it divides by how many. so you see m over z, not M
the arithmetic, do it once and you will never panic again
m/z = (M + n * 1.00728) / n n = number of added protons
semaglutide, average M = 4113.58 Da
[M+2H]2+ (4113.58 + 2.015) / 2 = 2057.80
[M+3H]3+ (4113.58 + 3.022) / 3 = 1372.20
[M+4H]4+ (4113.58 + 4.029) / 4 = 1029.40
[M+5H]5+ (4113.58 + 5.036) / 5 = 823.72
so your 1372 peak is the triply charged ion.
nothing is wrong. your instrument just did not
report the deconvoluted mass.🤯13🙏9
oh. ok that is very reassuring and also i feel stupid
everybody hits that once. it is a rite of passage in here
and the reverse direction is the useful skill. given a peak, work out M for each plausible n and see which one lands on something real
worked backwards from a real-ish peak
reverse it: unknown peak at 1605.5
n=1 M = 1605.5 - 1.007 = 1604.5
n=2 M = 1605.5*2 - 2.015 = 3208.99
n=3 M = 1605.5*3 - 3.022 = 4813.48
n=4 M = 1605.5*4 - 4.029 = 6417.97
n=3 lands on ~4813.5 which is tirzepatide.
the others do not correspond to anything in
the family, so n=3 is the reading.
and if you can see two charge states of the same
species the answer is forced, you can solve for n.so one peak is ambiguous and two peaks is basically an answer
two peaks plus an isotope spacing, yes. the spacing between isotope peaks is 1/n, so 0.33 spacing means triply charged and you never had to guess
if the instrument resolves isotopes at all. plenty of the printouts floating around this market do not
rough reference, approximate average masses only
| Compound | Approx. average M (Da) | [M+3H]3+ | [M+4H]4+ |
|---|---|---|---|
| semaglutide | ~4113.6 | ~1372.2 | ~1029.4 |
| tirzepatide | ~4813.5 | ~1605.5 | ~1204.4 |
| liraglutide | ~3751 | ~1251 | ~938.8 |
| (unknown at 3+ 1400) | ~4197 | 1400 | 1050 |
and the approximatelys are doing work there. quote those to one decimal at most in chat, the exact figures depend on whether you mean average or monoisotopic
whats the difference between those
monoisotopic uses only the lightest isotope of every atom. average uses the natural abundance mix.
for something small the two are nearly the same.
for a 4 kDa peptide with a couple of hundred carbons the average sits a few Da above the monoisotopic, because a fraction of your carbons are carbon-13.
low resolution instruments report an average-ish number. high resolution ones let you pick the monoisotopic peak. they are not the same number and people compare them as if they were
which produces the endless it is 2 Da off, is it degraded panic
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guilty. did exactly that with a Bachem reference figure last year
worth pinning the charge state block, this question comes up monthly
21:47pinned the charge state arithmetic
one more. my printout has peaks at 1372 and also 1377ish. is the second one an impurity
5 Da apart on a 3+ ion means about 15 or 16 Da apart on the neutral. that is the oxidation signature
16 divided by 3 is 5.33. so yes, oxidised species, sitting right where you would expect it
how bad is that
depends entirely on how much of it there is, and a mass spectrum is a poor way to judge how much. intensity in ESI is not proportional to amount across different species
that is another thing people get wrong constantly. the tall peak is not necessarily the abundant one
*not reliably. ionisation efficiency differs between species and a modified peptide can ionise better or worse than the parent
so mass spec for identity, HPLC for how much
roughly that, yes. and neither of them for how many milligrams are in the vial, that is the assay against a standard
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the SSA sheet i have does exactly this properly, deconvoluted mass plus the charge states listed. it is the best MS page i have seen off a reseller
post it, i want to see what good looks like
ssa-lot-24-0908-ms-page.pdf
1 page · 96 KB · not retained in the public archive