i was wrong about tailing factor. it is usually the injection or the pH, not the column
#hplc-massspec 2025-05-13
Tuesday44 messages11 participantstimes are UTC
Highlights from this day
- noct.titrate — baseline is drifting badly, is that the column or the mobile phase 18:20
- tb500_tobias — relative retention time against a standard is comparable between labs. absolute minutes are not, anyway 18:54
- mcg_not_mg — has anyone ever had a result where the mass was right and the purity was wrong 21:47
coming back after 1 months, what changed in how you read these
genuine question whats the void volume peak, is that always solvent
does ms/ms actually prove identity or just make it very likely
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a warm transit can put a visible degradant peak on a chromatogram. that is what 24 days at ambient does
baseline is drifting badly, is that the column or the mobile phase
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 -> consistentshallow gradient
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i asked VendorInvestigate for the raw trace and they sent it. that is the right answer to that question
*that should say weekly
while im here two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
whats a plus one dalton shift usually
why do two labs integrate the same peak differently
relative retention time against a standard is comparable between labs. absolute minutes are not, anyway
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whats a normal gradient for a peptide this size
how do you spot a deletion in a sequence from mass alone
thats co-elution
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
thats a charge state
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
[edited]plus 0.98 is deamidation
why does integration change the number so much
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
is monoisotopic or average mass the one on most certificates
a warm transit can put a visible degradant peak on a chromatogram. that is what 20 days at ambient does
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
chromatogram-overlay.png
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integration difference
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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
ms/ms or it isnt identity
thats the solvent peak
run a blank
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
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do the arithmetic
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
sorry to jump in plus sixteen, is that always oxidation
[edited]how would a truncation sequence show up
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
[edited]baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column, thats one data point
the first peak is the solvent. it is always the solvent
plus 16 is oxidation
how much sample does a lab actually need for identity as well as purity
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can you tell a dimer from the chromatogram alone
has anyone ever had a result where the mass was right and the purity was wrong
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you need more sample for identity than for purity. plan for that when you post it