right so i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
#hplc-massspec 2025-11-16
Sunday43 messages7 participantstimes are UTC
Highlights from this day
- vialkeeper — integration choices move the number by a few tenths easily. that is most of your inter-lab variance 17:44
- eat_more_please — deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant 18:50
- verify_or_dont — oxidation is about plus 15.99 Da and is usually methionine or tryptophan 19:17
- provincial_pat — how do you spot a deletion in a sequence from mass alone if the lab reports area percent without stating the wavelength, the number is less useful than it looks 20:29
need more sample
ms/ms or it isnt identity
whats a normal gradient for a peptide this size
is uv at 214 or 280 better for peptides
integration difference
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nobody runs enough blanks, including me
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column, from memory
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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retention time is a hint
do the arithmetic
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within inter-lab range
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, ymmv
why do two labs integrate the same peak differently
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update on the earlier thing relative retention time against a standard is comparable between labs. absolute minutes are not
fridge-temps.csv
92 rows · not retained in the public archive
thats co-elution
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*that should say weekly
plus sixteen, is that always oxidation
you need more sample for identity than for purity. plan for that when you post it
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial, n of 1 obviously
has anyone ever had a result where the mass was right and the purity was wrong
whats a plus one dalton shift usually
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
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a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
show me the spectrum
i was wrong about tailing factor. it is usually the injection or the pH, not the column
[edited]shallow gradient
thats the solvent peak
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a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
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run a blank
plus 0.98 is deamidation
transit-times.csv
264 rows · not retained in the public archive
plus 16 is oxidation
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
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
how do you spot a deletion in a sequence from mass alone
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
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is a tailing factor of anything specific a problem
thats a charge state
is there any point running a blank if the sample is the question
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
the first peak is the solvent. it is always the solvent