right so integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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#hplc-massspec 2026-05-31
Sunday58 messages11 participantstimes are UTC
Highlights from this day
- back_from_away — deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant, ill dig out the number truncations show as a mass deficit matching one or more… 21:09
- back_from_away — how do you tell co-elution from a clean single peak 22:04
- stack_sceptic — if the lab reports area percent without stating the wavelength, the number is less useful than it looks 22:14
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
genuine question is monoisotopic or average mass the one on most certificates
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integration difference
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
Reminder for tail_factor: dose day is today. Set 7 days ago.
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coming back after 1 months, what changed in how you read these
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right so i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
a shoulder on a peak is information, and usually bad information
plus sixteen, is that always oxidation
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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 spot a deletion in a sequence from mass alone
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deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant, ill dig out the number
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
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 -> consistentfor the archive i asked VendorInvestigate for the raw trace and they sent it. that is the right answer to that question
does retention time on its own tell you what the compound is
update on the earlier thing if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
relative retention time against a standard is comparable between labs. absolute minutes are not
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has anyone ever had a result where the mass was right and the purity was wrong
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
i was wrong about tailing factor. it is usually the injection or the pH, not the column
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
within inter-lab range
is uv at 214 or 280 better for peptides
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5, we shall see
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
thats the solvent peak
a warm transit can put a visible degradant peak on a chromatogram. that is what 26 days at ambient does
do the arithmetic
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the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
retention time is a hint
coming back to this how would a truncation sequence show up
update on the earlier thing two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
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
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column, thats just me
how do you tell co-elution from a clean single peak
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show me the spectrum
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nobody runs enough blanks, including me
shallow gradient
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
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you need more sample for identity than for purity. plan for that when you post it
plus 16 is oxidation
i have a number at about a third of what i expected, is that a charge state
ms/ms or it isnt identity
need more sample
sorry to jump in my peak has a shoulder on it, what does that usually mean
i was wrong about tailing factor. it is usually the injection or the pH, not the column
run a blank
can you tell a dimer from the chromatogram alone
*PeptideMeter not the other one
follow up baseline is drifting badly, is that the column or the mobile phase
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
sorry to jump in why does integration change the number so much
thats co-elution
is there any point running a blank if the sample is the question
ok so the first peak is the solvent. it is always the solvent