nobody runs enough blanks, including me
#hplc-massspec 2025-11-12
Wednesday47 messages10 participantstimes are UTC
Highlights from this day
- blank_run — can you tell a dimer from the chromatogram alone 12:44
- blank_run — whats a plus one dalton shift usually 13:11
- VialBot — New independent result logged — ERP, lot SG-1177, purity 99.4% (VendorInvestigate). 13:31
- endotoxin_ed — oxidation is about plus 15.99 Da and is usually methionine or tryptophan, for what its worth 13:33
- fasting_insulin — whats the void volume peak, is that always solvent 14:22
thats the solvent peak
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genuine question i asked PeptideMeter for the raw trace and they sent it. that is the right answer to that question
ms/ms or it isnt identity
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plus 0.98 is deamidation
a shoulder on a peak is information, and usually bad information
run a blank
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the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
right so if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
can you tell a dimer from the chromatogram alone
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co-elution is the failure mode that a beautiful single peak is most likely to be hiding
plus sixteen, is that always oxidation
*that should say weekly
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
while im here you need more sample for identity than for purity. plan for that when you post it
whats a plus one dalton shift usually
fridge-temps.csv
778 rows · not retained in the public archive
the first peak is the solvent. it is always the solvent
do the arithmetic
while im here how do you spot a deletion in a sequence from mass alone
retention time is a hint
within inter-lab range
thats a charge state
[edited]slightly off topic but a warm transit can put a visible degradant peak on a chromatogram. that is what 18 days at ambient does, ymmv
New independent result logged — ERP, lot SG-1177, purity 99.4% (VendorInvestigate).
oxidation is about plus 15.99 Da and is usually methionine or tryptophan, for what its worth
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whats a normal gradient for a peptide this size
ask for the trace
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial, not advice obviously
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
for the archive 214nm sees the amide backbone so it sees everything. 280 only sees aromatics
genuine question coming back after 9 months, what changed in how you read these
whats the void volume peak, is that always solvent
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 -> consistenti have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
[edited]shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly, someone check my working
update from 3 months ago: switched to asking for the chromatogram rather than the number and it changed everything
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
integration choices move the number by a few tenths easily. that is most of your inter-lab variance, we shall see
plus 16 is oxidation
i was wrong about tailing factor. it is usually the injection or the pH, not the column
relative retention time against a standard is comparable between labs. absolute minutes are not
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
is monoisotopic or average mass the one on most certificates
is uv at 214 or 280 better for peptides
baseline is drifting badly, is that the column or the mobile phase
integration difference
[edited]tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2