whats a normal gradient for a peptide this size
#hplc-massspec 2026-03-30
Monday43 messages10 participantstimes are UTC
Highlights from this day
- dexa_or_bia — how much sample does a lab actually need for identity as well as purity 01:54
- hair_month_four — i asked Janoshik for the raw trace and they sent it. that is the right answer to that question 04:19
- marrow.mod — shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly 05:17
for the archive co-elution is the failure mode that a beautiful single peak is most likely to be hiding, from memory
ms/ms or it isnt identity
whats the void volume peak, is that always solvent
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retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
relative retention time against a standard is comparable between labs. absolute minutes are not
[edited]tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
genuine question is monoisotopic or average mass the one on most certificates
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2, we shall see
do the arithmetic
shallow gradient
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
how much sample does a lab actually need for identity as well as purity
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update on the earlier thing plus sixteen, is that always oxidation
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
thats a charge state
ask for the trace
my peak has a shoulder on it, what does that usually mean
why do two labs integrate the same peak differently
can you tell a dimer from the chromatogram alone
[edited]how would a truncation sequence show up
is uv at 214 or 280 better for peptides
i have a number at about a third of what i expected, is that a charge state
plus 0.98 is deamidation
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
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if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
hair_month_fouri asked Janoshik for the raw trace and they sent it. that is the right answer to that…
does ms/ms actually prove identity or just make it very likely
does retention time on its own tell you what the compound is
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
transit-times.csv
821 rows · not retained in the public archive
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within inter-lab range
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you, ymmv
plus 16 is oxidation
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nobody runs enough blanks, including me
07:19marrow.mod pinned a message to this channel
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
thats the solvent peak
how do you tell co-elution from a clean single peak
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
i was wrong about tailing factor. it is usually the injection or the pH, not the column
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
integration difference
is a tailing factor of anything specific a problem
you need more sample for identity than for purity. plan for that when you post it