thats the solvent peak
#hplc-massspec 2026-01-03
Saturday49 messages13 participantstimes are UTC
Highlights from this day
- blank_run — is uv at 214 or 280 better for peptides 15:44
- tokyo_taper — a warm transit can put a visible degradant peak on a chromatogram. that is what 16 days at ambient does 16:01
- LC_MS_Lena — for the archive why does integration change the number so much 17:00
- cold_chain_cmdr — i was wrong about tailing factor. it is usually the injection or the pH, not the column 21:25
why do two labs integrate the same peak differently
plus 16 is oxidation
do you trust an area percent from a shallow gradient
ok so i have a number at about a third of what i expected, is that a charge state
does retention time on its own tell you what the compound is
need more sample
whats a normal gradient for a peptide this size
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
[edited]a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
the first peak is the solvent. it is always the solvent
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update from 1 months ago: switched to asking for the chromatogram rather than the number and it changed everything
is uv at 214 or 280 better for peptides
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ask for the trace
a warm transit can put a visible degradant peak on a chromatogram. that is what 16 days at ambient does
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do the arithmetic
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily, thats just me
does ms/ms actually prove identity or just make it very likely
retention time is a hint
shallow gradient
a shoulder on a peak is information, and usually bad information
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial, ask me again in a month
co-elution is the failure mode that a beautiful single peak is most likely to be hiding, anyway
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
for the archive why does integration change the number so much
peptidemeter-summary-kp-0925.pdf
2 pages · 323 KB · not retained in the public archive
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
right so truncations show as a mass deficit matching one or more residues. you need the sequence to say which
show me the spectrum
for the archive 214nm sees the amide backbone so it sees everything. 280 only sees aromatics
for the archive coming back after 10 months, what changed in how you read these
i was wrong about tailing factor. it is usually the injection or the pH, not the column
baseline is drifting badly, is that the column or the mobile phase
thats a charge state
relative retention time against a standard is comparable between labs. absolute minutes are not
has anyone ever had a result where the mass was right and the purity was wrong
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
i asked Janoshik for the raw trace and they sent it. that is the right answer to that question
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, someone check my working
how would a truncation sequence show up
nobody runs enough blanks, including me
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semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
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within inter-lab range
whats the void volume peak, is that always solvent
fridge-temps.csv
907 rows · not retained in the public archive
ms/ms or it isnt identity
thats co-elution
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
plus 0.98 is deamidation
i was wrong about tailing factor. it is usually the injection or the pH, not the column
vial-b-0114-front.png
900 × 1200 · 557 KB · not retained in the public archive