coming back after 6 months, what changed in how you read these
#hplc-massspec 2026-03-08
Sunday27 messages7 participantstimes are UTC
Highlights from this day
- VialBot — Recon calculator: 30mg in 3ml = 4mg/ml. 14:30
- VialBot — On this day 4 years ago this channel logged 65 messages. 14:47
- ollie_orders — two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly 15:16
- plus_sixteen — deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant 16:12
- hk_hplc — i was wrong about tailing factor. it is usually the injection or the pH, not the column 16:15
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
whats a normal gradient for a peptide this size
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
Recon calculator: 30mg in 3ml = 4mg/ml.
On this day 4 years ago this channel logged 65 messages.
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unrelated but is there any point running a blank if the sample is the question
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
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plus 0.98 is deamidation
do the arithmetic
within inter-lab range
while im here does ms/ms actually prove identity or just make it very likely
shallow gradient
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
thats the solvent peak
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
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show me the spectrum
peak RRT area% note
1 0.42 0.31 solvent front
2 0.88 0.44 related substance
3 1.00 98.72 main
4 1.14 0.53 related substanceif 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
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truncations show as a mass deficit matching one or more residues. you need the sequence to say which
thats co-elution
ok so the first peak is the solvent. it is always the solvent
[edited]a warm transit can put a visible degradant peak on a chromatogram. that is what 14 days at ambient does
nobody runs enough blanks, including me, happy to be corrected
follow up is uv at 214 or 280 better for peptides
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer