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#hplc-massspec 2025-08-23

Saturday44 messages8 participantstimes are UTC
Highlights from this day
  • two_four_ceiling — i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer 19:42
  • ms_ms_mira — co-elution is the failure mode that a beautiful single peak is most likely to be hiding 19:50
  • deadspace — if the lab reports area percent without stating the wavelength, the number is less useful than it looks, i have it written down somewhere 20:11
  • deadspace — truncations show as a mass deficit matching one or more residues. you need the sequence to say which, for what its worth 20:41
  • two_four_ceiling — nobody runs enough blanks, including me baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column 21:20
LN
lot_number_lou19:10

a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you

LN
lot_number_lou19:16

you need more sample for identity than for purity. plan for that when you post it

is uv at 214 or 280 better for peptides

is monoisotopic or average mass the one on most certificates

*VendorInvestigate not the other one

run a blank

need more sample

💀11

integration difference

🎉8💀10

i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer

[edited]
😂3❤️10
MM
ms_ms_miralab19:50

co-elution is the failure mode that a beautiful single peak is most likely to be hiding

📉2❤️9🧪13

baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column

DE
deadspace20:11

if the lab reports area percent without stating the wavelength, the number is less useful than it looks, i have it written down somewhere

DE
deadspace20:41

truncations show as a mass deficit matching one or more residues. you need the sequence to say which, for what its worth

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 substance
A1
a1c_lag21:00

update from 17 months ago: switched to asking for the chromatogram rather than the number and it changed everything

deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant

TF
two_four_ceiling21:20

nobody runs enough blanks, including me
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column

if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result, happy to be corrected

show me the spectrum

ok so integration choices move the number by a few tenths easily. that is most of your inter-lab variance

214nm sees the amide backbone so it sees everything. 280 only sees aromatics, for what its worth

A1
a1c_lag21:41

do you trust an area percent from a shallow gradient

quick one the honest position is that hobby-level interpretation of a chromatogram is worth something but not much

ms/ms sequencing is the only thing i would call identity. everything else is circumstantial

retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily

within inter-lab range

LU
luerlok22:15

the first peak is the solvent. it is always the solvent

DE
deadspace22:30

i asked Janoshik for the raw trace and they sent it. that is the right answer to that question

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