the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
[edited]#hplc-massspec 2025-03-10
Monday40 messages9 participantstimes are UTC
Highlights from this day
- steady_state_sue — a certificate with a number and no chromatogram is asking you to trust the integration you cannot see 18:24
- homa_ir_hugo — how much sample does a lab actually need for identity as well as purity 18:39
- hiccup_hollis — my peak has a shoulder on it, what does that usually mean semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2 18:51
- peak_split — i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer 19:17
- peak_split — how do you tell co-elution from a clean single peak update from 8 months ago: switched to asking for the chromatogram rather than the number and it changed everything 20:57
i was wrong about tailing factor. it is usually the injection or the pH, not the column, ymmv
why does integration change the number so much
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
[edited]*VendorInvestigate not the other one
whats a normal gradient for a peptide this size
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
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thats a charge state
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly, from memory
how much sample does a lab actually need for identity as well as purity
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ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
my peak has a shoulder on it, what does that usually mean
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
a shoulder on a peak is information, and usually bad information
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
you need more sample for identity than for purity. plan for that when you post it
follow up nobody runs enough blanks, including me
right so a warm transit can put a visible degradant peak on a chromatogram. that is what 15 days at ambient does
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
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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
run a blank
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
is uv at 214 or 280 better for peptides
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
while im here the first peak is the solvent. it is always the solvent, someone check my working
need more sample
can you tell a dimer from the chromatogram alone
thats the solvent peak
shallow gradient
is monoisotopic or average mass the one on most certificates
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
do the arithmetic
is a tailing factor of anything specific a problem
why do two labs integrate the same peak differently
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oxidation is about plus 15.99 Da and is usually methionine or tryptophan
how do you tell co-elution from a clean single peak
update from 8 months ago: switched to asking for the chromatogram rather than the number and it changed everything
update on the earlier thing 214nm sees the amide backbone so it sees everything. 280 only sees aromatics
genuine question coming back after 3 months, what changed in how you read these
truncations show as a mass deficit matching one or more residues. you need the sequence to say which