retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
#hplc-massspec 2024-11-17
Sunday37 messages9 participantstimes are UTC
Highlights from this day
- rotate_the_site — shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly 15:57
- VialBot — Reminder set. I will post here in 4 days. 16:14
- month_six_me — if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result 16:15
- area_percent — how much sample does a lab actually need for identity as well as purity if the number you are staring at is a third of what you expected, do the charge state… 16:39
- salt_bridge — tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5 214nm sees the amide backbone so it sees everything. 280 only sees aromatics 17:03
need more sample
does ms/ms actually prove identity or just make it very likely
ms/ms or it isnt identity
within inter-lab range
[edited]retention time is a hint
integration difference
[edited]a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
run a blank
M = 4113.6
[M+2H]2+ = (4113.6 + 2.02) / 2 = 2057.8
[M+3H]3+ = (4113.6 + 3.02) / 3 = 1372.2
observed 1372.3 -> consistentshallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
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deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
shallow gradient
plus 0.98 is deamidation
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
Reminder set. I will post here in 4 days.
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
can you tell a dimer from the chromatogram alone
how much sample does a lab actually need for identity as well as purity
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
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ok so update from 23 months ago: switched to asking for the chromatogram rather than the number and it changed everything
thats a charge state
truncations show as a mass deficit matching one or more residues. you need the sequence to say which
show me the spectrum
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i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
coming back after 6 months, what changed in how you read these
thats co-elution
plus sixteen, is that always oxidation
relative retention time against a standard is comparable between labs. absolute minutes are not
a shoulder on a peak is information, and usually bad information, happy to be corrected
why does integration change the number so much
genuine question does retention time on its own tell you what the compound is
my peak has a shoulder on it, what does that usually mean
how do you tell co-elution from a clean single peak
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
for the archive is there any point running a blank if the sample is the question
is uv at 214 or 280 better for peptides