i was wrong about tailing factor. it is usually the injection or the pH, not the column
#hplc-massspec 2025-04-26
Saturday42 messages10 participantstimes are UTC
Highlights from this day
- deamidation — the first peak is the solvent. it is always the solvent, someone check my working 08:29
- fake_tracking — retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily 09:32
- LC_MS_Lena — relative retention time against a standard is comparable between labs. absolute minutes are not 10:56
- deamidation — is there any point running a blank if the sample is the question 11:48
does retention time on its own tell you what the compound is
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
the first peak is the solvent. it is always the solvent, someone check my working
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update on the earlier thing a warm transit can put a visible degradant peak on a chromatogram. that is what 18 days at ambient does
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
does ms/ms actually prove identity or just make it very likely
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial, from memory
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
how much sample does a lab actually need for identity as well as purity
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
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show me the spectrum
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
you need more sample for identity than for purity. plan for that when you post it
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
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retention time is a hint
i have had a lot where the mass was exactly right and the purity was 96.8. wrong question, right answer
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
is monoisotopic or average mass the one on most certificates
plus 0.98 is deamidation
within inter-lab range
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5, from memory
i asked VendorInvestigate for the raw trace and they sent it. that is the right answer to that question
ms/ms or it isnt identity
unrelated but oxidation is about plus 15.99 Da and is usually methionine or tryptophan
is uv at 214 or 280 better for peptides
unrelated but how do you spot a deletion in a sequence from mass alone
whats a plus one dalton shift usually
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thats the solvent peak
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
nobody runs enough blanks, including me
relative retention time against a standard is comparable between labs. absolute minutes are not
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 number you are staring at is a third of what you expected, do the charge state arithmetic before you panic
whats a normal gradient for a peptide this size
Purity check: no report on file for lot B-0329. Nothing logged either way.
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whats the void volume peak, is that always solvent
the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
unrelated but coming back after 2 months, what changed in how you read these
is there any point running a blank if the sample is the question
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shallow gradient