coming back to this does retention time on its own tell you what the compound is
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
#hplc-massspec 2026-05-30
Saturday43 messages10 participantstimes are UTC
Highlights from this day
- nordic_price_nils — a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you 15:04
- lead_time_lars — slightly off topic but truncations show as a mass deficit matching one or more residues. you need the sequence to say which 16:39
- sharps_bin_sid — integration choices move the number by a few tenths easily. that is most of your inter-lab variance 17:20
- salt_bridge — co-elution is the failure mode that a beautiful single peak is most likely to be hiding 17:37
- nordic_price_nils — coming back to this the honest position is that hobby-level interpretation of a chromatogram is worth something but not much 18:35
can you tell a dimer from the chromatogram alone
the first peak is the solvent. it is always the solvent
do you trust an area percent from a shallow gradient
you need more sample for identity than for purity. plan for that when you post it
need more sample
whats a normal gradient for a peptide this size
ms/ms or it isnt identity
a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you
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shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
relative retention time against a standard is comparable between labs. absolute minutes are not
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
ask for the trace
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
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
semaglutide is about 4113.6 Da. [M+2H]2+ lands near 2057.8, [M+3H]3+ near 1372.2
update on the earlier thing a certificate with a number and no chromatogram is asking you to trust the integration you cannot see, i could be wrong
coming back after 14 months, what changed in how you read these
nobody runs enough blanks, including me
how do you tell co-elution from a clean single peak
a warm transit can put a visible degradant peak on a chromatogram. that is what 26 days at ambient does
tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
slightly off topic but truncations show as a mass deficit matching one or more residues. you need the sequence to say which
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 substanceis a tailing factor of anything specific a problem
plus 16 is oxidation
update on the earlier thing why does integration change the number so much
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coming back to this i asked PeptideMeter for the raw trace and they sent it. that is the right answer to that question
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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 -> consistentupdate from 5 months ago: switched to asking for the chromatogram rather than the number and it changed everything
co-elution is the failure mode that a beautiful single peak is most likely to be hiding
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genuine question a shoulder on a peak is information, and usually bad information
does ms/ms actually prove identity or just make it very likely
is there any point running a blank if the sample is the question
i was wrong about tailing factor. it is usually the injection or the pH, not the column
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if the lab reports area percent without stating the wavelength, the number is less useful than it looks, your mileage will differ
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do the arithmetic
coming back to this the honest position is that hobby-level interpretation of a chromatogram is worth something but not much
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shallow gradient, for what its worth
the first peak is the solvent. it is always the solvent
baseline drift on a gradient is normal. baseline drift on an isocratic hold is your column
run a blank