nobody runs enough blanks, including me, someone check my working
#hplc-massspec 2026-05-07
Thursday44 messages11 participantstimes are UTC
Highlights from this day
- hydrate_hana — sorry to jump in monoisotopic and average diverge more the bigger the molecule. say which one you are quoting 15:16
- sgp_subq — is uv at 214 or 280 better for peptides monoisotopic and average diverge more the bigger the molecule. say which one you are quoting 15:50
- nordic_price_nils — can you tell a dimer from the chromatogram alone 16:12
- hydrate_hana — whats the void volume peak, is that always solvent 17:31
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly
right so update from 24 months ago: switched to asking for the chromatogram rather than the number and it changed everything, from memory
[edited]why do two labs integrate the same peak differently
integration choices move the number by a few tenths easily. that is most of your inter-lab variance
sorry to jump in monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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tirzepatide is about 4813.5 Da, so 2+ is near 2407.8 and 3+ near 1605.5
how do you spot a deletion in a sequence from mass alone
need more sample
is uv at 214 or 280 better for peptides
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting
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ask for the trace
show me the spectrum
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
a certificate with a number and no chromatogram is asking you to trust the integration you cannot see, for what its worth
shallow gradients hide related substances behind the main peak. a steeper one separates and looks worse, honestly
i asked Medutest for the raw trace and they sent it. that is the right answer to that question
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can you tell a dimer from the chromatogram alone
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 -> consistentplus 0.98 is deamidation
a warm transit can put a visible degradant peak on a chromatogram. that is what 21 days at ambient does
integration difference
does ms/ms actually prove identity or just make it very likely
follow up relative retention time against a standard is comparable between labs. absolute minutes are not
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
[edited]slightly off topic but 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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thats a charge state
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co-elution is the failure mode that a beautiful single peak is most likely to be hiding
if the lab reports area percent without stating the wavelength, the number is less useful than it looks
oxidation is about plus 15.99 Da and is usually methionine or tryptophan
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whats the void volume peak, is that always solvent
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how much sample does a lab actually need for identity as well as purity
why does integration change the number so much
the first peak is the solvent. it is always the solvent
shallow gradient
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
has anyone ever had a result where the mass was right and the purity was wrong
i was wrong about tailing factor. it is usually the injection or the pH, not the column, i have it written down somewhere
run a blank
do the arithmetic
*VendorInvestigate not the other one
while im here is monoisotopic or average mass the one on most certificates
*Janoshik not the other one
Purity check: no report on file for lot B-0329. Nothing logged either way.