coming back to this the first peak is the solvent. it is always the solvent
#hplc-massspec 2025-05-16
Friday35 messages6 participantstimes are UTC
Highlights from this day
- sharps_bin_sid — coming back to this relative retention time against a standard is comparable between labs. absolute minutes are not 12:32
- gradient_greg — whats a normal gradient for a peptide this size 12:46
- gradient_greg — if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result 13:36
i have a number at about a third of what i expected, is that a charge state
nobody runs enough blanks, including me
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i asked Medutest for the raw trace and they sent it. that is the right answer to that question
within inter-lab range
if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic, i have it written down somewhere
thats a charge state
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has anyone ever had a result where the mass was right and the purity was wrong
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right so a certificate with a number and no chromatogram is asking you to trust the integration you cannot see, n of 1 obviously
run a blank
how do you spot a deletion in a sequence from mass alone
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coming back to this relative retention time against a standard is comparable between labs. absolute minutes are not
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whats the void volume peak, is that always solvent
why does integration change the number so much
ms/ms or it isnt identity
214nm sees the amide backbone so it sees everything. 280 only sees aromatics
retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily
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plus 16 is oxidation
whats a normal gradient for a peptide this size
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 substancedo you trust an area percent from a shallow gradient
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial
plus 0.98 is deamidation
whats a plus one dalton shift usually
if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result
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 substanceyou need more sample for identity than for purity. plan for that when you post it, still working it out
thats the solvent peak
deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant
show me the spectrum
integration difference
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integration choices move the number by a few tenths easily. that is most of your inter-lab variance
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right so oxidation is about plus 15.99 Da and is usually methionine or tryptophan
a warm transit can put a visible degradant peak on a chromatogram. that is what 24 days at ambient does
thats co-elution
coming back to this how would a truncation sequence show up
is monoisotopic or average mass the one on most certificates
ms/ms sequencing is the only thing i would call identity. everything else is circumstantial