#hplc-massspec 2026-06-17

has anyone ever had a result where the mass was right and the purity was wrong
monoisotopic and average diverge more the bigger the molecule. say which one you are quoting

is uv at 214 or 280 better for peptides

quick one do you trust an area percent from a shallow gradient

if the number you are staring at is a third of what you expected, do the charge state arithmetic before you panic

i was wrong about tailing factor. it is usually the injection or the pH, not the column

need more sample

the first peak is the solvent. it is always the solvent, i have it written down somewhere

slightly off topic but how do you tell co-elution from a clean single peak

truncations show as a mass deficit matching one or more residues. you need the sequence to say which, i have it written down somewhere

coming back after 23 months, what changed in how you read these

integration difference

a dimer usually shows up late and at roughly double the mass. the chromatogram alone will not tell you, take that with a pinch of salt

214nm sees the amide backbone so it sees everything. 280 only sees aromatics

i have a number at about a third of what i expected, is that a charge state

update from 13 months ago: switched to asking for the chromatogram rather than the number and it changed everything

thats co-elution

does ms/ms actually prove identity or just make it very likely

two labs a point or two apart on the same lot is normal and gets mistaken for one of them being wrong constantly, your mileage will differ

co-elution is the failure mode that a beautiful single peak is most likely to be hiding

ask for the trace

how much sample does a lab actually need for identity as well as purity

shallow gradient

integration choices move the number by a few tenths easily. that is most of your inter-lab variance

if somebody says they ran a mass spec and does not show you a spectrum, that is a claim, not a result

the honest position is that hobby-level interpretation of a chromatogram is worth something but not much

quick one ms/ms sequencing is the only thing i would call identity. everything else is circumstantial

while im here nobody runs enough blanks, including me

deamidation is about plus 0.98 Da. it is not a rounding error, it is a degradant

a certificate with a number and no chromatogram is asking you to trust the integration you cannot see
a warm transit can put a visible degradant peak on a chromatogram. that is what 6 days at ambient does

plus 0.98 is deamidation

retention time is a hint, not a fingerprint. two different peptides can co-elute perfectly happily, ymmv

how would a truncation sequence show up