Why the theoretical mass on the certificate might be the wrong theoretical mass
A worked example: one peptide, five charge states, five mass-to-charge ratios, one molecular weight.
HPLC
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The wavelength decides which impurities exist
Peptide bonds absorb strongly near 214 nm; aromatic side chains absorb near 280 nm. A method reading at 280 is blind to any fragment lacking an aromatic residue.
Six lines that would make an identity statement checkable
None of what a checkable identity statement requires is commercially sensitive, and all of it is known to whoever produced the document.
Why VendorInvestigate may run electrospray where another laboratory runs MALDI
The two ionisation techniques in general use produce different charge distributions, different adducts and different failure modes. Certificates almost never say which was…
Size exclusion sees the thing reversed-phase dissolved
Where two methods disagree, the conservative convention is to report the lower figure. It is not universal, and whether a laboratory follows it belongs on the report.
Two analysts, one chromatogram, two answers
Chromatographic software does not integrate every fluctuation in the baseline. It applies a threshold, and the threshold changes the reported purity by amounts that matter…
What column temperature does to selectivity
We set out the parameters a method disclosure would contain, all of which are known to whoever ran the sample and none of which is commercially sensitive.
The word “confirmed” is doing an enormous amount of work on this certificate
An intact mass measurement establishes elemental composition, at best. The number of distinct sequences consistent with a given composition is astronomically large.
Six lines that would make an identity statement checkable
What the Journal asks for when it writes to a supplier about an identity claim, and how often it gets it.
What column temperature does to selectivity
A shallow gradient resolves impurities that a steep one runs into the parent peak. Both methods are legitimate; only one of them can see the small stuff.
Assay against a standard, or area per cent: two different questions
We asked the four independent testing services what standards they run against and what suitability criteria they apply. The answers are printed.
Resolving power and mass accuracy are two different specifications
Parts per million sound impressive until they are converted into daltons at the molecular weight of the thing being measured.
Deamidation, oxidation, and the two shifts worth memorising
What would be needed to catch each of these, and what it would cost.
The column is a choice, and it is not a small one
We set out the parameters a method disclosure would contain, all of which are known to whoever ran the sample and none of which is commercially sensitive.
The instrument was calibrated on Tuesday. Your sample ran on Friday.
Parts per million sound impressive until they are converted into daltons at the molecular weight of the thing being measured.
Nought point nine eight of a dalton: the modification that hides in plain sight
Stereochemical inversion changes the molecule, changes its biology, and changes its mass by exactly nothing.
Resolution, tailing, repeatability: the arithmetic before the result
Matching a retention time against a standard is evidence of consistency, not proof of identity. Two different species can elute at the same time on one method.
What counts as total, and who decided
We work through a single chromatogram twice, under two integration conventions, and show where the difference comes from.
Calibrated, qualified, accredited: three words that are not synonyms
An assay against a quantitative reference standard measures how much is present. Area per cent measures what proportion of the visible material is the parent. Only the first…
The specification sheet says 500,000. The report says conforms.
Calibration drift is real, unremarkable, and the reason serious laboratories run internal standards.