How do I reconcile 99.1% from Janoshik with 96.4% from the supplier?

What I have: 99.1% · Janoshik · 96.4%.

I have a result I cannot explain, and I would rather diagnose it than guess.

I have checked the obvious explanations and eliminated the two easiest ones.

What is the differential here, and which test discriminates between the options?

2.7 percentage points, and the direction is the informative half. Janoshik reports 99.1 per cent and the certificate 96.4, so the independent figure is higher. As impurity that is 0.9 per cent against 3.6 — 0.25 times as much unassigned area, which is the unusual direction and worth a second look at the integration. Method differences usually make the independent number the lower one, so an independent result above the certificate points at integration limits, a different wavelength, or a different definition of the main peak rather than at a better vial. Ask both parties for the method section before you decide which figure is wrong, because the answer is often neither.

Purity is a method-dependent figure, and that is not a limitation of the measurement, it is a property of what the measurement actually answers.

Detection wavelength matters because 214 nm sees the peptide backbone while 280 nm sees only aromatic side chains — so truncation impurities lacking a tryptophan are invisible at 280 nm.

Mass on column affects detector linearity and peak overlap — overloading broadens peaks and hides neighbours, while underloading improves resolution but loses sensitivity.

Inter-laboratory studies on identical peptide material routinely find half-a-per-cent to a full-per-cent spreads in reported purity on the same sample.

The practical summary: ask for the chromatogram and the method, and ignore the headline number until you have both.

The method matters more than the vial, which is why specifying a method buys you far more than changing suppliers does.

Gradient slope controls resolution, and gentler slopes resolve co-eluting impurities into separate peaks — so the better method reports the worse purity number.

Specifically, mobile phase additive choice affects ionisation and peak shape — TFA gives sharp peaks but suppresses mass spectrometry signal, formic acid gives worse peaks but preserves signal.

The ICH Q3A impurity thresholds and the relevant pharmacopoeial chapters all specify method validation requirements that almost no research-grade certificate claims to meet.

The caveat is that purity without identity is only half an answer — a high purity does not mean the peak is actually what you think it is.

If you are ranking vendors, specify a method and have all samples tested at the same place.

edited 11 Sept 2025 by a_lindgren — added the citation requested in comments

Start from what the detector sees, because that tells you what the number means.

Buffer versus acid in the mobile phase changes the ionisation state of basic and acidic residues, shifting retention and selectivity — same vial, potentially different separation.

The part that matters: the fraction of your main peak that is actually your target versus isomers, fragments or related sequences is invisible without complementary identity testing.

Published side-by-side method comparisons show that a two-point difference in purity on the same vial is easily explained by method choice alone.

Compare purity within a single laboratory on the same method, never across laboratories.