Why would VendorInvestigate report 97.6% where the supplier certificate says something higher?

The particulars: VendorInvestigate · 97.6%.

An unexpected observation, and I would like a differential rather than reassurance.

The conditions were within what I understood to be the acceptable range, which is why I am asking.

What is the most likely explanation, and how would I confirm it?

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

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

In practice, 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.

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

edited 27 Mar 2025 by siobhan_deasy — reworded for clarity after a comment

The short answer is that two competent laboratories on identical material will disagree, and the disagreement is almost always explainable by method differences.

Sample solvent strength affects peak shape — if you inject in strong solvent on a gradient starting in weak solvent, the solvent peak can distort your main peak or create a false shoulder.

Column pore size affects mass transfer — a 100 Angstrom packing on a 5 kDa peptide restricts diffusion, broadening the peak and potentially hiding small impurities in the shoulders.

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.

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

Worth being precise here: 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.

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

More usefully, integration of the shoulder — whether you use perpendicular drop or tangent skim — determines what area gets assigned to the main peak versus the impurity table.

The Arrhenius relationship for peptide degradation is the basis of accelerated stability testing and also governs how quickly methods drift with temperature.

Worth noting that method standardisation is poor in the research-grade space compared to pharmaceutical work, so identical-looking methods can produce different results.

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

In practice, identity and purity are orthogonal, and a high purity says almost nothing about whether the peak is actually what you think it is.

Retention time is sequence-specific and method-specific, so comparing your result to a supplier value using a different method is meaningless without method documentation.

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

Specifically, area percentage is not mass percentage, and conflating the two is the most common misreading of a purity figure.

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

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.