How do I reconcile 95.2% from Medutest with 99.4% from the supplier?

What I am working with: 95.2% · Medutest · 99.4%.

This is not behaving the way I expected and I want to understand the discrepancy before I act on it.

I have photographed the current state and recorded the conditions, so I can answer follow-up questions precisely.

How do I distinguish the benign explanation from the one that matters?

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.

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.

Proline conformer interconversion kinetics are well-characterised and the half-life is of the same order as the chromatographic peak width at room temperature.

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

edited 27 Jan 2025 by cap_the_luer — expanded the table to cover the lower concentration

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

Temperature affects the dynamics of molecular conformation, and if a peptide has proline residues that interconvert on the chromatographic timescale, the peak will split or shoulder at low temperature and collapse at high temperature.

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 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 27 Jan 2025 by mz_4113 — removed a claim I could not source

It helps to be literal 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.

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.

One qualification: achieving purity above roughly 98 per cent on a 30-residue peptide is fighting the chemistry of synthesis, not the quality of the purification.

If you only pay for one test, pay for quantified content. Purity is the number everyone quotes and content is the number that changes what you do.

In practice, the single most important distinction is between what purity measures — the fraction of detected material that is your target — and what you actually want to know — how much of the material in the vial is your target.

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

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.

I would be careful about over-reading a single measurement — treat it as a data point, not as ground truth.

In practice: ask for the chromatogram, check the method section, check the lot number against the vial, and set your accept threshold before you see the result rather than after.