How do I reconcile 98.2% from Medutest with 99.1% from the supplier?

Setup, so nobody has to ask: 98.2% · Medutest · 99.1%.

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.

Should I be treating this as a failure or as noise?

0.9 percentage points, and the direction is the informative half. Medutest reports 98.2 per cent and the certificate 99.1, so the independent figure is lower. As impurity that is 1.8 per cent against 0.9 — 2 times as much unassigned area. A gentler gradient resolves impurities that a steeper one hides beneath the main peak, so the better method routinely reports the worse number; 0.9 points is comfortably inside what method choice alone produces on identical material. Ask both parties for the method section before you decide which figure is wrong, because the answer is often neither.

On the detail: 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.

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.

Specifically, 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.

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.

edited 16 Jul 2024 by Dr_Signe_Baldursdottir — updated for the 2026 guidance change

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

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

To be exact about it, 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.

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

The part that matters: understanding purity requires separating the chemistry from the method from the reporting convention, and the three are not independent.

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.

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.

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.

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

The underlying point is that 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.

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 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.

The relevant detail is that gradient slope is the most powerful parameter and almost nobody mentions it, which is why two reports on the same material disagree by a point.

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.

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 limitation is that single-digit micro-impurities become invisible at typical reporting thresholds, so "no impurities detected" means "none above one in two thousand."

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.