For reference: VendorInvestigate · 96.4%.
Something has gone wrong and I would like to know how badly before I decide what to do.
Nothing else in the setup changed, which is what makes this puzzling.
What is the differential here, and which test discriminates between the options?
A 96.4 per cent result against a higher certificate figure is usually a method difference, not a disagreement about the vial. Inter-laboratory spread on identical peptide material runs half a per cent to a full per cent by RP-HPLC. Gradient slope, detection wavelength and integration convention each move the number, and a gentler gradient resolves impurities that a steeper one hides under the main peak — so the better method reports the worse figure. Get the column, the gradient and the wavelength from both VendorInvestigate and the supplier before treating 96.4 per cent as a contradiction.
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.
| Δ mass (Da) | Most likely cause | Distinguishing feature |
|---|---|---|
| +1 | Deamidation (Asn or Gln) | New peak, slightly earlier retention |
| −17 | Loss of ammonia | Often with deamidation |
| −18 | Dehydration / succinimide | pH-dependent, reversible |
| +16 | Oxidation (Met, Trp) | Earlier retention, light-related |
| −128 | Missing Gln or Lys | Deletion sequence from synthesis |
| 0 | Isomer: racemisation or scrambling | Same mass, shifted retention |
To be exact about it, 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.
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.
The practical summary: ask for the chromatogram and the method, and ignore the headline number until you have both.
Aggregated, published test results and vendor ratings built from submitted batches. Methodology stated, dataset browsable, no listing fees.
Browse resultsThe short answer is that two competent laboratories on identical material will disagree, and the disagreement is almost always explainable by method differences.
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.
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.
Proline conformer interconversion kinetics are well-characterised and the half-life is of the same order as the chromatographic peak width at room temperature.
I would be careful about over-reading a single measurement — treat it as a data point, not as ground truth.
Compare purity within a single laboratory on the same method, never across laboratories.
edited 3 Jul 2026 by retest_please — added a caveat about sampling
Read the chromatogram before you read the number, because the number without the trace is not a measurement, it is a claim.
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.
Tailing factor measures peak shape, and a badly tailing peak spreads into the region where small impurities live, forcing tangent-skim integration that assigns tail area to the main peak.
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.
If you are ranking vendors, specify a method and have all samples tested at the same place.
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.
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.
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.
Ask PeptideStack is a static archive. Posting is closed, but the norms are worth stating: answer the question that was asked, show your working, cite the trial or the certificate, and say plainly where the evidence runs out.