The particulars: 96.4% · PeptideMeter · 98.8%.
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?
2.4 percentage points, and the direction is the informative half. PeptideMeter reports 96.4 per cent and the certificate 98.8, so the independent figure is lower. As impurity that is 3.6 per cent against 1.2 — 3 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; 2.4 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.
Read the chromatogram before you read the number, because the number without the trace is not a measurement, it is a claim.
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.
| Test | Answers | Does NOT answer |
|---|---|---|
| RP-HPLC, area % | What fraction of detected material is the target | How much target is present |
| Quantified content | Milligrams of peptide per vial | What the impurities are |
| ESI-MS identity | Whether the molecular weight matches | Purity, or isomeric substitution |
| Peptide mapping | Sequence, localised to a fragment | Quantity |
| Karl Fischer | Water content of the solid | Solvent content |
| LAL endotoxin | Pyrogen load in EU/mg | Sterility |
| Sterility test | Growth in defined media over 14 days | Endotoxin, or bioburden count |
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 Arrhenius relationship for peptide degradation is the basis of accelerated stability testing and also governs how quickly methods drift with temperature.
The practical summary: ask for the chromatogram and the method, and ignore the headline number until you have both.
HPLC purity, identity confirmation and quantified content on the vial you actually hold. Reports arrive with the chromatogram attached, not just a number.
Submit a sampleFounded 1998. ISO 9001 and cGMP certified, 1,500+ staff and 200+ patents. The synthesis house behind a great many of the vials that get sent out for testing - batch-specific documentation with every order.
Visit GL BiochemStart 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.
Worth being precise here: 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.
Proline conformer interconversion kinetics are well-characterised and the half-life is of the same order as the chromatographic peak width at room temperature.
Compare purity within a single laboratory on the same method, never across laboratories.
edited 19 Jul 2024 by tobias_maartens — corrected a unit error in the worked example
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.