Details up front: 98.4% · Janoshik · 95.2%.
I have a result I cannot explain, and I would rather diagnose it than guess.
I have checked the obvious explanations and eliminated the two easiest ones.
What is the most likely explanation, and how would I confirm it?
It helps to be literal here: start from what the detector sees, because that tells you what the number means.
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 |
Stated carefully, 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.
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 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.
The practical summary: ask for the chromatogram and the method, and ignore the headline number until you have both.
Analytical standards and reagents with traceable certificates. Every quantitative result you read inherits the accuracy of the standard behind it.
Shop standardsIn practice, the method matters more than the vial, which is why specifying a method buys you far more than changing suppliers does.
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.
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.
If you are ranking vendors, specify a method and have all samples tested at the same place.
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.
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.
On the detail: 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.
Worth noting that method standardisation is poor in the research-grade space compared to pharmaceutical work, so identical-looking methods can produce different results.
Compare purity within a single laboratory on the same method, never across laboratories.
Understanding purity requires separating the chemistry from the method from the reporting convention, and the three are not independent.
The fraction of your main peak that is actually your target versus isomers, fragments or related sequences is invisible without complementary identity testing.
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."
The practical summary: ask for the chromatogram and the method, and ignore the headline number until you have both.
Area percentage is not mass percentage, and conflating the two is the most common misreading of a purity figure.
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.
If you are ranking vendors, specify a method and have all samples tested at the same place.
edited 17 Apr 2025 by pierce_count — clarified the distinction between purity and content
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.