Purity by reverse-phase HPLC (% area)
The index reference method RG-2, what it measures, what it does not, and why a purity figure without a stated gradient is uninterpretable.
Chromatographic purity
Gradient design for peptides
Why 25 minutes is the index minimum for the acylated incretins, and how gradient slope trades against run time and against the impurities you can see.
A gradient separates by changing the mobile-phase organic fraction over time. The controlling variable is slope — percentage points of organic per minute — and it sets both how long the run takes and how much of what is in the vial you will see.
The trade
| Gradient | Slope (%B/min) | Run time | Diastereomer separation | Suitable for |
|---|---|---|---|---|
| 5→65 % over 15 min | 4.0 | ~19 min | None | Screening; confirming presence |
| 5→65 % over 25 min | 2.4 | ~29 min | Partial (valley 40–60 %) | Index minimum for incretins |
| 5→65 % over 30 min | 2.0 | ~35 min | Resolved for most pairs | Index reference method RG-2 |
| 5→65 % over 45 min | 1.33 | ~52 min | Resolved, plus regioisomers | Disputed results, method development |
| Focused: 30→45 % over 30 min | 0.5 | ~38 min | Best available | A single compound, once its window is known |
The last row is the one nobody runs and the one that answers the hardest questions. A focused gradient spanning only the organic window in which the compound of interest elutes gives four times the resolving power of a general-purpose gradient over the same run time. It requires knowing the window in advance, which is exactly what a compound fingerprint provides.
Why the incretins set the minimum
Semaglutide, tirzepatide, retatrutide, survodutide and mazdutide all carry α-aminoisobutyric acid residues introduced specifically to resist enzymatic cleavage. Aib is achiral, but the couplings around it are hindered and epimerisation at neighbouring residues is common. The resulting diastereomers are mass-identical and differ in retention by 0.19–0.26 min on RG-2 — a separation that scales with gradient duration and vanishes below about 25 minutes.
The index therefore treats gradient duration as part of the result rather than as metadata. Reports below the class minimum are marked METHOD LIMITED and their purity figures are excluded from class aggregates, not because they are wrong but because they measure a different thing.
Practical notes
- A 2-minute initial hold below 5 % B is required for compounds eluting under 6 minutes, or the main peak merges with the injection disturbance.
- A wash step to 95 % B is required for the lipidated analogues, or the C18 and C20 diacid species accumulate on-column and appear as ghost peaks in the following injection.
- Re-equilibration for at least 10 column volumes; short re-equilibration is a common cause of retention drift across a sequence.
- Trifluoroacetic acid at 0.1 % gives sharper peaks than formic acid for peptides but suppresses electrospray ionisation. Where LC-MS and purity are run on the same injection, 0.05 % TFA or a formic-acid method is the usual compromise, and it costs peak shape.
Normative references
Cited because they are the documents the acceptance criteria above are taken from. The index applies them as written and states every deviation.
- USP <621> — Chromatography, allowable adjustments
- Ph. Eur. 2.2.46 — Chromatographic separation techniques
Related methods
Retention-time normalisation across gradients
How the index compares a retention time measured on a 15-minute gradient with one measured on a 35-minute gradient, and where the approximation breaks.
Chromatographic purity
Diastereomers: the impurity no mass spectrum will find
Epimerisation during synthesis, why the resulting impurity is functionally significant and analytically invisible, and what it takes to see it.
Chromatographic purity