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
System suitability
The injections that establish a chromatographic system was fit to produce the result, and the parameters the index checks before accepting a purity figure.
System suitability is the check that the instrument, column and method were performing acceptably at the moment the sample was injected. Without it, a result describes an unknown system. It is required by every pharmacopoeia and it is documented on almost no research-peptide certificate.
Parameters
| Parameter | Definition | Index requirement |
|---|---|---|
| Plate count N | N = 5.54 (t/w½)² | ≥ 20,000 for the main peak |
| Asymmetry As | width at 10 % height, ratio of rear to front segment | 0.80 ≤ As ≤ 1.50 |
| Tailing factor T | (a + b) / 2a at 5 % height | T ≤ 2.0 |
| Resolution R | R = 2(t₂ − t₁) / (w₁ + w₂) | ≥ 1.20 to nearest named impurity; ≥ 1.5 preferred |
| Repeatability | RSD of peak area, replicate injections | ≤ 1.0 % (n = 5) |
| Signal-to-noise at LOQ | peak height / peak-to-peak baseline noise | ≥ 10 |
| Retention repeatability | RSD of retention time | ≤ 1.0 % |
Sequence design
- Blank injection — diluent only. Establishes that no peak in the sample chromatogram originates in the diluent, the vial, or a carry-over from a previous injection.
- System-suitability solution — five replicate injections of a reference or of the sample itself. Establishes area and retention repeatability.
- Resolution solution — the sample spiked with a known related substance, or a degraded sample, where a resolution criterion applies.
- Sample injections, bracketed by a repeat of the system-suitability injection at the end of the sequence. Bracketing detects column degradation across a long run.
Worked plate count and resolution
Main peak: t = 18.42 min, w½ = 0.212 min
N = 5.54 × (18.42 / 0.212)²
= 5.54 × (86.89)²
= 5.54 × 7 550
= 41 830 ✓ above 20 000
Nearest impurity: t = 17.60 min, w½ = 0.204 min
Convert half-height widths to baseline widths (w ≈ 1.70 × w½ for a Gaussian):
w₁ = 0.361 min, w₂ = 0.347 min
R = 2 × (18.42 − 17.60) / (0.361 + 0.347)
= 2 × 0.82 / 0.708
= 2.32 ✓ well resolved
Same impurity on a 15-minute gradient (retention gap scales down):
gap = 0.82 × 0.5 = 0.41 min, widths roughly halved to 0.181 / 0.174
R = 2 × 0.41 / 0.355 = 2.31
— resolution is approximately preserved for a resolved pair.
The pairs that fail on a short gradient are those separated by
diastereomeric selectivity, which is not preserved.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, system suitability
- Ph. Eur. 2.2.46 — Chromatographic separation techniques
- ICH Q2(R2) — Validation of analytical procedures
Related methods
Peak integration practice
Drop-line versus valley-to-valley, tangent skim, baseline placement, and the reporting threshold — where a purity figure is actually decided.
Chromatographic purity
Method validation to ICH Q2(R2)
The eight validation characteristics, what each one demonstrates, and which of them the reports in this index actually document.
Quality system