System suitability

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

Resolution is the one row in that table with no number beside it, and the omission is deliberate. Resolution to whichever named impurity happens to elute closest is a property of the compound's own impurity profile and of the gradient the laboratory chose — a compound whose nearest related substance is a diastereomer 0.2 min away can never reach the figure a compound with a clean 2-minute gap reaches, and penalising it for that would be scoring chemistry rather than chromatography. The index therefore computes R, prints it in the system-suitability block of every record that has one, and attaches no verdict to it. Rule AX-R12 was drafted with a resolution limb and that limb was withdrawn before it adjudicated anything; the rule is an asymmetry rule and nothing else. What the index does ask for where separation is poor is the integration convention, which is a documentation requirement rather than an acceptance criterion.

Sequence design

1. 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.
2. System-suitability solution — five replicate injections of a reference or of the sample itself. Establishes area and retention repeatability.
3. Resolution solution — the sample spiked with a known related substance, or a degraded sample, where the pharmacopoeial monograph being followed sets a resolution criterion. The index sets none of its own.
4. 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.