HPLC sequence review: worked decisions
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Synthetic data and fictional teaching criteria. No laboratory acceptance limit is supplied.
Each case is a retrospective, fictional excerpt: injection 01 is a solvent blank, 02 a two-component reference mixture and 03 a sample. Earlier injection history is not supplied. The recorded sample in case B is part of the exercise; it is not permission to continue a real sequence after a failed check.
Reference inputs and blank observations — all synthetic
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| Sequence | tᵣ₁ / min | tᵣ₂ / min | wb1 / min | wb2 / min | Blank component |
|---|---|---|---|---|---|
| A | 4.00 | 5.60 | 0.80 | 1.20 | None modeled |
| B | 4.00 | 5.20 | 0.80 | 1.20 | None modeled |
| C | 4.00 | 5.60 | 0.80 | 1.20 | U1: 4.02 min; 0.060 AU |
A · The two specified checks are met
Rₛ = 2 × (5.60 − 4.00) / (0.80 + 1.20) = 1.60
The reference reaches the fictional 1.50 criterion. The model contains no blank component, so no component triggers the separate blank flag. These two teaching checks are met. The excerpt does not establish that all requirements of a real system suitability test, or all conditions for reporting this sample, were met.
B · The reference resolution is insufficient
Rₛ = 2 × (5.20 − 4.00) / (0.80 + 1.20) = 1.20
The reference gives Rₛ = 1.20, below 1.50. An unflagged blank and a visually plausible sample do not repair that failed reference check. The recorded sample result is not supported by the stated sequence checks. Document the failure and follow the applicable procedure to investigate and re-establish suitability; the model does not prescribe reinjection or release.
C · Resolution is met; the blank remains unresolved
Rₛ = 2 × (5.60 − 4.00) / (0.80 + 1.20) = 1.60
The reference gives Rₛ = 1.60, but the blank’s 4.02 min apex lies within 3.80–4.20 min and its 0.060 AU height exceeds 0.020 AU. Flag the unexplained blank signal and leave its impact on the sample open. Retention-time proximity does not identify the signal. A passing reference resolution does not resolve it.
Keep the cause open
The blank was recorded before the reference and sample shown here. Neither later injection can explain it as carryover from that later injection. Earlier history is missing. Prior carryover, reagents or preparation, and system contributions are investigation possibilities, not established causes. Review the actual sequence history, preparation records and method-defined blank controls; choose follow-up work from that evidence. Do not silently subtract this blank from the sample or turn the signal ratio into an impurity percentage.
Record the boundary of the conclusion
Only one reference injection per case is supplied. There is no repeatability series, sensitivity or noise test, tailing assessment, full bracketing sequence, validated sample matrix or reporting range. Thus no case demonstrates complete system suitability, sample identity, purity or fitness for use. A real decision needs the current procedure and its complete criteria, observations and investigation record.