HPLC sequence review: worksheet
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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.
Here wb is the tangent-base width, not the width at half height. For the supplied Gaussian components, wb = 4σ. The curve’s tails never actually reach zero; the width comes from the baseline intersections of tangents at the two inflection points.
Rₛ = 2 × (tᵣ₂ − tᵣ₁) / (wb1 + wb2)
Use minutes for both retention times and widths. Both unequal widths belong in the denominator. Do not insert a half-height width into this baseline-width formula.
Teaching rule 1: the reference pair meets the resolution check when Rₛ ≥ 1.50, including equality. This number is a fictional exercise choice, not a general HPLC requirement.
Teaching rule 2: flag a modeled blank component whose apex lies from 3.80 to 4.20 min inclusive and whose height is ≥ 0.020 AU. This simplified component rule is not a detection limit or a real blank acceptance policy.
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 |
All signals are noise-free sums of Gaussian components in arbitrary signal units (AU). A flat modeled blank means no components were included; it is not a measured “not detected” result. Sample component labels S1/S2 carry no chemical identity, purity or concentration claim.
Write a decision for each case
- Calculate Rₛ from the reference pair, keeping both baseline widths in the denominator. Compare it with the fictional ≥ 1.50 rule.
- Assess the blank separately using the stated window and height rule. Record the observation without assigning a chemical identity or cause.
- State which teaching check is met or unresolved. Then name evidence still missing before a real analytical result could be accepted.
Record your calculation and qualified decision
This table scrolls horizontally. Focus the table region and use the left and right arrow keys.
| Sequence | Rₛ calculation | Resolution check | Blank observation / flag | Decision and missing evidence |
|---|---|---|---|---|
| A | ||||
| B | ||||
| C |
In C, why can the blank signal not be attributed to the reference injection shown after it? What earlier records or targeted checks would help investigate it?