What it does
In HPLC and GC, two numbers decide whether a method works: how far apart two peaks are, and how wide they are. This calculator turns retention times and peak widths into the standard figures, and helps you move a method to a modern column.
Two neighbouring peaks
- Retention factor k = (tR − t₀)/t₀: how long each compound is held, relative to an unretained one.
- Selectivity α = k₂/k₁: how differently the column treats the two.
- Resolution Rs, by the USP formula from baseline widths, 2(t₂ − t₁)/(W₁ + W₂), or the Ph. Eur. formula from half-height widths, 1.18(t₂ − t₁)/(W½₁ + W½₂). An Rs of 1.5 is baseline separation; 2 is the usual target for robust methods.
- Plate number N and plate height (HETP), which measure column efficiency.
- The Purnell equation checks the figures against each other, and gives the plates needed for the resolution you want.
Method transfer
Going from a 250 × 4.6 mm, 5 µm column to a 100 × 2.1 mm, 1.8 µm one can save 90 % of the time and solvent. The flow, injection volume and every gradient time must be scaled together so the separation stays the same. The tool does that, as in USP chapter <621>, and checks the change in column length to particle size (L/dp) against the −25 % to +50 % window.
How to use it
- Enter t₀, the two retention times and the two peak widths, and say how the widths were measured (most software reports W½).
- Add the column length for HETP, and the resolution you need for the plate target.
- For a transfer, enter the old and new column sizes, the flow, the injection volume and the gradient table.
Worked example
Peaks at 6.40 and 7.10 min with t₀ = 1.20 min and half-height widths 0.150 and 0.160 min:
- k₁ = (6.40 − 1.20)/1.20 = 4.33, k₂ = 4.92, so α = 1.135.
- Rs = 1.18 × (7.10 − 6.40)/(0.150 + 0.160) = 2.66, fully separated.
- N₂ = 5.54 × (7.10/0.160)² ≈ 10,900 plates. In a 150 mm column, HETP = 13.7 µm.
Moving a method from 250 × 4.6 mm, 5 µm at 1.0 mL/min to 100 × 2.1 mm, 1.8 µm gives a flow of 1.0 × (2.1/4.6)² × (5/1.8) = 0.58 mL/min and an injection of 20 µL × 0.083 = 1.7 µL. Gradient times are multiplied by 0.144, so a 26 min gradient takes 3.7 min, using about 8 % of the solvent. L/dp goes from 50,000 to 55,600 (+11 %), inside the USP window.
Questions
Why does doubling the column only give 41 % more resolution? Resolution grows with the square root of the plate number. Changing the selectivity, through the mobile phase, pH, temperature or column chemistry, is usually a far stronger lever.
Do I need to revalidate after a transfer? Changes inside the USP <621> or Ph. Eur. 2.2.46 limits are allowed without full revalidation for compendial methods, but system suitability must still pass. Check your monograph and local rules. Nothing you type leaves your browser.
Where the numbers come from
- Formulas and method transfer rules: USP general chapter <621> and the European Pharmacopoeia.
Standard scientific values, such as constants, atomic weights and melting points, are facts found in every textbook; we name where ours were checked. If you are an author or publisher and want a value, a credit or a table changed or removed, tell us and we will act quickly.
Last updated 2026-11-08.