HPLC · Calibration procedure
Gradient composition accuracy
Checks that the pump mixes solvents in the set proportions, using a step gradient with a UV-absorbing tracer (acetone or caffeine) in one channel.
PURPOSE
Why this is checked
Gradient methods rely on the pump delivering the right mobile-phase composition at each moment. A proportioning error changes retention times and resolution, and can make related-substances methods fail system suitability.
Frequency
Typically every 6 months, and after major repair or relocation, as defined in your calibration SOP.
Learn how the HPLC worksYOU WILL NEED
Standards & tools
- Channel A: HPLC-grade water
- Channel B: water containing a UV tracer, e.g. 0.1–0.5% v/v acetone (or about 10–20 µg/mL caffeine)
- Restriction capillary in place of the column
BEFORE YOU START
Prerequisites
- Remove the column and fit a restriction capillary that gives a stable back-pressure
- Purge both channels and make sure lines are free of air
- Set the detector to a suitable wavelength (e.g. 254 nm for acetone, 273 nm for caffeine)
PROCEDURE
Step by step
- 1Program a step gradient, e.g. 0% B, 10% B, 50% B, 90% B and 100% B, holding each step long enough for a flat plateau (e.g. 5 minutes).
- 2Run the programme at the routine flow rate (e.g. 1.0 mL/min).
- 3Measure the detector signal (height) of each plateau: 0% B gives the baseline, 100% B gives the full-scale signal.
- 4Calculate the actual % B for each intermediate step from the plateau heights.
- 5Compare actual with set composition. Repeat for other channel pairs (e.g. A/C, A/D) on a quaternary pump if your SOP requires.
RECORD
Example observation table
| Set % B | Plateau height (mAU) | Actual % B | Difference (% absolute) |
|---|---|---|---|
| 0 | 0.4 | — | — |
| 10 | 50.9 | 10.10 | +0.10 |
| 50 | 251.8 | 50.28 | +0.28 |
| 90 | 449.6 | 89.84 | −0.16 |
| 100 | 500.4 | — | — |
CALCULATION
How to calculate the result
Actual % B = (H step − H 0%) ÷ (H 100% − H 0%) × 100; Difference = Actual % B − Set % B
- H 0% = 0.4 mAU, H 100% = 500.4 mAU, H 50% step = 251.8 mAU
- Actual % B = (251.8 − 0.4) ÷ (500.4 − 0.4) × 100 = 251.4 ÷ 500.0 × 100 = 50.28%
- Difference = 50.28 − 50 = +0.28% absolute
ACCEPTANCE CRITERIA
Pass if
- Each step typically within ±1–2% absolute of the set composition, as defined in your SOP and the manufacturer's specification.
IF IT FAILS
What to do
- Check proportioning valves, check valves and pump seals; purge lines and repeat.
- Check that the tracer solution is homogeneous and that both solvents are degassed.
- Label the pump out of calibration for gradient methods and assess the impact on gradient results since the last passing check.
- Repeat the calibration after repair.
MORE
Other HPLC calibrations
In LimsPulse · Calibration Management
LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.