UHPLC · Calibration procedure
Pump flow rate accuracy
Checks that the UHPLC pump delivers the set flow by weighing the water collected over a timed interval.
PURPOSE
Why this is checked
UHPLC methods often run at low flows (0.2–0.6 mL/min) where a small absolute error is a large percentage error. Flow errors shift retention times and change peak areas, so flow is verified gravimetrically across the working range.
Frequency
Typically every 6 months, and after major repair or relocation, as defined in your calibration SOP.
Learn how the UHPLC worksYOU WILL NEED
Standards & tools
- Calibrated analytical balance (0.1 mg readability)
- Calibrated stopwatch or the CDS timer
- Calibrated thermometer
- HPLC-grade water
- Tared, capped collection vial
BEFORE YOU START
Prerequisites
- Replace the column with a restriction capillary that gives a back-pressure of roughly 100–200 bar (or as the manufacturer specifies)
- Purge the pump and check that there are no leaks
- Allow the flow to stabilise for at least 5 minutes at each setting
- Record the water temperature to choose the correct density
PROCEDURE
Step by step
- 1Set 100% water at 0.2 mL/min.
- 2Place the outlet into the tared vial and collect for exactly 10.00 minutes; cap the vial to prevent evaporation.
- 3Weigh the vial and record the net mass of water.
- 4Convert mass to volume using the density of water at the measured temperature (e.g. 0.9970 g/mL at 25 °C).
- 5Repeat at 0.5 mL/min and 1.0 mL/min, or at the flows your methods use.
RECORD
Example observation table
| Set flow (mL/min) | Time (min) | Mass (g) | Volume (mL) | Actual flow (mL/min) | % error |
|---|---|---|---|---|---|
| 0.2 | 10.00 | 1.991 | 1.997 | 0.1997 | −0.15 |
| 0.5 | 10.00 | 4.982 | 4.997 | 0.4997 | −0.06 |
| 1.0 | 5.00 | 4.997 | 5.012 | 1.0024 | +0.24 |
CALCULATION
How to calculate the result
Volume = Mass ÷ Density; Actual flow = Volume ÷ Time; % error = (Actual − Set) ÷ Set × 100
- Set 0.2 mL/min; 1.991 g collected in 10.00 min at 25 °C (density 0.9970 g/mL)
- Volume = 1.991 ÷ 0.9970 = 1.997 mL
- Actual flow = 1.997 ÷ 10.00 = 0.1997 mL/min
- % error = (0.1997 − 0.2) ÷ 0.2 × 100 = −0.15%
ACCEPTANCE CRITERIA
Pass if
- Actual flow typically within ±2.0% of the set value at each flow (some SOPs use ±1.0%, or the manufacturer's specification).
IF IT FAILS
What to do
- Label the instrument 'Out of calibration' and stop using it.
- Check for leaks, worn pump seals and faulty check valves; arrange service.
- Assess the impact on results generated since the last passing calibration.
- Repeat the calibration after repair.
MORE
Other UHPLC 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.