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HPLC · Calibration procedure

Pump flow rate accuracy

Checks that the pump delivers the flow rate that is set, by timing the collection of a known volume of water.

Photo of a HPLC
Photo: Fernando Frick, CC0 (cropped), via Wikimedia Commons
PURPOSE

Why this is checked

Retention times and peak areas depend directly on flow rate. An inaccurate pump shifts retention times and changes results, so flow is verified at low, medium and high settings.

Frequency

Typically every 6 months, and after major repair or relocation, as defined in your calibration SOP.

Learn how the HPLC works
YOU WILL NEED

Standards & tools

  • Calibrated stopwatch
  • Calibrated 10 mL volumetric flask (Class A) or a calibrated analytical balance
  • HPLC-grade water
BEFORE YOU START

Prerequisites

  • Remove the column and connect a restriction capillary or union
  • Purge the pump and make sure there are no leaks
  • Let the flow stabilise for at least 5 minutes at each setting
PROCEDURE

Step by step

  1. 1Set the flow rate to 0.5 mL/min with 100% water.
  2. 2Place the outlet tubing into the 10 mL volumetric flask and start the stopwatch when the first drop falls.
  3. 3Stop the stopwatch when the meniscus reaches the mark. Record the time.
  4. 4Repeat at 1.0 mL/min and 2.0 mL/min (and at any other flow your methods use).
  5. 5Alternatively, collect into a tared vial for a fixed time and convert the mass to volume using the density of water at the measured temperature.
RECORD

Example observation table

Set flow (mL/min)Volume (mL)Time (min)Actual flow (mL/min)% error
0.510.020.120.497−0.6
1.010.010.040.996−0.4
2.010.04.992.004+0.2
CALCULATION

How to calculate the result

Actual flow = Volume collected ÷ Time; % error = (Actual − Set) ÷ Set × 100
  1. Set 1.0 mL/min, 10.0 mL collected in 10.04 min
  2. Actual flow = 10.0 ÷ 10.04 = 0.996 mL/min
  3. % error = (0.996 − 1.0) ÷ 1.0 × 100 = −0.4%
Use the Flow rate accuracy calculator
ACCEPTANCE CRITERIA

Pass if

  • Actual flow within ±2.0% of the set value at each flow rate (some SOPs use ±1.0%).
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; get the pump serviced.
  • Assess the impact on results generated since the last passing calibration.
  • Repeat the full 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.

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