LimsPulse
Chromatography

High Performance Liquid Chromatography (HPLC)

Separates the components of a liquid sample on a packed column and measures each one as it leaves. The workhorse of pharmaceutical QC for assay, impurities and dissolution.

  • Pharmaceutical
  • Biotech
  • Food & beverage
  • Environmental
  • Cosmetics
  • Chemicals
Photo of a High Performance Liquid Chromatography (HPLC)
Photo: Fernando Frick, CC0 (cropped), via Wikimedia Commons
Follow your site SOP
This is a typical operating sequence for learning. Your instrument model and approved SOP take precedence.
STAGE 1

Before you start

  1. 1Check the instrument is within its calibration due date and has no 'out of order' label.
  2. 2Prepare the mobile phase as per the method, filter it through a 0.45 µm (or 0.22 µm) membrane and degas it.
  3. 3Fit the column specified in the method, in the direction of the flow arrow, and record its ID in the column usage log.
  • Never let buffer dry in the lines; buffer salts crystallise and damage seals and check valves.
STAGE 2

Start-up

  1. 1Switch on the modules and the detector lamp; allow the lamp to warm up (commonly about 30 minutes).
  2. 2Purge each pump line to remove air bubbles.
  3. 3Start the flow at a low rate and increase it gradually to the method flow rate.
  4. 4Set the column oven temperature and detector wavelength from the method.
  5. 5Equilibrate until pressure and baseline are stable (often 10–20 column volumes).
  • Watch the pressure while increasing flow. Stop if it exceeds the column's limit.
STAGE 3

Running samples

  1. 1Create the sequence: blank, system suitability injections (e.g. five replicate standards), then samples with bracketing standards.
  2. 2Run the blank and confirm there are no interfering peaks at the analyte retention time.
  3. 3Check system suitability results meet the method limits before processing any sample results.
  4. 4Process the chromatograms with the approved integration method; never re-integrate without a documented reason.
  • If system suitability fails, stop and investigate. Results from a failed run cannot be reported.
STAGE 4

Shutdown

  1. 1Flush buffer out of the column and system with water or a low-organic mixture (e.g. 10% methanol in water).
  2. 2Then flush and store the column in a high-organic solvent (e.g. 70–80% methanol or acetonitrile) as the column supplier recommends.
  3. 3Remove the column if it will not be used soon, cap both ends and store it with its log.
  4. 4Switch off the lamp, then the pump and modules; complete the instrument usage log.
In LimsPulse · Calibration Management

Keep every HPLC in control: usage logs, calibration and maintenance schedules, and automatic blocking of overdue instruments.

See how