LimsPulse
Chromatography

Ultra-High Performance Liquid Chromatography (UHPLC)

A high-pressure form of HPLC that uses columns packed with very small particles to give faster runs, sharper peaks and less solvent use. Used in pharma QC for fast assays, impurity profiling and high-throughput testing.

  • Pharmaceutical
  • Biotech
  • Contract research (CRO / CDMO)
  • Food & beverage
  • Environmental
  • Clinical research
Photo of a Ultra-High Performance Liquid Chromatography (UHPLC)
Photo: Groep29 BFW, Public domain (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 calibration status label and the system's normal pressure trace in the logbook.
  2. 2Prepare mobile phases with high-purity solvents; filter buffers through 0.2 µm and use them within their stated shelf life.
  3. 3Install the UHPLC column in the flow direction, using fittings rated for UHPLC pressure and seating the tubing fully to avoid dead volume.
  4. 4Filter or centrifuge all samples; use vials and filters that do not shed particles.
  • Never fit an HPLC column on a UHPLC system above that column's pressure rating.
  • A poorly seated fitting adds dead volume and causes split or broad peaks.
STAGE 2

Start-up

  1. 1Prime all solvent lines and the needle wash and seal wash solutions.
  2. 2Start the flow at a low rate and ramp up to the method flow while watching the pressure.
  3. 3Set the column temperature and allow the column to equilibrate until pressure and baseline are stable.
  4. 4Check the detector data rate and filter time constant match the method.
  • Set a maximum-pressure limit in the method so the pump stops before the column or system is damaged.
STAGE 3

Running samples

  1. 1Run a blank, then system suitability injections, then samples with bracketing standards.
  2. 2Confirm resolution, plate count, tailing and %RSD meet the method limits before processing samples.
  3. 3Watch the pressure trace for steady rises that indicate a blocking column or in-line filter.
  • Very small injection volumes are sensitive to air in the syringe or loop; investigate any outlying peak area.
STAGE 4

Shutdown

  1. 1Flush buffers out with water or a low-organic mixture, then store the column in high-organic solvent as the supplier recommends.
  2. 2Reduce the flow before stopping to avoid pressure shocks to the column.
  3. 3Switch off the lamp and complete the usage and column logs.
In LimsPulse · Calibration Management

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

See how