20 Jan

Ultra High Performance Liquid Chromatography: UPLC

Ultra Performance Liquid Chromatography is related to HPLC (High Performance or High Pressure Liquid Chromatography) but uses smaller particle sizes (less than 2 μm.) and smaller diameter columns.  The efficiency of HPLC separation is inversely related to particle size.  Unfortunately the smaller the particle size the higher the back pressure.  UPLC Systems are now being designed with columns and mobile phase pumps to accommodate this higher pressure (typically 20,000 psi vs. 6000 psi for HPLC).   The advantages of UPLC is shorter run times (4 minutes vs. 30 minutes)  and increased separation with taller and narrower peaks.  The short run times indicate how small the column volumes are.  The instrumentation must also be designed with much smaller volumes; for example injection volumes go from 20µl to less than 2µl.  Specialty fittings (ferrules, nuts) have been developed to withstand the higher pressures in UPLC.  The high pressures require extremely high quality tubing with specially cut and polished ends.  The detectors – UV, fluorescence, evaporative light scattering (ELS) – must be designed with smaller volumes.  In HPLC peak widths are typically 20 seconds so a data sampling rate of 1 point per second is suitable; with UPLC peak widths of only 1 0r 2 seconds the data sampling  rates must be increased to up to 40 points per second for reproducible integration.  The UPLC systems price is 40 to 100% more than HPLC systems but the increase in sample throughput is 10 times.  For a lab considering another HPLC system to accommodate higher sample loads the UPLC may be the most cost effective instrumentation.  In labs where HPLC is used only occasionally or the detection limits of conventional systems are adequate the UPLC system would be not be the cost effectiveChromatogram choice.

Robin Prymula