HPLC (high performance liquid chromatograph ) Instrument Operation

  • General components of a high performance liquid chromatograph.
  • HPLC solvent delivery systems.
  • How automatic injectors work.
  • Common HPLC detectors.

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HPLC (high performance liquid chromatograph ) Instrumentation

Main components in an HPLC system include the solvent reservoir, or multiple reservoirs, a high-pressure pump, a column, injector system and the detector.

The reservoir holds the solvent, which is referred to as the mobile phase because it moves. There are usually a minimum of two reservoirs in a system, with each holding up to 1000 cc of solvent and usually fitted with a gas diffuser through which helium can be bubbled. A pump is used to generate a specified flow of the mobile phase. Although manual injection of samples is still possible, most HPLCs are now fully automated and controlled by computer. The injector, or auto sampler, introduces the solvent into a phase stream that carries the sample into the high pressure (up to 400 bar) column, which contains specific packing material needed to effect separation. The packing material is referred to as the stationary phase because it is held in place by the column hardware. 

A detector is needed to see the separated compound bands as they elute from the high pressure column. The information is sent from the detector to a computer which generates the chromatogram. The mobile phase exits the detector and is either sent to a waste, or collected, as desired.

Helium sparging is an effective method of degassing the mobile phase to avoid unstable baselines caused by dissolved air. Nitrogen is used as a nebulisation gas in Evaporative Light Scattering Detector (ELSD) where the solvent is evaporated from the sample leaving a mist as is measured.