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HPLC Systems

The use of HPLC systems for analysis of compounds

HPLC machines are used in laboratories as analytical tools to separate the component parts of a mixture of compounds in order to analyse, identify, quantify or purify them.  HPLC refers to High Performance Liquid Chromatography also known as High Pressure Liquid Chromatography.  In traditional liquid chromatography a mixture of compounds is dissolved in a fluid referred to as the mobile phase.  This fluid is passed through a matrix such as a silica gel or a porous membrane known as the stationary phase as it is often fixed either in a column or planar format.  The component parts of the mixture can be separated by various means depending on the characteristics of the compounds.  For example, size exclusion chromatography separates mixtures by virtue of the size of the components whereas ion exchange chromatography is used to separate analytes based on their respective charges.

After the separation is completed, the mobile phase is eluted off the stationary phase often by the use of solvents and passed through a detector, which records information about the eluted liquid or effluent as it passes through.  The most common solvents used are methanol or acetonitrile. In liquid chromatography, the detection system is usually UV detection in which the effluent passes through a UV light and the amount of absorbance is recorded.  Fluorescence detection is also used in liquid chromatography where the effluent is irradiated with light at a set wavelength and the amount of fluorescent light emitted is recorded.

The interval between the liquid phase passing into the stationary phase and the moment of detection is called the retention time and this is recorded as a graph called a chromatogram.  Each retention time is recorded as a peak and each peak represents a different component of the separated mixture.   As these components are often unique in their composition, the component can often be uniquely identified by the retention time.

A HPLC system uses a pump which causes the mobile phase to move at high pressure through the stationary phase.  The pressurised movement of the mobile phase means that the stationary phase can be packed with much smaller particles giving a greater surface area for interaction with the molecules flowing past it.  This allows for much better separation of the mixture and increases sensitivity of the technique.  HPLC means that the chromatography can be automated and produce highly accurate results over a short period of time.  Another advantage of an HPLC system is that the solvent used to elute the mobile phase can be introduced on a gradient, controlling the elution and therefore allowing better separation and analysis of the peaks.

HPLC is used in many laboratories for analytical purposes, and is used to look for metal ions and organic compounds in solutions.  It has particular applications in forensic science for investigative purposes.  The technique is also used for pollution testing, where for example, water samples can be tested and the pollutants separated and identified.  Chromatography is also used in toxicology, separating and identifying different drugs of abuse.  Synthesised products can also be tested for purity using chromatography and therefore you will find an abundance of HPLC systems in pharmaceutical companies who will use them to prepare large amounts of pure substances for use in medicines.

HPLC systems are provided by a number of manufacturers, some of the major suppliers include:

  • Agilent technologies have a range of LC systems.  In particular, the Agilent 1220 infinity HPLC system is an affordable system for routine standard analysis.  The 920-LC HPLC system is a higher spec model for more in depth analyses.
  • Dionex Corporation produce a wide range of HPLC and Ultra HPLC systems to meet the demands of a busy analytical laboratory
  • GE Healthcare formerly Amersham biosciences produce the AKTA systems which meet GLP and cGMP systems suitable for use in drug development.
  • Hitatchi produce the popular LaChrom elite HPLC system
  • JASCO produce the XL-C system range of HPLC systems
  • Perkin Elmer offer a range of comprehensive preconfigured systems
  • Shimadzu are another popular supplier of HPLC machines, and have a wide range suitable for a variety of analyses, including the LC-2010, the Prominence HPLC and the Nexera UHPLC system

All of these systems and more can be found new or reconditioned at www.oxfordlabs.co.uk


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