High Performance Liquid Chromatography

In liquid chromatography the mobile phase is a liquid. The stationary phase is a solid contained inside a long narrow tube called "the column." Separation of mixtures occurs during passage through the column. The separation can occur by different mechanisms depending on the type of material in the mixture and the column, for example, adsorption, exclusion, ion exchange, partition. Whatever the mechanism, the volume of mobile phase at which a component elutes is constant and characteristic of that component, for a given chromatographic system. Thus the retention volume (time) can be used for qualitative identification. If the detector response is related to the amount of component in the sample, then the area under the chromatographic peak gives a measure of the quantity present.

The components in a mixture separate in the column and exit from the column at different times (retention times). As they exit, the detector registers the event and causes the event to be recorded as a peak on the chromatogram. A wide range of detector types are available and include ultraviolet adsorption, refractive index, thermal conductivity, flame ionization, fluorescence, electrochemical, electron capture, thermal energy analyzer, nitrogen-phosphorus. Other less common detectors include infrared, mass spectrometry, nuclear magnetic resonance, atomic absorption, plasma emission.

Chromatography is a very versatile technique offering a wide range of solid phase materials and detector types which can deal with very complex mixtures. In practice all materials and conditions used in the instrument are carefully chosen to match the type of sample mixture involved. This includes selection of stationary phase (chemical and physical properties); column type and length; sample pretreatment, operational temperatures, pressures, and flow rates; physical and chemical nature of mobile phase; detector type; and so forth. Detection to nanogram level is quite common and some systems can detect to picogram level using very small volumes of sample.

One of the major differences between normal liquid chromatography and HPLC is the column. In liquid chromatography the solid phase consists of large porous particles (75 to 200 ^m) packed into columns with internal diameters of 1 to 5 cm. Very low pressures are required to permit solvent flow (mobile phase) through the large particles in the column. Flow rates are very slow and separation times are long. HPLC uses narrower column diameters and solid phase particles that are much smaller and more uniform (3 to 10 ^m). This leads to much larger back pressures than with liquid chromatography and high pressure pumps are required. However, the column efficiency is increased 10- to 100-fold and separation times decreased compared to liquid chromatography. Separation methods employed in HPLC include normal phase, steric-exclusion, ion exchange, ion pair, and reversed phase.

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