High Performance Liquid Chromatography (HPLC)
is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known
as the mobile phase) at high pressure through a column
with chromatographic packing material (stationary phase). The sample is carried by a moving carrier
gas stream of helium or nitrogen. HPLC has the ability to separate, and identify
compounds that are present in any sample that can be dissolved in a liquid in trace concentrations
as low as parts per trillion. Because of this versatility, HPLC is used
in a variety of industrial and scientific applications, such as pharmaceutical, environmental,
forensics, and chemicals. Sample retention time will vary depending
on the interaction between the stationary phase, the molecules being analyzed, and
the solvent, or solvents used. As the sample passes through the column it
interacts between the two phases at different rate, primarily due to different polarities
in the analytes. Analytes that have the least amount of interaction
with the stationary phase or the most amount of interaction with the mobile phase will
exit the column faster. 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. High-Performance Liquid Chromatography - Other
HPLC Types Ultra High Performance Liquid Chromatography
(uHPLC): Where standard HPLC typically uses column
particles with sizes from 3 to 5�m and pressures of around 400 bar, uHPLC use specially designed
columns with particles down to 1.7�m in size, at pressures in excess of 1000 bar. The main advantage of an uHPLC is speed. These systems are faster, more sensitive,
and rely on smaller volumes of organic solvents than standard HPLC, resulting
in the ability to run more samples in less time. However, if the systems are run at typical
pressures greater than 800 bar, the columns age, or degrade quicker. Newer technology is being developed for uHPLC
units to use column particles with 1�m size, and pressure potentials up to 6,800 bar. uHPLC is also known as UPLCTM, a trademark
of the Waters Corporation. Fast protein liquid chromatography (FPLC):
FPLC is a system similar to high-performance liquid
chromatography that is used to separate or purify proteins and other
biomolecules from complex mixtures. The main difference between FPLC and HPLC
is the
standard working pressure. FPLC columns can only be
used up
to maximal pressures of 3-5 MPa. If the
pressure of the HPLC can be limited, nearly every FPLC column may also be used in an HPLC system.
