High Performance Liquid Chromatography (HPLC)
is an analytical process utilizing special instruments designed to separate,
quantify and analyze components of a chemical mixture. Samples of interest
are introduced to a solvent flow path; carried through a column packed with
specialized materials for component separation; and component data is obtained
through the combination of a detection mechanism coupled with a data recording
system. All this occurs under pressures which may reach or exceed 6,000
The basic components of an HPLC system include a solvent reservoir, pump,
injector, analytical column, detector, recorder and waste reservoir. Other
important elements are an inlet solvent filter, post-pump inline filter,
sample filter, precolumn filter, guard column, back-pressure regulator and/or
solvent sparging system. The function of each of these components is briefly
An HPLC system begins with the solvent reservoir, which contains
the solvent used to carry the sample through the system. The solvent should
be filtered with an inlet solvent filter to remove any particles
that could potentially damage the system's sensitive components.
Solvent is propelled through the system by the pump. This often includes
internal pump seals, which slowly break down over time. As these
seals break down and release particles into the flow path, an inline solvent
filter prevents any post-pump component damage.
The next component in the system is the sample injector, also known
as the injection valve. This valve, equipped with a sample loop of the appropriate
size for the analysis being performed, allows for the reproducible introduction
of sample into the flow path. Because the sample often contains particulate
matter, it is important to utilize either a sample filter or a precolumn
filter to prevent valve and column damage.
Following the injector, an analytical column allows the primary sample
separation to occur. This is based on the differential attraction of the
sample components for the solvent and the packing material within the column.
However, a sacrificial guard column is often included just prior
to the analytical column to chemically remove components of the sample that
would otherwise foul the main column.
Following the analytical column, the separated components pass through a
detector flow cell before they pass into the waste reservoir.
The sample components' presence in the flow cell prompts an electrical response
from the detector, which is digitized and sent to a recorder. The
recorder helps analyze and interpret the data.
As a final system enhancement, a back pressure regulator is often
installed immediately after the detector. This device prevents solvent bubble formation
until the solvent is completely through the detector. This is important
because bubbles in a flow cell can interfere with the detection of sample
components. Alternatively, an inert gas sparging system may be installed
to force dissolved gasses out of the solvent being stored in the solvent
Each of the components described above requires fittings to couple it into
It is important to note that improper selection or installation of these
fittings can lead to leaks or the formation of dead volume, both of which
can result in poor HPLC performance.
Please contact one of our local dealers or Upchurch
Scientific directly if you have any questions regarding fittings
or the various components that make up an HPLC system. Also, please visit
the HPLC Recommendations for suggestions
regarding the system components discussed above.
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