High-performance liquid chromatography (HPLC)
formerly referred to as high-pressure liquid chromatography is
used to separate the chemical components in a mixture, to identify and quantify
each component of the mixture based upon the retention time. HPLC contains
pumps, temperature module, injection loop, sampler, column, photo diode array
detector and fraction collector. The pumps are used to pass a pressurized solvent (water, acetonitrile and/or methanol) containing the sample mixture and runs through a column
filled with a solid adsorbent
material (granular material made of silica, polymers, etc.). Each component in
the sample interacts differently with the adsorbent material, causing different
retention time for the different components and leading to the separation of
the chemical components as they flow out the column and detected by a detector.
The interactions observed between the component and adsorbent material may be hydrophobic,
dipole–dipole and ionic, most often a combination. The information from the
detector is sent to the computer; each component will be observed as a peak
with retention time and can be collected by fraction collector for further use.
By comparing the retention time with reference we can identify the specific
chemical compound. The various applications of HPLC include medical, legal, research
and manufacturing purposes.