Chromatography and High Performance Liquid Chromatography (HPLC)

Liquid chromatography-mass spectrometry analysis of small molecules from biofluids requires
sensitive and robust assays. Because of the very complex nature of many biological samples,
efficient sample preparation protocols to remove unwanted components and to selectively extract
the compounds of interest are an essential part of almost every bioanalytical workflow. High-
performance liquid chromatography (HPLC) is a separation technique that can be used for the
analysis of organic molecules and ions. HPLC is based on mechanisms of adsorption, partition
and ion exchange, depending on the type of stationary phase used. HPLC involves a solid
stationary phase, normally packed inside a stainless-steel column, and a liquid mobile phase.
Separation of the components of a solution results from the difference in the relative distribution
ratios of the solutes between the two phases. HPLC can be used to assess the purity and/or
determine the content of many pharmaceutical bioprocessing substances. It can also be used to
determine enantiomeric composition, using suitably modified mobile phases or chiral stationary
phases. Individual separation mechanisms of adsorption, partition and ion exchange rarely occur
in isolation since several principles act to a certain degree simultaneously in a very
environmental monitoring, hydrophilic molecules will tend to associate with each other (like
water drops on an oily surface). The hydrophilic molecules in the mobile phase will tend to
adsorb to the surface on the inside and outside of a particle if that surface is also hydrophilic.
Increasing the polarity of the mobile phase will subsequently decrease the adsorption and
ultimately cause the sample molecules to exit the column. This mechanism is called Normal
Phase Analytical Chromatography. It is a very powerful technique that often requires non-polar
solvents. Due to safety and environmental concerns this mode is used mostly as an analytical
technique and not for process applications.

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