Fast performance liquid chromatography
or Fast Protein Liquid Chromatography (FPLC) is a type of liquid chromatography used for protein purification. The main components of FPLC are pumps,
injection loop, injection valve, column, flow cells (conductivity, pH,
absorption), display and fraction collector. The mobile phase is water or
buffer and it is controlled by a pump. The stationary phase contains resin composed of beads, packed into a cylindrical column.
FPLC resins of various bead sizes are available depending upon the size of the
proteins and wide range of surface ligands depending on the nature of the
protein. In case of size exclusion chromatography we use columns packed with
resins of desired pore size whereas in ion exchange chromatography we use DEAE
or carboxy methyl cellulose. A mixture containing one or more proteins of
interest is loaded onto the column. The proteins of interest bind to the resin
while other components are removed in the buffer due to non specific binding. After
some time each of the bound proteins dissociates from the column and appears in
the effluent. The effluent passes through two detectors and each protein eluted
in the effluent is observed as a peak in the computer. The eluted protein can
be collected for further use using the fraction collector.