Fast Protein Liquid Chromatography (FPLC)

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.