Write a short notes on Isoelectric Focusing and Ion Exchange Chromatography.
Isoelectric focusing is a type of electrophoresis carried out in a pH gradient created in a column by mixing different buffers. Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein. A pH gradient is established by allowing a mixture of low molecular weight organic acids and basis (ampholykes) to distribute themselves in an electric field generated across the gel, when a protein mixture is applied, each protein migrates until it reaches the pH that matches its isoelectric point (pI) proteins with different isoelectric points are thus distributed differently throughout the gel.
Ion exchange chromatography.
Ion exchange chromatography Ion exchange chromatography is the most widely used method for separating, identifying and qualifying the amounts of each amino acid in a mixture.
This technique primarily exploits differences in the sign and magnitude of the net electric charges of amino acids at a given pH value, which are predictable from their pka values or their filtration curves. Cation exchange and anion, exchange are two, important features of Ion exchange chromatography. The chromatography column consists of a long tube filled with particles of a synthetic resin containing fixed charged groups those with fixed anionic groups are called cation exchange resins and those with fixed cationic groups, anion exchange resin.
The affinity of each amino acid for the resin is affected by pH (which determines the ionization state of the molecule) and the concentration of other salt ions that may complete with the resin by associating with the amino acid. Separation of amino acids can therefore be optimized by gradually changing the pH and / or salt concentration of the solution being passed through the column. so as to create a pH or salt gradient.
A modern enhancement of this and other chromato-graphic techniques is called High Performance Liquid Chromatography (HPLC). This takes advantage of stronger resin material and is improved apparatus designed to permit chromatography at high pressure, allowing better separation in a shorter time.