Introduction
lon exchange chromatography is a separation technique where ion exchangers are used as the stationary phase. Ion exchange is a process in which insoluble substances exchange ions in a surrounding solution. This process has also been called base exchange and exchange adsorption. The insoluble substances are called ion exchangers. Proteins, synthetic resins, cellulose, bone, living cells, silicate minerals and sulfonated coals are good examples of ion exchangers.
Principle of Ion Exchange Chromatography
- Ion exchange resins are organic polymerized resins and act as cation or anion exchangers.
- The resins are insoluble and have one of the ions attached to the polymer and the other is free to move away.
- In a cation exchanger, the fixed ion is negative and the free ion is positive.
- The anion exchanger has a fixed positive charge and free negative charge.
- The net ionic forces vary.
- The proteins and amino acids may carry positive and negative charges and have ionizable Groups.
- The cationic exchangers are negatively charged molecules and so they can bind the Positively charged molecules.
- The anionic exchangers are positively charged molecules And so they can bind the negatively charged molecules.
- Using such reactions, the charged Molecules can be separated.
- The biological molecules exist in electrically charged forms under different conditions And these are separated by ion exchange chromatography.
- Aminoacids have both the coo group and the NH, groups depending upon the pH.
- They can remain either+vely or-vely charged and they can be separated on either a cation or anion exchanger.
- The 20 different amino acids that occur normally in a protein can be separated in an automatic amino acid analyzer which uses both cation exchanger (for acidic, hydroxy and neutral amino acids) and anion exchanger (to separate basic amino acids).
Procedure of Ion Exchange Chromatography
- The ion exchanger (either cation exchanger or anion exchanger) is packed in a column Of glass tube.
- The ion exchangers are in the form of beads.
- The samples to be separated is prepared with a suitable buffer and is passed through The column.
- Molecules containing charges opposite to that of the ion exchanger, bind with the Molecules, containing charges similar to that of the ion exchanger, will pass through Ion exchanger. Without any binding.
- The bind molecules can be eluted by increasing the concentration of the buffer.
- Anionic exchangers need cationic buffers like tris, pyridine, alkylamines, etc.
- The cationic exchangers need anionic buffers such as acetate, barbiturate, phosphate, etc..
Applications
- Used in water softening process in industries.
- lon exchange chromatography is useful to find the sequence of amino acids in pro Desalting process of the amino acids
- Used for insulin purification. Used for plasma fractionation
- Used for the purification of enzymes.
