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The ion-exchange chromatography process allows the separation of ions and polar molecules based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids, with the experimental solution to be separated collectively known as the analyte. It is often used as a first step in protein purification.
Principle
Ion exchange chromatography retains analyte molecules based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography:
- Cation exchange chromatography retains positively charged cations because the stationary phase displays a negtively charged functional group such as a phosphonic acid
- Anion exchange chromatography retains negatively charged anions using positively charged functional group such as a quaternary ammonium cation
Technique
The stationary phase is a resin or gel matrix consisting of either agarose or cellulose beads and containing covalently bound positive or negative functional groups. It resides within a column and the mobile phase is placed on top. The mobile phase is a buffered aqueous solution which carries the solution to be separated. The analytes can be eluted by a by increasing the concentration of similarly charged species to displace those bound to the stationary phase. For example, in cation exchange, the positively charged analyte could be displaced by the addition of positively-charged sodium ions.
For proteins, this technique separates according to their net charge, as the constituent amino acids will have differing charges at various pH's. By adjusting the pH or the ionic concentration, various protein molecules can be separated by changing the the charge on the analyte or the stationary phase. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively-charged beads, whereas a negatively charged protein would not. By changing the pH so that the net charge on the protein is negative, it too will be eluted.
See also
External links
"Ion exchange chromatography"