Ion exchange chromatography
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Ion-exchange chromatography (or ion chromatography) is a process that 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.
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[edit] History
Ion-exchange methods have been in use since 1850, when H. Thompson and J. T. Way, researchers in England, treated various clays with ammonium sulfate or carbonate in solution to extract the ammonia and release calcium. In 1927, the first zeolite mineral column was used to remove interfering calcium and magnesium ions from solution to determine the sulfate content of water. The modern version of IEC was developed during the wartime Manhattan Project. A technique was required to separate and concentrate the radioactive elements needed to make the atom bomb. Researchers chose adsorbents that would latch onto charged transuranium elements, which could then be differentially eluted. Ultimately, once declassified, these techniques would use new IE resins to develop the systems that are often used today for specific purification of biologicals and inorganics. In the early 1970s, ion chromatography was developed by Hamish Small and co-workers at Dow Chemical Company as a novel method of IEC usable in automated analysis. IC uses weaker ionic resins for its stationary phase and an additional neutralizing stripper, or suppressor, column to remove background eluent ions. It is a powerful technique for determining low concentrations of ions and is especially useful in environmental and water quality studies, among other applications.
The Dow Chemical Company technology was acquired by Durrum Instrument Corp., which later formed a separate business unit for its new IC products, naming it Dionex (Dow Ion Exchange). Dionex Corporation was incorporated in Sunnyvale, California in 1980, and, led by A. Blaine Bowman, purchased the Dionex assets.
[edit] 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 negatively charged functional group such as a phosphoric acid
- Anion exchange chromatography retains negatively charged anions using positively charged functional group such as a quaternary ammonium cation
[edit] Separating Proteins
Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. 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.
Elution by changing the ionic strength of the mobile phase is a more subtle effect - it works as ion from the mobile phase will interact with the immobilized ion in preference over those on the stationary phase. This "shields" the stationary phase from the protein, (and vice versa) and allows the protein to elute.
[edit] Typical Technique
A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance.
