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Ion Exchange Chromatography
Ion exchange chromatography (IEC) is applicable to the separation of almost any type of charged molecule, from large proteins to small nucleotides and amino acids.
It is very frequently used for proteins and peptides, under widely varying conditions. However, for amino acids standardized conditions are used. In protein structural work the consecutive use of gel permeation chromatography (GPC) and IEC is quite common.
Basic Principles of Ion Exchange Chromatography
The attractive forces between molecules carrying charged groups of opposite signs are used in FPLC in two different ways. One is called Ion Pairing and concerns primarily rather small molecules. This technique will be treated later in connection with reversed phase chromatography. The second is called Ion Exchange and charged particle (matrix) bind reversibly to sample molecules (proteins, etc..). Desorption is then brought about by increasing the salt concentration or by altering the pH of the mobile phase. Ion exchange containing diethyl aminoethyl (DEAE) or carboxymethyl (CM) groups are most frequently used in biochemistry.
The ionic properties of both DEAE and CM are dependent on pH, but both are sufficiently charged to work well as ion exchangers within the pH range 4 to 8 where most protein separations take place. Except for the addition of a gradient device, the instrument set-up is the same as for GPC. The ion exchange mechanism may be expressed as in the figure 2.
pH and Selectivity in Ion Exchange Chromatography
The property of a protein which govern its adsorption to an ion exchanger is the net surface charge. Since surface charge is the result of weak acidic and basic groups of protein; separation is highly pH dependent. Going from low to high pH values the surface charge of proteins shifts from a positive to a negative charge surface charge. The pH vs. net surface curve is a individual property of a protein, and constitutes the basis for selectivity in IEC. At a pH value below its isoelectric point a protein (+ surface charge) will adsorb to a cation exchanger (-) such as one containing CM - groups. Above the isoelectric point protein (-surface charge) will adsorb to a anion exchanger (+), e.g. one containing DEAE-groups.
The pH vs. net surface charge curves for three different proteins are shown. Schematic chromatograms for a CM and a DEAE ion exchanger are shown at the top and bottom respectively. At the most acidic pH value, all three proteins are positively charged and adsorb only to the CM ion exchanger. They are then eluted in the order of net charge. At the next pH value chosen, the protein has passed its isoelectric point and is now negatively charged, while the other two still retain positive charges. The blue protein will consequently adsorb to a DEAE ion exchanger, but not to a CM ion exchanger the other two do. At the next highest pH value the only one positively-charged protein still adsorbs to the CM ion exchanger. Because of their negative net charges, the two other proteins adsorb to the DEAE ion exchanger. At the most alkaline pH, all three proteins are adsorbed to the DEAE ion exchanger only. Thus, by varying the pH of the mobile phase, one can greatly influence the selectivity in ion exchange chromatography.
Ionic Strengths and Selectivity in Ion Exchange Chromatography
As in all forms of liquid chromatography, conditions are employed that permit the sample components to move through the column with different speeds. At low ionic strengths, all components with affinity for the ion exchanger will be tightly adsorbed at the top of the ion exchanger and nothing will remain in the mobile phase. When the ionic strength of the mobile phase is increased by adding a neutral salt, the salt ions will compete with the protein more of the sample components will be partially desorbed and start moving down the column. Increasing the ionic strength even more causes a larger number of the sample components to be desorbed, and the speed of the movement down the column will increase. The higher the net charge of the protein, the higher the ionic strength needed to bring about desorption. At a certain high level of ionic strength, all the sample components are fully desorbed and move down the column with the same speed as the mobile phase. Somewhere in between total adsorption and total desorption one will find the optimal selectivity for a given pH value of the mobile phase. Thus, to optimize selectivity in ion exchange chromatography, a pH value is chosen that creates sufficiently large net charge differences among the sample components. Then, an ionic strength is selected that fully utilizes these charge differences by partially desorbing the components. The respective speed of each component down the column will be proportional to that fraction of the component which is found in the mobile phase.
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