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Ion-exchange chromatography operating principle
https://www.youtube.com/watch?v=q3fMqgT1do8
Separation of molecules by ion-exchange (IEX) chromatography relies on differences between the net surface charges on the solute molecules. Proteins, for example, contain numerous groups which can ionize to varying extents depending on the pH of the solution. The ionic state of these groups is highly dependent on the pH, and as a result, the net surface charge of a protein will undergo a change as the pH of their environment varies.
At the isoelectric point (pI) of the protein, the protein will have little or no tendency to bind either to a cationic stationary phase (that is, one which has positively charged groups) or to an anionic stationary phase (one that has negatively charged groups).
At pH value below the pI, the protein will have a net positive charge, and will tend to bind reversibly with to the surface of a cation-exchange resin, that is, one that has negatively charged groups at that pH.
Note that a cation-exchange resin is anionic, having negatively charged groups, while an anion exchange resin is cationic, since it has positively charged groups.
Binding to the matrix requires that buffer ions that are bound to the matrix during the equilibration process be displaced by the solutes of interest. Thus, and exchange of ions takes place at the surface of the matrix.
On applying the sample, conditions are chosen so that as many as possible of the unwanted solutes do not bind to the resin, leaving the molecules of interest bound to the top of the column. When all the unwanted solutes have been eluted, the composition of the buffer is gradually altered, either by increasing the ionic strength, Γ, or, less commonly, by changing the pH of the eluting buffer. This process is called gradient elution. The bound solutes are eluted at different values of the ionic strength, as buffer ions compete with the bound molecule for cationic or anionic sites on the resin.
Types of matrix material
There is a variety of commercially available materials which can be classified as follows:
Anion exchangers |
Type |
Functional group |
---|---|---|
Quaternary ammonium (Q) | Strong | -OCH2N+(CH3)3 |
Diethylaminoethyl (DEAE) | Weak | -OCH2CH2NH+(CH2CH3)2 |
Diethylaminopropyl (ANX) | Weak | -OCH2CHOHCH2NH+(CH2CH3)2 |
Cation exchangers |
Type |
Functional group |
---|---|---|
Sulfopropyl (SP) | Strong | O-CH2CHOHCH2OCH2CH2CH2SO3– |
Methyl sulfonate (S) | Strong | O-CH2CHOHCH2OCH2CHOHCH2SO3– |
Carboxymethyl (CM) | Weak | O-CH2COO– |
Strong ion-exchangers do not show any marked change in their ionic states with changes in pH. They remain fully charged over a broad range of pH values.
Advantages of strong ion-exchangers
- the development and optimization of separations is fast and easy since the charge characteristics of the medium do not change with pH;
- the mechanism of interaction is simple since there are no intermediate forms of charge interaction;
- sample loading (binding) capacity is maintained at high or low pH since there is no loss of charge from the ion exchanger.
Most proteins can be separated on either strong or weak ion-exchangers. If strong ion-exchangers do not give the required results, try a weak exchanger. These offer a different seletcivity to strong exchangers, but their exchange capacity changes with pH.
Gradient elution
If the elution process is carried out using the same buffer that was used in applying the sample, the elution is said to be ISOCRATIC.
Alternately, the elution may be carried out for a while with that buffer, and then with another buffer. This is called STEP-WISE ELUTION.
For the best results, the composition of the eluting buffer is changed as the elution progresses. The change is usually a change in ionic strength, from a less dilute to a more concentrated buffer, or, less frequently, a change in pH. This elution process is called GRADIENT ELUTION, and this generally gives a much better separation of solutes.
In order to carry out gradient elution, one must use specially designed pumps, which, under software control, can provide a wide variety of gradients. These pumps are very expensive, and deserve the greatest care in their operation.