Technique and application of ion exchange principle
- Ion Exchange Chromatography is a method used for the separation as well as purification of ionically charged biomolecules like proteins, polynucleotides, nucleic acids etc.
- The process of ion exchange is described as reversible exchange of the ions that are present in a solution or a mixture, such that the ions are electrostatically bound to the inert support medium
The choice of the ion exchanger used in ion exchange chromatography depends upon factors like:
- Stability of sample components.
- Molecular weight of sample components.
- Specific requirements of separation etc.
Stability of sample components:
- Most biological components, especially proteins, are stable in a fairly narrow pH range. Therefore, the exchanger that is selected must be able to operate within these conditions.
- Based on the above stated point, thus, if the sample is more stable at a pH below its isotonic point, use of a cation exchanger is advocated.
- Conversely, if a sample exhibits stability above the isotonic point, an anion exchanger might be more useful.
- Lastly, compounds which exhibit a wide range of pH can be separated using either of the type of exchanger.
Molecular weight of the compounds
- It is necessary to take into account the molecular weights of the sample components because the degree of cross-linkage of gel material depends on the molecular weight of the sample components.
- When cross-linked gel material is used as an exchanger, the diameter of the pores is controlled.
- Molecular sieving effects of the gel material also play a role in separation.
- Due to these reasons, it is important to keep in mind the molecular weights of the sample as the cross linkage of the gel, which affects separation, is directly dependent on it.
Other specific requirements of separation
- Volume of the exchanger: Volume of the exchanger used for separation is usually 2-5 folds greater than needed to bind all of the sample. Excess is usually avoided.
- Columns used: The columns that are used affect the resolution obtained. Columns that have a high diameter to height ratio give better resolution. However, long and narrow columns impose a peak broadening effect because during elution, the free ions have to traverse a longer distance before the fractions can be collected. Due to this, the free ions have enough time and opportunity to diffuse and therefore be eluted in larger volumes which leads to peak broadening.
- pH: The pH of the buffer is usually carefully maintained at one pH degree above or less than the isoelectric point of the sample components.
- Buffer: Cationic buffers like Tris, pyridine and alkylamines are used when anion exchange chromatography is conducted. In cation exchange chromatography, anionic buffers such as acetate, barbiturate and phosphate are used. The initial buffer pH and anionic strength of it must be adjusted so that the sample components bind to the exchanger.
- Amount of sample: The amount of sample to be applied depends upon the size of the column and the capacity of the exchanger.
- In isocratic separation, the sample volume is about 1-5% of the bed volume.
- However, sample volume is not an important consideration when gradient elution is to be performed. Gradient elution is more commonly preferred than isocratic elution because it gives better resolution.
Application of ion exchange principle and example
The principles of ion exchange chromatography also apply to other macromolecules such as proteins and nucleic acids which are capable of showing the presence of both positive and negative ions. The type of macromolecules, being so large in size can then bind to both anionic and cationic exchangers because they possess both the types of charges.
The macromolecules can be modified to possess the desired (negative or positive) charge by:
- Negative charge: The macromolecules can be made to bear negative charges by increasing the pH. This increase in pH results in a stronger binding to anion exchanger.
- Positive charge: The macromolecules can be made to bear a positive charge by reducing the pH. This reduction in the pH results in a stronger binding to the cation exchanger.
Therefore, the amphoteric nature possessed by these macromolecules can be exploited.
Separation of protein by sequential use of cation and anion exchange:
- If a protein is to be separated from a mixture, the desired protein can be made to behave as a cation.
- This can be done by lowering the pH (as mentioned above) of the protein mixture. The pH is lowered to such a limit where most of the other proteins that are present, behave as anions.
- When this preparation is chromatographed on a cation exchanger, the exchanger will remove most of the anionic species present due to ion exchange mechanism as discussed.
- This process will remove most undesired proteins from the initial mixture.
- Therefore, the resultant mixture will majorly contain the desired protein to be separated.
- Now, if the pH of the resultant mixture is increased, the desired protein to be separated will predominantly exist as an anion.
- The pH must be increased in such a way that all but the desired proteins still behave as cations in the mixture and therefore do not get separated and reduce the specificity of separation.
- When this preparation (where all but the desired protein exist as cation) is chromatographed, most of the cationic species will be lost.
Therefore, this is how one can use cation and anion exchange chromatography sequentially to attain a large degree of purification of compounds.
Reference and Sources