In this ion exchange chromatography principle and instrumentation post we have briefly explained about ion exchange chromatography principle, instrumentation of ion chromatography, components, procedure, applications, advantages and limitations.
Ion exchange chromatography is a versatile, high resolution chromatography technique to purify the protein from a complex mixture. In addition, this chromatography has a high loading capacity to handle large sample volume and the chromatography operation is very simple.
Principle of Ion Exchange
This chromatography distributes the analyte molecule as per charge and their affinity towards the appositively charged matrix. The analytes bound to the matrix are exchanged with a competitive counter ion to elute. The interaction between matrix and analyte is determined by net charge, ionic strength and pH of the buffer.
For example, when a mixture of positively charged analyte (M, M+, M-1, and M-2) loaded onto a positively charged matrix, the neutral or positively charged analyte will not bind to the matrix whereas negatively charged analyte will bind as per their relative charge and needed higher concentration of counter ion to elute from matrix.
Affinity of analytes (M, M+, M-1, M-2) towards positively charged matrix
Types of Ion Exchange
The matrix used in ion-exchange chromatography is present in the ionized form with reversibly bound ion to the matrix. The ion present on matrix participates in the reversible exchange process with analyte. Hence, there are two types of ion-exchange chromatography:
Cation and Anion exchange chromatography
In cation exchange chromatography, matrix has a negatively charged functional group with an affinity towards positively charged molecules. The positively charged analyte replaces the reversible bound cation and binds to the matrix. In the presence of a strong cation (such as Na+) in the ion exchange chromatography mobile phase, the matrix bound positively charged analyte is replaced with the elution of analyte.
In anion exchange chromatography or anion chromatography, matrix has a positively charged functional group with an affinity towards negatively charged molecules. The negatively charged analyte replaces the reversible bound anion and binds to the matrix. In the presence of a strong anion (such as Cl-) in the ion exchange chromatography mobile phase, the matrix bound negatively charged analyte is replaced with the elution of analyte.
Isoelectric point in Ion Exchange
Protein is a polymer made up of amino acids with ionizable side chain. At a particular pH, this amino acid side chain ionizes differentially to give a net charge (positive/negative) to the protein.
The pH at which the net charge on a protein is zero is called as isoelectric point (pI). The protein will have a net positive charge below the pI where as it has net negative charge above the pI value.
Choice of Matrix for Ion Exchange
Before starting the isolation and purification of a substance, a choice for a suitable ion-exchange chromatography is important. There are multiple parameter which can be consider for choosing the right column matrix.
The information of a pI will be allowing you to calculate the net charge at a particular pH on a protein. As discussed above, a cation exchange chromatography can be use below the pI where as an anion exchange chromatography can be use above the pI value.
3-D structure of a protein is maintained by electrostatic and vander waal interaction between charged amino acid, Π-Π interaction between hydrophobic side chain of amino acids. As a result, protein structure is stable in a narrow range around its pI and a large deviation from it may affect its 3-D structure.
Similar to structural stability, enzymes are active in a narrow range of pH and this range should be consider for choosing an ion-exchange chromatography.
Exchange Chromatography Instrumentation
Typical Ion Exchange Chromatography instrumentation includes: pump, injector, column, suppressor, detector and recorder or data system.
Ion Exchange Chromatography instrumentation Diagram
The IC pump is considered to be one of the most important components in the system which has to provide a continuous constant flow of the eluent through the IC injector, column, and detector.
Sample introduction can be accomplished in various ways. The simplest method is to use an injection valve. Liquid samples may be injected directly and solid samples need only to be dissolved in an appropriate solvent. Injectors should provide the possibility of injecting the liquid sample within the range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000 psi).
Depending on its ultimate use and area of application, the column material may be stainless steel, titanium, glass or an inert plastic such as PEEK. The column can vary in diameter from about 2mm to 5 cm and in length from 3 cm to 50 cm depending on whether it is to be used for normal analytical purposes, microanalysis, high speed analyses or preparative work.
Guard column is placed anterior to the separating column. This serves as a protective factor that prolongs the life and usefulness of the separation column. They are dependable columns designed to filter or remove particles that clog the separation column.
The suppressor reduces the background conductivity of the chemicals used to elute samples from the ion-exchange column which improves the conductivity measurement of the ions being tested. IC suppressors are membrane-based devices which are designed to convert the ionic eluent to water as a means of enhancing the sensitivity.
Electrical conductivity detector is commonly use.
In routine analysis, where no automation is needed, a pre-programmed computing integrator may be sufficient. For higher control levels, a more intelligent device is necessary, such as a data station or minicomputer.
Procedure in Ion Exchange
Column material should be chemically inert to avoid destruction of biological sample. It should allow free low of liquid with minimum clogging. It should be capable to withstand the back pressure and it should not compress or expand during the operation.
The ionic strength and pH are the crucial parameters to influence the property of the ion exchange chromatography mobile phase.
The sample is prepared in the ion exchange chromatography mobile phase and it should be free of suspended particle to avoid clogging of the column. The most recommended method to apply the sample is to inject the sample with a syringe.
There are many ways to elute a analyte from the ion-exchange column. (1) Isocratic elution (2) Step-wise gradient (3) Continuous gradient either by salt or pH (4) affinity elution (5) displacement chromatography.
After the elution of analyte, ion-exchange chromatography column require a regeneration step to use next time. Column is washed with a salt solution with a ionic strength of 2M to remove all non-specifically bound analytes and also to make all functional group in a iononized form to bind fresh analyte.
Applications of Ion Exchange
1. Protein Purification: In the previous paragraph we have already discussed how ion exchange chromatography for protein purification can be used.
2. Protein-DNA interaction: Ion-exchange column is used as a tool to study interaction between DNA and a particular protein.
3. Softening of water: Ground water has several metals such as Ca2+, Mg2+ and other cationic metals. Due to presence of the metal, hard water creates problem in industrial settings. Ion-exchange chromatography is used to remove the metals present in the water through an exchange of matrix bound Na+.
4. Protein kinase assay: Protein kinase are class of enzyme responsible for transfer of phosphate group on the substrate molecule.
5. Purification of Metals: Ion-exchange matrix is used to isolate and purify rare earth metals such as uranium or plutonium. The first process to isolate uranium in large quantities was developed by frank spedding.
6. Concentrating a sample: An ion-exchange bead can be used to bind the analyte from a diluted solution and then sample can be eluted in smaller volume to increase the concentration.
Advantages of Ion Exchange
1. Ion-exchange is one of the most powerful methods of separating charged particles.
2. Using this method the inorganic ions can also be separated.
3. It can use more commonly for both analytical and preparative purposes.
4. Small amino acids, nucleotides and large proteins can separate using this method.
5. This is very effective and powerful water softening method.
6. Resins have a long life.
7. It has cheap maintenance an major ion exchange chromatography advantages.
Disadvantages of Ion Exchange
1. The buffer requirement is the major disadvantage of ion-exchange chromatography.
2. It has a high working cost since the buffer is used for the separation of components.
3. This method can only be used to isolate charged molecules.
4. Sodium ions entering the soft water will increase the acidity level in the water.