In analytical and clinical research, chromatography techniques are the gold standards for the separation of components in any given sample. The basic chromatography principle is the segregation of the constituent components present in the sample based on their interaction with two phases, i.e., the inner matrix (stationary phase) and the mobile phase.
Ion exchange chromatography is one of the chromatography methods that is widely used for the separation and purification of ions and polar molecules. This separation relies on the affinity of the sample components to ion exchangers.
Ion exchange chromatography is a successful method for separation due to the following key points:
- It has a very high resolution
- Heavy capacity
- Comparatively simpler mechanism
- Applicable for a wide range of chemical and biological components
- Ideal for the separation of charged biomolecules, including proteins, polypeptides, nucleic acids etc.
What is Ion Exchange Chromatography?
Ion exchange chromatography refers to the separation of charged ionisable molecules in a sample depending on their interaction with the inner matrix. This method of separation exploits the phenomenon of association between the ionisable molecule present in the sample and oppositely charged resins attached to the stationary phase.
Here, the underlying principle is the reversible exchange of ions between the target ion in a sample and the oppositely charged ions on the ion exchanger (Stationary phase). Mostly it is performed in the form of column chromatography; however, sometimes, even TLC methods can also work based on the ion exchange principle.
This chromatography technique is capable of separating similar types of molecules that are difficult to separate otherwise. The charge carried by the target molecule facilitates the process of separation.
This content discusses Ion exchange chromatography with its definition, principle, types, applications, procedure, advantages, disadvantages and diagrams.
Content: Ion Exchange Chromatography
- Principle of Ion Exchange Chromatography
- Instrumentation of Ion Exchange Chromatography
- Ion Exchange Chromatography Procedure
- Types of Ion Exchange Chromatography
- Applications of Ion Exchange Chromatography
- Advantages of Ion Exchange Chromatography
- Disadvantages of Ion Exchange Chromatography
Principle of Ion Exchange Chromatography
Ion exchange chromatography is based on the attraction and interaction between the ionised molecule in the sample and the oppositely charged resin. The resin is nothing but a stationary phase that provides the platform to interact. We refer to this charged stationary phase as an ion exchanger. As the charge it bears, we can classify them as cationic exchangers and anionic exchangers.
These ion exchangers comprise a charged moiety covalently linked to the surface of the insoluble inner matrix. The charged group is either positive or negative. These charged entities remain surrounded by oppositely charged functional groups.
The crude sample comprising the charged molecules is mixed along with the buffer. When this solution passes through the chromatographic column, the charged molecules exchange their place with the functional group and attach to the resin.
Example
For instance, if a protein sample contains positively charged moieties while the cationic exchanger possesses negative resins and a positive functional group. Then, the positive ions of the sample replace the functional group and bind to the resin.
Later, the attached molecules are eluted by using an elution buffer of varying ionic strengths.
Instrumentation of Ion Exchange Chromatography
Pump
A high-pressure pump is important to deliver and maintain a constant flow of eluent in the injector, column, and detector.
Injection
You can inject the sample into the eluent stream and to the column in various ways, but the simplest way is through an injection valve. You may inject the liquid samples directly, while the solid samples need proper dissolution before injection.
Columns
A column selection depends on the area of application. The columns for laboratory purposes are of glass, while those for industries are of stainless steel, titanium, inert plastic or high-quality polymer.
The separating column comprises a guard column at the anterior side. This acts as a protective layer that extends the life of the column.
Suppressor
The suppressor reduces the background conductivity of the chemicals used in elution. They are membranes-based devices designed to change the ionic eluent to water so as to enhance sensitivity.
Detectors
Mostly, electric conductivity detectors are used. They estimate the analyte peaks as soon as the eluent comes out of the column.
Data System
A computer remains attached to the ion exchange system that guides the process. There is a pre-programmed computing integrator for the routine analytical tests, as automation is unnecessary. Whereas more complex detections need more advanced devices like data stations, minicomputers etc.
Ion Exchange Chromatography Procedure
You can perform the basic Ion exchange chromatography procedure in 5 fundamental steps:
- Eluent loading: Equilibration
- Sample Injection: Loading
- Binding of desired molecules in the sample
- Washing of the unattached waste molecules
- Elution of the desired ions
1. Equilibration: This involves running the equilibrated buffer via the column. This is done to make the resins attached firmly to the surface of the column.
2. Loading: This chromatography is based on the attraction between oppositely charged ions, i.e., the sample molecule and the ion exchanger. You can use the ion exchanger as per your desired product where the matrix will be positively or negatively charged, respectively. The aqueous solution containing an impure sample mixed with charged ions is suspended in the ion exchange column.
3. Binding: Sooner, the oppositely charged molecules will start replacing the covalently attached functional group from the matrix. And after this, the ions of the ion cloud exchange their places with the functional group without altering the properties of the matrix.
4. Washing: In order to remove the attached free molecules that remain unbonded are washed off using a washing buffer. This removes all the unwanted elute out while the desired ions still remain intact in the ion exchanger. As the washing starts, we obtain the first peak on the graph. From the time where the graph starts to form in the graph- we will collect the washouts materials in a different place.
5. Elution: After washing, there is a time of elution. This last step is to get the desired product out as eluents. Elution buffer is used with gradually increasing concentrations to elute the bonded sample.
Types of Ion exchange Chromatography
Based on the type of resin used, we can classify this chromatography into two categories: cationic and anionic.
- Cationic Exchange Chromatography: Here, the cationic exchangers (acidic ion exchangers) are used. In this type of exchanger, the resins attached to the stationary matrix are negatively charged. The functional group associated with the resin is positively charged. This functional group gets replaced by the positive ions of the sample.
- Anionic Exchange chromatography: Here, the anionic exchanger (basic ion exchange) is used. In this type of exchanger, the resin attached bears a positive charge along with negatively charged functional groups. Thus, the resins attract the negative anions present in the sample, which will get attached to it by replacing the functional group.
Application
- A critical tool for pharmaceutical analysis.
- Ideal for estimating, separating, and purifying biomolecules, including proteins, amino acids, peptides, carbohydrates, vitamins, enzymes, etc.
- Used for the separation and purification of organic molecules from a natural resource.
- Vital in modern-day drug discovery methods.
- Highly preferred in routine analysis of amino acid mixtures.
- Used in an analysis of lunar rocks and rarely found grace elements.
- The chelating resin in the ion exchange column is used to help collect the trace metals from seawater.
- Used for analysing the hydrolysis products of nucleic acids.
- Most effective and trustworthy tool for water purification.
Advantages
- Ion exchange chromatography is one of the most effective tools for the separation of charged molecules.
- Actively used for clinical, analytical and preparative purposes.
- It can also separate the inorganic ions.
- It is easy to alter the separation by changing the pH of the buffer and salt gradient or by changing the type of exchanger resins.
- Highly selective techniques since it uses a slight difference in the molecular charges.
- Low maintenance, cheap and comparatively simpler working mechanism.
- Separation time is less. Thus, you can separate bulk sample volume in a short time.
Disadvantages
- It remains limited to the separation of charged molecules only.
- Requires a large volume of buffer.
- Instrumentation is expensive and requires a skilled expert to handle the apparatus.
- The resin can easily get damaged, which will hamper the whole procedure.
- Expensive chemicals
- The efficiency of the column diminishes after repeated use. Thus, it becomes difficult to predict the accuracy of results.
- Often, it becomes difficult to monitor selectivity and resolution.
Just like the other chromatographic techniques, Ion exchange chromatography is also a great tool that assists various research protocols, forensic cases, clinical studies etc.
This content will help you get all the necessary information about ion exchange chromatography in detail.
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