The chromatographic method works on the basis of four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.
What is ion-exchange chromatography?
Ion exchange chromatography is a separation technique that allows ion exchangers to separate ions and polar molecules based on their affinity. It is the most powerful type of liquid chromatography since this method allows the analysis of a broad range of molecules. This method allows the isolation of related types of analytes which would be not possible to isolate by other techniques, as the charge carried by the interested molecule can be easily controlled by adjusting the pH of the buffer.
Separation mechanism of ion-exchange chromatography:
Ion exchange is a separation mechanism depends on the relative ionic strength of the solutes. The stationary phase is a charged substance with the opposite charge, which would preferentially retain species. A positively charged stationary phase in anion exchange-HPLC is maintaining a negatively charged species. For a cation exchange, the opposite is true. In ion chromatography elution of analytes is influenced by the pH of the mobile phase, ionic-strength, and working temperature of the system. Ion exchange columns have distinct advantages over silica-based support; they can use the full pH range and high temperatures. Ion chromatography reduces the pre-treatment of the sample mixture and enhances the precision and accuracy of results.
Principle of ion-exchange chromatography:
The ion-exchange chromatography depends on the attraction amongst the oppositely charged stationary phase, which is called ion exchange. The ion exchange consists of an inert support medium attached covalently to the functional groups which can be positive or negative. The oppositely charged ions are bound to these covalently bound functional groups, which would be exchanged with charged ions in the sample mixture with a charge magnitude greater than ions bounded to the matrix. Consequently, if anion exchange chromatography is executed, the negatively charged compounds can more interact with the stationary phase and be exchanged for the charged ions, which are already bound to the matrix.
The procedure of ion-exchange chromatography:
- The separations in ion-exchange chromatography are generally performed in a column which is packed with an ion-exchanger and they are prepared of divinyl-benzene and styrene.
- The selection of exchanger relies on the particle to be isolated. The anionic exchanger is used to separate anions and to separate the cations the cationic exchanger is used.
- The first step in the IEC, the ion exchanger is filled in the column and then followed by buffer a sample is applied.
- Tris-buffer, acetate buffer, pyridine, phosphate buffer, and citrate buffer are broadly used.
- The analytes that have a more affinity towards the ion-exchanger, they will move down with buffer. In the next step in IEC, the tightly bound particles are separated using a compatible buffer.
- After the separation of these particles, they are analyzed spectroscopically.
- In the last step, the system has to re-equilibrate for the next purification in initial conditions.
Two types of exchangers can be used in this method, namely, cationic and anionic exchangers.
Cationic exchangers: It has negatively charged groups and this would attract positively charged groups.
Anionic exchangers: It consists of positively charged groups and this would attract negatively charged molecules.
Commonly asked questions on chromatography are as follows.
What are the applications of ion-exchange chromatography?
Ion-exchange chromatography is the most commonly used in pharmaceutical, agricultural, biotechnology, environmental, and some other industry for the purification of different proteins, nucleic acids, polypeptides, antibiotics, and other charged bio-molecules.
What is the use of suppressor in ion chromatography?
The Suppressor is located between a detector and the column and serves to reduce the eluent’s background conductivity and increase the conductivity of the molecules.
What is the mobile phase in ion-exchange chromatography?
The mobile phase of ion-exchange chromatography is composed of an aqueous solution of an appropriate salt or a mixture of salts and some percentage of organic solvent.
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