Monday, March 2, 2020

Principle and Procedure of Ion-exchange Chromatography

What is Ion-Exchange Chromatography?
Ion-exchange chromatography is a type of chromatography, which is commonly used in the purification of proteins and other charged molecules. In this technique, the molecules are separated based on their charge. In anion exchange chromatography, negatively charged molecules are attracted to solid supports with a positive charge. Conversely, positively charged molecules are drawn into a negatively charged solid support in cation exchange chromatography.

Ion-Exchange Chromatography Principle:
The charged bio-molecules are separated by ion-exchange chromatography. The samples with charged analytes will be used as a liquid phase. In the stationary phase, the analyte is opposite to the charged sites when it passes through the chromatography column. The analytes isolated based on their charge are eluted, utilizing a solution of different ionic strengths. Through moving such a mobile phase through the chromatography column, the analytes are very selectively separated according to their different charges.

Types of Ion-Exchange Chromatography:
Ion-exchange chromatography has two types, cation exchange, and anion exchange chromatography.
Cation-exchange chromatography: It separates molecules based on their net surface charge. It uses a negatively charged ion-exchange resin with an affinity for molecules having net positive surface charges.
Anion-Exchange Chromatography: 

Ion-Exchange Chromatography Procedure:
Before beginning an Ion-exchange Chromatography experiment, we must recognize the various stages essential to perform the process. Many studies on ion-exchange are conducted in five main stages.
  1. The first stage in the IEC is the equilibrium where the ion-exchanger is brought into a starting state.
  2. The second stage in the IEC is the application of samples and adsorption, in which 
  3. In the third stage of IEC, the molecules are separated from the chromatographic column by transferring them to elution conditions adverse to the ionic bonding of the molecules.
  4. The fourth and fifth stages of the IEC are the elimination from the chromatographic column of analytes not eluted under the earlier conditions and the re-equilibration for the next purification in the initial conditions.
Experimental Procedure of Ion-Exchange Chromatography:
  • The separation of ion-exchange is done mainly in columns packed with an ion-exchanger. These ion exchangers are made up of styrene and divinylbenzene and available commercially.
  • Next, the column is loaded with an ion-exchanger then a sample is applied followed by the buffer. In which the acetate buffer, pyridine, Tris-buffer buffer, phosphate buffer,      and citrate buffers are commonly used.
  • The higher affinity particles for the ion exchanger will come down the column as well as under the buffer. The next step is tightly bound particles separates using a compatible buffer.
  • These particles are then spectroscopically analyzed.
Ion-Exchange Chromatography Applications:
  • Ion-exchange chromatography is highly commonly used in the amino acid analysis.
  • Proteins are also separated using IEC.
  • For monitoring fermentation, the cation exchange resins are used.
  • It is the mainly helpful method for water purification.
  • It is used in the applications of food and clinical research.
  • This is used in analytical applications including quality control and monitoring of processes.
  • The ion-exchange chromatography is used for the separation and purification of blood components.
  • This technique is often used to isolate certain vitamins, and organic acids, and bases. 
The advantages of ion-exchange chromatography are as follows.
  • This is the most powerful way to separate charged particles.
  • This can be used for almost any charged molecule like large proteins, small amino acids, and nucleotides. 
  • It is performed in the laboratory for both analytical and preparative reasons, uses analytical to be more general.
  • Inorganic ions can also be isolated by this technique.
  • It is a useful and efficient technique for softening water.
The disadvantages of ion-exchange chromatography are as follows.
  • One of the main disadvantages of ion-exchange chromatography is the buffer requirement. 
  • Only charged molecules can be isolated by this method.
  • It requires a buffer for the separation of components.
  • It has a high operating cost.
  • The acidity level in the water can be raised by sodium ions entering the softened water. 
Commonly asked questions on chromatography are as follows.

What is ion-exchange chromatography?
Ion-exchange chromatography is the most commonly used chromatographic technique in chemistry for the separation and purification of nucleic acids, polypeptides, proteins, and other charged bio-molecules.

What is the basic principle of ion-exchange chromatography? 
The exchange of ions is the basic principle of ion-exchange chromatography. Cationic and anionic exchangers are two forms of ion-exchange chromatography are used in this process. 

What type of chromatography is ion-exchange chromatography?
The ion-exchange chromatography is a type of adsorption chromatography

What is the major advantage of ion-exchange chromatography?
The major advantage of ion-exchange chromatography is a powerful way to separate charged particles.

What is the major difference between affinity and ion-exchange chromatography?
The major difference between ion-exchange and affinity chromatography is that we can use ion-exchange chromatography for the separation of charged molecules, while we can use affinity chromatography to separate charged or uncharged molecules in a sample mixture.

What is the major difference between anion and cation exchange?
The major difference between anion and cation exchange is that the stationary phase is positively charged in the anion exchange, whereas in cation exchange it is negatively charged.


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