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, nucleotides, amino acids 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. 

Types of Ion-Exchange Chromatography:

Cation-exchange chromatography: 

negatively charged group, and these will attract positively charged cations. These exchangers are also called “Acidic ion exchange” materials

Anion-Exchange Chromatography: 

positively charged groups that will attract 

negatively charged anions. These are also called “Basic ion exchange” materials.

Ion-Exchange Chromatography Procedure:

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 anion-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.

 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.

 

 It has a high operating cost.