Chromatography is a separation and purification technique which is used for the separation of solutes in a mixture, biomolecules etc. on the basis of distribution of the sample to be separated between stationary phase (phase which is not mobile and is usually mounted against a support like a chromatographic column) and the mobile phase (which is continuous/is poured or passed over the stationary phase)

Ion Exchange Chromatography is a method used for the separation as well as purification of ionically charged biomolecules like proteins, polynucleotides, nucleic acids etc. 

This technique finds a wide array of applications in the scientific world because of its simplicity and high resolution. 


The process of ion exchange can be defined as the reversible exchange of the ions present in a solution, with the ions electrostatically bound to the inert support medium.

The main factor which governs the process of ion exchange chromatography is the electrostatic force of attraction present between the ions. This electrostatic force of the ions depends on their relative charge, radius of the hydrated ions and the degree of non-bonding


Usually, ion exchange separations are carried out in columns packed with an ion-exchanger. The ion-exchanger is a support medium which is inert and insoluble. The medium may be capable of covalently binding to positive (anion exchanger) or negative (cation exchanger) functional groups. The ions which do bind to the exchanger electrostatically are called counterions. 

The conditions of separation can be manipulated in such a way that some compounds are electrostatically bound to the ion exchanger while some are not, therefore, helping in the separation of the desired compound.

The sample which contains the sample to be separated is allowed to percolate through the exchanger for a certain amount of time that will be sufficient for the equilibrium of the ions to be achieved. 

E–   Y  E–   X+ + Y+

In the equation mentioned above, 

  • E–  : Charged cation exchanger.
  • Y+ : Counterion of the opposite charge associated with the exchanger matrix.
  • X+ : Charged molecule bearing a charge similar to the counterion to be separated. This molecule is capable of exchanging sites with the counterion as shown above.

Once the exchange of the counterion with the sample has been achieved, the rest of the uncharged and like charged species is washed out of the column.

The ions that did bind can then be eluted out by either percolating the medium with increasing concentration of Y+ (works by increasing the possibility that the Y+ will replace the X+ in the above-mentioned equation due to it being present in a higher concentration). The elution can also be carried out by increasing the pH of the solvent and hence converting X+ to an uncharged species.

Simply put, once the sample containing the specific ions to be separated it passed through the ion exchanger column, the sample ions (which act as counterions to the ions of the exchanger column) bind with the ions on the exchanger column and form associations. However, the ions of the same charge as the exchanger column, present in the sample solution, repel each other and, therefore, do not bind and pass through the column.

The principles which have been mentioned above also apply to other macromolecules such as proteins and nucleic acids which are capable of showing the presence of both positive and negative ions. The type of molecules can bind to both anionic and cationic exchangers. 


The two main types of materials used to prepare ion exchange resins are:

  1. Polystyrene, and
  2. Cellulose

Polystyrene resins are prepared by the polymerization reaction of styrene and divinyl benzene. These resins are very useful for separating compounds with a small molecular weight. 

Cellulose based resins have a much greater permeability to macromolecular polyelectrolytes as compared to polystyrene resins and they also possess a much lowers charge density. 

Based on the type of charge carried by these ion exchangers and their strength, the ion exchange resins can broadly be classified into four types:

Strong cationic exchange resins:Weak cationic exchange resins:
Sulphonated polystyreneSulphopropyl cellulose Condensed acrylic acidCarboxymethylcellulose
Strong anionic exchange resins:Weak anionic exchange resins:
Polystyrene with -CH2NMe3ClDiethyl (2 hydroxypropyl)quaternary amino celluloseDiethylaminoethyl celluloseDiethylaminoethyl agarose

The buffer is that component of the chromatographic column that helps in the maintenance of the pH. The choice of these buffers is usually dictated by the compounds to be separated and whether the ion exchange is anionic or cationic.

  • Anion exchange chromatography should be carried out with cationic buffers.
  • Similarly, cation exchange chromatography should mostly be carried out with only anionic buffers for satisfactory separation and results. 

      Examples of some buffers used in this technique are:

  • Ammonium acetate
  • Ammonium formate
  • Pyridinium formate
  • Ammonium carbonate etc.
  1. The most significant application of ion exchange chromatography is in amino acid analysis.
  2. This technique is used to determine the base composition of nucleic acids.
  3. Ion exchange chromatography is used as a method of purification of water. Water is completely deionized using this technique.
  4. This technique is used for the ultra-purification of metal ion free reagents.
  5. It can also be used for the separation of a varied number of vitamins, biological amines, organic acids as well as bases. 


Biophysical Chemistry principles and techniques – Upadhyay and Nath


BY: Ria Fazulbhoy (MSIWM031)


  • Ion exchange chromatography is based on the principle of reversible exchange of ions and polar molecules by retaining the sample molecules on a column (inert support medium).
  • It is based on electrostatic force of attraction or ionic interactions between the ions and the surface of the stationary phase which has ionic functional groups (R-X)
  • Ion exchange chromatography is carried out in columns packed in an ion exchanger, which is an inert insoluble support medium. Ions bound electrostatically to the column are known as counterions.
  • The stationary phase can be ion exchange resins that carry charged functional groups to interact with oppositely charged groups in the sample.
  • It can be employed on charged molecules like ions, amino acids, small nucleotides, large proteins, etc.
  • The sample containing the ionic species which has to be separated is allowed to percolate through the exchanger for a sufficient amount of time so that equilibrium can be achieved.

Types of exchanges in ion exchange chromatography

  1. Based on charge:
  1. Anion-exchange chromatography:

This uses ion exchange resins containing positively charged groups like diethyl-aminoethyl groups. In solution, resins are coated with positively charged counter ions, which have an affinity for molecules with net negative charges on the surface. Also known as “Basic ion exchange” materials.

  1. Cation-exchange chromatography

Cation exchange chromatography is a technique that uses a negatively charged ion exchange resin which has an affinity for molecules having net positive charges on their surface. Also known as “Acidic ion exchange” materials.

The total number of equivalents of replaceable protons per unit volume of resin determines the exchange capacity of the resin.

Based on this there are two kinds of exchangers:

  1. Strong exchangers

Strong ion exchangers show no variation in ion exchange capacity with changes in pH. They are prepared with a tertiary amine, yielding a strongly basic quaternary ammonium group.

  1. Weak exchangers

They are ionized over only a limited pH range. Weak anion exchanger is prepared with secondary amines which yield a weakly basic tertiary amine.

What is the Isoelectric point in ion exchange chromatography?

Ion exchange chromatography is based on the different charges of ions and the electrostatic force between the ionic charges and that of the column of chromatography.

Isoelectric point is the pH at which the overall number of negative and positive charges is equal to zero. Thus, no ion exchange takes place at the isoelectric point.

Resins used in ion exchange chromatography:

Applications of ion exchange chromatography

  1. Used in in amino acid analysis. Amino acids are known as “autoanalyzer” and this is based on ion exchange principle
  2. Ion exchange has also been extensively used to determine the base composition of nucleic acids. Treatment with DNAses and RNAses which results in a mixture of nucleotides can be readily separated by ion exchange chromatography.
  3. In Biological applications, ultrapure, metal ion free reagents are needed. This is commercially performed by ion exchange chromatography.
  4. Ion exchange chromatography has been used for the separations of many vitamins, other biological amines, and organic acids and bases.