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
- Based on charge:
- 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.
- 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:
- 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.
- 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
- Used in in amino acid analysis. Amino acids are known as “autoanalyzer” and this is based on ion exchange principle
- 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.
- In Biological applications, ultrapure, metal ion free reagents are needed. This is commercially performed by ion exchange chromatography.
- Ion exchange chromatography has been used for the separations of many vitamins, other biological amines, and organic acids and bases.