Ion Exchange Chromatography- Definition, Principle, Parts, Steps, Uses

What is Ion Exchange Chromatography?

Considering their preference for ion exchangers, ions and polar compounds may be separated using ion exchange chromatography(also referred to as ion chromatography).

By comparing how well a combination of chemicals interacts with an inert matrix, chromatography may separate the components of the mixture into their distinct parts.

The isolation principle is therefore the reversible exchange of ions among the target ions in the sample solution and the ions on the ion exchangers.

This method is compatible with both cationic as well as anionic exchangers.

  1. Cationic exchangers:Positively charged cations will be drawn to cationic exchangers with negatively charged groups. Due to the fact that their negative charges result from the ionisation of acidic groups, these exchangers are often termed as “Acidic ion exchange” materials.
  2. Anionic exchangers:Positively charged anions will be drawn to negatively charged anions by anionic exchangers. Also referred as “Basic ion exchange” materials, these.
  • Column chromatography is the method most frequently used for ion exchange chromatography. However, there are additional thin-layer chromatographic techniques that essentially operate on the ion exchange principle.

Working Principle of ion exchange chromatography

  • This form of chromatography relies on the interaction between an analyte and a stationary phase or ion exchanger with an oppositely charged stationary phase.
  • The key ion exchanger components are charged groups covalently linked towards the surface of an insoluble matrix.
  • The charged groups in the matrix might be either positively or negatively charged.
  • In an aqueous solution, the charged groups of the matrix would be accompanied by ions with the opposite charge.
  • In this “ion cloud,” ions can exchange reversibly without changing the matrix’s composition or physical properties.

Instrumentation of ion exchange chromatography

Pump, injector, column, suppressor, detector, and recorder or data system are examples of common IC instrumentation.

  1. Pump

One of the most crucial parts of the system, maintaining a constant supply of eluent via the IC injector, column, and detector is the responsibility of the IC pump.

  1. Injector

Various techniques can be used to complete a sample introduction. Utilizing an injection valve is the most basic technique. Hard samples need only be dissolved in the appropriate solvent; liquid samples could be added directly. A liquid sample must be able to be injected with high pressure and repeatability utilising injectors with a volume range of 0.1 to 100 ml (up to the 4000 psi).

  1. Columns

The column may be made of stainless steel, titanium, glass, or an inert plastic such as PEEK, depending on its intended function and location of use. Based on whether it will be utilised for normal analytical operations, microanalysis, high-speed analyses, or preparative work, the column’s width and length can vary between 2 mm and 5 cm and 3 cm and 50 cm, respectively.

In front of the dividing column is the guard column. This serves as a precaution, extending the longevity and use of the separation column. They are sturdy columns created to filter or get rid of debris that clogs the separating column.

  1. Suppressor

By reducing the background conductivity of the chemicals utilized to elute samples from the ion-exchange column, the suppressor improves the conductivity measurement of the studied ions. IC suppressors are membrane-based devices designed to boost sensitivity by turning ionic eluent into water. 

  1. Detectors

Commonly used is an electrical conductivity detector.

  1. Datasystem

A computational integrator with pre-programmed instructions may be adequate for routine analysis in which automation is not required. A more sophisticated device, such as a data station or minicomputer, is required for greater control levels.

Procedure of ion exchange chromatography

  • Many ion exchange separations are accomplished in ion-exchanger-packed columns.
  • These ionic exchangers are marketed commercially. It consists of divinyl benzene and styrene. Example. DEAE-cellulose is an anionic exchanger.This substance is a cationic exchanger.
  • The exchanger employed is determined by the charge of the particles to be separated. “Anionic exchanger” is utilised to isolate anions, whereas “Cationic exchanger” is utilised to separate cations.
  • First, ion exchanger is introduced to the column, followed by the sample and buffer. Tris-buffer, pyridine buffer, acetate buffer, citrate buffer, and phosphate buffer are often used.
  • Having particles with high attraction for the ion exchanger, buffers will drop the column.
  • The closely bonded particles are separated in the following step using the appropriate buffer.
  • Then, spectroscopic examination is done on these particles.

Applications of ion exchange chromatography

  • Ion-exchange chromatography is utilised extensively for the routine analysis of amino acid combinations.
  • The 20 main amino acids are isolated and employed in clinical diagnosis, whether they come from protein breakdown or blood serum.
  • The best way for purifying water is this one. By swapping solute cations for hydrogen ions and solute anions for hydroxyl ions, water (or a non-electrolyte solution) is completely deionized. This is often accomplished using a technique for drinking water softening.
  • while examining the byproducts of nucleic acid breakdown. This is how the shape of these molecules and its relationship to their biological function as carriers of genetic information are discovered.
  • To extract trace metals from saltwater, chelating resins are employed.
  • To examine rare traces of elements on Earth and lunar materials.

Advantages of ion exchange chromatography

  • It is among the best techniques for separating charged particles.
  • Almost any charged molecule can be utilised, including big proteins, tiny nucleotides, and amino acids.
  • In the laboratory, ion exchange is utilised for both analytical and preparative applications, with analytical usage being more prevalent.
  • Inorganic ions can also be separated via ion-exchange chromatography.

Limitations of ion exchange chromatography

  • Only charged molecules may be isolated.
  • Need for Buffers


  • Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular Biology (8 eds.). Cambridge University Press: New York.
  • https://www.biochemden.com/ion-exchange-chromatography/
  • https://www.britannica.com/science/ion-exchange-reaction/Applications-of-ion-exchange
  • http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch06s02.html
  • http://cdn.intechopen.com/pdfs/43603/InTech-Ion_exchange_chromatography_an_overview.pdf
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