Everything You Need to Know About Ion exchange chromatography

Everything You Need to Know About Ion exchange chromatography


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  • Source: Microbioz India

  • Date: 18 Mar,2023

The separation of ionizable molecules according to their aggregate charges is the goal of the chromatographic technique known as ion exchange chromatography. Because the charge carried by the molecule of interest can be easily changed by changing the buffer pH, this technique permits the separation of comparable types of molecules that would be difficult to separate using other techniques. This would be the case if other techniques were used.

Ion exchange chromatography is frequently utilised for the separation of inorganic anions (such as chloride, nitrate, and sulphate), as well as inorganic cations (e.g., lithium, sodium, and potassium). It is also possible to utilise it for organic ions, although this use is becoming less prevalent as a result of the development of technologies for reversed phase liquid chromatography, which will be discussed further on. Ion-exchange chromatography is used as a stage in the process of purifying proteins, which is another notable application of this technique. Because some of the substituent groups of amino acids are charged (the total charge for a given protein is a function of the pH of the solution), ion exchange is an effective technique for purifying proteins. This is because the total charge of a protein is dependent on the pH of the solution.

Ion exchange chromatography Vs Ion Chromatography

Ions in the sample can be “paired” and separated as the ion pair in ion pair chromatography, whereas in ion exchange chromatography, ions in the sample can be separated as cations and anions separately. This is the primary distinction between ion pair chromatography and ion exchange chromatography. Ion pair chromatography is more commonly used.

Ion Exchange Chromatography: Schematic Diagram

Chromatography is an important technology that may separate various components that are present in a mixture. We can use analytical techniques such as ion pair and ion exchange chromatography to separate ions and polar molecules in a mixture depending on the electrical charge that they carry with them. These techniques are called ion pair chromatography and ion exchange chromatography, respectively.

What is an ion exchange column?

Chromatography columns that use ion exchange can separate and identify substances based on the ionic or electrostatic interactions between those components. The purification of nucleic acids, proteins, and peptides, as well as other biomolecules, is a common application for these columns. Whereas cation exchange columns will trade positively charged molecules for negatively charged ones, anion exchange columns will trade negatively charged molecules for positively charged ones. The effects of buffer pH are what differentiate weak ion exchangers from strong ion exchangers among chromatography resins; this differentiation is made based on the strength of the ion exchange.

Workflow of ion exchange chromatography (How it works?)

The separation of charged biomolecules is achieved by the use of ion exchange chromatography. The liquid phase is comprised of the crude sample, which is known to include charged molecules. Molecules will attach themselves to sites in the stationary phase that have opposing charges as they travel through the chromatographic column.

Eluting the molecules that were separated based on their charge requires the utilisation of a solution that varies in its ionic strength. When a solution of this kind is put through the column, a highly selective separation of molecules can take place based on the differences in the charges they carry.

Key steps in the ion exchange chromatography procedure are listed below

  1. At a specific pH, a sample of impure protein is injected into the ion exchange chromatography column.
  2. Positively charged proteins will bind to the negatively charged functional groups found in the resin if they are of the same type.
  3. The separated proteins are eluted with the use of a salt gradient. At low salt concentrations, proteins that have few charged groups are eluted, but at higher salt concentrations, proteins that have multiple charged groups are eluted.
  4. Washing the ion exchange column removes unwanted proteins and contaminants.

A pH gradient can also be applied to elute individual proteins on the basis of their isoelectric point (pI) i.e. the point at which the amino acids in a protein carry neutral charge and hence do not migrate in an electric field.

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