HOW DOES ION EXCHANGE CHROMATOGRAPHY WORK?

Chromatography is a technique of separating a mixture of different compounds, such that the respective components of the mix, based on their relative interaction, do so with the inert matrices.

IEC (Ion Exchange Chromatography) is a technique used frequently to separate and determine the polar molecules and ions, depending on their affinity towards the ion exchanger. In this process, the stationary phase is a resin, while the mobile phase is an eluent.

Ion Exchange Chromatography is an integral part of ion chromatography, ion exclusion chromatography, and ion interaction/ partition chromatography. The ion-exchange chromatography (IEC) process is applicable for many types of charged molecules, for instance, amino acids, large protein, and small nucleotides.

Principle of Working:

  • Ion exchange chromatographydepends on the attraction between stationary phases that have opposite polarities. The stationary phases are known as the ion exchanger and analyte, respectively.
  • The stationary layer ion exchanger has charged groups that are linked covalently to an insoluble matrix surface.
  • The matrix has charged groups, and these groups can either be positively charged or negatively charged.
  • When the matrix is suspended in an aqueous solution, the charged groups present on the matrix surface will be covered by ions of opposite polarity.
  • These surrounding ion particles form a ‘cloud’; hence it is termed an ion cloud. In the ion cloud surrounding the charged group, the ions can be exchanged reversibly without tampering with the properties of the matrix.

Instrument Setup of IEC:

The basic IEC setup will have a pump, suppressor, injector, column, detector, and a recorder.

  • Pump: The pump is one of the most critical components in the instrument setup, as it helps provide a continuous flow of the mobile phase (the eluent) through the column, detector, and IC injector.
  • Injector:Samples can be introduced in the system with the help of an injector, as it is one of the easiest ways to do so. The liquid samples are injected directly into the system, whereas the solid samples need to be dissolved in a solvent that is appropriate and then injected into it.
  • Column:Columns used in the instrument vastly depend on the area of application and its use. They can vary from glass, titanium, or even stainless steel. The column diameter can also be a variable, it could either be 2mm – 5cm, and the length of the column could also vary from 3cm – 50cm.
  • Suppressors: The background conductivity of any chemical used in the process is reduced using a suppressor. These chemicals used could either be for eluting the samples from the ion exchanging column. It will ensure that there will be a significant improvement in the conductivity measurement of the ions.
  • Detectors:The detectors are used to detect and measure the ions, and the data is then sent into a data system which will help assess the ions further. The detector used is primarily electrical conductivity detectors.

Working Procedure:

  • The first step is to introduce an impure mixture sample into the setup. The loading of the impure sample is done so in the chromatography column, at a particular specified pH value.
  • When the process begins, the charged proteins will start to travel towards the charged group in the mixture and will react and bind to the resin.
  • After the reaction is complete, to elute the separated proteins, a salt gradient is introduced.
  • At lower salt concentrations, the proteins with a significantly lower charge value are eluted, whereas, at a higher salt concentration, proteins with several charged particles intact are eluted.
  • The residue of unwanted protein and salts and other impurities are removed by simply washing the columns.
  • pH gradient can be applied; such elution can be done for the proteins based on their pH(l) (isoelectric point). An isoelectric point is when the amino acids present in the protein carry a neutral charge and hence do not migrate towards any electric field.

Application of IEC:

  • One of the main applications of IEC is to analyze the amino acid mixture routinely.
  • In clinical diagnosis, IEC plays a vital role, as the 20 principal amino acids present in blood serum or even from the hydrolysis process of proteins are separated.
  • This chromatography process is found to be the best for the purification of water. The total deionization of water, also known as the non-electrolyte solution, is done by exchanging solute anion with hydroxyl ion, and the solute cations are exchanged with hydrogen ion.
  • One interesting IEC application is to analyze the lunar rocks and the rare trace elements on planet Earth.
  • Traces of metals from seawater are collected by using chelating resins.
  • Products made from nucleic acid are analyzed using this process. The information about the molecular structure is gained, but biological functions and other hereditary information are also found.

 

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