Ion chromatography (also known as ion-exchange chromatography) is a technique for separating ions and polar molecules based on their affinity for an ion exchanger. It acts on large proteins, small nucleotides, and amino acids, among other charged molecules. Ion chromatography, on the other hand, must be performed in conditions one unit away from a protein's isoelectric point.
This article will study ion exchange chromatography principle and application, ion exchange protein purification, and anion exchange chromatography protein purification in detail.
Use of Ion Exchange Chromatography
Anion exchange and cation exchange chromatography are the two methods of ion chromatography. When the molecule of interest is positively charged, cation-exchange chromatography is used. Since the pH for chromatography is less than the pI, the molecule is positively charged. The stationary phase is negatively charged in this form of chromatography, and positively charged molecules are loaded to be attracted to it. Anion-exchange chromatography is when the stationary phase is positively charged and negatively charged molecules are loaded to be drawn to it (meaning the pH for chromatography is greater than the pI).
Exchanger In Ion Exchange Chromatography
Cation exchangers (which exchange positively charged ions) and anion exchangers (which exchange negatively charged ions) are the two types of ion exchangers (anions).
Amphoteric exchangers will swap cations and anions at the same time. The simultaneous exchange of cations and anions, on the other hand, is often done in mixed beds ion exchangers, which contain a mix of anion- and cation-exchange resins, or by passing the solution through several different ion-exchange materials.
Examples of Cation Exchanger in Ion-Exchange Chromatography
Ion exchange resins (functionalized porous or gel polymers), zeolites, montmorillonite, clay, and soil humus are examples of common ion exchangers.
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Ion Exchange Chromatography Principle and Application
Ion-exchange chromatography divides molecules into groups based on their charged groups. Ion-exchange chromatography uses coulombic (ionic) interactions to keep analyte molecules on the column. Positively and negatively charged ions make up the ion exchange chromatography matrix. On the stationary phase matrix, molecules interact electrostatically with oppositely charged molecules.
An immobile matrix containing charged ionizable functional groups or ligands makes up the stationary phase. Ionic functional groups (R-X) on the stationary phase surface interact with analyte ions of opposite charge. These inert charges couple with exchangeable counterions in the solution to achieve electroneutrality. Purifiable ionizable molecules contend with these exchangeable counterions for binding to the stationary phase's immobilised charges.
The charge of these ionizable molecules determines whether they are retained or eluted. The molecules that do not bind to the stationary phase or bind only weakly wash away first. The molecules that bind to the stationary phase must be eluted under different conditions. The pH can be modified or the concentration of exchangeable counterions, which compete with the molecules for binding, can be increased. A change in pH influences the charge on individual molecules, causing binding to change.
The molecules then begin to elute out as their charges shift as a result of the adjustments. Such tweaks may be used to unlock the protein of interest in the future. To differentiate ionised molecules, the concentration of counterions can be steadily increased. Gradient elution is the name for this form of elution.
Since the stationary phase has a negatively charged functional group, cation exchange chromatography preserves positively charged cations.
Using a positively charged functional group, anion exchange chromatography preserves anions.
Method of Ion Exchange Chromatography Protein Purification
Since proteins contain charged functional groups, ion exchange chromatography may be used to separate them. On a charged solid help, the ions of interest (in this case, charged proteins) are exchanged for another ion (usually H+). The solutes are usually found in a liquid form, which is usually water. Take proteins in water, for example, which is a liquid process that is passed through a column. Since it is filled with porous synthetic particles with a specific charge, the column is generally referred to as the solid phase. These porous particles, which are also known as beads, maybe animated (containing amino groups) or charged with metal ions.
These porous particles, which are also known as beads, maybe animated (containing amino groups) or charged with metal ions. Porous polymers can be used to make the column; the optimum size of the porous particle for macromolecules over 100,000 is around 1 m2. This is due to the fact that the slow diffusion of solutes inside the pores has no effect on separation efficiency. Anion exchange resins are beads with positively charged groups that attract negatively charged proteins.
For proteins that do not have a charge at pH 7, using buffers instead of water is a good idea because it allows you to manipulate the pH to change the ionic interactions between the proteins and the beads. If the pH is high enough or low enough, weakly acidic or basic side chains may have a charge. Separation can be accomplished using the protein's natural isoelectric point.
Ion-exchange chromatography can also be used to isolate individual multimeric protein assemblies, allowing for the purification of complexes based on the number and location of charged peptide tags.
Applications of Ion Exchange
Here are Some Applications of Ion Exchange Given:
The food and beverage industry, hydrometallurgy, metals finishing, chemical, petrochemical, pharmaceutical technology, sugar and sweetener manufacturing, ground- and potable-water treatment, nuclear, softening, industrial water treatment, semiconductor, steam, and many other industries all use ion exchange.
Preparation of high-purity water for the power engineering, electronic, and nuclear industries is a common example of application; polymeric or inorganic insoluble ion exchangers are commonly used for water softening, purification, and decontamination, among other things.
Ion exchange is a technique for producing soft water in household filters, which is beneficial to laundry detergents, soaps, and water heaters. Exchanging divalent cations (e.g., calcium Ca2+ and magnesium Mg2+) with highly soluble monovalent cations (e.g., Na+ or H+) does this (see water softening). The removal of nitrate and natural organic matter is another application for ion exchange in domestic water treatment.
Application of Ion Exchange Chromatography
Here is Some Application of Ion Exchange Chromatography Given:
Ion-exchange chromatography for industrial and analytical applications is another field worth mentioning. Ion-exchange chromatography is a chromatographic tool for chemical analysis and ion separation that is commonly used. It is commonly used in biochemistry to isolate charged molecules such as proteins, for example. Extraction and purification of biologically derived substances such as proteins (amino acids) and DNA/RNA are essential aspects of the application.
Water treatment is a major industrial application for ion exchange. Hard water is softened by exchanging calcium and magnesium ions with sodium ions. Hard water is caused by the presence of calcium and magnesium ions, which form insoluble precipitates with soaps. To do so, the hard water is passed through a sodium-ion-containing cation exchanger column. Calcium and magnesium begin to appear in the water leaving the column after it has been in use for a while.
The column must then be regenerated by slowly passing a concentrated solution of common salt through it; the excess sodium ions displace the ions that cause stiffness, and the bed of the exchanger is ready to use again after flushing with water. Natural aluminosilicates were initially used as heat exchangers, but synthetic resins were eventually substituted.
Cation exchange separates rare earth elements on an industrial scale using a displacement technique in which each element displaces elements bound less strongly as it progresses down the column. The elements appear in high purity one after the other (the one with the weakest bond first).
Catalysts can be used with ion exchangers. Certain chemical reactions in the liquid phase, such as hydrolysis and esterification, are catalysed by strong-acid cation-exchange resins filled with hydrogen ions (ester formation).
Did You Know?
Membrane exchange chromatography is a form of ion exchange chromatography that was developed to overcome the limitations of using columns packed with beads. Unlike other chromatography devices that need maintenance and time to revalidate, Membrane Chromatographic devices are inexpensive to mass-produce and disposable. When it comes to separating liquids, there are three types of membrane absorbers. Flat sheet, hollow fibre, and radial flow are the three varieties. Multiple flat sheets are the most popular absorber and are better suited for membrane chromatography since they have more absorbent volume.
It is particularly useful for isolating and purifying viruses, plasmid DNA, and other large macromolecules because it can resolve mass transfer limitations and pressure drop. The column is made up of microporous membranes with internal pores that contain adsorptive moieties capable of binding the target protein. Adsorptive membranes come in a variety of geometries and chemistry, allowing them to be used for purification, fractionation, concentration, and clarification with ten times the efficiency of beads.