Introduction to Ion Exchange Chromatography

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Introduction to Ion Exchange Chromatography

For decades ion exchange chromatography has been used across numerous disciplines - from water purification and softening to food and clinical research to fermentation. In this Purolite How to Series, we'll explore the basics of ion exchange chromatography for the purification of monoclonal antibodies (mAbs) and recombinant proteins.
 
The purification procedure of monoclonal antibodies can be broken down into three stages: capture, intermediate purification, and polishing. Ion exchange chromatography is the separation and purification of molecules based on charge; specifically, the separation of molecules that are similar in character to the target molecule identified for purification and thus difficult to separate using other chromatography techniques.
 
Ion exchange resins are concentrated, highly selective, and have a high capacity. While ion exchange is one of many techniques used to separate and purify proteins and biomolecules, it can be used in all three stages of purification.

 

Ion Exchange Types & Structure

To perform ion exchange chromatography, a resin is coupled with a positively or negatively charged molecule which interacts with the target molecule selected for purification.
 
The resin attached to the ligand should possess three key characteristics. The resin should be:
 

1. Macro porous to flow efficiently through the column.
2. Hydrophilic or neutral to prevent non-specific protein interactions.
3. Chemically and physically stable to withstand multiple rounds of the purification procedure.

 
Due to the unique properties required for chromatography resins, Purolite resins for both affinity and ion exchange chromatography purposes are manufactured from Agarose.

 

Cation vs Anion Exchange Chromatography 

Two types of ion exchange chromatography are applied during the purification procedure: anion exchange chromatography and cation exchange chromatography.
 
Cation exchange resins, such as Purolite&#;s SP product line, carry a negatively charged ligand and bind to positively charged molecules. In contrast, anion exchange resins, such as Purolite's Q product line, carry a positively charged ligand, thus binding to negatively charged molecules.

 

The Basic Steps of Ion Exchange Chromatography

Five key steps are required during ion exchange, as outlined below:
 
1. Equilibration
 
Begin by equilibrating the column with a starter buffer. Typically, equilibration requires 5-10 column volumes of buffer to reach a baseline. The purpose of this step is to prepare the column to bind to the sample, so the buffer selected should possess all the conditions that will encourage the sample to bind to the column.
 
2. Column Loading
 
Load the sample in the column, ensuring it has the same conditions as the binding buffer selected so it will bind to the column too.
 
3. Washing
 
Wash the column to return to baseline.
 
4. Gradient Elution
 
Gradually increase the salt concentration within the column. As salt concentration increases, proteins with opposing charges will elute. Gradient elution, the process of slowly increasing salt concentration, helps to ensure that proteins with the highest interaction with the column will elute last.
 
5. Wash & Re-Equilibration
 
Complete the process by washing the column once again with a high salt solution to remove any remaining molecules bound to the column and begin re-equilibration to perform another purification.

 

For decades ion exchange chromatography has been used across numerous disciplines - from water purification and softening to food and clinical research to fermentation. In this Purolite How to Series, we'll explore the basics of ion exchange chromatography for the purification of monoclonal antibodies (mAbs) and recombinant proteins.The purification procedure of monoclonal antibodies can be broken down into three stages: capture, intermediate purification, and polishing. Ion exchange chromatography is the separation and purification of molecules based on charge; specifically, the separation of molecules that are similar in character to the target molecule identified for purification and thus difficult to separate using other chromatography techniques.Ion exchange resins are concentrated, highly selective, and have a high capacity. While ion exchange is one of many techniques used to separate and purify proteins and biomolecules, it can be used in all three stages of purification.To perform ion exchange chromatography, a resin is coupled with a positively or negatively charged molecule which interacts with the target molecule selected for purification.The resin attached to the ligand should possess three key characteristics. The resin should be:Due to the unique properties required for chromatography resins, Purolite resins for both affinity and ion exchange chromatography purposes are manufactured from Agarose.Two types of ion exchange chromatography are applied during the purification procedure: anion exchange chromatography and cation exchange chromatography.Cation exchange resins, such as Purolite&#;s SP product line, carry a negatively charged ligand and bind to positively charged molecules. In contrast, anion exchange resins, such as Purolite's Q product line, carry a positively charged ligand, thus binding to negatively charged molecules.Five key steps are required during ion exchange, as outlined below:1. EquilibrationBegin by equilibrating the column with a starter buffer. Typically, equilibration requires 5-10 column volumes of buffer to reach a baseline. The purpose of this step is to prepare the column to bind to the sample, so the buffer selected should possess all the conditions that will encourage the sample to bind to the column.2. Column LoadingLoad the sample in the column, ensuring it has the same conditions as the binding buffer selected so it will bind to the column too.3. WashingWash the column to return to baseline.4. Gradient ElutionGradually increase the salt concentration within the column. As salt concentration increases, proteins with opposing charges will elute. Gradient elution, the process of slowly increasing salt concentration, helps to ensure that proteins with the highest interaction with the column will elute last.5. Wash & Re-EquilibrationComplete the process by washing the column once again with a high salt solution to remove any remaining molecules bound to the column and begin re-equilibration to perform another purification.

Chromatography is the separation of a mixture of compounds into their individual components based on their relative interactions with an inert matrix.

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What is Ion Exchange Chromatography?

Ion exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their affinity to ion exchangers.

The principle of separation is thus by reversible exchange of ions between the target ions present in the sample solution to the ions present on ion exchangers.

In this process, two types of exchangers i.e., cationic and anionic exchangers can be used.

1. Cationic exchangers possess negatively charged group, and these will attract positively charged cations. These exchangers are also called &#;Acidic ion exchange&#; materials, because their negative charges result from the ionization of acidic group.

2. Anionic exchangers have positively charged groups that will attract negatively charged anions. These are also called &#;Basic ion exchange&#; materials.

• Ion exchange chromatography is most often performed in the form of column chromatography. However, there are also thin-layer chromatographic methods that work basically based on the principle of ion exchange.

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Working Principle of ion exchange chromatography

This form of chromatography relies on the attraction between oppositely charged stationary phase, known as an ion exchanger, and analyte.

• The ion exchangers basically contain charged groups covalently linked to the surface of an insoluble matrix.

• The charged groups of the matrix can be positively or negatively charged.

• When suspended in an aqueous solution, the charged groups of the matrix will be surrounded by ions of the opposite charge.

• In this &#;ion cloud&#;, ions can be reversibly exchanged without changing the nature and the properties of the matrix.

Instrumentation of ion exchange chromatography

Typical IC instrumentation includes: pump, injector, column, suppressor, detector and recorder or data system.

1. Pump

The IC pump is considered to be one of the most important components in the system which has to provide a continuous constant flow of the eluent through the IC injector, column, and detector.

2. Injector

Sample introduction can be accomplished in various ways. The simplest method is to use an injection valve. Liquid samples may be injected directly and solid samples need only to be dissolved in an appropriate solvent. Injectors should provide the possibility of injecting the liquid sample within the range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the psi).

3. Columns

Depending on its ultimate use and area of application, the column material may be stainless steel, titanium, glass or an inert plastic such as PEEK. The column can vary in diameter from about 2mm to 5 cm and in length from 3 cm to 50 cm depending on whether it is to be used for normal analytical purposes, microanalysis, high speed analyses or preparative work.

Guard column is placed anterior to the separating column. This serves as a protective factor that prolongs the life and usefulness of the separation column. They are dependable columns designed to filter or remove particles that clog the separation column

4. Suppressor

The suppressor reduces the background conductivity of the chemicals used to elute samples from the ion-exchange column which improves the conductivity measurement of the ions being tested. IC suppressors are membrane-based devices which are designed to convert the ionic eluent to water as a means of enhancing the sensitivity.

5. Detectors

Electrical conductivity detector is commonly use.

6. Data system

In routine analysis, where no automation is needed, a pre-programmed computing integrator may be sufficient. For higher control levels, a more intelligent device is necessary, such as a data station or minicomputer.

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Procedure of ion exchange chromatography

• Ion exchange separations are carried out mainly in columns packed with an ion-exchanger.

• These ionic exchangers are commercially available. They are made up of styrene and divinyl benzene. Example. DEAE-cellulose is an anionic exchanger, CM-cellulose is a cationic exchanger.

• The choice of the exchanger depends upon the charge of particle to be separated. To separate anions &#;Anionic exchanger&#; is used, to separate cations &#;Cationic exchanger&#; is used.

• First the column is filled with ion exchanger then the sample is applied followed by the buffer. The tris-buffer, pyridine buffer, acetate buffer, citrate and phosphate buffers are widely used.

• The particles which have high affinity for ion exchanger will come down the column along with buffers.

• In next step using corresponding buffer separates the tightly bound particles.

• Then these particles are analyzed spectroscopically.

Applications of ion exchange chromatography

• An important use of ion-exchange chromatography is in the routine analysis of amino acid mixtures.

• The 20 principal amino acids from blood serum or from the hydrolysis of proteins are separated and used in clinical diagnosis.

• This is most effective method for water purification. Complete deionization of water (or) a non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and solute anions for hydroxyl ions. This is usually achieved by method is used for softening of drinking water.

• In the analysis of products of hydrolysis of nucleic acids. In this way, information is gained about the structure of these molecules and how it relates to their biological function as carriers of hereditary information. 

• Chelating resins are used to collect trace metals from seawater.

• To analyze lunar rocks and rare trace elements on Earth.

Advantages of ion exchange chromatography

1. It is one of the most efficient methods for the separation of charged particles.

2. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.

3. Ion exchange is used for both analytical and preparative purposes in the laboratory, the analytical uses being the more common.

4. Inorganic ions also can be separated by ion-exchange chromatograph.y

Limitations of ion exchange chromatography

• Only charged molecules can be separated.

• Buffer Requirement

References

1. Wilson, K., Walker, J. (). Principles and Techniques of Biochemistry and Molecular Biology (8 eds.). Cambridge University Press: New York.

2. https://www.biochemden.com/ion-exchange-chromatography/

3. https://www.britannica.com/science/ion-exchange-reaction/Applications-of-ion-exchange

4. http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch06s02.html

5. http://cdn.intechopen.com/pdfs//InTech-Ion_exchange_chromatography_an_overview.pdf

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