Anion Exchange Chromatography: Principle, Procedure and Application

Anion exchange chromatography, a specialized technique within ion exchange chromatography (IEX), is employed for the separation of molecules based on their net surface charge. In this method, a positively charged ion exchange resin is utilized, showcasing an inherent attraction to molecules possessing a net negative surface charge. This chromatographic approach finds versatile applications in both preparative and analytical contexts, facilitating the separation of a diverse range of molecules – from small entities like amino acids and nucleotides to larger and more complex proteins.

What is Anion Exchange Chromatography?

An effective method for purifying a variety of negatively charged compounds is anion exchange chromatography, which is utilized in biochemistry and biotechnology. The technique of ion-exchange chromatography uses an ion-exchange resin with positively charged groups, like diethyl-aminoethyl groups (DEAE), to separate compounds according to their charges.

• Initially, positively charged counter-ions (cations) cover the resin in a solution. The negatively charged molecules in the sample interact with the positively charged groups on the resin when the sample containing the target compounds is applied to the resin.
• The negatively charged molecules bond to the resin and the counter-ions are displaced as a result of this interaction.
• The strength of the negative charge on the molecules controls the binding between the negatively charged molecules and the resin.
• Substances possessing a greater negative charge will exhibit stronger binding to the resin, whereas those with a lower negative charge will experience a less robust interaction, potentially resulting in easier elution. The manipulation of the system’s pH allows for the modulation of the negative charge strength on the target substances, consequently impacting their affinity and binding strength to the resin.
• Under some circumstances, such as at higher pH levels, proteins can have a net negative charge. By leveraging this negative charge, anion exchange chromatography allows for the selective separation of distinct proteins present in a mixture.
• Protein preparative anion exchange chromatography is usually carried out in a column configuration. The positively charged ion exchange resin-filled column is filled with the sample containing the protein combination.
• The proteins in the sample, carrying a negative charge, attach to the resin, with unbound molecules being rinsed away. Subsequently, the adhered proteins can be specifically released from the resin by altering the pH or ionic strength of the elution buffer. This elution procedure facilitates the isolation and gathering of individual proteins, guided by their distinct charge characteristics.
• There are several uses for anion exchange chromatography in the purification of different biomolecules, such as proteins, amino acids, sugars carbohydrates, and other acidic compounds. When it comes to purifying and separating proteins according to their net surface charge, it is especially helpful.
• Anion exchange chromatography is used not only for preparative but also for analytical applications. Tracking the elution profiles of proteins and utilizing analytical methods like mass spectrometry or spectrophotometry to examine the eluted fractions, makes protein characterization and quantification possible.
• Anion exchange chromatography is crucial in the realms of biochemistry and biotechnology, offering a means to separate and purify substances characterized by negative charges. Its adaptability and efficiency render it a fundamental method for scientists engaged in diverse fields such as protein purification, pharmaceutical research, and biochemical analysis

Anion Exchange Chromatography Principle

The protein’s isoelectric point, or pI, determines how a protein’s net surface charge varies with pH. A protein has no net charge when its pH is equal to its partial charge. The protein has a net positive charge at pH values lower than the pI. The buffer has a net negative charge if the pH is increased above the pI of the protein.

A buffer that ensures a known net charge for a protein of interest can then be selected because a protein’s pI can be estimated based on its primary amino acid sequence. Thus, when the protein of interest has a net negative charge at the operating pH, a positively charged anion exchange resin is selected. Different proteins will bind to the resin with varying strengths, facilitating their separation, because they will have different affinities for the positively charged surface groups on the particles of the anion exchange media at a given pH due to their differing degrees of charge.

The resin will bind to all suitably charged proteins at a specific pH of the loading buffer. For instance, any proteins with a pI less than 7.5 will generally carry a net negative charge and bind the positively charged anion exchange resin when the resin is utilized at a pH of 7.5. The protein of interest is then separated from other bound proteins using a salt gradient; the order in which proteins elute is determined by their net surface charge. Proteins eluting at a high salt concentration will elute at a lower ionic strength, while those with pI values closer to 7.5 will do so.

Anion Exchange Chromatography Procedure

The procedure below is a generalized ion exchange chromatography protocol and specific running conditions for anion exchange chromatography can be changed to best suit your protein of interest, the buffer system, and the anion exchange resin chosen. This is because all ion exchange chromatography is dependent on electrostatic interactions between the resin functional groups and proteins of interest. Buffer pH must be precisely titrated, and the right counterions must be utilized, as these factors have a significant impact on protein binding to the column resin.

• Buffer preparation
  • Ionic strength and buffer pH are essential for all types of ion exchange chromatography.
  • After altering the salt concentration, it is best to correct the pH of the buffer and make sure the buffer counterions are compatible.
  • Buffer counterions should have the same charge as the resin; for positively charged anion exchange resins, Tris buffers are an ideal choice.
• Column Equilibration
  • Once the pH and conductivity values settle, connect the column to the chromatography system and equilibrate it with the buffer.
  • This usually necessitates sending the buffer through the column at least five times.
• Sample Loading
  • Load the protein sample into the beginning buffer whenever it is feasible.
  • The ionic strength and pH have an impact on the protein’s ability to bind to the anion exchange resin, so putting the sample in the right buffer increases the binding efficiency.
• Column washing
  • Wash the column with the loading buffer (0% Buffer B) until no protein is identified in the flowthrough.
  • Three to five column volumes of the loading buffer are usually enough to wash the column.
• Elution
  • Protein can be extracted using a step isocratic elution method or a linear gradient elution method.
  • To improve elution conditions, a gradient elution is frequently employed.
  • A step elution can be utilized to expedite the purification process once the protein of interest’s elution profile has been determined and the ionic strength or pH at which a protein elutes is known.
  • For ion exchange elution, pH gradients are often ineffective because it is very difficult to produce consistent linear pH gradients without changing the ionic strength. Step gradients might yield findings that are more consistent when pH is utilized for elution.
• Column Stripping and Equilibration
  • Proteins that are still attached to the column resin are eluted by raising the ionic strength or changing the pH of the elution buffer after the target protein has been eluted.
  • Once every last bit of protein has been extracted from the resin, equilibrate the column using a buffer with a low ionic strength.
  • If the column is going to be used soon, then the starting buffer for purification would be a wise decision.
  • To stop microbiological growth during long-term storage, it is usual practice to replace the column buffer with 20% ethanol in water.

Advantages of Anion Exchange Chromatography

• Applicable to a broad spectrum of molecules like proteins, nucleic acids, and other negatively charged compounds, anion exchange chromatography provides flexibility in the separation and purification of diverse analytes.
• Because anion exchange chromatography is easily scalable, it can be used for both large-scale industrial production and laboratory study. Effective purification at diverse scales is possible thanks to the technique’s flexibility in accommodating varying sample quantities and flow rates.
• High-resolution separation is made possible by anion exchange chromatography, which makes it possible to purify complicated mixtures containing several constituents. It makes it possible to isolate target molecules with exceptional selectivity and purity.
• As the anion exchange resin used in chromatography columns can be recycled and regenerated several times, it is more economical and produces less waste.
• It is possible to conduct anion exchange chromatography under mild environments, such as room temperature and almost neutral pH. This helps maintain the structure and activity of sensitive molecules and makes them compatible with them.

Disadvantages of Anion Exchange Chromatography

• Selectivity issues may arise when attempting to separate compounds that are closely related and have similar net negative charges using ion exchange chromatography. In these situations, selecting the right resin and fine-tuning the buffer conditions are essential to attaining the best possible separation.
• The greatest binding capacity for target molecules may be restricted by the resin’s ion exchange capacity. To process high sample quantities or concentrations, this may need the use of bigger columns or numerous purification stages.
• Anion exchange chromatography’s efficacy is reliant upon the pH range that corresponds to the selected resin. Reduced separation efficiency can result from a decrease in the analyte-resin binding affinity outside of the ideal pH range.
• Anion exchange chromatography’s adsorption and elution conditions occasionally cause fragile proteins to become denatured or lose their biological activity. To reduce protein damage, careful buffer condition and elution strategy optimization is required.
• Excessive levels of salt present in the sample or buffers may impede the process of separation, resulting in decreased binding effectiveness or non-specific interactions with the resin. It could be necessary to perform buffer exchanges or desalting properly either before or during the chromatographic procedure.

Application of Anion Exchange Chromatography

The capacity of anion exchange chromatography to separate and purify compounds depending on their net negative charge makes it useful in a variety of industries. The following are some important uses for anion exchange chromatography:

• Separation of Protein: Proteins are often purified and separated using anion exchange chromatography. Positively charged anion exchange resins are an effective way to separate proteins that have a net negative charge at the operating pH. This method is useful in the manufacture of biopharmaceuticals, protein research, and other fields that require the isolation and purification of particular proteins.
  • Proteins are frequently purified and separated from crude mixes made from blood serum using ion exchange chromatography. A complex mixture of proteins, including enzymes, antibodies, and other macromolecules, can be found in blood serum. Proteins with a net negative charge at the working pH can be selectively bound to the resin by utilizing the right anion exchange resin, while other constituents, such as salts and impurities, are washed away. The bound proteins can then be released from the resin and separated and purified by modifying the buffer conditions and using elution methods. This makes it possible for researchers to separate particular proteins of interest from the blood serum, which makes it easier to do subsequent analyses including enzyme activity tests, protein characterization, and the synthesis of therapeutic proteins.
• Purification of Water: Anion exchange chromatography plays a significant part in water purification procedures. By substituting the hydroxyl ions (OH^–) on the anion exchange resin for the hazardous anions in the water, it can be used to eliminate them. By using this method, pollutants including nitrates, sulfates, and arsenic ions are eliminated from drinking water, ensuring its safety for human consumption.
• Separation of Metal: Metals can be efficiently extracted and recovered from complicated combinations using anion exchange resins. Metals commonly create negatively charged complexes that can bind to the anion exchangers. Certain metals can be attached to the resin selectively and then eluted independently by varying the pH and ionic strength. This process enables the concentration and purification of the metals. This is useful for several areas, such as recycling metal, environmental analysis, and mining.
• Amino Acid Analysis: Analyzing and quantifying amino acids is a beneficial application of ion exchange chromatography. This method makes use of the various affinities that amino acids have for the anion exchange resin to separate and identify individual amino acids within a mixture. It is extensively utilized in nutritional research, metabolic studies, and protein sequencing.
• Nucleic Acid Analysis: Negatively charged nucleic acids, like DNA and RNA, are separated and purified using ion exchange chromatography. It helps isolate particular nucleic acid fragments, which makes it easier to do further investigation using methods like genetic engineering, PCR, and sequencing.
  • Anion exchange chromatography can be applied to extract nucleic acids, such as DNA and RNA, from complicated mixtures acquired following cell death. During this process, other elements in the mixture, such as proteins and cell debris, do not adhere and are washed away, while the negatively charged nucleic acids bond to the positively charged anion exchange resin. The nucleic acids can be purified and separated from the original mixture by selectively eluting them and carefully changing the buffer’s pH and ionic strength. Following purification, this nucleic acid fraction can be examined further for a range of downstream uses, such as genetic engineering, PCR, and sequencing.

In summary, anion exchange chromatography holds wide-ranging applications encompassing protein purification, amino acid analysis, nucleic acid research, water purification, and metal separation. Its versatility, grounded in the capacity to segregate substances based on their negative charge, positions it as a valuable asset in diverse scientific, industrial, and environmental contexts. These instances underscore the efficacy of anion exchange chromatography in isolating nucleic acids from cell lysates and purifying proteins from intricate biological mixtures like blood serum. Leveraging the electrostatic interactions between negatively charged molecules and the positively charged anion exchange resin, anion exchange chromatography emerges as a potent tool in various biochemical and biotechnological applications.

References

• Khan HU. The Role of Ion Exchange Chromatography in Purification and Characterization of Molecules, Chapter 14. Ion Exchange Technologies, Ayben Kilislioğlu. IntechOpen.
• Acikara ÖB. Ion-Exchange Chromatography and Its Applications. Chapter 2. Column Chromatography, Dean F. Martin and Barbara B. Martin. IntechOpen. 2013.
• https://www.bio-rad.com/en-np/applications-technologies/anion-exchange-chromatography?ID=MWHAZ4C4S
• https://microbiologynote.com/anion-exchange-chromatography/