Chromatography is one of the most important techniques in the study of biological molecules. Thanks to chromatographic separation techniques, the unique properties of proteins, amino acids, and other molecules can be isolated, analyzed, and used to create new scientific developments.
What is ion exchange chromatography?
Ion exchange high-performance liquid chromatography is a scientific analysis technique that separates molecules based on their charge. This chromatographic technique is effective for the analysis of ionizable groups.
In ion exchange chromatography, the interaction between a sample’s charged molecules and an oppositely charged stationary phase of the chromatography matrix is observed and exploited. The charge can be easily manipulated by the pH of the buffer being used, giving this technique a functional simplicity not found in similar analytical methods.
Likewise, ion exchange chromatography is different from other chromatographic separation techniques because there is only one interaction involved. This gives ion exchange chromatography the potential to have a higher matrix tolerance. Another notable advantage of this chromatographic method is the predictability of its elution patterns (source).
There are two distinct types of ion exchange chromatography:
- Anion-exchange chromatography
- Cation-exchange chromatography
The purpose of anion-exchange chromatography is to study negatively-charged particles. This is done by using a positively-charged stationary phase. Once the stationary phase is at the right polarity, negatively-charged particles are loaded. Anion-exchange chromatography is commonly used in protein purification, water analysis, and quality control.
In contrast to anion-exchange chromatography, cation-exchange chromatography is used when the molecule being studied is positively charged. To achieve cation-exchange chromatography, a negatively-charged stationary phase is used to attract positively charged molecules.
What is the difference between ion chromatography and ion exchange chromatography?
There is no difference between ion chromatography and ion exchange chromatography. Both terms can be used to refer to the same procedure.
What is the importance of ion exchange chromatography?
Purified molecules are vital to the development of scientific research. Protein purification, for instance, plays a crucial role in the development of assay reagents, therapeutic candidates, and other biochemical studies.
Principles of ion exchange chromatography
Ion-exchange chromatography can be used to separate proteins or any other kind of biomolecule. The way this is achieved is through the analysis of the differences in the net charge of a molecule at a particular pH.
The charge of a protein is determined by the number and type of ionizable amino acid side chain groups. Every protein has a point where the overall number of its negative and positive charges equals zero. This is known as the isoelectric point (pI).
If a buffered solution below a protein’s isoelectric point is being observed, then the protein will be protonated, or positively charged. This will cause it to bind to negatively charged functional groups of a cation exchange resin. On the other hand, a buffered solution above a protein’s isoelectric point will display a deprotonated, or negatively charged, molecule.
Ion exchange resins
An ion-exchange resin attracts biomolecules of the opposite charge. Negatively charged resins are known as cation exchange resins, while positively charged resins are known as anion exchange resins.
The stability of proteins is only found within a narrow pH range. While a protein should be able to bind to either cation or anion exchange resin, in reality, the interactions with proteins must be handled with a resin corresponding to the stability of the protein at a given pH.
Resins are categorized as strong and weak ion exchangers. The words “strong” and “weak” in this context do not relate to the strength of the ion binding but the variations in pH caused by the ionization state of the functional groups:
- Weak exchange resins: Weak exchangers are only ionized over a limited pH range. Weak exchange resins can react to changes in buffer pH by losing or gaining protons. This variation creates different parameters upon which binding and elution can be observed.
- Strong exchange resins: The ion exchange capacity of strong exchangers remains constant when the pH changes. This means that strong exchanges stay fully charged over a large pH range. Naturally, this makes the optimization of a workflow using strong exchange resins simpler than when using their weaker counterparts.
Another pivotal aspect of ion exchange resins is their physical properties. The maximum operating pressure, flow rate, and resolution of the separation will be affected by the size, material, and porosity of the resin beads.
Larger resin beads allow for fast flow rates. In addition, the resolution they provide is appropriate for the early and intermediate stages of protein purification. On the other hand, smaller resin beads offer an improved resolution and can be used to achieve the best purity during the later stages of purification.
The isoelectric point
The isoelectric point of a molecule is measured by the charge of the amino acids in the protein chain. The in-silico determination of the isoelectric point of a molecule should be the first step in ion-exchange chromatography.
The charge of a protein at a particular pH must be iteratively computed until a net protein charge of zero is found. The preferred method to calculate this is the Henderson-Hasselback equation.
How does ion exchange chromatography work?
A successful ion exchange chromatographic separation is achieved by binding the target molecules to a chromatography column by using their electric charge. This makes ion exchange chromatography rather simple compared to other separation methods.
Step 1: The preparation of the sample
The charge of proteins, amino acids, and nucleic acids is dependent on the pH of the solution they are found in. Because of this, a solution that buffers the mixture at a specific pH must be used to contain the sample before further work can be done. The solvent or mixture of solvents used to carry a sample is known as the mobile phase.
Step 2: Ion exchange chromatography columns
An ion exchange chromatography column is used to separate biological molecules taking advantage of their ionic or electrostatic interactions. The charge of the molecule being targeted for separation determines the functional group that will be used. Molecules with a positive charge are extracted using a column with a negative charge, and vice versa.
Step 3: The ion exchange separation process
Once the sample and the column are ready, the former must be slowly poured into the latter. The fluid will drain down the gel matrix, causing the ions to bind to the functional groups in the column. Meanwhile, the rest of the solution goes through the column and can be collected as it exits through its bottom.
Step 4: Collecting the sample
Once the molecules are bound to the column, they must be collected. To do this, they must be freed from their respective functional groups. The process used to achieve this is known as elution:
- Forcing the charge of the molecules in the sample to change. This can be done by running a buffer of a different pH through the same column. The shape of the bound molecules will be affected by this, changing which parts of them are exposed and altering their overall charge in the process. A high salt concentration can be used to elute molecules with stronger ionic interactions.
- Using another substance to displace the bound molecules. The displacement of molecules already bound to an ion exchange chromatography column can be accomplished by running ions that are more attracted to the functional groups in the gel matrix through the column.
Applications of ion exchange chromatography
The vast applicability, high capacity, high resolution, and simplicity of ion exchange chromatography make it ideal for the separation of molecules that present a charge. This makes the technique useful in the field of biotechnology, as well as in food and clinical research.
In a lab setting, ion exchange chromatography is useful for both analytical and preparative purposes. Nonetheless, it is mostly used for analysis of biotechnological products to determine charged heterogeneity of protein products. For gene therapeutic products, it has been used to determine topoisomeric variants of supercoiled DNA. In another example, ion exchange chromatography is routinely used to analyze amino acid mixtures. A less common, but very important, application of ion exchange chromatography is the analysis of the products of the hydrolysis of nucleic acids.
Even though this type of chromatography is mostly reserved for biological molecules, inorganic outliers exist. Some rare earth elements, for example, can be separated on columns of cation-exchange resin. Examples of this phenomenon occur in Lanthanoids. In turn, anion exchange in hydrochloric acid is a functional method to separate metal ions (source).
More examples of the applications of ion exchange chromatography include:
- The analysis of drinking water for pollution and other potential hazards.
- The analysis of water in aquatic ecosystems to determine water chemistry.
- The observation and analysis of sugar and salt content in processed foods.
- This type of analysis is common in nutrition research. The effects of individual food components on health are investigated by separating proteins from food.
- The separation of highly valuable materials from other compounds. In hydrometallurgy, for instance, ion exchange allows valuable metals like gold, silver, and copper to be recovered from wastewater.
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