The key difference between affinity and ion exchange chromatography is that we can use affinity chromatography to separate charged or uncharged components in a mixture whereas we can use ion exchange chromatography to separate charged components in a mixture.
Chromatography is a technique that we can use to separate the desired components in a mixture. There are different types such as liquid chromatography, gas chromatography, etc. The affinity chromatography and ion exchange chromatography are two subcategories of liquid chromatography. Also, in these techniques, there are two phases. Namely, they are the stationary phase and mobile phase. The purpose of these techniques is to separate the components, depending on the binding of the components, in the mobile phase on to the surface of the stationary phase.
1. Overview and Key Difference
2. What is Affinity Chromatography
3. What is Ion Exchange Chromatography
4. Side by Side Comparison – Affinity vs Ion Exchange Chromatography in Tabular Form
What is Affinity Chromatography?
Affinity chromatography is a biochemical technique that we use to separate components in a mixture depending on the interactions between these components.
The interactions that we use in this case include the followings:
- Antigen-antibody interactions
- Enzyme-substrate interactions
- Receptor-ligand interactions
- Protein-nucleic acid interactions
In this technique, we use the molecular properties of molecules for this separation technique. Here, we allow the desired compound to interact with a stationary phase via hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interaction, etc. The molecules that do not interact with the stationary phase will elute first. Thus, we can separate it from the mixture. The desired compound will remain attached to the stationary phase. Therefore, we can detach it using an eluting solvent and make it elute to separate it as well.
Affinity chromatography is useful in purification and concentrating a substance from a mixture using a buffer solution. Also, it is helpful in reducing the unwanted substances in a mixture. When considering the apparatus that we use for this process, we should use a column filled with our stationary phase. Then, we should load the mobile phase which contains the biomolecules that we are going to separate. Next, allow them to bind with the stationary phase. Thereafter, using a wash buffer, we can separate the non-target biomolecules, but the target molecules should have a high affinity for the stationary phase in order to success the separation process.
What is Ion Exchange Chromatography?
Ion chromatography is a form of liquid chromatography in which we can analyze ionic substances. Often, we use it to analyze inorganic anions and cations (i.e. chloride and nitrate anions and potassium, sodium cations). Though it is less common, we can analyze organic ions as well. Moreover, we can use this technique for the purification of proteins because proteins are charged molecules at certain pH values. Here, we use a solid stationary phase to which the charged particles can attach. For example, we can use the resin polystyrene-divinylbenzene copolymers as the solid support.
To explain this further, the stationary phase has fixed ions such as sulfate anions or quaternary amine cations. Each of this should associate with a counterion (an ion with opposite charge), if we are to maintain the neutrality of this system. Herein, if the counterion is a cation, then we name the system as a cation exchange resin. But, if the counterion is an anion, the system is an anion exchange resin.
There are five major phases in an ion exchange process;
- Initial stage
- Adsorption of target
- Starting of elution
- End of elution
What is the Difference Between Affinity and Ion Exchange Chromatography?
Affinity chromatography is a biochemical technique that we use to separate components in a mixture depending on the interactions between these components whereas ion chromatography is a form of liquid chromatography in which we can analyze ionic substances. Therefore, the major difference between affinity and ion exchange chromatography is that we can use ion exchange chromatography only for the separation of ionic substances while the affinity chromatography is capable of separating both charged and uncharged particles. When considering the working principle, the difference between affinity and ion exchange chromatography is that the affinity chromatography proceeds due to the fact that target molecules have a high affinity for the stationary phase. However, for ion exchange chromatography, target molecules have an opposite charge to that of the stationary phase surface.
The below infographic presents the difference between affinity and ion exchange chromatography as a side by side comparison.
Summary – Affinity vs Ion Exchange Chromatography
In summary, affinity and ion exchange chromatography are two forms of liquid chromatographic techniques. The key difference between affinity and ion exchange chromatography is that we can use affinity chromatography to separate charged or uncharged components in a mixture whereas we can use ion exchange chromatography to separate charged components in a mixture.