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Chromatography is a versatile tool used in bioprocessing to purify and analyse biomolecules. This blog will explore the basics of chromatography, its properties, and its different types.
Principles of Chromatography
Chromatography is a technique used to separate components of a mixture. In the process of separation, a mixture is prepared in a fluid solvent, which is either gaseous or liquid and is called the mobile phase. This solvent is then carried through the device – in our case, a monolithic column – where it interacts with the fixed stationary phase. The technique works on the principle that different components of the mobile phase interact differently with the stationary phase, allowing for the separation of the individual components.
Types of Chromatography
The special properties of the mixture components that enable us to separate them include several unique characteristics that we can utilize.
First off, we can consider the different sizes of the components. Larger molecules cannot enter the small pores of the stationary phase and therefore pass through the column faster than smaller molecules. Size exclusion chromatography is based on the difference in size of our components.
Alongside size and charge, we can also use hydrophobicity, where molecules with higher hydrophobicity interact more strongly with a non-polar stationary phase.
Ion exchange chromatography separates molecules based on their positive or negative charge. The stationary phase can be either positively charged (anion exchange) or negatively charged (cation exchange), and it attracts and retains molecules of the opposite charge. By gradually changing the pH or ionic strength of the buffer, the bound molecules can be eluted and collected separately.
Another characteristic we can use is ligand specificity. This is when we use the specific interactions between molecules and the stationary phase, like how certain keys can only open certain locks. Affinity chromatography relies on specific interactions between ligands. Similarly, metal affinity uses metallic bonds between the components and the stationary phase to trap the components in the mixture.
For the final property, we can use hydrogen bonding, where some functional groups of the stationary phase can form complementary hydrogen bonds with the components in the mixture, thus binding onto them.
Last but not least, we can learn about ion pair reversed-phase chromatography, where we use ion-pairing reagents, which bind to the components in the mixture and enable their binding to strongly hydrophobic chromatography supports.
Understanding the principles and types of chromatography is essential for various scientific applications. In the next part of this blog series, we explore the practical applications of these techniques in bioprocessing.
