Chromatography is a process which is widely used for the separation, purification and identification of the components of the mixture for quantitative and qualitative analysis. Owing to the convenience and effectiveness of the process, it is carried out largely across a wide range of industries. Chromatographic separation is mainly based on the differential affinities of the components of mixture towards the stationary and mobile phase leading to the separation of the components. Because of the factors that affect separation; each component of the mixture moves differently in the chromatographic system, some stay longer and move slowly through the system while others pass rapidly into mobile phase leaving the system faster.
Some of the chromatographic techniques that are widely used for the separation of targeted molecules are:
Hydrophobic Interaction Chromatography: Hydrophobic Interaction Chromatography is one of the most widely used methods for the separation and purification of proteins in their original form. It also helps to study the folding and unfolding in proteins apart from isolating the complex protein molecules. Protein molecules are separated using the properties of hydrophobicity where proteins containing both hydrophilic and hydrophobic regions are applied to the HIC column under high salt buffer conditions and because of this, HIC is popularly known as ‘Salting Out’.
Proteins exhibiting different degrees of surface hydrophobicity are separated using this method. The protein molecules are allowed to bind to the hydrophobic ligand on the HIC resin in a binding buffer with a high salt concentration for an enhanced interaction. As we know, the interaction between HIC resin and hydrophobic proteins is greatly impacted by the running buffer and changing the salt concentration changes the interaction. Higher salt concentration enhances the interaction and lower salt concentration weakens the interaction and interactions can anytime be reversed by reducing the strength of the buffer.
Normal Phase Chromatography: It is the technique that uses columns packed with polar adsorbent surface as stationary phase along with the non-polar or moderately polar mobile phase which is allowed to run through the column leading to the separation of components of the mixture. Here, less polar solutes are first detected as they move through the column faster than polar solutes. Normal phase chromatography is the first choice for those separations in which water solubility of sample compound is limited and it is the best technique for the separation of isomers in particular.
Reversed Phase Chromatography: Reverse phase chromatography uses non-polar adsorbent surface as stationary phase and water‐based polar mobile phase. Here, more polar solutes move the fastest as elution order of solutes is based on their hydrophobicity unlike polarity in normal phase. It is useful for separation of mixtures containing components that differ in molecular weight and solubility in water.