What are the Classification of Chromatography?

Chromatography is an important analytical technique used to separate components or solutes from a mixture. Based on the chromatography type, there are different parameters based on which the separation is achieved. The basic principle of chromatography is based on the distribution of solutes or components from a mixture of organic compounds between the stationary and mobile phases. As the mobile phase, which is the mixture of the solute components, slowly moves over the stationary phase, it separates the components through adsorption and partition. The topic helps in understanding the importance of mobile and stationary phases as well as various classifications of chromatography.

Importance of Stationary and Mobile Phase 

• The mobile phase is the mixture of the solute or components dissolved in a suitable medium. It runs through the stationary phase along with the same. 
• The stationary phase is fixed and attached to either a column or a plate. Some common examples of it are silica gel, activated charcoal, calcium carbonate, alumina etc. 
• As the components in the mobile phase migrate through the stationary phase, they are adsorbed on the surface. 

As the components move at a different rate and are eluted using different solvents, it helps with supporting the separation mechanism.

Chromatography Classification based on the Chromatographic Bed

Chromatography mechanisms and processes are classified based on various parameters. The first parameter for chromatographic separation is the shape of the chromatographic bed. According to the shape, chromatography mechanisms are classified into the following two categories:

• Column chromatography – The main vessel used for the chromatographic process is a column. The stationary phase is set up inside the tube and filled with solid stationary components. The mobile phase is passed along the stationary solid phase, which adsorbs the solute particles from the mixture. Examples include size-exclusion chromatography, ion-exchange chromatography and affinity chromatography. 
• Planar chromatography – In this type, a planar apparatus is used, and a stationary solid phase is applied to it. The mobile phase is passed through the planar surface, which separates the solute from the mixture based on capillary action. Two examples of such chromatography are thin-layer chromatography and paper chromatography. 

Classifying Chromatography Based on the Physical State 

There are two types of chromatography based on the state of the matter:

• Liquid chromatography – In this type of chromatography mechanism, the mobile phase is liquid consisting of solute or components mixed in a solvent. It is carried out on both column and planar chromatography. 
• Gas chromatography – The mobile phase in this category exists in a gaseous state. It is also referred to as gas-liquid chromatography and applies to packed columns. 

Classifying Chromatography Based on Chromatographic Mechanism 

The three types of chromatography that fall under this category are as follows:

Ion exchange Chromatography 

• It helps in separating ions and polar molecules based on their difference in charge properties. 
• It helps to separate inorganic anions like nitrate, chloride, sulphate etc., or inorganic cations like sodium, lithium, potassium etc. 
• Ion exchangers are attached to the stationary phase, and ions or polar molecules are separated based on their affinity for ion exchangers. 
• The cationic exchanger consists of negatively charged groups and attracts positively charged cations in the mobile phase.
• The anionic exchanger consists of positively charged groups and attracts negatively charged anions. 

Size Exclusion Chromatography

• It separates the molecules based on their size by filtration through a gel. It is also known as gel-filtration chromatography. 
• It is used for separating larger molecules like proteins, antibiotics, drugs, industrial polymers etc. 
• The chromatography column is packed with porous beads mainly composed of dextran polymers, agarose or polyacrylamide. The pore size of the beads is varied and different. 
• The mobile phase is passed through the column where the smaller molecules are trapped in gel pores, whereas larger molecules are released out. The small molecules are later eluted out through an elution process. 

Affinity Chromatography 

• It helps in separating biomolecules from a mixture in a mobile phase based on specific macromolecular interactions between the biomolecule and another substrate. 
• Some examples of affinity chromatography are antigen and antibody, receptor and ligand, enzyme and substrate etc. 

Use of Retention Factor in Thin Layer Chromatography 

The retention factor stands for Rf and is important for thin-layer chromatography. The mobile phase moves along the plate on the stationary solid phase through capillary action. The mixture separation leaves several fine spots. The components are identified using the Rf value, which expresses the relative adsorption of each component of a mixture. 

The retention factor defines the ratio of the rate of movement of a substance on the planar to that of the solvent. Rf  value depends on the nature of the substance, adsorbent, solvent, and temperature.

It is represented by 

Rf  = Distance moved by the substance from baseline / Distance moved by solvent from baseline

Conclusion 

Chromatography is classified into different categories depending on various parameters. The fundamental parameter for classification is the chromatography bed, which is column and planar chromatography. Each chromatography mechanism makes use of the mobile phase and stationary phase. The stationary solid phase is immovable and is fixed in a column or plate surface. The mobile phase is a mixture which consists of solute dissolved in a solvent, which is passed through the stationary or fixed phase that aids adsorption and ultimate separation.