Process of Chromatography

Chromatography is a biophysical technique that allows us to separate, identify, and purify components of an unknown mixture. It helps us perform both qualitative and quantitative analysis. For example, the process of adsorption chromatography, where the relative rates of adsorption of the mixture components onto the stationary phase and then desorption off that phase, allows the different components to be separated. Few other examples are ion-exchange chromatography, column chromatography, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), affinity chromatography etc. Chromatography has three basic components, the mobile phase, stationary phase, and separated molecules from the unknown mixture. 

Process of Chromatography

A sample is first dissolved in a solvent in chromatography, known as the mobile phase. The mobile phase then carries the sample via a second substance known as the stationary phase. A stationary phase may be a column, plate, capillary tube, or sheet. Now, as the different constituents of the sample possess different affinities towards the stationary phase, they will travel via the stationary phase at different speeds. Such different travel times via the column are called the retention time. Retention time allows the mixture’s components to be separate from each other. 

While analysing a mixture, it is crucial to focus on the specific characteristics of the mobile and the stationary phase, which will determine which of the components of the mixture will travel faster or slower than the rest of the components. 

Components of the Chromatography process

The process of chromatography technique has three basic components. 

• Stationary phase: This phase comprises either a solid component or a liquid adsorbed onto a solid support surface that stays fixed inside the chromatography column. In the stationary phase, the unknown mixture to be separated gets selectively adsorbed, which allows their separation. The stationary phase can be of different types, such as silica, glass, gel beads, paper, gas, liquid components etc.

• Mobile phase: This phase is either a liquid or a gaseous component that flows through the column. It helps the different components of the mixture to be carried on via the column. Different kinds of mobile phases are pure water, alcohol, acetone, acetic acid, gas etc.

• Separated molecules: The different separated components of the unknown mixture.

Types of Chromatography

Different chromatography processes are performed based on different aspects of the mixture particles, like particle size, charge, binding affinities of molecules etc. 

Column chromatography 

• This adsorption chromatography technique separates different components of a mixture based on their different adsorption capacity toward the absorbent present in the stationary phase. 

• The components move at different speeds while passing through the column and are separated into different fractions.

• Here the stationary phase is a solid component (mostly silica), and the mobile phase is either a liquid or a gas.

• It is generally used for separating impurities from biological samples, their purification, isolation of metabolites, detection of drugs from crude samples, etc.

Ion-exchange chromatography

• It is based on the principle of ion exchange mechanism, where mixture components get separated based on their respective charges.

• The stationary phase is an ion-exchange resin column that carries charged functional groups that separate charged components from a mixture, such as anions, cations, proteins, amino acids, peptides etc. 

• When a sample mixture is passed through that column, it interacts with the oppositely charged groups of the resin and gets retained in the column. 

• Two kinds of ion-exchange chromatography are available, cation exchange and anion exchange. 

• Cation-exchange chromatography has a negative charged stationary phase that attracts cations from the mixture to be separated. 

• Anion-exchange chromatography has a positively charged stationary phase that attracts the anions from the mixture to be separated.

• Generally used for water purification , proteins, separate amino acids, metals from mixtures etc.

Affinity chromatography 

• Different components of a mixture get separated based on their affinity towards the stationary phase of the chromatography system.

• The stationary phase is composed of ligands with reactive sites for the different components of a mixture. 

• When the mixture gets passed through the mobile phase, its components that have binding sites for the ligands bind to them in the stationary phase and get retained. 

• The rest of the components with no affinity towards the stationary phase get eluted out with the mobile phase.

• Components attached to the stationary phase are then eluted via changing conditions like the pH, ionic strength, or other properties of the solution, favouring the separation of the ligands and components of the mixture.

• Generally used for separating components, removing impurities from a mixture, or purification processes. 

Thin-layer chromatography 

• A thin layer of stationary phase is applied on a solid plate with the help of a liquid mobile phase.

• Mixture components with an affinity towards the stationary phase get bound to that, while the other components get eluted via the mobile phase.

• After a successful separation, different components can be seen in the stationary phase at different locations, like spots.

• Helps analyse fibres in forensic samples, identify components of different pharmaceutical products, medicinal plants etc.

Gas chromatography

• Molecules are separated based on their retention time in the column. 

• Different molecules show different affinity toward the stationary phase and have different retention times.

• Molecules with a higher affinity towards the stationary phase show higher retention time and take longer to get eluted from the column.

• Gases, like helium, are used as the mobile phase.

• Helps determine different concentrations of air pollutants, oil spills etc.

High-performance liquid chromatography (HPLC)

• Different components show different interactions with the absorbents present in the stationary phase and get separated.

• Components with a higher affinity towards the absorbents take longer to move via the column, whereas components with lower affinity move faster.

• The difference between this process and the column chromatography is that the solvent gets forced under high pressures to move along the column instead of waiting for it to move down via gravity.

• Helps maintain product purity and quality and can separate biological molecules such as proteins, nucleic acids etc.

Other than the above mentioned chromatography techniques, several other kinds of chromatography are also available: paper chromatography, reverse-phase chromatography, liquid chromatography, gel-permeation chromatography, hydrophobic interaction chromatography, lash chromatography etc.

Conclusion

Chromatography is a popular separation technique used in laboratory settings for separating different ionic species, inorganic or organic compounds, molecular components of a sample etc. This analysing procedure is generally used in the industries such as pharmaceuticals, food and beverage, forensics etc. Several kinds of chromatographic techniques are available with improved technologies. Components with different ionic strengths, inorganic or organic in nature, with different sizes etc., can be easily separated by chromatography techniques. The principles behind different chromatographic techniques range from components’ molecular characteristics and their kind of interaction, such as ion exchange, surface adsorption, particle size, etc. It can also be based on the stationary phases used, such as column, thin layer, paper chromatography etc.