Correct Answer - Option 4 : All of the above
HPLC is a method of chromatography in which it includes partition chromatography (liquid/liquid), Adsorption chromatography (liquid/solid), ion exchange, and size exclusion chromatography. It can show the functioning of all these chromatography techniques in one method.
Partition Chromatography
- It is based on the partition coefficient.
- Here both the stationary phase and mobile phase are liquids.
- The partition coefficient is the ratio of the concentration of a compound in a mixture of two immiscible phases at equilibrium.
- Polar molecules get partitioned into the polar phase to a maximum extent while a non-polar component gets partitioned in the non-polar liquid phase.
- The mobile phase moves from the stationary phase and components get separated.
- A common application is in the identification of carbohydrates, alkaloids, tannins, amino acids, etc.
Adsorption Chromatography:
- Adsorption chromatography is a process of separation of a mixture based on the relative differences in adsorption of components to the stationary phase present in the column.
- Here, the molecules or components of the mixture travel at different rates due to the differences in their affinity towards the stationary phase.
- When the stationary phase is polar, During the separation of components, the non-polar components come out of the column first while the polar component elutes out last due to greater adsorption. Vice-versa in the non-polar stationary phase.
- Common applications include isolation of antibiotics, peptides, and proteins, etc.
Ion Exchange Chromatography:
- Separation occurs based on ion displacement theory (exchange of ions).
- The charged molecules in the sample are separated by the electrostatic forces of attraction when passed through the ionic resin at particular pH and temperature.
- The separation occurs by reversible exchange of ions between the ions present in the mobile phase and those present in the ion exchange resin (stationary phase) by electrostatic forces.
- The process of separation of molecules from the mixture is dependent on the type of ion exchange resin used (cation or anion exchange resin).
- High-Performance Liquid Chromatography is a modern analysis technique that is used in various fields like analysis of biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin.
- This technique yields perfect results in the separation, identification, and quantification of the analyte to be analyzed.
- HPLC instrumentation includes a pump, injector, column, detector, and integrator or acquisition and display system.
- The heart of the system is the column where separation occurs.
