An Insight into Techniques of Column Chromatography
Column chromatography is a widely carried out technique for the Separation of components of the mixture. Due to the easy setup of the process, it purifies drug metabolites and compounds of industrial importance. As this method provides high accuracy, it is also used to analyze complex organic mixtures. Chromatography is a physical separation method that helps separate the components based on their distribution between the stationary and mobile phases. The stationary phase can be in the form of a porous bed, bulk liquid, or a thin layer of the adsorbent and the mobile phase is the fluid that runs through the stationary phase.
Column chromatography involves the preparation of a column using silica gel or aluminium oxide. The mobile phase is then allowed to run through the column to cause the Separation of the desired component. The Separation of the compound is based on its affinity toward the mobile and stationary phases. 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 leave the system faster by rapidly moving into the mobile phase.
Column chromatography has gained much importance over the years due to its easy setup, low cost operations, and effective results in less time. Below are the three main divisions of chromatography differing in the mechanism of Separation and are used for various purposes like separation, identification, purification, qualitative and quantitative analysis of compounds:
1) Adsorption Chromatography
Adsorption chromatography is also known as liquid-solid chromatography. Components are retained based on their affinity with the stationary phase, and retention of any component wholly depends on the competition of the analyte with compounds of the mobile phase for the binding sites on the stationary phase.
Retention in adsorption chromatography depends on the following factors:
- How strongly the compound can bind to the stationary phase
- the surface area of the stationary phase
- the volume of the mobile phase being displaced from the stationary phase by the compound to be separated
- How strongly the mobile phase binds to the stationary phase.
- Also, interactions via hydrogen bonding, van der Waals forces, and dipole-dipole interactions can influence retention in this chromatography.
Adsorption chromatography is further classified into:
I. Column Chromatography
In column chromatography, the stationary phase is packed into a column that is run over by the mobile phase to separate the desired component from the mixture. Here, silica gel is the most commonly used adsorbent for packing the columns. They are available in different particle sizes ranging from 40 μm to 63 μm as the appropriate particle size is used for different molecules. The results of column chromatography depend hugely on how well the column is prepared and developed with the mixture of solvents or a particular solvent (solvents are selected based on the molecule's polarity to be separated) which leads to successful elution of the component.
In this chromatography, a thin layer of adsorbent is smeared onto the slide and kept aside to let air dry. Then, a sample is applied to the plate and put inside the container containing the mobile phase. The plate should be kept so that the level of the mobile phase stays below the spotted sample. Solvent travels up the plate by capillary action, moving the components of the sample at different rates depending upon their degrees of interaction with the stationary phase and solubility in the mobile phase. The thickness of the silica gel layer varies between 200 μm to 250 μm.
2) Partition Chromatography
It is the process in which mixtures are separated in columns or on the filter paper, and Separation occurs due to the differences in the partition coefficient of two solvents. Here, both stationary and mobile phases are liquid, so it requires support material to hold the stationary phase in place. Other separation techniques like High-performance liquid chromatography and gas chromatography also work on the basic principle of partition chromatography. The mixture's components get distributed into either of the two liquid phases differing in their partition coefficients. Partition chromatography is further classified into:
a. Liquid-Liquid chromatography:
In Liquid-liquid chromatography, columns are prepared with liquid by absorbing it to the surface of the column as a stationary phase. This chromatography is not as popular as the other methods because the prepared column offers limited stability and is inconvenient to use. Here, Separation occurs based on the differences between partition coefficients of liquids of the stationary and mobile phases.
b. Gas-Liquid Chromatography:
In gas-liquid chromatography, inert gas moves the substance (to be separated) along the tube filled with finely powdered silica gel in a diatomaceous structure is further protected by the coating of waxy polymer.
3) Ion Exchange Chromatography
This chromatography is used to separate ions and molecules that are susceptible to ionization. Here, Separation depends on the affinity of ions towards the stationary phase, which results in the generation of an ion exchange system. Separation happens with electrostatic interactions between the analytes, the mobile, and stationary phases. Only charged complexes can interact with their respective charged ion exchangers.
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