Saturday, May 2, 2020

Principle and applications of column chromatography

Column chromatography is a basic and simple type of chromatography technique used for separating and purifying the compounds. It is a kind of adsorption chromatography in which the compounds are separated based on their different affinity to the stationary phase. In column chromatography, generally, silica gel is used as a stationary phase (adsorbent), and solvent or a mixture of solvent used as a mobile phase. Column chromatography has a polar stationary phase. The separation of the compounds based on its polarity since the polar compounds elute slowly and nonpolar compounds travel rapidly through the column.
The principle of column chromatography:
The adsorption is the basic principle involved in the separation process of column chromatography. The liquid mobile phase is allowed to travel through a solid stationary phase with the sample mixture under the force of gravity. The analytes of the sample become adsorbed in the stationary phase throughout their travel and they get separated due to differences in affinity of individual analytes. The analytes have low affinity to the stationary phase they rapidly eluted, while those analytes have greater affinity they elute slowly from the column. The solute reversibly adsorbs to the stationary phase. Therefore, with a constant input of the solvent from above all the samples flow out.
Various essential components involved in the column chromatography process.
Tubular glass column, stationary phase, solvent, stand with clamp and funnel, etc.
Major steps involved in the process of column chromatography.
1. Preparing the column
2. Selecting the suitable solvent to run the column
3. Loading the column
4. Fractions Collection
 
The applications of column chromatography are as follows.
  • One of the major applications of column chromatography is that it can be used for isolation and purification of many classes of components.
  • Essential components can be separated from plant extracts, formulations, active components, from plant extracts.
  • It is very useful in the removal of impurities from the sample.
  • It can be used to isolate amino acids, glycosides, and alkaloids.
  • This method is used in the purification of reaction mixtures in chemical synthesis.
  • Metabolites from biological fluids such as blood, serum, etc. are isolated using column chromatography.
  • The impurities of the sample can be separated by using the appropriate mobile and stationary phases.
  • This is used for the determination of drugs from formulations.
  • Pharmaceutical manufacturers typically used to purify compounds.
  • It is used for the purification of proteins (biomolecules) for pharmaceutical research.
  • The column chromatography is used to isolate any type of sample mixture.
  • The column chromatography is used to analysis of environmental samples.
Commonly asked questions on column chromatography are as follows.

What are the types of column chromatography?
Based on the stationary phase there are four types of column chromatography are adsorption chromatography, ion-exchange chromatography, partition chromatography and, gel chromatography
 
What is the most common absorbent in column chromatography?
The silica gel (SiO2) and alumina (Al2O3) are the most widely used adsorbents in column chromatography. The calcium phosphate, calcium carbonate, starch, and magnesia, are also used as stationary phase in column chromatography.

What are the factors affecting column chromatography?
The dimension of the column, the particle size of the adsorbent, the temperature of the column, flow rate of solvent, nature of the solvent used, and pressure on the system, etc.

How do you pack columns for column chromatography?
Usually, two methods are used to prepare a column i.e. dry method and wet method.

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