Column chromatography

Introduction

The technique of separating a mixture of chemicals into their constituents based on their interactions with an inert matrix is known as chromatography. However, it is much more than a basic procedure; it is an essential aspect of research that encompasses bio and physical chemistry, chemical engineering, or other domains. The IUPAC definition of chromatography is "the separation of sample components following their dispersion between two phases." It is an important biophysical technique for separating, identifying, and then purifying the components of a mixture for identification but also quantification. There are several types of chromatography, which differ in the mobile as well as stationary phases utilised.

What is column chromatography?

This chromatography is the often-used extraction or filtration method. It assists in the purification and separation of both solid-liquid materials. It sorts specimens based on differential adsorption of materials to the adsorbent because the substances travel at different speeds through the column, enabling the separation of infractions. In the separation of liquid and solid from a solution, 5 kinds of column chromatography are utilised.

Column chromatography principle

Adsorption is the basis for column chromatography. A combination of materials is dissolved & put into the column in the mobile phase, as well as the components move based on their relative accordance. The component with the lowest adsorption plus highest affinity for the stationary phase travels faster than the component with the lowest adsorption plus highest affinity for the stationary phase. Elements that move swiftly are removed first, whereas components that move slowly are removed afterwards. The rate at which the elements travel is −

Rf = the distance covered by solute/ the distance covered by the solvent

Where Rf is termed as the retardation factor.

Column chromatography procedure

Column Preparation

• The column is mostly made up of a glass container.

• The stationary phase is filled after filling the column's end with glass/cotton wool.

• Following that, a paper disc is put on top to prevent the stationary phase from being disturbed during sample insertion.

• A disturbance in the stationary phase causes unequal separation bands.

The packing processes are given as follows

• Dry − The stationary phase is filled after filling the end of the column with glass/cotton wool. Following that, a paper disc is put on top to prevent the stationary phase from being disturbed during the sample introduction.

• Wet − An adsorbent slurry is made along with the mobile phase as well as put into the column. It is recognised as the best packing method.

  Before using the column, it is well cleaned and then dried.

Sample introduction

• The sample is mixed in the least amount of the mobile phase.

• The mixture is pumped into the column at one time including absorbed in the top section of the column.

• The particular sample may be extracted from this domain using the elution procedure.

Elution technique

The constituent elements are isolated from the column using this procedure. Two strategies can be used to carry out the elution procedure −

• Isocratic elution − A solvent with the same polarity or concentration is used throughout the operation. Use of chloroform alone as an example

• Gradient elution − During the extraction process, solvents with slowly increasing polarity or elution intensity are used. For instance, benzene chloroform ethyl acetate chloroform

Component Detection

• Assessing the separation process is straightforward if the mixture separated in a column chromatography method is coloured.

• If the chemicals being separated are colourless, tiny portions of the eluent are collected consecutively in labelled tubes.

Column chromatography experiment

• Consider a chromatographic tube with a filter glass disc with glass wool at the bottom.

• Magnesium oxide should be added to the chromatographic tube.

• Fasten the chromatographic tube to the entrance of the filter flask and a suction pump to the flask's sidearm.

• Cover the top of the magnesium oxide tube with a petroleum ether solution of leaf pigments.

• Wait a while and notice how each pigment develops a separate colour band.

• After running the entire pigment solution through the magnesium oxide, develop the chromatogram by adding approximately 50 ml of an equal mixture of petroleum ether plus benzene.

Column chromatography applications

• To extract active compounds, column chromatography is utilised.

• It is quite useful for separating chemical mixtures.

• It is used to estimate drug concentrations from medication formulations.

• The mobile phase is more diverse in column chromatography.

• Automation is a possibility.

• For the isolation of active ingredients from plants, column chromatography is the most favoured and sole separation technique in phytochemistry.

• Metabolites are isolated from biological fluids using this method.

• It is used to clean contaminants.

• Because the flow of the mobile phase is influenced by gravity force, no specific mechanical equipment is required to perform the process.

Types of column chromatography

• Column chromatography

• Adsorption column chromatography

• Gel column chromatography

• Partition column chromatography

• Gas & High-Performance Liquid Chromatography

Conclusion

It is a process of sorting chemical mixtures into their constituent molecules using chromatography. Chromatography comprises two phases: one mobile phase plus a continuous stationary phase. To make the column, silica is combined with a suitable solvent, which is then put into a glass column. Depending on its polarity, the chemical composition carries a solvent system through the column. This chromatography is used to separate proteins during protein synthesis. It may also be used to explore the kinetics of an enzyme reaction, as well as to analyse or characterise various materials and/or processes.

FAQs

1. In column chromatography, what are some examples of stationary and mobile phases?

Organic solvents are examples of mobile phases, while silica gel and alumina are examples of stationary phases.

2. Which compounds elute first in the column chromatography process?

Non-polar compounds are the solution. Polar chemicals will actively interact with silica as compared to non-polar ones.

3. In this chromatography procedure, which chemicals elute first?

The answer is non-polar molecules. In contrast to non-polar compounds, polar substances actively interact with silica (SiO₂).

4. What thickness is a chromatography column?

A chromatography column is a glass or plastic tube that is vertically orientated. The column size can range from a few centimetres (as in a Pasteur pipette) to metres (such as with industrial columns). The width of the column is controlled by the quantity of material to be separated, while the length is determined by the difficulty of separation.

5. What is the distinction between partition column chromatography versus gel column chromatography?

Former is the variation in partition coefficients of the distinct components of a combination in which both the phases are liquids. While the latter is a separation that employs a gel-packed column with a porous stationary phase.

6. A solvent's elution power is determined by

The solvent's overall polarity determines its elution power. The polarity of the stationary phase as well as the nature of the sample components.

• Column chromatography is used to extract one of the following?

• Sugar byproducts

• Hydrolysis of a protein molecule produces amino acids.

• Cations with complexes that are inorganic.

• Carotenoids and chlorophyll

Ans: Sugar byproducts

Explanation: The primary use of column chromatography is to identify plant pigments such as chlorophyll but also carotenoids.