Column chromatography Principle

Introduction

M. S. Tswett, a Russian scientist, introduced the chromatographic technique. The technique that has been used for the separation of particles present in a given mixture is chromatography. In which the mobile phase moves through the stationary phase and the separation of particles present in the mixture takes place according to the affinity with the solvent used. There are distinct chromatographic techniques and are based on the different mobile and stationary phases. Column chromatography is one such chromatographic technique. The stationary phase is a column of adsorbent coated with the mixture and the mobile phase is a solvent. The separation of particles present in the mixture takes place according to the affinity of components with the solvent used.

Column Chromatography Definition

A purification technique used in analytical chemistry. In which the separation of components in a mixture takes place by using a column of adsorbent as the stationary phase. Through which the liquid or gas phase flows through the stationary phase and separation of components takes place based on the affinity with the solvent.

Column Chromatography Principle

The principle of column chromatography is. This chromatographic technique includes a stationary phase as a column of adsorbent which contains small solid particles coated on a glass or a metal column. This small solid particle may apply inside the column as a thin film too. The mobile phase or eluent and mixture of analyte is then enacted to the column with the help of a pumping system or with the use of gas pressure. Then the solvent will flow through the column and the components present in the mixture based on its distribution coefficient. Thus the separation of distinctive components present in the mixture will take place.

Segments of column chromatography

The important components used in the column chromatography process are

• Stationary phase − The general stationary phase used in column chromatography is silica gel and alumina.

• Mobile phase − It is a solvent or eluent that can be used for the separation of components present in the mixture. And the selection of solvents depends on the stationary phase. For silica gel and alumina organic solvents are used as a mobile phase.

• Column − It will depend on the type of column chromatography. If it is liquid chromatography the column used is stainless steel approximately 25cm long and 4mm in diameter. While gas column chromatography is glass or stainless steel with 2cm long and an internal diameter of 3mm.

• Injector system − It will help in the delivery of samples on the top of the column used.

• Detector or recorded − To obtain a continuous record of the components as they emerge from the column. Usually, UV-Visible spectroscopy is used.

• Fraction collector − It is used for the collection of separate components which can further be used for studies.

Procedures in column chromatography

The steps involved in column chromatography are discussed below.

• Preparation of column − The first step is the preparation of the column with the stationary phase. And they may be dry packing and wet packing. In dry packing dry powder of adsorbent is used in the column. In wet packing, a slurry of adsorbent is used in the column.

• Introduction of sample − The sample of mixture which is dissolved with the mobile phase is applied to the column. Individual components in the sample are then separated by elution.

• Elution − The separated components separated from the column with the help of solvents are elution.

Advantages And Disadvantages Of Column Chromatography

The advantages and disadvantages of column chromatography are formulated in the following table.

Advantages	Disadvantages
The choice of the mobile phase is wider.	The method is time seizing.
It can be used for removing impurities present in a given compound.	Constant attention is required in the entire process of separation.
The separation of mixtures can be done	The amount of solvent used for the
with any amount of mixture no particular measurement is there.	processing of this chromatographic technique is more.
Automation is feasible.	Automation present in the instrument makes it more expensive and confused.
Solvents can be changed during the process of chromatography.	The collection vessels used in the separation process must be changed continuously.
The sample used for the separation can then be reused.	Some technical skills are required for the packing columns.
Efficient separation since the use of different stationary phases at different solvents is possible.

Applications of Column chromatography

It is an excellent technique that has been found in applications in different sectors. The advantage of using this technique is wide. Some of the applications of this method are,

• Separation of biological metabolites from biological fluids is possible with this method.

• Analysis of drugs during formulation is possible.

• It can be used for the purification of natural compounds obtained from plants.

• It can be used for the separation of functional constituents.

• It can be used for the determination of fluocinolone, dexamethasone, etc. in formulations.

• This technique can separate many hundreds of components that are unknown too.

• Some amounts even in the parts per billion ranges or picogram can be detected with this technique.

• It is also used for the separation of end products in organic synthesis.

Conclusion

One of the important types of chromatographic technique in which the separation of components takes place in a particular mixture takes place with the help of a column of adsorbent as the stationary phase. The components present in the mixture will separate according to the adsorption power of particulates present in the mixture. The instrument of column chromatography contains a stationary phase, mobile phase, column, injector system, detector, and fraction collector. There are mainly three steps in the column chromatographic process. The advantages of column chromatography are wide even though it has some disadvantages too. And the application of this chromatography is found in many fields for example organic synthesis, drug estimation, plant component identification, biological component identification in biological fluids, etc.

FAQs

1. What is the Rf value?

Rf value, retention factor is a term used in chromatographic technique in which it is the ratio of distance travelled by a particular constituent to the distance travelled by the solvent front. This value can be used for the identification of a particular component present in a mixture.

2. Why is silica gel employed in column chromatography?

Silica gel is commonly employed as a stationary phase in column chromatography and is because of the high adsorption power of these compounds. And also these compounds are easily available and cost-effective making them more useful. Silica is a polar adsorbent.

3. What are the factors influencing column chromatography?

The factors that affect column chromatography are

• Temperature.

• Nature of solvent used in the technique.

• The pressure of the column.

• The size of the adsorbent.

• Dimension of the column.

4. What category of mixtures is separated by column chromatography?

Some of the examples of mixtures that can be easily separated using column chromatography are the separation of amino acids, carbohydrates, drugs, separation of narcotic components, coloured ink components, etc.

5. How does particle size influence column chromatography?

The size of particles used in column chromatography has an important role. If the size of particle used in the column chromatography is small the rate of separation with great efficiency is higher. And the process can be done without increasing the time of separation, length of the column, and rate of flow.