Chromatographic Techniques: At many instances in our daily lives, we have to separate components that are mixed together. Depending on the nature of the components present in the mixture, we use various techniques of separation such as – Crystallization, Filtration, Evaporation, Sublimation, Distillation, Chromatography and so on.

For instance, if you are provided with an ink solution, using the techniques of Evaporation will only result in obtaining Solute. A better technique to separate the components of the solution in this case would be to use the technique of Chromatography. In this article, we will explore more about chromatography and its types, with procedure and separation principle.

What is Chromatography?

Chromatography was first developed by M. Tswett, a botanist, in \(1906\) in Warsaw for the separation of coloured substances into their components. In Greek, chroma means colour, and graphy means writing. Hence the name chromatography means colour writing.

What is the Basic Principle of Chromatographic Techniques?

Chromatography is based on the general principle of distribution of components of a mixture of organic compounds between two phases, i.e., the stationary phase and the mobile phase.

• 1. Stationary phase: The material on which the various components are adsorbed is called the stationary phase. It is porous, and the commonly used stationary phases are alumina, silica gel, calcium carbonate, or activated charcoal.
• 2. Mobile phase or moving phase: The mixture to be separated is dissolved in a suitable medium maybe, liquid or gas. This constitutes the mobile phase.

The moving phase is made to run on a stationary phase, and the separation is based on the principle that the components of a mixture present in the moving phase move at a different rate through the stationary phase.

The chromatography is based on the two general phenomena, adsorption, and partition. When the stationary phase is solid, the basis of separation is adsorption. But when the stationary phase is liquid, the basis is partition.

Depending upon the principles, chromatography is classified into two categories, i.e., adsorption chromatography and partition chromatography.

• a. Adsorption chromatography: It is based on the principle that different components are adsorbed to different extents. The two important types of adsorption chromatography are column chromatography and thin-layer chromatography (TLC).
• b. Partition chromatography: It is based on the continuous differential partitioning of components of a mixture between stationary and moving phases. Paper chromatography is the common type of partition chromatography.

Types of Chromatography

The various types of chromatography which are commonly used are:

• 1. Column (adsorption) chromatography
• 2. Thin-layer chromatography
• 3. Paper chromatography
• 4. High-pressure liquid chromatography (HPLC): HPCL is the automation of traditional liquid chromatography. It depends on the principle of adsorption as well as partition chromatography based on the nature of the stationary phase. In the solid stationary phase, it follows the adsorption principle, and in the liquid stationary phase, it follows the partition principle. This technique is commonly used in the identification and purification of organic compounds.
• 5. Gas chromatography: It is applicable for the separation of gaseous and volatile substances. It is based on the principle of partition of volatile compound between the liquid phase and gaseous phase. These are used for the analysis of organic compounds like fatty acids, drugs and their metabolites, etc.
• 6. Ion exchange chromatography: In this method, ions and polar molecules are separated based on their affinity to the ion exchangers. These are used for the separation of proteins, demineralisation of water, softening of hard water, etc.

Column (Adsorption) Chromatography

Column chromatography was developed by D.T. Day, American chemistry. Principle: The chromatography is based on the principle of selective absorption and desorption.

Procedure:

• 1. The stationary phase consists of a solid packed in a glass tube. Alumina, activated charcoal, silica gel, magnesia, calcium carbonate, starch, etc., can be used as a stationary phase.
• 2. The mixture to be separated is dissolved in a suitable solvent like petroleum ether. The mixture to be separated is allowed to run through the stationary phase.
• 3. The constituents of the moving phase all adsorbed at different parts of the adsorbent column depending upon their nature. This is called selective or differential absorption.
• 4. The adsorbed components are recovered by dissolving the components in suitable solvents, and this process is called elution. The commonly used eluent are petroleum, ether, benzene, chloroform, etc. The eluent act as a moving phase. The eluent dissolves the components and tries to take them down the column. The weekly adsorb component is eluted more rapidly than the more strongly absorbed components. The components thus get separated from one another and come out of column one by one. They are collected in separate receivers.
• 5. The components from the eluent are recovered by evaporation or distillation.

Thin Layer Chromatography (TLC)

Adsorption chromatography, or thin-layer chromatography, is a form of adsorption chromatography. With the use of an applicator, a thin layer of an adsorbent, such as silica gel formed from a slurry in a suitable solvent, is spread across a glass plate of size \({\rm{5 \times 2 \; cm}}{\rm{.}}\)

The separated mixture is dissolved in a suitable solvent and placed on a glass plate using a fine capillary at a distance of about \({\rm{2 \; cm}}\) from the bottom. The dry plate is then placed in a development chamber containing a suitable solvent or a mixture of solvents in a vertical position.

It is important to keep in mind that the spot marked on it should not be dipped in the solvent during the operation. The solvent will progressively increase due to capillary action once the compartment is closed.

A number of fine spots form when the mixture separates. Finally, the plate is removed and dried. Colored components can be found using the naked eye, while colourless components can be found using UV light, iodine, sulfuric acid mixed with an oxidising agent such as potassium permanganate, nitric acid, and other methods. The components can be identified using the \({{\rm{R}}_{\rm{f}}}\) value or by using a suitable procedure to elute each component separately.

The relative adsorption of each component of a mixture is expressed by a retention factor known as the \({{\rm{R}}_{\rm{f}}}\) value. \({{\rm{R}}_{\rm{f}}}\) value is defined as the ratio of the rate of movement of a substance under the investigation to that of the solvent.

\({{\rm{R}}_{\rm{f}}}{\rm{ = \;}}\frac{{{\rm{Distance\;moved\;by\;the\;substance\;from\;base\;line}}}}{{{\rm{Distance\;moved\;by\;solvent\;from\;baseline}}}}\)

The \({{\rm{R}}_{\rm{f}}}\) value of a substance depends upon the nature of the substance, solvent, adsorbents, and temperature.

Paper Chromatography

Paper chromatography was introduced by Schonbein \((1865)\) under the name capillary analysis. In paper chromatography, a strip of specially designed filter paper is known as chromatography. Here paper acts as a stationary phase and a pure solvent or a mixture of solvent is used to act as the mobile phase.

The mobile phase is also called an irrigant or developing solvent. The mobile phase travels through the filter paper by the capillary action. Paper chromatography is based on a mechanism that is partly partition and partly adsorption.

The stationary phase, i.e., filter paper, is made up of cellulose fibre with molecules of water adsorbed on them. Drops of solution of the mixture to be separated are placed on the strip of filter paper, and the solvent is allowed to travel along the strip.

The mixture undergoes partly adsorption on the filter paper and partly partition between the water molecule attached to the cellulose fibre and the solvent. Based on the amount of adsorption and partition, the components in the mixture travel at different rates on the filter paper and get separated from one another.

After some time, the paper strip is taken out, dried, and the position of the different components is noted. If the position of different components is not visible on the filter paper, then it can be located with the help of a suitable reagent like ninhydrin that is used to identify different types of \({\rm{\alpha }}\)-amino acids present in a mixture.

The various components present in a mixture are identified by comparing the calculated \({{\rm{R}}_{\rm{f}}}\) value with the reference substance or with the standard values given in the databook.

Chromatographic Techniques in Pharmacognosy

Pharmacognosy is the study of the crude drugs obtained from natural sources like animals, plants, minerals, etc. To study this, thin layer chromatography (TLC) is a simple and efficient technique. In this method, silica gel is act as a stationary phase, and natural sources to be studied act as a mobile phase. Separated components are identified based on \({{\rm{R}}_{\rm{f}}}\) value. Paper chromatography can also be used for this purpose.

Applications of Chromatography Techniques

The uses of chromatography are:

• 1. Chromatography is used for characterizing and isolating organic compounds such as amino acids, alcohol, amines, acids, antibiotics, etc.
• 2. Chromatography is used to find the amount of chemicals in each pharmaceutical product.
• 3. It is also used in hospitals to detect the alcohol level in the patient’s bloodstream.
• 4. It is used in clinical tests like urine analysis, antibiotic analysis, etc.
• 5. The level of pollutants in water can be identified using chromatography.
• 6. Chromatography is used in forensic labs to identify components in the samples collected from the suspects.
• 7. Chromatography is also used in the food industry to identify the nutritive value of the food additives and their components in the food.

Summary

In this article, you have studied the principle, procedure of three basic types of chromatography technique, i.e., column chromatography, paper chromatography, and thin-layer chromatography. You have also studied about uses of chromatography.

FAQs on Chromatographic Techniques

Q. What is the chromatographic technique used for?
Ans: The chromatographic technique is used for the separation and purification of the components in the given mixture with high efficiency. 

Q. What do all chromatographic techniques have in common?
Ans: All chromatographic techniques have two common phases, i.e., stationary phase and mobile phase.

Q. What is the first step in chromatographic technique?
Ans: The first step in the chromatographic technique is the preparation of column or chromoplate. It acts as a stationary phase.

Q. What are the four types of chromatography?
Ans: The four types of chromatography are column chromatography, paper chromatography, thin-layer chromatography, and ion-exchange chromatography.

Q. What are the techniques used in the chromatography method?
Ans: The chromatographic method based is on adsorption and partition of components on the stationary phase. The separation of components is based on the desorption of the component during the elution.

Q. What are the Moving and Stationary phases in Paper Chromatography ?
Ans: Water absorbed on cellulose constituting the paper serves as the stationary phase and organic solvent serves as the moving phase.

Q. What does the term “developing” mean in Chromatography ?
Ans: During Chromatography, if the components to be separated are colourless, these separated components on Chromatogram are not visible. Their presence is detected by development, which involves spraying a suitable reagent (which is called Developing Reagent) on the Chromatogram, or placing the Chromatogram in Iodine chamber, when various components become visible. This process is called developing of Chromatogram.

Q. What is R_f value ?
Ans: R_f (retention factor) of a substance is defined as the ratio of the distance moved up by the solute from the point of its application to the distance moved up by the solvent from the same point.

Q. How does liquid rise through filter paper ?
Ans: Liquid rises through filter paper by means of capillary action.

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