17 General Chromatography : Distribution Coefficient and its implications

Dr. Varinder Kaur

  1. Description and background

A Russian botanist, Mikhail Tswett was working on the separation of plant pigments i.e. chlorophylls and carotenoids. He packed a glass tube with powdered calcium carbonate and poured finely ground plant extracts dissolved in different solvents. After adding mixture of solvent from the upper end of glass tube, he observed separation of different colors in the column with the downward movement of solvents due to gravity. The appearance of different bands was due to separation of colored compounds in column, so, Tswett coined term ‘chromatography (Greek word ‘chroma’ means color and ‘graphy’ means writing). Afterwards, various scientists worked on the chromatographic methods and it established foundation of separation science.

 

Chromatography is a technique which is used to separate various closely related chemical components of mixture. It is divided into two categories on the basis of their usage; Analytical chromatography and preparative chromatography (given below).

 

 

Some common terms used in chromatography

 

Before understanding the principle of chromatography, it is important to know some basic terms related to chromatography.

 

  1. Stationary phase: It is the phase which remains fixed at its position. It provides active sites for interacting the components of sample. It is usually a solid, viscous liquid or a thin coating bonded with a surface.
  1. Mobile phase: It is the phase which moves through the stationary phase and is responsible for the movement of components of sample through the stationary phase. It may be a liquid or a gas.
  1. Analyte: It is the component (or components) of interest which is (are) under observation or to be analyzed. It may be a liquid, solution, gas or volatile species.
  1. Elute: The mobile phase after passing through the stationary phase bringing the components out of the system and is termed as elute. The process of getting components out from the system with the use of appropriate medium is called as elution.
  1. Eluent: The medium which is introduced to the system to bring the components out of the system is called as eluent. It may be same of different as the mobile phase.

 

Basically in chromatographic techniques, sample having mixture of various components (or analyte) is exposed to the mobile phase running through the stationary phase; where the components interact with the active sites and adhere to the stationary phase. These components get released again in the mobile phase (or eluent) to come out of the system. However, the stay of components on the stationary phase depends upon the physico-chemical properties of the components, therefore, they release/move at different time intervals. This leads to the separation of components.

 

Example: Suppose a mixture with three colored components A, B, and C is analyzed using a glass tube filled with silica. The diagrammatical procedure for the separation of components is given below.

 

In this case, glass tube (column) is filled with silica (stationary phase) and the sample mixture (analyte) is poured from the top of the tube. After running solvent through the column, components move down and get separated. With the continuous addition of solvent they get separated into bands. The component B has weak interaction (than A and C), whereas component A adheres to the stationary phase strongly, therefore, their movement follow the order; component B followed by C and then A. In this way, the band due to component B comes out and is collected in a flask. Similarly, components A and C are collected as they elute with the solvent.

 

Overall, the separation of components depends upon the different affinities of components to adhere the stationary phase and solubilization of components in the mobile phase. If the affinity of a component to interact with the stationary phase is more, it will stick for longer period and come out slowly, and vice versa. In contrast, if the solubility of a component is more mobile phase it will come out rapidly and vice versa. Hence, the separation of components can be achieved by choosing a best combination of stationary phase and mobile phase.

 

Distribution of components between two phases

 

As discussed above, the components of a mixture are distributed between stationary phase and mobile phase. This distribution of a particular component in the two phases achieves equilibrium as given below

 

 

Overall, the chromatographic techniques can be categorized into the following forms as given below.

 

Advantages of chromatography over other separation techniques

 

  • It can be used to separate multi-components of a mixture.
  • It can be used for separation without knowing about the exact identity of the component.
  • It can be used in every field such as chemical, biological, biochemical, pharmaceutical and industrial applications.
  • It can be used for all the sizes i.e. sub-nanograms to kilograms.

 

Applications of chromatography

 

  • It is used for the purification and testing of various components of medicines in pharmaceutical industries.
  • It is used for the separation of hydrocarbons in petroleum industry.
  • It is used in the food industry.
  • The environmental testing laboratories mainly employ chromatographic techniques to monitor the amounts of pollutants like pesticides, polyaromatic hydrocarbons, wastes etc in the environment.
  • It is used to test the drinking water.
  • It is used for the separation of insulin purification, plasma fractionation and enzyme purification.
  • It is also used by forensic laboratories, fuel industries and bioinformatic laboratories.

 

Implications of distribution constant

 

As discussed earlier, a component distributes between two immiscible phases and establishes an equilibrium governed by distribution law. The ratio of activities of the component between two phases is constant and the equilibrium constant may be termed as distribution constant. For example; a

 

Bibliography

 

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