CHROMATOGRAPHIC TECHNIQUES AND APPLICATION
Chromatography is a technique first invented by M.Tswett,a botanist in 1906. He was successful
in doing the separation of chlorophyll, xanthophylls and several other coloured substances by
percolating vegetable extracts. He termed this system of coloured bands as chromatogram and
the method as chromatography. Therefore chromatography may be regarded as a method of
separation in which separation of solutes occur between a stationary phase and mobile phase.
Definition:
Chromatography may be defined as a method of separating a mixture of components into
individual components through equilibrium distribution between two phases. The technique is
based on the difference in the rate at which the components of a mixture move through a porous
medium (called stationary phase) under the influence of some solvent or gas (called mobile
phase). Chromatogram is the pattern formed on an adsorbent medium by the layers of
components separated by chromatography
TYPES OF CHROMATOGRAPHY:
In chromatography, stationary phase may be a solid or a liquid and the mobile phase may bea
liquid or a gas. Depending on stationary and mobile phase used, separation occurs because of
combination of two or more factors such as rates of migration, capillary action, extent of
adsorption etc. Chromatographic methods can be classified on the basis of stationary and mobile
phase used.
TECHNIQUE STATIONARY PHASE MOBILEPHASE
Column chromatography Solid Liquid
Paper Chromatography Liquid Liquid
Thin Layer Chromatography (TLC ) Liquid or Solid Liquid
Gas Liquid Chromatography (GLC ) Liquid Gas
Gas Solid Chromatography (GSC ) Solid Gas
Ion Exchange Chromatography Solid Liquid
Types of chromatography: (based on principles)
i. Adsorption chromatography
This is based on the fact that different components of a mixture are adsorbed on an adsorbent
(stationaryphase) to different degrees. Commonly used adsorbents are silica gel, alumina,
calcium carbonate, magnesium carbonate, zinc carbonate, magnesium oxide etc. When the
stationary phase used is a solid and the mobile phase used is a liquid or a gas, the process taking
place is adsorption. The mixture to be separated first gets adsorbed on the stationary phase
because of the active sites present on the adsorbent. Later based on the affinity of the mixture to
the mobile phase the different components get separated to different extents. Ex. TLC, Column
chromatography.
ii. Partition chromatography
This is based on continuous differential partitioning of components of a mixture between
stationary and mobile phase. When the stationary phase used is a liquid and mobile phase used is
a liquid or a gas, the process taking place is partition. The components of a mixture get
distributed between the stationary phase and the mobile phase.Ex: Paper Chromatography.
iii. Ion exchange chromatography
It is a process that allows the separation of ions and polar molecules based on their affinity to
ion exchanger (usually resins). An ion exchange resin or ion exchange polymer is a resin or
polymer that acts as a medium for ion exchange. Anion resins and cation resins are the two most
common resins used in the ion exchange process. While anion resins attract negatively charged
ions, cation resins attract positively charged ions.
Elution and eluent
The process of separating components of a mixture by allowing the mobile phase to run down
the column through the stationary phase is called as elution.
Eluent: Mobile phase
Eluate: Separated component
PAPER CHROMATOGRAPHY:
Here a chromatographic paper/ cellulose paper is used as a stationary phase. This paper is
hydrophilic and contains water trapped in it which acts as astationary phase. Therefore, it is a
type of liquid liquid chromatography d the principle involved is Partition chromatography
PRINCIPLE: It follows partition chromatography i.e substance gets distributed between two
phases, one is stationary liquid (usually water) which is held in the fibers of the paper and called
the stationary phase; the other is the moving liquid or developing solvent and called the moving
phase. Components of the mixture to be separated migrate at different rates and appear as spots
at different points on the paper.
PROCEDURE: Mixture of components to be separated is prepared in a suitable solvent and is
applied as spots on the filter paper (at the base) and the spot is dried. The paper is kept in a
closed chamber and the edge of the filter paper is dipped into a solvent called developing
solvent. As soon as the filter paper gets the liquid through its capillary axis and when it reaches
the spot of the test solution, various substances are moved by solvent system at various speeds.
When the solvent has moved these components to a suitable height, the paper is dried and
various spots are visualized by suitable reagents called visualizing reagents. The movement of
substances relative to the solvent is expressed in terms of Rf values.
