Chapter-2( experimental techniques)
The technique used to separate the component of mixture on the basis of different affinities of the components for stationary or mobile phase.
The word chromatography derived from Greek words Chromates meaning Colour Writing.
In this process components distribute itself between two phases i.e. stationary phase and mobile phase.
Stationary phase may be solid or liquid.
Mobile phase may be gas or liquid, which move over stationary phase. It is also called eluvant.
While the phase over which mobile phase flows in chromatography is called stationary phase.
Basic Principle of Chromatography:- The basic principle involved in chromatography depends on the relative solubilities of the components between two phases. This is governed by distribution Co-efficient K.
K = Concentration of component in mobile phase/ divded by
Concentration of components in stationary phase
Classification of Chromatography:- On the basis separation mechanism chromatography is classified into two classes.
(1) Adsorption Chromatography (2) Partition Chromatography
(1) Adsorption Chromatography:- In this type solid is used as stationary phase e.g. column chromatography & TLC (Thin Layer Chromatography).
(2) Partition Chromatography:- In this type liquid is used as stationary phase e.g. paper chromatography.
In 1944 Consden and Mortin introduced paper chromatography.
It is a type of partition chromatography.
In paper chromatography the H2O molecules bonded to the OH group of the cellulose of paper act as stationary phase.
The paper strip act as supporting agent.
Mobile phase is a solvent or mixture, but its polarity is less than H2O.
Partition of the solute occurs between the H2O in stationary phase & the solvent in the mobile phase.
In paper chromatography a filter paper strip of 2 – 3 cm wide & 15 – 20 cm long is suspended in chromatographic tank.
A drop of mixture to be analysed is placed (2 – 2.5cm) from the edge of paper by using a capillary tube.
The developing solvent is usually a mixture of organic liquids placed in chromatographic tank.
The solvent move upward by capillary action when solvent front rises about ¾ of the length of the paper. Then remove the paper strip from the chromatographic tank. You will observed that every component of the mixture gives coloured spot at different position on the paper strip.
Measure the distance covered by solvent front from the spot. Also measure the distance covered by each components.
Retention factor or ratio of front (Rf value) of each component is calculated by the following formula.
Rf = Distance covered by component/ divided by
Distance covered by the solvent
Retention factor is the ratio of distance traveled by a component to the distance traveled by solvent front.
The finished filter paper showing the various coloured spot and measurement is called chromatogram.
For colourless component some locating agent is sprayed, which give colour to the colourless spot. Such reagents are called locating agent. Examples are
(1)Strong ammonia solution (2) 0.1% alcoholic solution of robeanic acid (3)H2S–gas etc.
Application of Chromatography:-
(1) It is used for separation, identification and purification of substances from a mixture.
(2) It is used for qualitative and quantitative analysis.
(3) It is useful in the separation of coloured pigments.
(4) Substances produced by body like urine can also be analysed by chromatography.
(5) Medicine and protein which are complicated molecules can be identified.
(6) Separation of inorganic cations and radioisotopes.