chromatography
Chromatography
Chromatography is an analytical technique employed for the purification and separation of organic and inorganic substances. It is also very helpful for the fractionation of complex mixtures, separation of closely related compounds such as isomers, homologues as well as in the isolation of unstable compounds. In this technique the compounds are recovered without any change or alteration.
Classification: Chromatography can be classified into following types:
(1) Paper Chromatography
(2) Column Chromatography
(3) Gas Chromatography
(4) Ion-Exchange Chromatography
1. Paper Chromatography: (Liquid-liquid chromatography): The principle of paper chromatography is based on the fact that solutes have the capacity to migrate through filter paper at different rates as a solution is drawn into strip of paper by capillary action.
In paper chromatography the dissolved substance is applied as a small spot about 2-3 cm. from the edge of a strip.
paper chromotography
or square of filter paper and is allowed to dry. This strip is then suspended in a large close container where atmosphere is saturated with the solvent system whereas the end containing the sample is dipped into the mobile phase which has already been saturated with stationary phase. When the solvent front has reached the other end of the paper, the strip is removed and the zones are located by analytical methods.
However in descending order chromatography as shown in diagram given above the top end of the paper is dipped into the solvent and hung over an antisyphon rod while in ascending order method the lower end of paper is dipped directly into the solvent in the bottom in the container.
Precautions in Paper Chromatography
1. Heavy metals and complexing agents should not be used as they convert the solute into a complex with different properties resulting in multiple spots.
2. Neutral salts should not be used as they disturb the cellulose water complex which will result in separation of liquid water on paper.
2. Column Chromatography (Liquid partition chromatography):
The liquid partition chromatography consists of a glass tube at the bottom to retain the packing and permit the liquid to pass through. Inert silica or celite is mixed with 5—30% of the stationary phase and packed into column as a slury in mobile phase. Now the solvent is added until all the sample has passed from the column. The different fractions obtained are analysed by different suitable methods such as spectrophotometry, by changes in refractive index etc.
Thin Layer Chromatography
This is a special type of column chromatography and now-a-days it is generally used for the quantitative determination of the radioactive substances, separation of amino acids, fatty acids, mineral oils, glycerides and phenols. It is also used for the separation of essential oils and fats.
Experimental technique
In the experimental procedure, a glass plate is taken on which a solution consisting of the homogeneous mixture of the adsorbing agent with water is applied in a thin and uniform layer. Such pasted plates are then dried in open at 383 K for about 30-40 minutes. The sample to be analysed is spotted on the plate and the plate is placed vertically in the developing trough containing suitable solvent. After about 30-40 minutes the plate is taken out from the trough and dried in air. The substance becomes visible in ultra-violet light or turned to be visible by a colour reaction and hence the mixtures present in the same are detected.
3. Gas Chromatography
Gas chromatography is now-a-days used as an important analytical tool for the separation of gases and volatile substances.
Gas chromatography is defined as the method of separation in which gaseous or the vaporized components to be separated are distributed between two phases, a fixed stationary phase with large surface area and a moving gas phase.
Gas-solid chromatography is defined as a method of separation in which fixed phase is a solid adsorbent.
Gas-liquid chromatography is defined as a method of separation which has a fixed phase, a liquid distributed in an inert support. This is also known as vapour-chromatography.
Separation of constituents by gas-solid chromatography and gas-liquid chromatography involve the following technique:
(a) Flution Analysis
(b) Displacement Analysis
(c) Frontal Analysis.
(a) Flution Analysis: In this technique the sample is introduced into the column and is carried through the column by inert gas like helium or nitrogen. In gas-solid chromatography the constituents of the sample are selectively retarded by solid adsorbent while in gas-liquid chromatography the constituents are retarded by liquid phase.
(b) Displacement Analysis: Here the sample is introduced into the column and a vapour of the high adsorption capacity is used to displace the constituents of the sample from the column. The component which is least adsorbed emerges first followed by second least adsorbed and so on.
(c) Frontal Analysis: This analysis is generally used in industrial operations. In this technique the total sample is continuously fed into the column, the less adsorbed component passes through the column and continues to pass through until more strongly adsorbed constituents emerge.
4. Ion-Exchange Chromatography: If a mixture of two or more different cations X, Y etc. is added into an ion exchange column and suppose X is held more firmly by the exchange resin than cation, Y all the Y present will flow out of the bottom of the column before any of X is liberated, provided that the column is long enough and other experimental factors are favourable for the particular separation. This separation technique is called ion-exchange chromatography.
The term ion exchange is generally understood to mean the exchange of ions of like sign between a solution and a solid highly insoluble body in contact with it. The solid (ion exchanger) must contain ions of its own and must have an open, permeable molecular structure so that ions and solvent molecules can move freely in and out.
The process of removing adsorbed ions is known as elution, the solution employed for elution is known as eluant and the solution resulting from elution is called elutate.
Generally, ion exchangers are insoluble in water and in organic solvents. They contain active or counter ions that can exchange reversibly with other ions. In a cation exchanger the active ions are cations while in an anion exchanger the active ions are anions.