Aim
To identify, purify and/or separate constituents of a mixture that are present in very small amounts.
Principle
The principle behind this technique is the differential adsorption of the various components of a mixture between two different phases that are as follows:
Fixed or stationary phase
The adsorbent is termed as the stationary phase. Suitable adsorbents that are commonly used are magnesium oxide, alumina, cellulose, paper, silica gel etc.
Mobile or moving phase
The liquid in which the substance is dissolved is termed as the mobile phase or eluent. The eluents employed are petroleum ether, carbon tetrachloride, benzene, alcohol etc. Their selection depends upon the relative solubilities of the components of the mixture in them.
There are various types of chromatographic techniques depending upon the nature of the two phases.
Commonly Employed Chromatographic Techniques
Column chromatography
This is the simplest chromatography based on the differential adsorption of the constituents of a mixture. A suitable adsorbent like alumina (Al2O3), taken in the form of a slurry in petroleum ether, constitutes the stationary phase. It is packed in a column prepared in a long burette-like glass tube with a stop-cock near the bottom. A plug of cotton or glass wool is placed at the bottom of the column to support the adsorbent. One-fourth of the tube is left empty. A loose plug of cotton or glass wool is then placed at the top of the adsorbent column.
Fig: 16.8 Column chromatography
Process
The mixture to be separated is taken in a suitable solvent and the solution is poured on the top of the column of the adsorbent. It is allowed to pass slowly through it and as it passes through the column the different constituents of the mixture get adsorbed to different extent, forming bands in different parts of the column. The components are then eluted out by a suitable solvent (known as eluent and which acts as a mobile phase). More than one eluent may be used in certain cases because the eluents dissolve different compounds selectively.
Fig: 16.9 I, II and III show the progressive separation of the components a, b and c of a mixture with a solvent
The weakly adsorbed component will be eluted more rapidly than a more strongly adsorbed component. One may see separate bands in the column formed by different compounds of the mixture, if they are colored. Different components of the mixture are collected in the form of different fractions in separate conical flasks and distilled to get them in a pure form.Paper chromatography
Paper chromatography is the analysis of unknown substances by the flow of solvent on a filter paper.
Principle
This technique proceeds by a partly partition (distribution) and partly adsorption mechanism. The constituents of the mixture are distributed between the water held on the filter paper (water acts as a stationary phase) and organic solvent (mobile phase).
Procedure
In this procedure, a drop of the test solution is applied as a small spot near one edge of the filter paper and the spot is dried. Then, the end of the paper strip is dipped into a developing solvent. The liquid is sucked up through the capillaries of the paper; it reaches the mixture and removes its components at various speeds.
After the solvent front has reached a suitable height (15-20 cm), the paper is dried. The separated components are made visible with a suitable reagent called 'visualization reagent'.The movement of any compound relative to the solvent is determined by the measurement of RF values, viz.,
Then,
The symbol RF which is used to denote this ratio stands for the ratio of fronts (or Retention Factor). The RF value of a particular substance is characteristic of that substance and signifies the movement of the substance relative to the solvent front in a given chromatographic system.
Ascending and Descending Paper Chromatography
Depending upon the direction of the solvent travel, paper chromatography is termed as ascending or descending type. When the solvent travels upward on the paper, it is termed as ascending paper chromatography and descending paper chromatography when the solvent travels in the downward direction.
Fig: 16.11 An experimental setup for paper chromatography
Radial paper chromatography
In this method, a circular filter paper is taken and a cut is made in its center to yield a wick or tongue. The spot of the material is placed at the center of the circular filter paper. The paper is positioned horizontally on the petri-dish containing the solvent so that the tongue or the wick of the paper dips into the solvent. The paper is covered by another petri-dish. The solvent rises through the tongue (or wick) and percolates evenly through the horizontally positioned filter paper and as development proceeds, the components are separated in the form of concentric circular zones. Hence, this radial paper chromatography is also known as circular paper chromatography. This technique is very useful for substances having low RF values.
Fig:16.12 Radial or circular paper chromatography
Thin layer chromatography
In this chromatography, the stationary phase is a thin layer of an adsorbent (generally alumina) coated on a flat glass strip. The solvent (mobile phase) moves up the layer due to the capillary action and thus causes the separation of constituents of the mixture. The constituents are identified by measuring their RF values.
