Differential extraction: When the organic compound is present in an aqueous medium, then it is separated by shaking it with an organic solvent in which it is more soluble than in water .
- The organic solvent and the aqueous solution should be immiscible with each other .So, that they form two distinct layers which can be separated with separating funnel.
- The organic solvent is later removed by distillation or by evaporation.
Chromatography: It is a technique used to separate mixtures in to their components, purify. Compounds which can be solid or liquid and also test the purity of compounds.
- In this technique the mixture of substances are applied on to a stationary phase .Then a pure solvent or a mixture of solvents is allowed to move slowly over stationary phase .The components of mixture gets gradually separated from one another .The moving phase is mobile phase .
The chromatography is classified into two categories:
- Adsorption chromatography
- Partition chromatography
Adsorption chromatography: It is based on the facts that different compounds are adsorbed on adsorbent at different degrees.
The commonly used adsorbents are silica gel and alumina.
- When a mobile phase is allowed to move over a stationary phase the components of mixture move by varying distances over a stationary phase.
Depending upon differential adsorption the two types of techniques are:
- Column chromatography
- Thin layer chromatography
Column chromatography: It involves separation of mixture over a column of adsorbent packed in a glass tube.
- The column is fitted with a stop cock at its lower end .The mixture adsorbed on adsorbent is placed on the top of the adsorbent column packed in a glass tube.
- An appropriate eluant which is liquid or a mixture of liquids is allowed to flow down the column slowly.
- Depending upon the degree to which the compounds are adsorbed complete separation takes place.
- The most readily adsorbed substance is retained near the top and the other comes down to various distances in the column.
Thin layer chromatography: This is another type of adsorption which involves separation of substances of mixture over a thin layer of adsorbent coated on glass tube.
- A thin layer of adsorbent is spread over a glass plate of suitable size. The plate is known as thin layer chromatography plate.
- The solution of mixture to be separated is applied as a small spot about 2 cm above one end of the TLC plate.
- Then glass plate is then placed in a closed jar containing the eluant.
- As the eluant rises up ,the plate the different components of mixture move along with eluant to different distances depending on their degree of adsorption .Hence, separation takes place .
- The relative adsorption of each component of mixture is expressed in terms of retardation factor (Rf).
- The spots of the compounds are visible on TLC plate due to their original color. The spots which are not visible to eye but fluoresce in ultraviolet light can be detected by putting the plate under ultra violet light.
Position chromatography: It is based on the continuous differential partitioning of components of a mixture between stationary and mobile phases.
- Paper chromatography: Is a type of partition chromatography .In this a special paper called chromatography paper is used .It contains water trapped in it which acts as a stationary phase.
(i)In this we take a thin strip of filter paper (25cm x 5cm approx).
(ii) Draw a line on it using a pencil, approximately 3cm above the lower edge.
(iii) Put a small drop of solution of mixture .Lets say ink from a sketch pen or fountain pen) at the centre of the line. Let the ink dry.
(iv) Attach the paper strip on the thread with the help of cello tape.
(v) Lower the filter paper strip into a large size gas jar in such a way that the drop of ink on the paper is just above the water level as shown in the figure [2.8 (b)]. Adjust the thread and fix it on the sides of gas jar with the help of cello tape.
(vi)Cover the gas jar with a lid and leave it undisturbed.
(vii) Watch carefully as the water rises up on the filter paper.
(viii) Remove the filter paper strip dry it and observe.
The paper retains different components according to their differing partition in the two phases .the paper strip is called chromatogram. The spots of separated coloured compounds are visible at different heights from the position of initial spot.