The technique used in the laboratory for the separation of mixtures is called chromatography. The mixtures are usually dissolved in a fluid solvent like gas or liquid.
This technique has two phases. The stationary phase is one that is fixed and the mobile phase which moves over stationary phase and maybe gas or liquid.
The chromatography having solid stationary phase is adsorption chromatography and one that has a liquid stationary phase is partition chromatography.
For accurate and error-free analysis of food, drugs, chemicals, water samples chromatography is very helpful.
The word chromatography stems from the Greek word “Khromatos” meaning colour writing.
Chromatography is a technique utilized mostly for the separation of a sample of the mixture. It includes the distribution of a solute in between a stationary phase and a mobile phase. The fixed phase might be a solid or a liquid supported as a thin film on the surface of an inert solid. The mobile phase streaming over the surface of the fixed phase may be a gas or a liquid.
In chromatography, compounds are separated due to their relative affinities for the fixed and mobile phases. The distribution of the elements of a mix between the two phases is governed by circulation coefficient K.
K = Concentration of a component in the moving phase/ Concentration of that element in the Fixed phase
The part of a mixture with a small value of K primarily stays in the stationary phase as the moving phase streams over it. The part with a higher value of K remains largely dissolved in the mobile phase and passes over the fixed phase rapidly. Chromatography in which the stationary phase is solid is classified as adsorption chromatography. In this type, a substance leaves the mobile phase to end up being adsorbed on the surface of the solid phase. Chromatography in which the fixed phase is a liquid is called partition chromatography. In this type, the substances being separated are distributed throughout both the fixed and mobile phases.
It is a type of partition chromatography. Here the fixed phase is a liquid (say H2O) adsorbed on paper. The adsorbed water acts as an immiscible liquid towards the mobile phase, which passes over the paper. The mobile phase is normally an organic liquid. There are three typical methods of performing paper chromatography namely (i) ascending (ii) descending (iii) radial/circular. Just the ascending type will be discussed.
In this method, the solvent is in a pool at the bottom of a vessel in which the paper is supported and the solvent travels upwards by capillary action.
A solvent mixture especially made up in accordance with the sample to be separated, is poured into the chromatographic tank. Cover the tank to homogenize its inner environment. Take about 20 cm strip of Whatmann’s chromatographic paper No. 1 and draw on it a thin pencil line about 2.5 cm from one end. Spot a point, on the pencil line, with the sample mixture solution. To facilitate identification of the components of the mixture, spots of the known compounds may also be placed alongside.
When the areas have dried, suspend the paper with clips so that the impregnated end dips into the solvent mixture to a depth of 5-6 mm. Cover the tank. As the solvent front passes the spots, the solutes start to move upward. The rate at which they move depends on their circulation coefficients. When the solvent front has actually risen to about 3/4th of the length of the paper, remove the strip, mark the solvent front with a pencil and allow the strip to dry.
Once the paper is dried, the pattern on the paper is called a chromatogram.
The different components of the mix, if coloured, can aesthetically be identified. If colourless, the chromatogram needs to be established by chemical techniques or physical strategies used to determine the spots. Each component has a specific retardation factor called Rf value. The Rf value is connected to its circulation coefficient and is given by:
Rf= Distance travelled by a component from the original spot / Distance travelled by solvent from the original spot
The Rf values for B and C are given by:
Rf (C)= z/y
Uses of Chromatography
The methods of chromatography are really useful in natural synthesis for separation, seclusion and purification of the products. They are similarly crucial in qualitative and quantitative analyses and for determination of the purity of a substance.