CHROMATOGRAPHY

CHROMATOGRAPHY

Chromatography is an analytical technique, used for the separation, puri­fication and identification of the different chemical constituents of complex mixtures or solutions. It is also used for the separation of the different com­ponents of cytosol, after centrifugation

The first chromatographic separation was accomplished by the Russian Botanist Mikhail Tsewett (1906) for the separation of chlorophylls and other pigments from plant extracts. Accordingly, he named the technique chromatography, meaning 'coloured writing' (Gk. chroma = colour, graphein = to write).

Basic principle of chromatography

Chromatography relies on the phenomenon of differential distribution of the constituents of a mixture or solution between two immiscible phases due to their selective adsorption and differential migration. It requires a propel­ling force for moving the molecules, and a selective impedence for slowing down the movement of selected molecules. The propelling force is provided by a liquid or a gas, pumped through a column that contains the sample mixture. Selective impedence is provided by an insoluble ad­sorbent medium, which has different affinity for the molecules of different substances. So, when the mixture is allowed to percolate in an absorbent medium, different kinds of molecules move at different speeds and rates. This brings about their separation.

The sample to be analysed chromatographically is called solute. It is al­lowed to interact with two physically different and mutually immiscible phases, namely mobile phase and stationary phase. The former may be a gas or a liquid, and the latter a solid or a liquid. Stationary phase is contained in a region called sorbent. It has the ability to "bind" some types of solutes. It may be different in different types of chromatographic techniques. Mobile phase moves the solute through the sorbent. During this, the molecular components of the solute get distributed between the mobile and stationary phases. Some of them have high affinity for the stationary phase. So, they preferentially bind or interact with the stationary phase and spend more time with it. In other words, they get retarded in their movement through the chromatographic system. The other components, having only low affinity for the stationary phase, spend more time with the mobile phase. So, they get rapidly removed from the chromatographic system. This is called elu­sion. The differential interaction between the solute molecules and the sta­tionary phase brings about the separation of the different kinds of molecules from each other. The movement of the solute mixture through the chromato­graphic system is called development. The mobile phase, collected at the end of the system after elusion, is called effluent.

Types of chromatography

Chromatographic techniques are highly varied and are variously classi­fied. The most basic classification is to classify them on the basis of the nature of the mobile and stationary phases. Accordingly, there are four kinds of chromatography, namely (i) gas-liquid chromatography(GLC) in which mobile phase is a gas, and stationary phase is a liquid (ii) gas-solid chromatography (GSC) in which mobile phase is a gas and stationary phase is solid (iii) liquid-liquid chromatography (LLC) in which both the phases are liquids and (iv) liquid - solid chromatography (LSC) in which mobile phase is a liquid and stationary phase a solid.

An alternative scheme is based on the operational technique. It recog­nizes three types of chromatography, namely column chromatography, pa­per chromatography and thin-layer chromatography (TLC). In the first one an adsorbent column is used, in the second one a stationary liquid held on a paper strip is used, and in the third one a stationary liquid film, held on an adsorbent coating on a glass plate is used.

Column chromatography

This is the original and the simplest form of chromatography. In fact, it is a type of adsorption chromatography in which an inert adsorbent (such as calcium carbonate, aluminum oxide, cellulose, etc.), packed in a vertical glass tube or steel tube, forms the stationary phase. The molecular mixture to be analysed is dissolved in a suitable solvent (petroleum, ether, benzene, ac­etone, etc.) and then poured down and continuously washed through the column This is called elusion. Different components of the mixture adsorb on the column differently and drain down at different rates, depending upon their adsorption-desorption property. Consequently, they separate into dis­tinct bands. The most readily adsorbed molecules form the nearest band, and the least adsorbed ones form the farthest (lowest) band. LLC and LSC are examples of column chromatography.

Paper chromatography

This is a special type of liquid-liquid partition chromatography. In this, stationary phase is a film of immobilized water, adsorbed on a special type of paper mat or filter paper, usually made of highly purified cellulose. The paper mat serves as an inert support for the stationary phase. The mobile phase is an organic solvent (e.g., butanol, acetic acid) percolating over the stationary phase.

The apparatus for paper chromatography consists of a support for the chromatographic paper, a solvent trough and an air-tight glass cylinder. The sample solution to be analysed is spotted near the lower edge of the paper and then dried. The paper is then suspended in the glass cylinder with its lower end just dipping in a suitable solvent chosen as the mobile phase. The spotted points on the paper must always remain above the solvent level. The solvent ascends through the paper by capillary action, carrying along with it the various components of the sample. The components move at different rates on account of their differential distribution between the two immiscible liquid phases. After some time the paper strip is removed and dried. Coloured components form a number of coloured spots on the paper. Colourless com­ponents can be located by spraying a reagent which can form coloured com­plexes with them.