Column chromatography: principle, procedure & applications
Column chromatography is the actual or the basic type of chromatography procedure which was developed during early stages of chromatography discovery. From it the principle of chromatography is described and these are still followed in the most advanced modes of chromatography. Other types of chromatography methods were developed with column chromatography as a module and with slight variations.
Column chromatography is basically a type of adsorption chromatography techniques. Here the separation of components depends upon the extent of adsorption to stationary phase. Here the stationary phase is a solid material packed in a vertical column made of glass or metal.
Principle: When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates. Those with lower affinity and adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last.
The solute molecules adsorb to the column in a reversible manner. The rate of the movement of the components is given as follows
R= Rate of movement of a component / Rate of movement of mobile phase. i.e. it is the ratio of distance moved by solute to the distance moved by solvent.
Column chromatography animation
The column chromatography requires a vertical column (preferably glass column) with a knob at the bottom end. This is preferably a burette shaped cylindrical column without graduations or readings.
Stationary phase or adsorbent, mostly fine sand will do or that recommended for the experiment. The particles of stationary phase should be of uniform size and shape without contamination.
Mobile phase preferably solvents of chromatography grade either a single solvent or a mixture of solvents as required for the separation.
Cotton wool or asbestos pad to plug the exit of column at the bottom and there by hold the column of stationary phase and let only escape of solvent and sample.
The stationary phase material is suitably moistened with mobile phase and packed sufficiently in the column with a cotton or asbestos pad at the bottom. The extract material or sample to be separated is placed on the top of packed stationary phase with a second cotton or asbestos pad in between.
The mobile phase is poured into the column over the sample. A collecting beaker is placed at the bottom of column near the end to collect the elute.
1. Keep the column in a clean and dust free place.
2. Do not disturb the column till the separation is complete.
3. Avoid gaps within the stationary phase packing.
♦ Column chromatography is best suited to separate active principle from plant materials.
♦ In separation of compounds after organic synthesis to obtain desired molecule.
♦ To separate or purify natural compound mixtures like alkaloids, glycosides.
For more details refer to column chromatography basics.
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