Column chromatography is the prototype or the basic type of chromatography.
It was the first form of techniques developed in chromatography.
One can easily explain the principle and procedure of chromatography using it.
Other types of chromatography techniques like high performance liquid chromatography (HPLC), gas chromatography (GC), paper chromatography were developed with column chromatography as a module and making slight variations.
In-spite of many advanced methods of chromatography, still this method of chromatography is widely used in research and industry.
This 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 polar solid material packed in a vertical column made of glass or metal.
Column Chromatography 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.
Column Chromatography Procedure
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.
The mobile phase percolates through entire stationary phase reaches the bottom of the column. From there it is elutes out and gets collected in the beaker placed below.
This elution is drop by drop and the process may take few hours to days based on the sample size, length of the column, mobile phase used and the packing material used.
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 Applications
♦ Column chromatography is best suited to separate active principle from plant materials. Since plants contain many ingredients like alkaloids, resins, glycosides, tannins, flavonoids and other bio-molecules, the individual constituents are to be separated. Since the plant extract is bulk this method is best to separate them.
♦ In separation of compounds after organic synthesis to obtain desired molecule.
♦ To separate or purify natural compound mixtures like alkaloids, glycosides.
Reference: column chromatography basics.