Column Chromatography| Principle, Procedure & Applications

Column chromatography is the prototype of chromatography.

It was the basic form of chromatography developed initially.

Many modifications and improvements were made to column chromatography to derive advanced chromatography techniques.

The advanced forms of column chromatography are high-performance liquid chromatography (HPLC), Ultra Performance Liquid Chromatography (UPLC), gas chromatography (GC), etc.

One can easily demonstrate the principle and procedure of chromatography using it.

Despite many advanced methods of chromatography, still, this model of chromatography is widely used in science, research, and industry.

This chromatography is 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. column chromatographyThose 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

Column chromatography instrument

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 (mixture column) reaches the bottom of the column. From there it is elutes out and gets collected in the beaker placed below.

When the mobile phase flows through, different components of the sample travel with different rates through the silica gel. This rate of travel is decided by the adsorption and affinity of molecules towards the stationary phase and mobile phase.

The fractional components of the mixture with greater affinity to mobile phase travels fast and reach the bottom early. Those with higher affinity to stationary phase travel slow and reach bottom late.

Thus the colored bands of the sample are formed.

Each color is an indicator of one particular set of compound in the sample mixture.

Then by differential mobile phase different components are taken out of column by further flow of solvents.

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.

Precautions:

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.

Also see HPLC chromatography.

Reference:  column chromatography basics.

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