Column chromatography is a useful separation technique in organic chemistry. The separation follows same principles as TLC, but is applied to separate larger quantities than TLC. The application of this technique is seen in many disciplines such as biology, biochemistry, microbiology and medicine.
As we know, chromatography consists of stationary phase and mobile phase. Alumina and silica are the two mostly used stationary phases in column chromatography. The molar polar sample will be retained on the stationary phase for a longer time. The least polar compound will elute from the column first, followed by each compound in order of increasing polarity. The order of elution should be decided very carefully. There are various factors which affect the separation such as type of absorbent, the size of column, the rate of elution as well as the polarity of the mobile phase.
The solvents used as mobile phase can be determined from the TLC experiments, the literature, or experimentally. Firstly, a non - polar or low polarity solvent is used which allows the compounds to adsorb on the stationary phase. Then the polarity of the solvent is increased to desorb the compounds and allow them to flow with the mobile phase. There are some solvent combinations such as hexane-ethyl acetate, hexane-toluene and ligroin-dichloromethane.
The size of the column can be thin as a pencil to a diameter of several feet in industrial processes. They can separate milligram to kilogram quantities of materials. There are different methods for packing a column. One is dry packing and the other is slurry method. In the slurry method, the column is filled with solvent and then the solid is poured into the column. In the dry method, the solid is filled in the column and then the solvent is poured over the solid which reduces the chance of channeling. The slurry method achieves the best packing results. Dry packing is best for a microscale column and is usually used for alumina as silica gel expands and does not pack well with this method.
The sample can be directly loaded to the top of the column once the packing is complete. Normally, the sample is dissolved in a polar solvent, 5-10 drops before introducing into the column.