Chromatography is one of the most useful and popular tools of biochemistry. It is an analytical technique dealing with the separation of closely related compounds from a mixture. These include proteins, peptides, amino acids, lipids, carbohydrates, vitamins and medicines.
Principles and classification
Chromatography usually consists of a mobile phase and a stationary phase. The mobile phase refers to the mixture of substances, dissolved in a liquid or a gas. The stationary phase is a porous solid matrix through which the sample contained in the mobile phase percolates. The interaction between the mobile and stationary phases results in the separation of the compounds from the mixture. These interactions include the physicochemical principles such as adsorption, partition, ion-exchange, molecular sieving and affinity.
The interaction between stationary phase and mobile phase is often employed in the classification chromatography e.g. partition, adsorption, ion exchange. Further, the classification of chromatography is also based either on the nature of the stationary phase, or on the nature of both mobile and stationary phases.
Partition chromatography
The molecules of a mixture get partitioned between the stationary phase and mobile phase depending on their relative affinity to each one of the phases.
Adsorption column chromatography
The adsorbents such as silica gel, alumina, charcoal powder and calcium hydroxyapatite are packed into a column in a glass tube. This serves as the stationary phase. The sample mixture in a solvent is loaded on this column. The individual components get differentially adsorbed on to the adsorbent. The elution is carried out by a buffer system. The individual compounds come out of the column at different rates which may be separately collected and identified. For instance, amino acids can be identified by ninhydrin colorimetric method. An automated column chromatography apparatus fraction collector is frequently used nowadays.