Chromatography Techniques

Chromatography Techniques

Chromatography techniques are used to separate a mixture of various chemicals into their individual forms. The process involves separation of a mixture, with the help of different mobile and stationary phases ...
Chromatography is the study of separation of molecules, based on differences in their composition and/or structure. It is the collective term used for a set of laboratory methods, used for the separation of mixtures.

It involves the preparation of a test solution, which will interact differently with the stationary support, thereby leading to separation of similar molecules. The molecules with tighter interactions with the stationary support will move slower than those molecules with weaker interactions. In this manner, different types of molecules in a solution can be separated.

The basic principle of chromatography is that different chemicals have different degrees of dissolving power in a liquid, and different powers of sticking to a solid surface. Thus, chromatography can identify chemical components in a mixture, and separate them by making them visible on a surface. Such separations using chromatography techniques can be done by using various supports like glass plates (thin layer chromatography), insoluble molecules (liquid chromatography), volatile gases (gas chromatography), etc.

Paper Chromatography
This technique involves the presence of a solvent that moves along blotting or filter paper. In this technique, the cellulose of the filter paper acts as an inert support. When a few drops of the mixture is applied to this filter paper and dipped into the container with water, the water begins to move upwards by capillary action. The water then spreads out the final color of the mixture into their constituent colors. Thus, interactions between the sample or mixture, water or solvent, and the filter paper brings about separation of the components. Paper chromatography is used to separate most colored compounds and is widely used in artificial and natural pigment research.

Thin Layer Chromatography (TLC)
This technique involves the use of an inert piece of glass, plastic, or metal. It is a simple and inexpensive technique used to judge the purity of a synthesized compound. It is also used to detect the extent of progress of a particular chemical reaction. A drop of solution to be analyzed is added to the TLC glass plate, which has already been coated with a thin layer of silica gel. This silica-coated glass plate acts as the stationary phase. The sheet is then placed in a glass chamber containing a solvent, which forms the mobile phase. Via capillary action, the solvent moves upwards and carries the molecules of the solution to be detected at different rates. The components of the solution will appear in the form of a series of spots at various locations on the plate. Calculations are made based on the ratio of the distance that the substance travels to the distance that the solvent travels up the plate.

Column Chromatography
This technique consists of a powdered adsorbent as the stationary phase, placed in a vertical glass column. The mixture to be analyzed is placed on top of this vertical glass column. The solvent forming the mobile phase is poured from the top of the column and flows down the column. In most cases, either silica (SiO2) or alumina (Al2O3) is added to the solvent which act as the stationary phase. As the solvent flows, the solvent elutes the sample through the column, and the components of the mixture begin to flow at their respective rates down the column. However, this technique needs large volumes of solvent and any careless additions can cause disturbance to the stationary phase, leading to poor separations.

Gas Chromatography
This technique involves the packing of a stationary phase in a long, narrow glass column. The stationary phase is a high-boiling liquid into which the mixture to be analyzed is loaded with the help of a syringe. An inert gas is used as the mobile phase, which flows through the column with the stationary phase. Once the gas is passed, the components of the mixture will begin to distribute between the stationary high-boiling liquid and the inert gas, and begin to reveal themselves in the form of different peaks on a recorder.

Besides these techniques, the other techniques used to separate compounds are as follows:
  • High pressure liquid chromatography
  • Ion exchange chromatography
  • Gel permeation chromatography
  • Hydrophobic interaction chromatography (HIC)
  • Affinity chromatography
Chromatography techniques are one of the most widely used chemical techniques for separation of contaminants from chemical plants. For example, chemical plants use these techniques for removal of pesticides from ground water. Moreover, pharmaceutical companies also use these techniques to prepare large quantities of pure compounds required to prepare medicines. Manufacturers also use these techniques to check the presence of any contaminants in their manufactured compounds.