Chromatography is a laboratory technique that is used for analyzing mixtures of colored chemicals. It involves separation of constituent elements of the mixture. Chromatography is derived from the Greek words, chroma meaning color, and graphein meaning to write. Thus, the word "chromatography" literally means color writing.
There are two phases in this technique, namely, the stationary phase and the mobile phase. The former refers to the column packing material, and is either solid or liquid, whereas the latter represents a mobile phase of liquid or gas. The basic principle of every method regarding chromatography is the separation of component molecules by distributing them into the two phases.
This method was invented in 1901, by a Russian botanist called Mikhail Semyonovich Tsvet, while researching on plant pigments. After him, many scientists have developed several types of chromatography following the same principle.
Chromatography methods are classified, based on the medium of the stationary and mobile phases, and the mechanism of separation. Among its many types, thin layer chromatography (TLC) is widely used, as it gives accurate results, and is easy to implement. The process of separation is similar to paper chromatography. However, TLC is advantageous due to its fast and better separation results. Also, there are many choices of the stationary phase, in case of TLC, like alumina and silica.
Stationary and Mobile Phases
The stationary phase is made up of a thin layer of absorbent, which is coated on an nonreactive plate of glass, metal, or plastic. Silica gel or alumina is mostly used as the stationary phase. Nowadays, TLC plates with standard particle sizes are available commercially. Depending upon its usage, the thickness of the absorbent layer varies. For analytical purposes, the recommended thickness is 0.1 - 0.2 mm., whereas for preparative usage, thickness should be about 1 - 2 mm. Regarding the mobile phase for TLC, hexane and/or ethyl acetate is commonly used, which is poured in a sealed chamber.
The technique of thin layer chromatography is very simple. Firstly, introduce a small spot of the sample (to be separated) on the TLC plate at about 1 cm. from the base. Then, put the plate into the chamber containing the mobile phase, and seal it. Make sure the plate touches this phase.
The solvent moves up the plate due to capillary action, and carries the sample upwards. Thus, the components of the sample mixture get separated, based on their attraction to the stationary phase, and the difference in the solubility in the mobile phase. Remove the TLC plate when the mobile phase almost reaches the top, and mark the level with a pencil. This level is called the solvent front. Allow the plate to dry, and mark the spots of varied colors. These spots represent the constituents of the mixture. In case the spots are not visible, you have to view the plate under UV light, or by spraying ninhydrin or iodine.
Calculation of Retention Factor (Rƒ)
For identification of the separated constituents of the mixture, retention factor (Rƒ) calculation is necessary. It is defined as the ratio of the distance traveled by the component to the distance traveled by the mobile phase.
For calculating Rƒ, measure the distance of the solvent front, and the distance traveled by each of the component molecules (take the center of the spot). Now, calculate the Rƒ values for all the component molecules, and compare with the Rƒ value table. This way, you can identify the constituents of the particular sample mixture.
Due to its fast and reliable separation, thin layer chromatography is used in detecting and identifying chemicals, which are of forensic concern. Hence, this technique is commonly followed in forensic science laboratories, clinical research, and chemical manufacturing industries.