Chromatography is a laboratory technique, used for analyzing
mixtures of colored chemicals. This technique 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 a chromatography 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 chromatography method is the separation of component molecules by distributing them into the two phases.
Chromatography method was invented by a Russian botanist, Mikhail Semyonovich Tsvet, in 1901, while researching on plant pigments. After him, many scientists have developed several types of chromatography following the same principle.
An Overview of Thin Layer Chromatography
Chromatography methods are classified, based on the medium of the stationary and mobile phases, and the mechanism of separation. Among the many types of chromatography, 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. There are also many choices of the stationary phase, in case of TLC, like alumina and silica.
Stationary and Mobile Phases
In thin layer chromatography, the stationary phase is made up of a thin layer of absorbent, coated on an unreactive plate of glass, metal or plastic. In general, silica gel or alumina is used as the stationary phase. Nowadays, TLC plates with standard particle size are available commercially. Depending upon the usage of TLC, 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.
Technique
The technique of thin layer chromatography is very simple. First, 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 the mobile 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 reaches to almost 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. Retention factor is 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.
Thin layer chromatography, due to its fast and reliable separation, is used in detecting and identifying chemicals, which are of forensic concern. Hence, TLC is commonly followed in forensic science laboratories, clinical research and chemical manufacturing industries
Read more at Buzzle: http://www.buzzle.com/articles/thin-layer-chromatography.html
There are two phases in a chromatography 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 chromatography method is the separation of component molecules by distributing them into the two phases.
Chromatography method was invented by a Russian botanist, Mikhail Semyonovich Tsvet, in 1901, while researching on plant pigments. After him, many scientists have developed several types of chromatography following the same principle.
An Overview of Thin Layer Chromatography
Chromatography methods are classified, based on the medium of the stationary and mobile phases, and the mechanism of separation. Among the many types of chromatography, 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. There are also many choices of the stationary phase, in case of TLC, like alumina and silica.
Stationary and Mobile Phases
In thin layer chromatography, the stationary phase is made up of a thin layer of absorbent, coated on an unreactive plate of glass, metal or plastic. In general, silica gel or alumina is used as the stationary phase. Nowadays, TLC plates with standard particle size are available commercially. Depending upon the usage of TLC, 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.
Technique
The technique of thin layer chromatography is very simple. First, 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 the mobile 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 reaches to almost 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. Retention factor is 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.
Thin layer chromatography, due to its fast and reliable separation, is used in detecting and identifying chemicals, which are of forensic concern. Hence, TLC is commonly followed in forensic science laboratories, clinical research and chemical manufacturing industries
Read more at Buzzle: http://www.buzzle.com/articles/thin-layer-chromatography.html
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