por The Thin layer chromatography It is a method that allows the separation and identification of substances present in a mixture. The so-called ‘thin layer’ is made up of silica gel (silicon oxide), alumina (aluminum oxide) and cellulose. However, other preparations can be used for certain applications.
The following are used as supports for the thin layer: sheets of glass, aluminum or plastic material. The material that forms the thin layer serves as the stationary phase of the chromatography, that is, the one that remains fixed; while the solvent or the mixture of solvents used constitutes the mobile phase, which moves with the substances to be separated.
Paper chromatography follows the same principle as thin layer chromatography. The paper becomes the «fine» stationary phase, whose resolution does not compare with that obtained using the other variants of this technique.
The distance reached by substances in a thin layer chromatography (see colored spots) depends on the polarity of the stationary phase, the polarity of the solvent (mobile phase) and the polarity of the substances.
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Characteristics of thin layer chromatography
Generalities
Thin layer chromatography is basically an analytical method. Therefore, very small amounts of the substances to be analyzed are used.
Thin-layer chromatography slides have a rigid support of glass, aluminum, or cellulose material, on which the material used for the stationary phase is placed. This depends on the objectives to be achieved with the chromatography.
Among the materials used in thin layer chromatography are: silica gel or silica gel, alumina (aluminum oxide), cellulose or magnesium silicate.
The chromatography chamber is made up of a beaker or a glass cylinder, to which a glass lid is placed that closes the chamber hermetically, thus preventing the escape of the vapors of the solvents used in the chromatography.
The chromatography slide is placed vertically in the chromatography chamber, resting on its base. The height of the solvents in the chromatography chamber is usually 1 cm.
Development
Thin layer chromatography consists of a stationary phase and a mobile phase. The stationary phase is made up of the material used to perform the chromatography, for example, silica gel. This material is polar and is used, among other uses, in the analysis of steroids and amino acids.
The mobile phase is made up of a mixture of solvents, generally volatile and organic in nature. A widely used solvent mixture is ethyl acetate and hexane.
The solvent rises by capillarity over the stationary phase, establishing a competition between the substances subjected to chromatography and the solvents of the mobile phase for sites in the stationary phase.
If (polar) silica gel is used as the stationary phase, polar substances interact with it and achieve a small displacement during chromatography. Meanwhile, non-polar substances will have a much higher displacement during chromatography as they do not effectively interact with the silica gel.
Revealed and analysis of the result
The chromatography is concluded when the solvent reaches an appropriate height that does not reach the height of the chromatography sheet. The chromatography slide is removed from the chamber and the solvent front is marked with a line.
The location of substances on thin layer chromatography can be visualized by several methods, including: direct visualization with ultraviolet light, the use of ultraviolet light on phosphor-treated slides, or iodine fuming of slides. , etc.
In the identification and characterization of the different substances, the so-called retention factor (RF) is used. RF = distance traveled by a substance / the distance traveled by the solvent.
The RF value is specific to each substance for a given mixture of mobile phase solvents, as well as for the type of stationary phase.
What is thin layer chromatography used for?
Thin layer chromatography is used to identify the different substances that are part of a mixture. For example: you can know the free amino acids present in milk or other material.
Thin layer chromatography can identify the type of lipids present in a food. It is also used to determine the degree of development of an organic chemistry reaction, establishing the presence of reactants and products at different stages of chromatography.
Procedure
First step
The solvent mixture is placed in the chromatography chamber, using a volume such that its height reaches approximately 1 cm.
Second step
It is convenient before starting the chromatography, to close the chamber hermetically and let the solvent vapors saturate the air from it.
Third step
A line is made with a graphite pencil at a height of 1.5 cm from one end of the chromatography slide. The samples to be used in the chromatography are placed on the line through a glass capillary.
Fourth step
Subsequently, the chromatography sheet with the samples to be analyzed is placed in the chamber and it is closed by placing the glass lid.
Fifth step
The solvent mixture is observed to rise until the solvent front reaches a height of approximately 2 cm below the height of the chromatography slide. The chromatography slide is then removed from the chamber and the solvent front is marked on it with a line.
sixth step
The slide is placed in an oven for drying and the substances present in the chromatography are analyzed using a general visualization method or a specific one for the chromatography material.
Seventh step
The Rf values for the different substances present are obtained, and based on the chromatography controls, as well as the bibliography in this regard, the identification of the substances is carried out.
The following video summarizes in a simple way what is explained here:
Applications
Thin layer chromatography allows the following analyses:
-Components of a drug
-Presence of different metabolites in body fluids
-Determination of the purity of a given substance
-Identification of colorants, flavorings and sweeteners in the food industry
-Determination of the state of development of an organic chemistry reaction
The development of the HPTLC (High Performance Thin Layer Chromatography) method has considerably increased the potential of thin layer chromatography by automating its use. For example: in the placement of the sample, development of it and in the analysis.
This has made HPTLC one of the most widely used methods in analyzes carried out in the areas of the pharmaceutical, biochemical, cosmetology, food, drug and environmental industries.
References
Whitten, Davis, Peck & Stanley. (2008). Chemistry. (8th ed.). CENGAGE Learning.
Wikipedia. (2020). Thin layer chromatography. Retrieved from: en.wikipedia.org
Jim Clark. (2019). Thin layer chromatography. Retrieved from: chemguide.co.uk
Chemistry LibreTexts. (December 5, 2019). Thin layer chromatography. Retrieved from: chem.libretexts.org
Attimarad, M., Ahmed, KK, Aldhubaib, BE, & Harsha, S. (2011). High-performance thin layer chromatography: A powerful analytical technique in pharmaceutical drug discovery. Pharmaceutical methods, 2(2), 71–75. doi.org/10.4103/2229-4708.84436
The Editors of Encyclopaedia Britannica. (2020). Thin layer chromatography. Recovered from: britannica.com