Glass, aluminum or plastic sheets are used as supports for the thin film. 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 constitute 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 is not compared to that obtained using the other variants of this technique.
Characteristics of thin layer chromatography
Thin layer chromatography is basically an analytical method. Therefore, very small amounts of the substances to be analyzed are used.
Thin-layer chromatography sheets have a rigid support made 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 sheet is placed vertically in the chromatography chamber, resting on its base. The height of the solvents in the chromatography chamber is usually 1 cm.
Thin layer chromatography consists of a stationary phase and a mobile phase. The stationary phase is made up of the material used to carry out 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 silica gel (polar) is used as the stationary phase, the polar substances interact with it and achieve a small shift during chromatography. Meanwhile, nonpolar substances will have a much greater displacement during chromatography as they do not interact effectively with the silica gel.
Development and analysis of the result
Chromatography is concluded when the solvent reaches a suitable 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, use of ultraviolet light on phosphor-treated slides, or subjecting slides with iodine vapors. , etc.
In the identification and characterization of the different substances the so-called retention factor (RF) is used. RF = distance reached by a substance / the distance reached by the solvent.
The RF value is specific to each substance for a certain mixture of solvents of the mobile phase, as well as the type of stationary phase.
What is thin layer chromatography 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 know the degree of development of an organic chemistry reaction, establishing the presence of reagents and products in different stages of chromatography.
The solvent mixture is placed in the chromatography chamber, using a volume such that its height reaches approximately 1 cm.
Before starting the chromatography, it is advisable to close the chamber hermetically and allow the solvent vapors to saturate the air in it.
A line is made with a graphite pencil at a height of 1.5 cm from one end of the chromatography sheet. The samples to be used in the chromatography are placed on the streak by means of a glass capillary.
Subsequently, the chromatography sheet with the samples to be analyzed is placed in the chamber and this is closed with the placement of the glass lid.
The rise of the solvent mixture is observed until the solvent front reaches a height approximately 2 cm lower than the height of the chromatography sheet. The chromatography sheet is then removed from the chamber and the solvent front is marked on it with a line.
The sheet is placed in an oven for drying and the substances present in the chromatography are analyzed using a general visualization method or one specific to the chromatography material.
The Rf values are obtained for the different substances present, and based on the chromatography controls, as well as the literature in this regard, the substances are identified.
The following video summarizes in a simple way what is explained here:
Thin-layer chromatography enables the following analyzes:
-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 greatly increased the potential of thin layer chromatography by automating its use. For example: in the placement of the sample, its development and in the analysis.
This has made HPTLC one of the most used methods in the analysis carried out in the areas of the pharmaceutical industry, biochemistry, cosmetology, food, drugs and in the environment.