Separation of black ink on a TLC plate |
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| Acronym | TLC |
|---|---|
| Classification | Chromatography |
| Other techniques | |
| Related | Agarose gel electrophoresis SDS-PAGE |
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.[1] Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.[2]
Thin layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics , assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents [3]
A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantization. This method is referred to as HPTLC, or "high performance TLC".
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TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 – 2.0 mm for preparative TLC.[4]
The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.
To run a thin layer chromatography, the following procedure is carried out: [5]
Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column.[6]
Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. It is commonly said that "strong" solvents (elutants) push the analyzed compounds up the plate, while "weak" elutants barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated plates the order is reverse. Practically this means that if you use a mixture of ethyl acetate and hexane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
Once visible, the Rf value , or retardation factor, of each spot can be determined by dividing the distance the product traveled from the initial spotting site by the distance the solvent front traveled from the initial spotting site. These values depend on the solvent used, and the type of TLC plate, and are not physical constants. Eluent on the thin layer is put on top of the plate
In organic chemistry, reactions are qualitatively monitored with TLC. Spots sampled with a capillary tube are placed on the plate: a spot of starting material, a spot from the reaction mixture, and a "co-spot" with both. A small (3 by 7 cm) TLC plate takes a couple of minutes to run. The analysis is qualitative, and it will show if the starting material has disappeared, i.e. the reaction is complete, if any product has appeared, and how many products are generated (although this might be under-estimated due to co-elution). Unfortunately, TLCs from low-temperature reactions may give misleading results, because the sample is warmed to room temperature in the capillary, which can alter the reaction—the warmed sample analyzed by TLC is not the same as what is in the low-temperature flask. One such reaction is the DIBALH reduction of ester to aldehyde.
As an example the chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.
In one study TLC has been applied in the screening of organic reactions[8] for example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method the alcohol and catalyst solution (for instance iron(III) chloride) are placed separately on the base line, then reacted and then instantly analyzed.