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Applications of Thin Layer ChromatographyBY: Lorato Lekgari | Category: Biology | Submitted: 2010-07-26 17:20:45
Article Summary: "Thin Layer Chromatography has proved to be such a strong tool in the study of natural products. The application of TLC in analysis of phytochemicals is discussed in this article.."
Thin Layer Chromatography (TLC) is a widely used separation technique used in life sciences and chemistry studies. It is easy, cheap and gives rapid results. It is used to isolate and analyse mixtures of compounds. Like any other chromatography technique, it has a stationary and mobile phase.
Different kinds of stationary phases are used. These stationary phases are made of adsorbent material which is coated on glass, aluminium or plastic and they can be cellulose, silica gel or aluminium oxide. These are called TLC plates. The mobile phase is made of solvents that are very variable. The solvents come at different polarities which are; polar solvents e.g. water, methanol, medium-polar solvents e.g. ethyl acetate and dichloromethane, and non-polar solvents such as hexane and toluene. The samples to be separated or analysed are applied onto the TLC plate. The TLC plate is then placed in a chromatography tank that contains an appropriate solvent depending on what is being separated or targeted for separation. The solvent then rises up the TLC plate and the components of the sample get separated depending on how strongly they bind to the adsorbent and how they dissolve in the solvent used, thus they move to different heights on the plate.
Visualisation of Separated Compounds
The different separated components of the sample can be visualised in different ways. The most usual way to visualise separated molecules is via staining. There are broad spectrum sprays that can be used to visualise almost or most compounds. However there are different stains that can be used to visualise particular molecules. The sulphuric acid in methanol spray is a common broad spectrum stain and an example of a specialised spray is ninihydrin spray, which is used to visualise amino acids. Compounds can also be visualised using ultra violet light after separation. TLC plates are normally sprayed and then oven dried for 10-15 minutes at 100°C, to develop them.
Applications in Phytochemistry
TLC has applications in different field of study such as in the pharmaceutical industry, insecticides and pesticides industry, medicine and other industries. In this article, the different applications of TLC in botanical or plant studies will be looked at briefly. Plants contain a mixture of diverse metabolites, primary or secondary, which are used for different applications.
In traditional medicines, plant extracts have been used to treat different ailments and conditions rather successfully. This spiked scientific interest into finding out what is in these plants extracts that makes them to be effective in the treatment of the different ailments and conditions. TLC thus becomes a very powerful tool to do this. If samples from a plant can be separated on TLC alongside metabolites that have been identified before, as standards, then the composition of the plant extract can be elucidated to a certain extent. Also plants of the same species that are exposed to different environmental conditions, produces different secondary metabolites at a given time. Therefore TLC can be used as the first guide to see whether there is any difference in the metabolites of a specific plant species in relation to its locality.
Biological activities of the separated compounds in medicinal plants extracts using TLC assays, can be tested. These can be anti-oxidant, antibacterial and antifungal TLC assays. In the antioxidant TLC assay, after separating the compounds, the TLC plate is sprayed with 2,2-diphenyl-picrylhydrazyl (DPPH) dissolved in methanol. The spray is a deep purple colour. DPPH have free radicals that antioxidant can scavenge and this reaction results in the production of a yellow kind of bleached colour on the TLC plate, and thus antioxidants can be identified for further analysis. For the antimicrobial and antifungal TLC assays, a method known as TLC direct bioautography can be used. This is whereby after running TLC, a particular bacteria or fungi, that is known as a plant pathogen, is inoculated onto the TLC plate with the separated compounds. The TLC plate with the microorganisms is then incubated for 2 days at 25°C, in a moist atmosphere. The antifungal compound are then identified by observing where the growth of the microorganism is inhibited or reduced and then further analysis using these compounds can be carried out.
In plant biotechnology, when plants have been genetically modified by a particular gene, different clones of the same plant transformed by the same gene, can lead to the production of different secondary metabolites or similar ones but at different levels. TLC can first be used to compare metabolites produced by the transgenic plants as compared to the wildtype, to see if there are any differences. Then different clones of the same gene can also be compared to see if there are any variations between the clones.
As old and simple as TLC is, it has found its way still in modern science as it can be used as a first indicator of the components of plant extracts and their biological roles, before more advanced techniques such as high performance liquid chromatography can be used. It is cheap and does not take a long time making it an ideal quick technique for basic analytical studies of phytochemicals.
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