Thin-layer chromatography (TLC) is the separation of non-volatile compounds from a mixture utilizing a stationary adsorbent phase coated on the surface of a plate and a mobile phase which carries out the separation process.
TLC fosters a wide range of applications ranging in fields from biological research, organic chemistry, pharmaceutical analysis to food and cosmetic industries.
Let us have an in-depth look at them for better comprehension.
Many countries around the world serve as hubs of traditional medicine, its development and practice. Usually, traditional medicines are derived from plants – extracts or purified compounds in some cases. However, in many cases, when the medicines are being developed or when the extracts are being studied, it is important to identify the active ingredients present in them so as to specifically focus on them in the future.
In such cases, thin layer chromatography is applied to not only separate the individual components constituting an extract but also it is further used to identify the active components by making further fractions of the compounds and their comparison with standards. Moreover, it can also be utilized to identify the location of metabolites a plant may produce e.g., by comparing different plant tissue samples.
Determining Biological Activities
TLC can be utilized to determine the anti-oxidant, antifungal or antimicrobial properties of a compound. For example, to determine the presence of antioxidant properties in a compound, the TLC plate is sprayed with a combination of chemicals containing free radicals (e.g., 2,2-diphenyl-picrylhydrazyl (DPPH) and methanol).
This mixture is purple in color initially but after reacting with antioxidants it turns yellow thereby providing a key indication of the compound’s anti-oxidative properties.
For antifungal or antibacterial properties’ determination, the TLC plate (containing the separated compounds to be checked) can be checked for the growth of respective microorganisms.
The lack of growth of microorganisms at a specific spot indicates the presence of the relevant activity of that compound. This can then be isolated and further proceeded. Enantiomeric Chiral Analysis – an application with specified conditions.
Often it so happens in the pharmaceutical industry that one enantiomer of a compound turns out to be more effective than the rest. Under such circumstances, it becomes extremely important for researchers to find out the more active enantiomer. For that reason, chromatographic techniques have been in continuous development for their use in enantiomer determination.
For the determination of optical resolution of a compound, special TLC plates are used which are impregnated with chiral compounds or solutions. In one type, the chiral stationary phase is used while in the other chiral mobile phase is utilized for the identification of drug enantiomers.
Since thin layer chromatography is a widely used method for the separation and analysis of compounds as well as determination of the purity of compounds, it is extensively applied in the field of medical diagnosis.
For example, porphyria is a disease in which a compound named porphyrin is excreted in the urine. Thin-layer chromatography is used to for the diagnosis of porphyria by extracting porphyrins from urine and then running them on TLC.
There is a wide variety of specific TLC applications in the pharmaceutical industry. Some of those most important in the field are: detection of impurities in the drugs, identification of related substances in drugs, detection for the presence of alkaloids and steroids, and testing of amino acids for ninhydrin positive substances.
In the pharmaceutical industry, drugs are tested for the presence of impurities using TLC. A number of pharmaceutical drugs contain organic substances as impurities and it is necessary to check them for these impurities as they can become serious health hazards later on.
For example, aminobenzoic acid contains 4-nitrobenzoic acid as an impurity. Similarly, benzotropine mesylate is another drug which contains a compound names tropine as an impurity.
Pesticide analysis constitutes an important application of both thin layer chromatography as well as high-performance thin layer chromatography (HPTLC). Some of the examples are discussed below:
- Reverse phase and normal phase TLC both are employed to determine the lipophilicity of pesticides. This is because it is a physiochemical property of a compound having relation to its biological activity and degradation as well.
- It is also employed in metabolic studies mainly done by performing TLC analysis of radiolabeled pesticides.
- It can be used for the separation, detection, and analysis (both qualitative and quantitative) of pesticide residues. For example, a research study employed this method for the detection and separation of the insecticide residues and found its putative importance for the environmental and forensic analysis of pesticide mixtures.
- Soils are often treated in different ways to improve its quality. In such cases, TLC can be applied to check the antifungal activity of soil before and after treatments.
- TLC also presents a cost-effective application for the determination of pesticides in various commercial products. For example, a head lice treatment shampoo was shown by a study to have contained contamination of a pesticide named cypermethrin which was determined using TLC.
- TLC is also applied in the studies of pesticide degradation. Pesticides pose important contamination hazards to crop fields so scientist try to find ways for their decontamination. For example, a study involved the utilization of bioremediation and degradation of cypermethrin present in the soil. Consequently, TLC is an important technique to analyze the end products of the pesticide degradation studies.
Just like the pesticide industry, thin layer chromatography also holds a wide range of applications in the food industry. Some of these are as follows:
- Just as TLC is used to determine impurities in pesticides, it can also be used to determine pesticides’ impurities in food too.
- Various methods are being utilized to determine which compounds in food impart a specific flavor. TLC is one of these, being utilized in food research and industry. For example, the compounds vanillin, ethyl vanillin, and coumarin are used as flavors and their analysis from different food sources can be easily carried out using TLC.
- Thin-layer chromatography is applied to determine the presence of organic acids in the food. For example, the analysis for the presence of sorbic acid, benzoic acid, and ascorbic acid can easily be done by this method.
- Mycotoxins are toxic metabolites produced by certain types of fungi. They can often serve as hazardous impurities of various food e.g., aflatoxins are present in groundnuts and mustard oil. These can be easily analyzed for their presence in food items using TLC.
- Analysis of the additives in various foods can be done using thin-layer chromatography. The examples of most common additives include aspartame and saccharin (sweeteners), and some antioxidants like vitamin C and E.
- Different food adulterants and food color impurities are also determined via TLC.
Thin-layer chromatography is applied in the cosmetic industry for the identification and separation of colors and constituent compounds. Moreover, it can also be used to determine the presence of sweetening and preservative agents in the cosmetics products which is important in deciphering their formulations.