Where is thin layer chromatography used

If you have a standard pure form of the compound , you can use TLC to look for your compound by running the standard and extract together, and checking if their Rf values match.

Similarly, you can run samples from different sources together to determine if both compounds are actually the same. Again, identical Rf values would be the key.

If you run TLC using a mixture of compounds, you can separate them as well as detect the number of compounds present in the mixture. You can use the number of spots developed on the TLC plate to approximate the number of compounds in the mixture.

You can use TLC to test different solvent systems for your compound of interest to help determine the solvent you should use for column chromatographic separation of that compound. The solvent system that gives the best resolution during TLC can also be used to purify the compound using column chromatography. You can monitor the progress and rate of reactions by performing TLC of the reaction samples at particular intervals.

For example, while performing biotransformation of compounds, you can collect reaction mixtures at different time intervals and use TLC to monitor the conversion of the reactants to products. You can test the efficiency of column chromatographic separations by obtaining samples of the purified compounds and performing TLC. Potassium permanganate. Works well for all compounds that can be oxidized.

Yellow on purple. Yellow or light brown on purple. Bromocresol Green. Yellow spot on blue background. Good general stain, very well with polyhydroxylated and carbonyl compounds. Blue or green spot.

Upon heating, very sensitive! Good general stain, particularly sensitive towards nucleophiles. Varying colors on light pink plate upon heating. Does not work with alkenes, alkynes or aromatic system unless functional groups are present.

Phosphomolybdic acid PMA. Very sensitive. Dark green spot on light green plate. Sensitivity can be enhanced by use of cobalt II chloride. Indoles, amines. Pink or red-violet. Dragendorff-Munier Stain. Amines even the ones that are low in reactivity. Various colors. Due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well.

TLC University of Colorado. TLC Macherey Nagel. TLC chamber for development e. Thin layer chromatography T LC is a chromatographic technique used to se parate the components of a mixture using a thin stationary phase supported by an inert backing.

It may be performed on the analytical scale as a means of monitoring the progress of a reaction, or on the preparative scale to purify small amounts of a compound. TLC is an analytical tool widely used because of its simplicity, relative low cost, high sensitivity, and speed of separation.

TLC functions on the same principle as all chromatography: a compound will have different affinities for the mobile and stationary phases, and this affects the speed at which it migrates. The goal of TLC is to obtain well defined, well separated spots. After a separation is complete, individual compounds appear as spots separated vertically. Each spot has a retention factor Rf which is equal to the distance migrated over the total distance covered by the solvent.

As stated earlier, TLC plates also known as chromatoplates can be prepared in the lab, but are most commonly purchased. Silica gel and alumina are among the most common stationary phases, but others are available as well. Many plates incorporate a compound which fluoresces under short-wave UV nm. The backing of TLC plates is often composed of glass, aluminum, or plastic.

Glass plates are chemically inert and best withstand reactive stains and heat, but are brittle and can be difficult to cut. Aluminum and plastic plates can be cut with scissors, but aluminum may not withstand strongly acidic or oxidizing stains, and plastic does not withstand the high heat required to develop many stains.

Aluminum and plastic plates are also flexible, which may result in flaking of the stationary phase. Never under any circumstances touch the face of a TLC plate with your fingers as contamination from skin oils or residues on gloves can obscure results.

Instead, always handle them by the edges, or with forceps. The properties of your sample should be considered when selecting the stationary phase. It is also important to note that silica gel is acidic. Therefore, silica gel offers poor separation of basic samples and can cause a deterioration of acid-labile molecules. This would be true for alumina plates in acidic solutions as well. It is important to note that there are differences between silica gel and alumina.

Alumina is basic and it will not separate sample sizes as large as silica gel would at a given layer thickness. Also, alumina is more chemically reactive than silica gel and as a result, would require more care of compounds and compound classes. This care would avoid decomposition and rearrangement of the sample. Chromatographic Columns is a good reference to learn more about the different types of columns and stationary phases.

Proper solvent selection is perhaps the most important aspect of TLC, and determining the best solvent may require a degree of trial and error. As with plate selection, keep in mind the chemical properties of the analytes. A common starting solvent is hexane:ethyl acetate. When performing your experiment, you do not want your values to be 0 or 1 because your components that you are separating have different polarities.

If the value is 0, you need to increase your solvent polarity because the sample is not moving and sticking to the stationary phase. If the value is 1, you need to decrease your solvent polarity because the compound was not able to separate. If you know that one component of a mixture is insoluble in a given solvent, but another component is freely soluble in it, it often gives good separations. How fast the compounds travel up the plate depends on two things:.

Acids, bases, and strongly polar compounds often produce streaks rather than spots in neutral solvents. Adding a few percent of acetic or formic acid to the solvent can correct streaking with acids.

Similarly for bases, adding a few percent triethylamine can improve results. For polar compounds adding a few percent methanol can also improve results. The volatility of solvents should also be considered when chemical stains are to be used. Any solvent left on the plate may react with the stain and conceal spots.

Many solvents can be removed by allowing them to sit on the bench for a few minutes, but very nonvolatile solvents may require time in a vacuum chamber. Volatile solvents should only be used once. If the mobile phase is used repeatedly, results will not be consistent or reproducible. Developing a TLC plate requires a developing chamber or vessel. This can be as simple as a wide-mouth jar, but more specialized pieces of glassware to accommodate large plates are available.

The chamber should contain enough solvent to just cover the bottom. It should also contain a piece of filter paper, or other absorbent material to saturate the atmosphere with solvent vapors. Finally, it should have a lid or other covering to minimize evaporation. If fluorescent plates are used, a number of compounds can be seen by illuminating the plate with short-wave UV.

Quenching causes dark spots on the surface of the plate. These dark patches should be circled with a pencil. For compounds which are not UV active, a number of chemical stains can be used. These can be very general, or they can be specific for a particular molecule or functional group.