Thin layer chromatography (TLC) is a chromatographic technique for separating mixture components that uses a thin stationary phase supported by an inert backing. It can be done on an analytical scale to track the development of a reaction, or on a preparative scale to purify small amounts of a product. TLC is a popular analytical tool because of its simplicity, low cost, great sensitivity, and rapidity of separation. TLC works on the same concept as all chromatography: a molecule has varying affinities for the mobile and stationary phases, which affects the rate at which it migrates. The purpose of TLC is to produce well-defined, well-separated spots.
As previously stated, TLC plates (also known as chromatoplates) can be created in the lab but are most commonly purchased. The most common stationary phases are silica gel and alumina; however, other choices are available. Many plates include a chemical that fluoresces in the short-wave UV (254 nm). TLC plates' backings are typically composed of glass, aluminum, and plastic. Glass plates are chemically inert and can withstand reactive stains and high temperatures; yet, they are delicate and difficult to cut. Aluminum and plastic plates can be cut using scissors. However, aluminum may not sustain highly acidic or oxidizing stains, and plastic cannot endure the high temperatures required to produce many stains. Because plastic and aluminum plates are pliable, the stationary phase may flake off of them. Under no circumstances should you touch a TLC plate's face with your fingers, as this could contaminate it with skin oils or leave residue on your gloves, which could skew the findings. Rather, always handle them with forceps or by their edges.
When establishing a stationary phase, consider the features of your sample. Silica gel is only compatible with amino acids and hydrocarbons. It is also vital to understand that silica gel is acidic. As a result, silica gel is ineffective in separating basic samples and has the potential to destroy acidic compounds. This also applies to aluminum plates in acidic liquids. It is critical to recognize that silica gel and alumina have separate properties. Alumina is a simple substance that cannot differentiate sample sizes as large as silica gel at the same layer thickness. Furthermore, because alumina is more chemically reactive than silicon gel, it is important to choose compounds and classes carefully. This care would avoid decomposition and rearrangement of the sample.
Proper solvent selection is perhaps the most critical component of TLC, and choosing the best solvent may involve some trial and error. Consider the chemical properties of the analytes, just as you would when selecting a plate. A typical starting solvent is 1:1 hexane: ethyl acetate. Varying the ratio can have a significant influence on Rf. Rf values vary from 0 to 1, where 0 indicates very low solvent polarity and 1 indicates very high solvent polarity. When doing your experiment, you do not want your values to be 0 or 1, because the components you are separating have opposite polarity. If the value is 0, you should increase the solvent polarity since the sample is not moving and is stuck in the stationary phase. If the value is one, you should reduce the polarity of the solvent since the compound did not separate properly. Acids, bases, and strongly polar chemicals frequently leave streaks rather than spots in neutral solvents. Streaks make it difficult to calculate Rf values and may obscure other areas. To correct acid streaking, add a few percent acetic or formic acid to the solvent. Similar to bases, adding a few percent triethylamine can boost outcomes. For polar chemicals, adding a few percent methanol can improve results.
When using chemical stains, it is also important to consider the volatility of the solvent. Any solvent remaining on the plate may react with the stain, concealing spots. Many solvents can be removed by leaving them on the bench for a few minutes, but extremely non-volatile solvents may require time in a vacuum chamber. Volatile solvents should be used just once. If the mobile phase is employed repeatedly, the outcomes will be inconsistent and unpredictable.
A developing chamber or vessel is required while producing a TLC plate. This might be as basic as a wide-mouth jar, but more specialist glassware designed to hold huge platters is available. The chamber should have just enough solvent to cover the bottom. It should also include a piece of filter paper or another absorbent substance to fill the atmosphere with solvent vapors. Finally, it should have a lid or another covering to reduce evaporation.
- Cut the plate to the appropriate size and use a pencil to draw a straight line about 1 cm from the bottom. When writing on a TLC plate, do not use too much effort because it will remove the stationary phase. It is critical to use a pencil rather than a pen since inks frequently migrate up the plate with the solvent. Use TLC pipettes to administer analyte to the line. Make sure there is enough sample on the plate. This can be accomplished by using short-wave UV. You should see a purple spot. More sample needs to be added to the plate if the spot is not visible. It is best practice to create a co-spot by shining the target compound standard onto a spot of the unknown combination if one is available. This guarantees the target compound's identification.
- Place the plate evenly in the chamber and lean against the side. Never let the bulk solvent climb above the line you drew. Allow capillary action to pull the solvent up the plate until it reaches about 1 cm from the end. Never let the solvent reach the end of the plate.
- Remove the plate and draw a pencil line on the solvent front.
- Using a short-wave UV light, circle the indicated components with a pencil.
TLC is extremely simple to use and affordable. Undergraduates can learn this approach and apply comparable principles to other chromatographic techniques. TLC requires relatively few materials. As a result, after the optimal solvent has been identified, it can be applied to other techniques, such as high performance liquid chromatography. A TLC plate can separate more than one compound as long as each compound has its own preferred mobile phase. The solvents for the TLC plate are simply changed, and you can use many solvents depending on the results you want. As previously stated, TLC can be used to determine the purity of a chemical. The purity can be easily checked with a UV light. Most compounds may be identified simply by verifying their Rf values. You can quickly change the chromatographic conditions to improve the optimization for resolution of a certain component.