Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures. It is performed on a TLC plate made up of a non-reactive solid coated with a thin layer of adsorbent material. This is called the stationary phase. The sample is deposited on the plate, which is eluted with a solvent or solvent mixture known as the mobile phase (or eluent). This solvent then moves up the plate via capillary action. As with all chromatography, some compounds are more attracted to the mobile phase, while others are more attracted to the stationary phase. Therefore, different compounds move up the TLC plate at different speeds and become separated. To visualize colourless compounds, the plate is viewed under UV light or is stained. Testing different stationary and mobile phases is often necessary to obtain well-defined and separated spots. TLC is quick, simple, and gives high sensitivity for a relatively low cost. It can monitor reaction progress, identify compounds in a mixture, determine purity, or purify small amounts of compound.
Procedure The process for TLC is similar to paper chromatography but provides faster runs, better separations, and the choice between different stationary phases. Plates can be labelled before or after the chromatography process with a pencil or other implement that will not interfere with the process. There are four main stages to running a thin-layer chromatography plate: Plate preparation: Using a capillary tube, a small amount of a concentrated solution of the sample is deposited near the bottom edge of a TLC plate. The solvent is allowed to evaporate before the next step completely. A vacuum chamber may be necessary for non-volatile solvents. The spotting procedure can be repeated to ensure sufficient compound to obtain a visible result. Depending on the application, multiple samples may be placed in a row, the same distance from the bottom edge; each sample will move up the plate in its own "lane."
Development chamber preparation: The development solvent or solvent mixture is placed into a transparent container (separation/development chamber) to a depth of less than 1 centimetre. A strip of filter paper (aka "wick") is also placed along the container wall. This filter paper should touch the solvent and almost reach the top of the container. The container is covered with a lid and the solvent vapors are allowed to saturate the atmosphere of the container. Failure to do so results in poor separation and non-reproducible results. Development: The TLC plate is placed in the container such that the sample spot(s) are not submerged into the mobile phase. The container is covered to prevent solvent evaporation. The solvent migrates up the plate by capillary action, meets the sample mixture, and carries it up the plate (elutes the sample). The plate is removed from the container before the solvent reaches the top of the plate; otherwise, the results will be misleading. The solvent front, the highest mark the solvent has travelled along the plate, is marked. Visualization: The solvent evaporates from the plate. Visualization methods include UV light, staining, and many more.
Separation process and principle The separation of compounds is due to the differences in their attraction to the stationary phase and because of differences in solubility in the solvent. Different compounds in the sample mixture travel at different rates due to the differences in their partition coefficients. Different solvents, or different solvent mixtures, give different separations. The retardation factor (RF) quantifies the results. It is the distance traveled by a given substance divided by the distance traveled by the mobile phase.
In normal-phase TLC, the stationary phase is polar. Silica gel is very common in normal-phase TLC. More polar compounds in a sample mixture interact more strongly with the polar stationary phase. As a result, more-polar compounds move less (resulting in smaller RF) while less-polar compounds move higher up the plate (higher RF). A more-polar mobile phase dissolves polar compounds more. As such, all compounds on the TLC plate move higher up the plate in polar solvent mixtures. "Strong" solvents move compounds higher up the plate, whereas "weak" solvents move them less. If the stationary phase is non-polar, like C18-functionalized silica plates, it is called reverse-phase TLC. In this case, non-polar compounds move less and polar compounds move more. The solvent mixture will also be much more polar than in normal-phase TLC.
Solvent choice An eluotropic series, which orders solvents by how much they move compounds, can help select a mobile phase. Solvents are divided into solvent selectivity groups. Using solvents with different elution strengths or different selectivity groups can often give very different results. While single-solvent mobile phases can sometimes give good separation, some cases may require solvent mixtures. In normal-phase TLC, the most common solvent mixtures include ethyl acetate/hexanes (EtOAc/Hex) for less-polar compounds and methanol/dichloromethane (MeOH/DCM) for more polar compounds. Different solvent mixtures and solvent ratios can help give better separation. In reverse-phase TLC, solvent mixtures are typically water with a less-polar solvent: Typical choices are water with tetrahydrofuran (THF), acetonitrile (ACN), or methanol.
Analysis
As the chemicals being separated may be colourless, several methods exist to visualise the spots:
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