Thin Layer Chromatography
Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O). Both of these adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in neutral, basic, or acidic forms. Thin Layer Chromatography (TLC) is a sensitive, fast, simple and inexpensive analytical technique. It is a micro technique; as little as 10-9g of material can be detected, although the sample size is from 1 to 100x10-6 g. TLC involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent the stationary adsorbent phase. The more strongly a given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the plate. The following are some common uses of Thin-Layer Chromatography:
The standards and commercial painkillers will be dissolved in a 50/50 Ethanol/Ethyl Acetate solution.
Prepare a developing chamber as indicated in the picture using a large beaker as the chamber, a half-piece of filter paper inside, and foil or plastic wrap to cover. Pour the eluting solvent, a 99/1 mixture of Ethyl Acetate/Glacial Acetic Acid, into the beaker to a depth of approximately 1 cm. Place the prepared TLC plates in the developing chamber.
After the solvent has risen to near the top of the plate (about 1 cm from the top), remove the plate and mark the solvent front with a pencil. Keep the plates in the hood until the majority of the eluting solvent has evaporated from the plates. Examine the plate under UV light to see the components as dark spots against a bright green-blue background.
Outline the spots with a pencil and note anything distinctive about any of the compounds. The spots should also be visualized by putting the plate in an iodine chamber. The iodine chamber is pre-made and contains a few crystals of iodine in the bottom of a capped jar. More than 2 plates can be placed in the iodine chamber at one time. Remove the plates when a definite change in appearence takes place on your plates. Note which compounds stained with iodine and to what intensity. The iodine stains will dissipate over time.
Wrap your TLC plates in plastic wrap and scan them into your e-lab.
Calculate the Rf values for each spot. Unknowns can be identified using Rf values, fluorescence in UV light, changes due to iodine exposure, the reference spot and Table 1.