Thin-Layer Chromatography

September 30, 2014

Thin-Layer Chromatography

Background: The purpose of this experiment is is to utilize thin-layer chromatography (TLC) in order to separate compounds based on their polarity and other properties. Chromatography indicates the number of compounds in a mixture and the characteristics of each component. TLC is also a very useful technique for identifying compounds in an unknown mixture by comparing them to known samples, and it can also be used to check the purity of a compound or examine the progress of a reaction or separation. Column chromatography and TLC are both forms of liquid chromatography; they utilize similar methods, but column chromatography is on a larger scale, and it enables the separation of purified compounds.

        When a mixture of compounds is separated using liquid chromatography, the components are distributed between a mobile phase and stationary phase. An organic solvent is used as a relatively non-polar medium to make up the mobile phase, and the solvent moves up the plate by capillary action, carrying the mixture along with it; the efficiency of the solvent depends on the polarities of the compounds in the mixture being separated.  The mobile phase passes over the stationary phase, moving in a definite direction. Each compound in the mixture migrates at a different speed, depending on its solubility in the mobile phase and the strength of its absorption to the stationary phase.  In the stationary phase, the compounds in the mixture are attracted to the polar inorganic substance by intermolecular forces. The structure of each molecule determines the amount of time it spends in each phase; the characteristics such as molecular size and shape and the amount and type of functional groups influence the bonding capabilities of the compound. Silica gel is most commonly used as the stationary phase, and its surface is covered in hydroxyl groups due to the presence of adhered water molecules. The intermolecular hydrogen bonding and dipole-dipole interactions occurring on the surface of the silica gel cause polar molecules to be attracted to the water-coated silica much more than non polar molecules. The stronger the intermolecular forces are, the longer the compounds will spend absorbed onto the surface of the stationary phase, and the slower the compound will move through the mobile phase. These different migration rates for each component in a mixture are what allow for the separation to occur in chromatography.

         TLC identifies compounds by analyzing the polarity (how quickly they move across the plate), and this is done by measuring the Rf (retention factor) values. The Rf value is the ratio of the distance a compound traveled on the plate relative to the distance the solvent traveled. Each compound has its own characteristic Rf value that is dependent upon the compounds used for the stationary and mobile phases.

Procedure:

To start this procedure, take a 10cm 3 5.3 TLC plate and with a pencil, lightly draw a line across the plate 1cm from the bottom. Then, vertically mark five spots 1cm apart and at least 0.5cm away from the edge of the plate and label them: fluorene, fluorenol, fluorenone, unknown, and reference mixture. Prepare 5 micropipets for each solution. Place a small drop, 1-2mm max, of each liquid to the appropriately marked spaces. Be sure to never touch the top of the TLC plate. In a large beaker, place a piece of filter paper that is cut to fit inside and must not wrap all the way around. Thoroughly moisten the filter paper with the development solvent then fill the beaker with the solvent to a depth of about 5 mm. Place the plate inside, don’t allow it to touch the filter paper at all, and seal the top with aluminum foil or saran wrap. Set it aside and allow it to develop. Once the solvent reaches about 2cm away from the top of the TLC plate, take the plate out and measure exactly how far the solvent traveled and record. Take the plate and place it under a UV light to be able to accurately mark the location of each spot. Then calculate the distance each spot traveled as well as the Rf  values and record all data.