TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (preferrably both run on the same TLC plate).
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.
As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluor which fluoresces everywhere except where an organic compound is on the plate.)
The procedure for TLC, explained in words in the above paragraphs, is illustrated with photographs on the TLC Procedure page.
The Indiana University site has an animated graphics movie on TLC procedure.
In the teaching labs at CU Boulder, we use silica gel plates (SiO2) almost exclusively. (Alumina (Al2O3) can also be used as a TLC adsorbent.) The plates are aluminum-backed and you can cut them to size with scissors. Our plates are purchased ready-made from EM Sciences or from Scientific Adsorbents. The adsorbent is impregnated with a fluor, zinc sulfide. The fluor enables most organic compounds to be visualized when the plate is held under a UV lamp. In some circumstances, other visualization methods are used, such as charring or staining.
Choosing a solvent is covered on the Chromatography Overview page. The charts at the bottom of that page are particularly useful.
The strength with which an organic compound binds to an adsorbent depends on the strength of the following types of interactions: ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the dominant interactive forces between the adsorbent and the materials to be separated are of the dipole-dipole type. Highly polar molecules interact fairly strongly with the polar Si—O bonds of these adsorbents and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules thus generally tend to move through the adsorbent more rapidly than the polar species. Roughly, the compounds follow the elution order given on the Chromatography Overview page.
Rf is the retention factor, or how far up a plate the compound travels. See the Rf page for more details:
If the compounds are colored, they are easy to see with the naked eye. If not, a UV lamp is used (see the Procedure page).
All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better. One thing you have to be careful Examples of common problems encountered in TLC:
The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run together and appear as a streak. Perhaps, the experiment did not go as well as expected.
Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates.
Likely, the adsorbent was disturbed during the spotting, causing the crescent shape.
Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crookedly run plates make it harder to measure Rf values accurately.
Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC plate and leave it laying on your workbench as you do the experiment, you might drop or splash an organic compound on the plate.
You might not have spotted enough compound, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or, spot it several times in one place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the plate. Or, perhaps you do not have any compound because your experiment did not go as well as planned.
If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to move up the plate by capillary action. Thus, you will not see spots after the plate is developed.
Perhaps, you used an ink pen instead of a pencil to mark the origin?
