CH 463 Experimental Chemistry II
Thin Layer Chromatography (tlc) to check purity of benzophenone reaction mixture
tlc is often used in addition to melting point determination to check the purity of a compound. This technique is frequently used by organic chemists to immediately gauge the success of a synthetic step since it is quick and informative and doesn’t require any expensive equipment.
There are many different types of chromatography but all involve a stationary phase and a mobile phase. In tlc it is common to use a thin film of silica gel as the stationary phase, ~Si(OH)2 O ~ units. The film is deposited on rectangular backing sheets of aluminum or plastic for support and is called a tlc plate. The mobile phase, designed to be more non-polar than silica gel, drives the compounds within the sample to separate and become distributed across the surface of the stationary phase through capillary wetting. In the experiment, each plate is loaded with sample by applying or spotting small amounts onto one end of the plate and the plate is then placed in a developing tank saturated with mobile phase. During the course of a few minutes, the compounds in the sample partition selectively between the two phases striking a balance between the intermolecular attractive forces of each distinctive compound with each phase component.
Often a standard compound is analyzed simultaneously on the same plate with the sample for easy comparison. The distance that a compound will move up a plate relative to the distance for the solvent, or mobile phase front, is known as the Rf, and depends not only on chemical composition, but also on the temperature.
If the compound is pure, one spot will develop; if it is not pure and a mixture of two or more compounds, separate spots will appear for each compound in the mixture. At least that is the idea. Sometimes it is difficult to get a clean separation between compounds of very similar chemical makeup. The polarity of the mobile phase is then adjusted and the experiment is repeated with a new plate. There are more analytical forms of chromatography that have very high resolution compared to tlc (e.g., gas chromatography, or GC, high performance liquid chromatography, or HPLC).
Using tlc to check purity of the Ketone mixture.
|
|
Prepare 10 mL of a mobile phase mixture that is 3:1 of
hexane:acetone2. Pour about 7 mL into a 4 oz screw cap bottle that has been
prepared with a piece of filter paper (the mobile phase layer should not be higher than
about 0.5 cm). This is to be the "developing tank". Cover the tank and let it
equilibrate while you prepare the tlc plate. |
|
|
Dissolve
a few mg of the ketone in about 0.5 mL of acetone (use 1 dram vial). Do the same for the
corresponding carboxylic acid. If you are checking the progress of the acylation reaction
by removing reaction mixture from the pot, decompose first before spotting plate.
Put a few drops of the rxn mix into a small vial (e.g., scintillation vial with screw cap)
and add a small amount of dil acid; add a small amount of ether or other solvent to
separate the ketone from the aqueous layer. |
|
|
Using
a pencil carefully draw a line about 1 cm from the bottom of one tlc plate and mark
three dots evenly spaced on the line (be careful not to scratch off the silica gel
coating). You will want to spot the plate with the ketone/organic layer, with the
corresponding carboxylic acid, and with a combination of both. |
|
|
To
apply the sample to the plate (i.e., "spot" it), partially fill a thin capillary
tube by capillary action and use it to load one drop of the acid solution onto each dots 1
and 2, being careful not to break through the silica gel coating. After the spots dry,
re-spot a couple of times. Use a second capillary tube and load one drop of the
ketone solution onto each dots 2 and 3. Again re-spot when dry to increase concentration. |
|
|
Carefully
hold the plate by the edges and lower it down into the tank, sample side down. The mobile
phase should not wash over the sample spots - if it does, remove the tlc plate, reload it
and replace the mobile phase in the tank. |
|
|
Once
the plate is in the tank, close the lid. As the mobile phase ascends the plate you will
see the "solvent front" line move up. Continue developing the plate until the
solvent front is about 1 cm from the top edge of the plate. Carefully remove the plate and
immediately mark the solvent front line with a pencil. Let the tlc plate air dry or
use a air gun. |
|
|
Observe the plate under short wavelength UV light and outline the spots with pencil (Caution: UV light is dangerous. Can cause blindness. Do not look into the light directly and use only under the supervision of the instructor.) Occasionally the spots are "comet" shaped - termed "tailing". This effect can be reduced by adding a couple of drops of acetic acid to the solvent system and repeating the experiment with a new plate. The relative distance that a spot of compound moves relative to the solvent from is called the "Rf" . Find and report the Rf for the ketone and for the acid, etc. For the optimum mobile phase, the Rf should be around 0.3. |
1. Aikens, David A., et al Principles for an Integrated Chemistry Laboratory; Waveland Press, Inc.: Illinois, 1984; pp 187-191. ISBN 0-88133-102-3
2. Mil'to, V.I.; Mironov, G.S.; Kopeikin, V.V. Zhur. Org. Khim. 1989, 25 (11) , 2372-2374.
last updated by C. Pastorek, Department of Chemistry, Oregon State University, pastorekc@chem.orst.edu
03/26/04