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Thin Layer Chromatography

Melting Point

Simple Distillation

Column Chromatography

Infrared Spectroscopy


Solubility Testing



Gas Chromatography-Mass Spectrometry

Classification Tests-Halides

Fractional Distillation


Thin Layer Chromatography

(Adapted from Mohrig, 1st ed., pp. 151-162.)

Chromatography is a sophisticated method of separating mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned.

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:

  1. To determine the number of components in a mixture.
  2. To determine the identity of two substances.
  3. To monitor the progress of a reaction.
  4. To determine the effectiveness of a purification.
  5. To determine the appropriate conditions for a column chromatographic separation.
  6. To monitor column chromatography.

In this experiment, you will use TLC to identify unknown analgesic painkillers using the table of analgesics and their components in the experimental section of this experiment.


Experimental Procedure for TLC Analysis of Analgesic Drugs
(adapted from Fieser & Williamson, pp. 128-129)
Obtain 2 TLC plates. Draw a light pencil line about 1 cm from the end of each chromatographic plate. Spot one plate with your 4 known standards (Acetaminophen, Aspirin, Caffeine, and Ibuprofen) and the other plate with the 5 unknown commercial painkillers. Both plates should also have a Reference spot that contains all 4 standards. Use a separate capillary tube for each standard and unknown solution. Make each spot as small as possible, preferably no more than 2-3 mm in diameter. Examine the plate under the ultraviolet (UV) light to see that enough of each compound has been applied; if not, add more.

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 or take a picture of them with your laptops so as to be able to insert their image 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.