The Identification of Drugs

The identification of Drugs

Frequently, a chemist is called on to identify a particular unknown substance. If there is no prior information to work on, this can be a formidable task. There are several million known compounds, both organic and inorganic. For a completely unknown substance, the chemist must often use every available method. If the unknown substance is a mixture, then the mixture must be separated into its components and each component identified separately. A pure compound can often be identified from its physical properties (melting point, boiling point, density, refractive index, etc…), and a knowledge of its functional groups (which can be identified by spectroscopy – IR, NMR, UV and mass spectroscopy).

One of the methods used to identify drugs is a method known as TLC (thin layer chromatography).

Under specified conditions, many drug substances can be identified by their Rf (order of elution) values and by the colors that their TLC spots turn when treated with various reagents or when they are observed under certain visualization methods.

In this experiment, TLC will be used to characterize certain analgesics. Standard reference solutions of aspirin, acetaminophen, caffeine and salicylamide will be provided. The goal of this experiment is to determine the Rf (order of elution) values of these known compounds for use in the identification of unknowns that will be provided later in the semester.

The reference substances will be available as solutions of 1 g of each dissolved in 10 mL of a 50:50 mixture of methylene chloride and ethanol. If you have to determine the Rf value(s) of an unknown, you will need to crush it if it is in tablet form and dissolve is in a 50:50 methylene chloride-ethanol mixture.

Two methods of visualization will be used to observe the positions of the spots on the developed TLC plates. First, the plates will be developed while under illumination from a short-wave UV lamp. Under these conditions, some of the spots will appear as dark areas on a plate, while others will fluoresce brightly. This difference in appearance under UV illumination will help to distinguish the substances from one another. You might find it convenient to outline very lightly in pencil the pots observed and to place a small X inside those pots that fluoresce. For a second means of visualization, iodine vapor will be used. Not all the spots will become visible when treated with iodine, but at least 2 will develop a deep brown color. The differences in the behavior of the various spots with iodine can be used to further differentiate among them.

It is possible to use several developing solvents for this experiment, but ethyl acetate with 0.5% glacial acetic acid added is preferred. The small amount of glacial acetic acid suppresses the ionization of the aspirin, allowing it to travel upward on the plates. Without the acid, aspirin does not move up the TLC plate.

Special Instructions: Develop plates under UV light first, and with iodine second. Iodine permanently affects some of the spots. If iodine is used first, you will need to run another TLC plate. Aspirin presents some special problems because it is present in a large amount in many analgesics and also hydrolyzes easily. For these reasons, the aspirin spots often show excessive tailing. Take special care to notice that, although they have similar Rf values, salicylamide spots have a different appearance when viewed under UV illumination. Waste Disposal: Dispose all development solvent in the container in the hood for non-halogenated organic solvents. Dispose of the ethanol-methylene chloride mixture in the container for halogenated organic solvents.

Procedure:

Spotting the plates. You will need at least 12 capillary micropipets to spot the plates. The procedure for the preparation of these pipets is described and illustrated in Technique 14, Section 14.4, page 759 ofyour handout.

After preparing the micropipets, obtain one 10 cm x 6.6 cm TLC plates (EM Silica Gel 60 F-254, No. 5554- 7) from your instructor. These plates have a flexible backing, but they should not be bent excessively otherwise the coating flakes off and the edges become uneven. Handle them carefully. Also, you should only handle them by the edges; the silica gel surface should not be touched. Using a lead pencil and not a pen, lightly draw a line across the plates (short dimension) about 1 cm from the bottom. Using a centimeter ruler, lightly mark off six 1-cm intervals on the line. These are the points at which the samples will be spotted.

On the plate, starting from left to right, spot acetaminophen, then aspirin, caffeine and salicylamide. Use different micropipets for each compound. Note that the compounds have been arranged in alphabetical order. This avoids confusion. Solutions of these compounds will be found in small bottles on the benches by the windows. The correct method of spotting a TLC place is described in Technique 14, Section 14.4, page 579 of your handout. It is important that the spots be made as small as possible and that the plates are not overloaded. If not, then the spots will tail and will overlap one another after development. The applied spot should be about 1-2 mm (1/16 in). in diameter. If scrap pieces of the TLC plates are available, it would be a great idea to practice spotting on these before preparing the actual sample plates.

Development of the TLC Plates. When the plate has been spotted, obtain a 16 oz. wide-mouth screwcap jar (or a beaker covered will Al foil) for use as a development chamber. Again the preparation of the development chamber is in Technique 14, Section 14.4, page 579 of your handout. Since the backing of the TLC plate is very thin, you may omit the filter paper liner.

When the development chamber has been prepared, obtain a small amount of the development solvent (0.5% glacial acetic acid in ethyl acetate) from the stock solution. Fill the chamber with the development solvent to a depth of about 0.5 cm. The pencil line should not be immersed in the solvent; neither should the solvent level be above the spots on the plate or the samples will dissolve off the plate into the reservoir instead of developing. Place the spotted plate in the chamber and allow the plate to develop.

Let the solvent rise up the plate until it reaches about 0.5 cm from the top edge. Remove the plate from the chamber and mark the position of the solvent front or level using a lead pencil. Set the plate ona piece of paper towel to dry. It may be helpful to place a small object under one end of the plate to allow optimum air flow around the drying TLC plate. When the plate is dry, observe it under a a short-wavelength UV lamp, preferably in a darkened hood. Lightly outline all the observed spots with a lead pencil. Carefully notice any differences in behavior. Make a sketch of the plate in your notebook and note the differences in appearance that you observed. Next, place the TLC plate in a jar containing a few iodine crystals, cap the jar and warm it gently on a steam bath or hot plate until the spots begin to appear. Notice which spots become visible and note their relative colors. Remove the plate from the jar and record your observations in your notebook. It is best to also make a sketch of the TLC plate in your notebook.

Using the millimeter side of your ruler, measure the distance that each spot traveled relative to the solvent front. Calculate the Rf values for each spot. The calculation for Rf can be found in Technique 14, Section 14.4, page 579 of your handout.

Analysis of Unknowns:

Obtain half a tablet of at least two (or all) of the unknown analgesics. Place the half-table on a clean piece of notebook paper and crush it with a spatula. Transfer each crushed half-tablet to a small labeled test tube or Erlenmeyer flask. Using a graduated cylinder, mix 15 mL of absolute ethanol and 15 mL of methylene chloride. Mix the solution well. Add 5 mL of this solvent to each of the crushed tablet and then heat each of them gently for a few minutes on a steam bath at about 100 oC. Not all the tablets will dissolve since the analgesics usually contain an insoluble binder. In addition, many of them contain inorganic buffering agents or coatings that are insoluble in this solvent mixture. After heating the samples, allow them to settle and then spot the clear liquid extracts on the plate. Develop the plate in 0.5% glacial acetic acid-ethyl acetate developing mixture. Observe the plate under UV illumination and mark the visible spots as you did before. Repeat the visualization using iodine crystals. Sketch the plates in your notebook and record your conclusions.