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Experiment 12: THIN-LAYER CHROMATOGRAPHY OF ANALGESICS

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Purpose: The identities of the components in an unknown containing one or more of five different analgesics are determined by thin layer chromatography. Introduction: Thin-layer chromatography (TLC) is a chromatographic technique that is useful for separating organic compounds in a mixture and identifying them. Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of organic reactions and to check the purity of products. TLC consists of a stationary phase immobilized on a glass or plastic plate, and a mobile phase (an organic eluting solvent). The sample, either liquid or solid, dissolved in a volatile solvent, is deposited as a spot on the stationary phase. The components of a sample can be identified by simultaneously running standards with the unknown. The bottom edge of the plate is placed in a solvent reservoir, and the eluting solvent moves up the plate by capillary action. When the solvent front reaches close to the other edge of the stationary phase, the plate is removed from the solvent reservoir, and the solvent front is marked on the plate.

The different constituents in the mixture move up the plate at different rates due to differences in their partitioning behavior between the stationary phase and the mobile liquid phase. Compounds that are attracted more to the eluting solvent than to the stationary phase will travel faster up the plate. Those that are attracted more to the stationary phase than to the eluting solvent will move much slower. The degree of attraction is generally based on the intermolecular forces such as dipole forces, hydrogen bonding, and London dispersion forces. Using the general rule of “Like Dissolves Like” one would try out stationary phases and eluting solvents of various polarities to find the appropriate combination to effect a separation. In this experiment the stationary phase is silica gel, which is polar. Polar compounds would be expected to be held more strongly and therefore move slower up the TLC plate than nonpolar compounds.

The separated spots are visualized with ultraviolet light or by placing the plate in iodine vapor. If you see more than one spot in a column it could mean the sample was a mixture. The mobility of a compound is measured by the Rf value, defined as the ratio of the distance traveled by the spot (with measurements made from the center of each spot) to the distance traveled by the solvent front. The distance can be measured in any unit of length as long as the same unit is used for the spot as for the solvent; however, usually it is measured in cm. Rf value is expressed in decimal notation, and as a ratio, it has no units.

where dspot is the distance traveled by the spot

dsolvent is the distance traveled by the solvent front

spot f

solvent

d R =

d

• • •

A B C

dspot

dsolvent

2 EXPERIMENT 12: THIN-LAYER CHROMATOGRPAHY OF ANALGESICS

You may have performed a paper chromatography analysis in the past to identify food dyes (as in candies). In this experiment, you are using chromatographic TLC plates with the solid silica gel coated on a plastic sheet. Each student has an unknown containing one or more of five analgesics:

acetaminophen, acetylsalicylic acid, caffeine, phenacetin, salicylamide These substances do not have color and therefore when spotted on the TLC, will not be visible to the eye like the food dyes. To view the location of these substances after the plates have been developed, you will place the plates in the UV viewing box. The multiple double bonds in these structures will make them glow under UV light. Equipment/Materials Two TLC plates, 400-mL beaker, pencil, metric ruler, glass rod, 50-mL graduated cylinder, toothpicks, ceramic well plate, ethyl acetate, aluminum foil, known analgesic samples, unknown analgesic sample, UV light box. WARNING: Materials provided in the lab are for experiments only. Never consume any of the materials provided for experiments. Procedure (Using a pen or pencil, record by hand all of your data and results on the Data Collection and Results Pages.)

Known solutions of the five possible analgesics have been prepared for you. Be very careful that you do not contaminate these solutions. 1. Obtain an unknown from your instructor and record your unknown number. 2. Obtain two TLC plates and draw a very light pencil line (Line of Origin) about 1 cm

from the end of each TLC plate. (Why can’t you use a pen?) You will be applying three spots on each TLC plate on the pencil line, evenly spaced out (see diagram below). Devise a code to label the spots with pencil at the top of the slide. Needless to say it is paramount that you do not get confused as to which spot corresponds to which solution. Record your code in your notebook and include it in your lab report.

With your pencil place a small mark (Removal Mark) 2 cm from the top end of the TLC plate at either the left or right edge. This is to show how far the solvent front should reach before the plate is to be removed from the solvent reservoir.

1 cm • • •

2 cm Removal Mark

Line of Origin

EXPERIMENT 12: THIN-LAYER CHROMATOGRPAHY OF ANALGESICS 3

3. Weigh 0.020 g of your unknown into a vial and use a minimum amount of

chloroform/ethanol solution to dissolve it. Stir with a glass rod until all solids have dissolved.

4. Place a clean and dry ceramic well plate on a piece of paper and label on the paper

the positions of the known solutions you are about to place in each of five wells. 5. Place a couple drops of each of the known solutions in separate wells according to

your label. Be careful you don’t contaminate the adjoining wells. You do not need to place your unknown solution in the well plate.

6. For each of the solutions of five knowns and your unknown, do the following: Dip a

clean toothpick (wide end) into the solution and then touch it briefly to the TLC plate as described below.

Use a separate toothpick for each of the five knowns and your unknown. Make each spot as small as possible by touching the tip of the toothpick to the plate only briefly. After the solvent has evaporated off, re-wet your toothpick and touch the tip of the toothpick on the same spot again. Do this a total of about 3 times, depending on the size of the tip of the toothpick. This way you would have a small but concentrated spot on the plate for each of the 6 solutions.

7. Ethyl acetate is the developing (eluting) solvent. First add 15 mL of the ethyl acetate

in a 400-mL beaker. Keep in mind that the depth of your solvent must be such that it does not touch the Line of Origin on the TLC plates.

8. You will be placing the two plates into the same beaker by leaning them against each other with the spots facing out, as shown in the diagram below. You might want to practice doing this on the bench top before you actually place them inside the beaker with the eluting solvent. Care must also be taken that the plates do not touch the sides of the beaker in order to keep the solvent going up the plate evenly. Once the plates are in, do not move the beaker!! Carefully cover the beaker with a piece of aluminum foil in a way that you can see the Removal Mark (2 cm from the top edge of the plate) and know when it is time to take the plates out.

• • • ethyl acetate

aluminum foil

where solvent front should end up (2 cm from top edge)

4 EXPERIMENT 12: THIN-LAYER CHROMATOGRPAHY OF ANALGESICS

9. Once the solvent has reached the Removal Mark, remove the plates and quickly trace

the solvent front with a pencil before the solvent evaporates. (The solvent front may not be exactly a straight line. Be sure you trace the actual solvent front with your pencil.) Leave the plates on a piece of paper towel and allow the solvent to dry.

10. Examine the plate under UV light to see the components. Use both the short-wave

and the long-wave UV light to distinguish between spots that may have similar Rf values. Outline the spots with a pencil. If any spots fluoresce under the long-wave UV, note it in your notebook! This information will help you identify your unknown.

11. Measure dspot for each spot, the distance from the center of each spot to the point of

origin (to 0.1 cm). Also measure dsolvent, the distance from the solvent front to the point of origin. (If the solvent front is not exactly horizontal, you will have different dsolvent for each spot.) Calculate the Rf values for the spots, and identify the components in your unknown. You should do this before you leave the lab.