IDENTIFICATION OF ACTIVE INGREDIENTS IN ANALGESIC DRUGS

 

The structures of the three active ingredients from common pain pills are shown below.

 

STRUCTURE
ACTIVE INGREDIENT	Acetylsalicyclic acid	Acetaminophen	Ibuprofen

 

 

Purpose of the experiment:  For this lab,  you will be introduced to a technique called thin layer chromatography (TLC).  This technique can be used to identify different components in a mixture.  In this experiment, you will use this technique to identify analgesic drugs by isolating the active ingredients from these drugs. 

 

Required reading:  Mayo 90-93 (focus on polarity), 125-127

 

Procedure:

 

1.  Isolation of active Ingredient

 

Your instructor will assign for you one of the three unknown tablets.  Record the exact weight of the tablet before proceeding.  Crush the tablet between a folded piece of weighing paper.  Place the powder in a 3 mL conical vial, add about 2 mL of methanol, cap the vial and shake vigorously.  Loosen the cap a few times during the mixing process to release the build up of pressure in the vial.  Allow the solution to sit and settle for a few minutes.  Transfer the cloudy solution (supernatant) to a 16 x 125 mm test tube (leave the white solid behind).  Repeat the extraction process by adding an additional 2 mL of methanol in the vial, and transfer the solution to the same centrifuge tube that contains the first extract.  Centrifuge the mixture for 2-3 minutes or until the supernatant liquid is clear.  Don't forget to balance your test tube in the centrifuge by using another lab mate's test tube of the same volume or a test tube containing water.

 

Next step is to set up an alumina Pasteur pipet column to filter out any white solid that left in the solution.  To do this, obtain a clean Pasteur pipet, and use a second pipet to insert a small piece of cotton or glass wool through the top and wedge gently in the constriction of the clean pipet.    Use a paper towel to cover the narrow part of the pipet tip and break off the tip to a length of about 2 cm.  Clamp the pipet in a vertical position so that the liquid from the column drains into a small beaker.  Add 2 cm of alumina into the pipet and tap the column with your finger gently to pack the alumina.  Obtain 3-5 mL of methanol and add about 2 mL of methanol to wet the alumina (you don't have to collect these 2 mL of methanol).   Allow the liquid to drain until the level of methanol reaches the surface of the alumina.  If necessary add more methanol but don’t let the column run dry  (in other words don’t let the methanol to drain below the surface of the alumina).  Carefully transfer the clear liquid from the test tube to the column using the Pasteur pipet and collect the liquid that passes through the column in a small beaker.    Again wait for the liquid to reach the surface of the alumina, add an additional 1 mL of methanol and allow the solution to drain into the same beaker.  This ensures that the entire active ingredient in the analgesic drug has been eluted from the column.  At this point, keep a small amount of sample for TLC (see section 2).  It will take a while to develop a TLC plate so set up your TLC plate as soon as you get some sample from the column.

 

For the rest of the sample, evaporate the solvent by placing the beaker in a warm sand bath or a steam bath at about 50-70ºC.  When the solvent has completely evaporated or until the remaining liquid is no longer evaporating remove the vial from the water or sand bath and cool it down to RT.  The volume of the solution should be less than 0.5 mL before you stop the evaporation process.  Ibuprofen has a low melting point so it might be melted during the evaporation.  It is essential to complete the evaporation within 10-15 minutes.  Aspirin will partially decompose if you heat it too long.  Place the beaker in an ice-bath if necessary you can scrape the inside of the beaker with a glass rod or spatula to induce crystallization.

If your crystals are wet after the evaporation filter the crystals using the Hirsch funnel connected to a vacuum, break up the lumps and allow the solid to dry for 5 minutes on the funnel.  When the crystals are completely dry, determine the isolated weight.  Calculate the % recovery and run an IR of your sample. 

 

2.  Thin Layer Chromatography:

 

Obtain a TLC plate and a few micropipets from the reagents bench.  Your instructor will show you how to make micropipets if they are not provided.  Don't use Pasteur pipets to spot TLC plates.   Use a pencil and lightly draw a line about 1 cm above the bottom of the plate.  Mark the plate with four points evenly spaced where the samples will be spotted, three knowns and one unknown.  Obtain a few drops of the knowns in small test tubes. 

 

Use a 400 mL or a bigger beaker (make sure the TLC plate completely fits in the beaker) as a developing jar by introducing about 10 mL of 0.5% acetic acid in ethyl acetate eluting solvent mixture to cover the bottom of the jar to about 0.5 cm.  Introduce a filter paper folded to fit in the jar on the side, and cover the beaker with a watch glass.  Allow the solvent to soak the filter paper and let the atmosphere in the jar equilibrate.  The sample can be applied on the plate by gently spotting the tip of the filled capillary to the plate, and repeat the spotting about 2-3 times depending on the concentration of the solution.  It is important to allow the solvent to evaporate between each spotting.  

 

Visualize the developed TLC plate under UV light and mark them with pencil, and label UV.  Sketch the plate in your notebook with any necessary comments.

 

Calculate the Rf values and identify your unknown. 

 

Run an IR spectrum of your sample and use this to confirm the identity of the unknown.    Label the important peaks in the IR spectrum that correspond to functional groups present in the compound.

 

Questions:

 

1)  Define R_f value.

 

2)  Which compound in each of the two pairs below will have a larger Rf value if they were both run on the silica gel TLC plate in 5% ethyl acetate/hexane:  benzene or benzoic acid,  dimethyl amine or cyclooctanone?  Explain your reasoning.

 

3)  If a 755 mg Tylenol tablet contains 81% acetaminophen how many moles of acetaminophen are present in the tablet?

 

4)  What will be the result of the following errors in TLC technique?

a)      Solvent of too high polarity

b)      Solvent pool in developing jar too deep

c)      Forgetting to remove the TLC plate when the solvent has reached the top of the plate

d)      Forgetting to mark the solvent front immediately after the removal of the plate from the developing chamber.

 

References:

 

1)      Mayo, D. W.; Pike, R. M.; Trumper, P. K.  Microscale Techniques for the Organic Laboratory; Wiley & Sons:  New York 2001.

 

2)      Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G.  Introduction to Organic laboratory Techniques, A Microscale Approach; Saunders College Publishing: Fort Worth 1999.

 

3)      Padias, A. B. Organic Chemistry, Laboratory Manual; Hayden McNeil Publishing:  Plymouth 2001.