Separation of the Components of Extra-Strength Excedrin II: Column Chromatography

Introduction
Column chromatography will be used to separate the components of extra -strength Excedrin, a combination pain reliever that contains three active ingredients: acetylsalicylic acid (aspirin), acetaminophen, and caffeine. The components will be isolated, analyzed for purity by thin-layer chromatography and their percent recoveries determined.1

Background
One tablet of extra-strength Excedrin contains aspirin (250 mg), acetaminophen (250 mg), and caffeine (65 mg) as well as a binding material. The combination of these three compounds is said to be effective against migraine headaches.2 Aspirin, an analgesic and anti-inflammatory, is the most used drug world-wide with an annual global consumption of 40,000 tons. 3 Caffeine, a stimulant, is the most consumed psychoactive substance in the world and one that 90% of North Americans consume on a daily basis. 3 Acetaminophen is an analgesic and antipyretic that is sold as the brand name Tylenol as well as being present in several combination products in addition to Excedrin. 2

Column and thin-layer chromatography separate compounds based on the polarities of the compounds, with more polar compounds adhering better to, and therefore moving more slowly through, the solid support. Column (liquid) chromatography will be used to separate and isolate the three compounds. Thin -layer chromatography will be used to determine which column fractions contain the three components and to assess the purity of the isolated compounds. The percent recovery of each component will be calculated based on the amount of that component that is alleged to be present in the tablet. According to the original source 1 the percent recoveries of aspirin, acetaminophen, and caffeine are typically >60%, 10-20%, and 40%, respectively. If you also do the acid-base extraction to separate the components of a tablet, your instructor may have you compare the two separation techniques with respect to the percent recoveries and purities of the three substances isolated.

Additional Reading
Techniques in Organic Chemistry, 3rd ed., by Mohrig, Hammond and Schatz: Essay on Intermolecular Forces starting on page 99 and Essay on Chromatography on page 219. Organic Chemistry, 6th ed., by Bruice: Sections 1.16-1.25

Techniques (from Techniques in Organic Chemistry, 3rd ed., by Mohrig, Hammond and Schatz
Measurements: Chapter 5. Recovering Products: Chapter 12 (no rotovap). Thin-Layer Chromatography: Chapter 17. Column (Liquid) Chromatography: Sections 18.1-18.8

Safety Information
(from www.sciencelab.com MSDS information, Mohrig loose-leaf pages, and common knowledge) Acetaminophen, N-(4-hydroxyphenyl)acetamide, is slightly hazardous in case of skin contact (irritant), of ey e contact (irritant), of ingestion, of inhalation.

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Acetic acid is very hazardous in case of skin contact (irritant), eye contact (irritant), ingestion, of inhalation. Hazardous in case of skin contact (corrosive, permeator), of eye contact (corrosive). Liquid or spray mist may produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may produce burns. Inhalation of the spray mist may produce severe irritation of respiratory tract. Acetone is hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation. Slightly hazardous in case of skin contact (permeator). Flammable with flash points of -20C (closed cup) and -9 (open cup). Acetylsalicylic acid is hazardous in case of skin contact (irritant), of eye contact (irritant). Slightly hazardous in case of skin contact (corrosive, permeator), of ingestion, of inhalation. Severe over -exposure can result in death. Caffeine is toxic and an irritant in its solid form. Avoid contact with skin, eyes, and clothing. Mutagenic for mammalian somatic cells. May be toxic to heart, gastrointestinal tract, central nervous system (CNS). Repeated or prolonged exposure to the substance can produce target organs damage. Diethyl ether, also known as ether, is very flammable and volatile with a low flash point (-45C, closed cup). Use in a hood if possible. Keep away from hot electrical devices. Do not use anywhere near an open flame. Hazardous in case of skin contact (irritant), of eye contact (i rritant), of ingestion, of inhalation. Slightly hazardous in case of skin contact (permeator). Ethyl acetate is hazardous in case of ingestion or of inhalation. Slightly hazardous in case of skin contact (irritant, permeator), of eye contact (irritant). Flammable with flash points of -4.4C (closed cup) and 7.2 (open cup). Hexanes are hazardous in case of skin contact (permeator), of ingestion, of inhalation. Slightly hazardous in case of skin contact (irritant), of eye contact (irritant). May be toxic to peripheral nervous system, skin, central nervous system (CNS). Flammable with a flash point of -22.5C (closed cup). Silica (silica gel, SiO2xH 2 O) is slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation.

Procedure
A. Thin-Layer Chromatography of Reference Compounds and Tablet. You may be able to omit this part if already done during the other Excedrin Separation Lab, as it is the same as the first TLC in part B of that lab. Consult with your instructor. 1. You will be provided with reference solutions of the three pure compounds and of the tablet. 2. Obtain a 2 x 5 cm TLC plate. Lightly draw a pencil line 1 cm from the bottom of the plate. Lightly draw 4 hatch marks, evenly spaced across the starting line. Label the hatch marks 1-4. (Technique 17.2) 3. Use a micropipettor to deposit a tiny amount of each reference solution on one of the three marks. On the last hatch mark, spot a tiny amount of the solution of the soluble components of the Excedrin tablet. Keep track of which is which! Look at the undeveloped plate under the UV lamp to make sure that there is enough of each material to see, but that the spots are not too large. If the spots are not visible, then spot over them a few times each, allow the spot to dry each time before spotting again 4. Using a small beaker, prepare a developing jar containing <1 cm depth of a solution of 1:2 hexanes:ethyl acetate with 1% acetic acid. Make sure you have a lid to cover the jar (a watchglass or stretchy plastic wrap work well). (Technique 17.3) 5. Using your forceps, place the TLC plate in the developing chamber. Allow it to remain undisturbed in the covered chamber until the solvent is nearly to the top of the plate (3-5 cm from the top). 6. Remove the TLC plate. Immediately lightly draw a pencil mark to indicate the location of the solvent front, then wait for about 20 seconds to allow the solvent to evaporate. 7. Use the UV light to visualize your spots (Figure 17.6). Use a pencil to trace the location and size of your spots. Record a sketch of your TLC plate in your notebook. Calculate the Rf of each spot (Technique 17.5). 8. Expected approximate Rf values are as follows: aspirin-0.6, acetaminophen-0.3, and caffeine-0.1 9. Cleanup: all solvents and TLC samples and plates go in the organic waste. Micropipettors go in the broken glass container. B. Column Chromatography (Part II) (Technique 18.4) ( 1. Crush one Excedrin tablet using a mortar and pestle. To the mortar, add 20 mL of diethyl ether and stir. While the three active components will dissolve, the starch binder will not. In order to remove this binder, decant the ethereal solution through a funnel equipped with filter pape r or a plug of glass wool in the stem into a small beaker and rinse the filtrate with 5 ml diethyl ether. Put a boiling stick in the ether solution and evaporate most, but not all, of the ether, in a warm water bath Then remove the beaker from the heat source, add a small amount of silica (approximately mL, or a scoopula tip -ful) and stir the slurry with the boiling stick to finish the evaporation, leaving behind a powder.

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2.

Obtain 30 mL of each of the solvents in labeled beakers: solvent A (1:1 hexanes:ethyl acetate), solvent B (1:2 hexanes:ethyl acetate), reagent-grade acetone (not from squirt bottle). Label six 15-mL capacity test tubes (your largest test tubes). Get a large-capacity (blue) pipet bulb. Also get 3 g of silica gel and a scoop of sand in separate containers. 3. Obtain a chromatography column and check to make sure that the frit is OK: if it isnt you will need to use a plug of cotton or glass wool. Pour the 3 grams of silica gel into it. Add the silica/Excedrin mixture from your beaker (step B1) to the top of the silica already in the column. This is the dry adsorbent method on page 240 of the Techniques book, coupled with the sample adsorbed on silica gel application method on page 242. Add the sand to the top. 4. General instructions: You will add solvents A, B, and acetone in succession to the top of the column while collecting the eluent in 15-mL fractions from the bottom of the column. Add the solvent carefully so as not to disturb the silica at the top. You will have to add each solvent in aliquots as the column wont be large enough to hold 30 mL of solvent and the silica. Apply sufficient pressure so it takes no more than 5 minutes to collect each fraction. Do not let the level of the solvent go below the top of the sand: add more solve nt carefully when the solvent level is 1-1.5 cm from the top of the sand. 5. Detailed instructions: a. Add 30 mL, or however much fits, of solvent A to the top of the silica column. Collect the eluent in two test tubes (fractions 1 & 2), changing to the new tube once about 15 mL have been collected. (See elution of the column on page 243 of the Techniques book). You may use a blue single-outlet pipet bulb to provide air pressure to push the eluent through the column at a faster rate than provided by gravity alone; however, you must be careful not to release the pipet bulb and suck the silica back up the column. Add more of solvent A when the solvent level is 1-1.5 cm from the top of the sand. b. When the top of solvent A is 1-1.5 cm from the top of the silica, carefully start adding the solvent B while continuing to collect fraction #2 until you have about 15 mL and then change to tube #3. c. Elute 30 mL of solvent B through the silica column, collect the eluent in two test tubes (fractions 3 & 4) d. Elute 30 mL of acetone through the silica column. Collect the eluent in two test tubes (fractions 5 & 6). 6. On a single extra-wide TLC plate (or use two regular width ones), draw a line with six hatch marks across the bottom. Spot a small amount from each tube on the hatch marks, and develop the TLC plate as in part A. Record a sketch of this TLC plate in your notebook. You do not need to calculate R f values. 7. Use the TLC to plan which tubes to combine to isolate each component and show your TLC and and your plan to the instructor before proceeding. Determine which tube(s) contain primarily the top spot, and empty these tubes into a pre-weighed beaker. Evaporate the solvent using a water or steam bath heated with a hot plate. Weigh the beaker plus solid, and determine the mass of the recovered material. 8. Repeat step 11 for the middle spot, and then for the lower spot. You should now have three solid samples, one for each active ingredient, in three separate beakers. 9. Report the mass and percent recovery of each of the three solids isolated. 10. Cleanup: Dump the silica and remaining solid out of the column, being sure to remove all of it and any cotton or glass wool used as a plug. You may need to apply pressure to the bottom to remove all of the solid. This can all go in the organic waste container. C. Thin-Layer Chromatography analysis of the separated components (optional) 1. In three separate test tubes, dissolve a small amount of each solid compound isolated from the column in a few drops of acetone. 2. Use TLC to analyze each using the procedure starting with step A.2, but using three hatch marks, one for each compound. 3. Draw a sketch of the TLC in your notebook and calculate R f values for each compound. Use the number of spots obtained for each component to assess its purity. 4. Expected approximate Rf values are as follows: aspirin-0.6, acetaminophen-0.3, and caffeine-0.1 5. Cleanup: all solvents and TLC samples and plates go in the organic waste. Micropipettors go in the broken glass container. References (1) Revell, K. D. J. Chem. Educ. 2011, 88, 1413-1415. This is the source of this laboratory experiment. (2) PubMed Health - http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0000521/ (3) Farmer,S.C.. J. Chem. Educ. 2011, 88, 1648-1650.

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