1
Professional Strength
ASPINOL®
Ingredients:
Aspirin ...... 1,200,000 g
Acetaminophen ...... 1,000,000 g
Inert Material ...... 800,000 g
Dist. by B&B Pharmaceuticals, Cape Girardeau, MO
“We Sell ‘em Any Way We Can!”
ANALYSIS OF ASPINOL
One of the responsibilities of the U.S. Food and Drug Administration (FDA) is to ensure that over-the-counter drugs are safe and effective. Testing drugs entails identifying and quantitating the amounts of each component in a preparation. In this experiment you are playing the part of a FDA chemist, and your job is to analyze a newly- marketed drug, Aspinol. B & B Pharmaceuticals recently introduced Aspinol, which is reported to contain aspirin, acetaminophen (Tylenol), and some filler, usually sucrose. B & B has been accused of marketing a product which does not meet the approved specifications. In some lots, acetanilide or phenacetin has been substituted for acetaminophen. Acetanilide costs less to manufacture and has similar analgesic properties to acetaminophen, but is somewhat more toxic. Phenacetin is similarly more toxic than acetaminophen. In other lots, the aspirin or acetaminophen, or both, is missing. Your job is to check a sample of Aspinol and determine its composition.
Aspirin / Acetanilide / Acetaminophen / PhenacetinSince Aspinol is a mixture of compounds you must first separate the components of the mixture before beginning any analysis. To obtain pure compounds from this mixture, you will take advantage of the physical and chemical differences of the individual components. Substances having different solubilities in a given solvent can be separated by extraction or filtration. Acidic or basic compounds can be converted into water-soluble salts, which can then be separated from water-insoluble components. It is this strategy that will be used to separate the components of Aspinol.
The separation is begun by dissolving the Aspinol in dichloromethane (CH2Cl2, sometimes called methylene chloride). The filler material is insoluble in the dichloromethane, and can be filtered away from any aspirin and acetanilide or phenacetin.
Since aspirin is a carboxylic acid it will react with dilute sodium bicarbonate solution and form a sodium salt. This salt (see reaction below) is soluble in aqueous solution and thus the aspirin can be separated from any acetanilide which might be present.
Since acetanilide and phenacetin do not react with dilute sodium bicarbonate to form a water-soluble salt and since they are water-insoluble, they remain in the dichloromethane. By evaporating or distilling off the dichloromethane any acetanilide or phenacetin present may be recovered.
Aspirin may be recovered by acidifying the aqueous solution, chilling, and then filtering the solid aspirin.
PROCEDURE
CAUTION: Dichloromethane may be harmful if ingested, inhaled, or absorbed through the skin. There is a possibility that dichloromethane may induce cancer, but animal tests have been inconclusive to date. Avoid prolonged or unnecessary contact with the liquid, and do not breathe its vapors.
SEPARATION OF THE FILLER
Obtain a sample of Aspinol, weigh it, and transfer it to a 150-mL beaker. Add 50 mL dichloromethane and stir the mixture thoroughly to dissolve as much of the solid as possible. Using a pre-weighed fluted filter paper, filter the mixture to separate the filler material. Set the filter paper aside (you can set it in the hood to minimize your exposure to the dichloromethane vapors - put your initials on the filter paper lightly in pencil) and reweigh it when all the dichloromethane has evaporated. You may wish to weigh this sample more than once to ensure that all the dichloromethane has evaporated. Record the weight of the filter paper and solid, and determine the weight of solid by subtracting the weight of the filter paper. This solid is the filler present in your sample.
SEPARATION OF ASPIRIN
Transfer the dichloromethane filtrate (liquid) to a separatory funnel and extract the aspirin from it with two 25-mL portions of aqueous sodium bicarbonate solution. Dichloromethane has a density greater than that of the sodium bicarbonate solution, so it will be the bottom layer. Separate the two layers. You will have to transfer each layer to a separate container and then return the dichloromethane to the separatory funnel for the second extraction.
Save the dichloromethane solution for isolation of acetanilide or phenacetin, below!
Combine the two sodium bicarbonate solutions in 250-mL beaker. Add 10 mL of 6 M hydrochloric acid in small portions with vigorous stirring: the solution will bubble as the acid is added. Test the pH of the solution with pH paper: be sure the pH is 2 or lower. If the pH is above 2, add more acid to bring it below 2. Cool the mixture in a ice bath and collect the aspirin by vacuum filtration. Wash the aspirin with only a few mLs of cold water. Let the aspirin dry on the Buchner funnel for a couple of minutes. Scrape out the crystals onto a watch glass and set it aside to air-dry until next laboratory period. The filtrate in the filter flask may be poured down the drain.
ISOLATION OF ACETANILIDE OR PHENACETIN
Pour the dichloromethane solution into a 100-mL round-bottom flask and remove the dichloromethane on the Rotary Evaporator. Scrape out the remaining solid onto a watch glass, allow it to dry, and weigh the dried material.
Take melting-points of any analgesic components, if instructed to do so.
IDENTIFICATION OF THE COMPONENTS BY THIN-LAYER CHROMATOGRAPHY.
Procedure:
Obtain a TLC plate, and draw a light pencil line across the width of the plate, about 1 cm from the edge. Place 5 light “tic marks” on the line, approximately equally spaced.
In separate test-tubes, dissolve tiny amounts of your aspirin and your unknown (acetanilide or phenacetin) in about 1 mL of acetone. Obtain two spotting capillaries. Using one of the spotting capillaries, spot a tiny drop of the acetone solution of your aspirin on the left “tic-mark” - you may want to practice spotting on a paper towel or tissue, to make small spots. Allow it to dry, then check the plate under a short-wave UV light, to see if you have a visible spot. If not, spot your solution again on the same “tic-mark”. Do not spot any more times than needed - too much material may give results that are hard to interpret! Using a different capillary, spot the solution of your unknown on the second to the leftmost “tic-mark”. After you have your samples spotted, take your plate to the standards in the hood, and spot acetanilide on the middle “tic-mark”, the phenacetin on the second to the rightmost “tic-mark”, and the aspirin on the rightmost “tic-mark”. Do not get the spotting capillaries contaminated! Check the spots under the UV light, to see if they are all visible.
To prepare a developing chamber, obtain a piece of 11 cm filter paper, and fold it about 1 inch from the edge, to obtain a flat edge. Place the filter paper, flat edge down, in your 400 mL beaker. Obtain 15 mL of the TLC solvent, methyl t-butyl ether, and pour it into the beaker, swirling the beaker to wet the filter paper with the solvent.
TLC PlateTLC Developing Chamber
Carefully place the TLC plate in the developing chamber, spotted side down, trying not to splash the solvent on the plate. The level of solvent must be below the pencil line. Cover the beaker with a watch glass. The solvent will rise up through the stationary phase on the plate. When the solvent has risen to 1-2 cm from the top of the plate, remove the plate, and draw a light pencil line across the plate, at the level to which the solvent rose. Allow the solvent to evaporate (waving the plate in the air will speed this up), then look at the plate under the UV light. Circle all of the spots.
You will identify your unknown by comparing the distances the components traveled up the plate with the distances the standards traveled. These distances are reported as Rf (retention factor) values.
XDistance the spot traveled
Rf = --- = ------
YDistance the solvent traveled
Measure the distance a spot moves from the center of the spot.
A developed plate may look like this:
From the locations of the spots, it would appear that the Your Aspirin (YA) contains Aspirin (A) and acetanilide (Ac). Perhaps you didn’t do a good job of separation in the extraction. Your unknown (YU) only contains Ac. The spot for Ac in the standard looks larger than the spot for Ac in YU: this is probably due to different amounts of material being spotted on the plate initially. The Rf value for the A spot would be X/Y: measure X from the middle of the spot. Separate Rf values are calculated for each of the spots in YA. Since the spots for the materials in the samples and in the standards are different sizes and shapes, they may have slightly different Rf values.
Cleaning Up
When you are finished with the experiment, pour the TLC solvent in the “Recovered TLC Solvent” container. The filter paper may be thrown away in the trash can. Used spotting capillaries should be placed in the “Clean Broken Glass” container. Your acetone solutions you used to spot with may be flushed down the sink with lots of water.
Report Format for Analysis of Aspinol
1. Title Page
a. A descriptive title containing 15 - 25 words.
b. Course and section numbers.
c. Dates that the experiment was performed.
d.Your name, and partner’s name, if any.
Tape your TLC plate to the bottom of the page, and label it clearly to explain what spots are what.
2. Aspinol Data and Calculations (New Page)
a.Unknown number.
b. Initial weight of your Aspinol sample.
c. Weight of fluted filter paper.
d. Weight of filter paper and sample after the methylene chloride filtration.
e. Weight of filler. (d-c)
f. Weight of aspirin.
g. Melting point range of aspirin
h. Weight of unknown.
i. Melting point range of unknown
j. Calculate your individual percent recoveries of filler, aspirin, and unknown, plus the total percent recovery.
k. Calculate Rf values for all of the spots on your TLC plate.
(Show your calculations for parts j. & k.)
3. Questions
a. What was your total percent recovery? Of which components do you think you might have lost some? Explain where these losses most likely occurred. If your percent yield was >100%, give two likely possibilities to explain this.
b. What does the thin-layer chromatography tell you about the purity of each of the components of Aspinol that you isolated? Explain your reasoning.
c. What do your melting point ranges tell you about the purity of each of the components of Aspinol that you isolated? Explain your reasoning.
Aspinol Operations Handout
Gravity Filtration
BeakerStemless Funnel Fan-Folded Filter PaperComplete Assembly
Suction Filtration
Assembled Apparatus Filter Flask Büchner Funnel Neoprene Adapter
1.Clamp your filter flask to a support rod.
2.Place a neoprene adapter in the neck of the flask.
3.Place the Büchner funnel on the neoprene adapter.
4.Place a piece of 5.25 cm filter paper in the Büchner funnel, and wet it with a few mLs of water.
5.Connect the filter flask to the water aspirator with your thick tubing, and turn on the aspirator to start the vacuum.
6.Pour the aspirin solution into the funnel. You may need to press down on the funnel to initiate the vacuum. Scrape the crystals out of the beaker with your spatula. You may rinse the container with a few mLs of cold water solvent to get the last little bit out.