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Jimmy Frisbie
Experiment 23: Three Component Separation of Excedrin by Column Chromatography
Introduction
Although often expensive and time-consuming, column chromatography is a vital and popular
purifying technique for organic chemists. Unlike extraction of liquids or recrystallization of solids,
column chromatography can help separate both liquids and solids of different polarities.1 From this basic
technique of column chromatography, the scientific world has expanded its use for this separating
procedure. Today, different forms of chromatography are used to help separate proteins, such as amino
acids, lipids, and even chiral molecules.1,2,3 In this experiment, column chromatography is utilized to
separate Excedrin’s three key ingredients: aspirin, acetaminophen, and caffeine.
In column chromatography, a column is constructed using silica or alumina gel. After the
sample is added, the mobile phase is run through the column to help elute the different components.
Since the stationary phase is extremely polar, the least polar compounds elute first due to their inability
to interact with the silica or alumina. Then, as the polarity of the mobile phase is increased, the more
polar compounds elute.1
Although column chromatography can be conducted using dry column packing, the most
common method of constructing a column is a slurry pack. In this method, the solid stationary phase is
combined with the beginning mobile phase. After mixing these compounds into a paste, the
homogenous mixture is poured into the column. Additional mobile phase and solid stationary phase can
be added and flashed with nitrogen through the column to help achieve the desired column length.4
Excedrin is a very common analgesic, but many people fail to realize why this medication is
effective. In the body, arachidonic acid is metabolized to yield multiple prostaglandins.5 Many of these
metabolites cause inflammation. Excedrin contains three key ingredients: aspirin, acetaminophen, and
caffeine.6 Aspirin and acetaminophen are two important non-steroidal inflammatory drugs (NSAID’s).7
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Aspirin and acetaminophen help prevent inflammation by blocking COX-2, a key enzyme involved in
the conversion of arachidonic acid to prostaglandins, including PGJ2.8 Studies have shown that aspirin
may have advantages over other NSAID’s due to its potential to prevent cancer and increase
cardiovascular health.9 Acetaminophen’s mechanism pathway has been shown to also act on the
prostaglandin pathway through the central nervous system. Thus, acetaminophen does not effectively
curb inflammation in the periphery.10 In addition to curbing PGJ2 production, NSAID’s competitive
binding of COX-1 and COX-2 affect other prostaglandins, such as PGJE2. PGJE2 helps regulate blood
pressure and maintain balance in the gastrointestinal tracts.8
Figure 1: The three ingredients in Excedrin.
Caffeine, unlike aspirin and acetaminophen, is a stimulant and analgesic. It helps accomplish
this physiologic effect by blocking the adenosine receptor.9 Caffeine has been shown to not only help
increase focus and prevent headaches, but may also act as a therapeutic in different diseases, such as
Parkinson’s disease.9
The purpose of this experiment was to verify the quantity and presence of the ingredients in
Excedrin through flash chromatography. After running flash chromatography, it was necessary to use
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liquid extraction to help extract caffeine from the acetaminophen-caffeine fractions. By adding a strong
acid, acetaminophen was separated from caffeine by deprotonating the phenol of acetaminophen. Since
acetaminophen became a charged species, it was drawn into the aqueous layer, thus separating the
caffeine and acetaminophen. After the liquid extraction, 1H NMR was used to characterize all three
purified compounds.
Figure 2: The extraction process used to extract caffeine from the acetaminophen-caffeine fractions.
Experimental
Aspirin, Acetaminophen, and Caffeine. One Excedrin tablet containing aspirin (250 mg, 1.39 mmol),
acetaminophen (250 mg, 1.65 mmol), and caffeine (65 mg, 0.335 mmol) was crushed and added to
dichloromethane (20 ml). This solution was analyzed by TLC (75% ethyl acetate in hexanes) and then
evaporated using a warm water bath. A column was then slurry-packed (50% ethyl acetate in hexanes)
halfway up the column using silica gel. The first mobile phase (50% ethyl acetate in hexanes) was used
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to collect fractions (12 x 9.5 mL) containing aspirin. The second mobile phase (75% ethyl acetate in
hexanes) was used to collect fractions of acetaminophen (20 x 9.5 mL). The third mobile phase (100%
acetone) was used to collect caffeine (4 x 9.5 mL) fractions. After similar fractions were combined, the
solvent in the combined fractions was evaporated. The combined caffeine fractions were further
purified using liquid extraction. Potassium hydroxide (1M, 10 mL) was added, and then the aqueous
layer was drained. The organic layer was dried with sodium chloride (10 ml). The organic layer was
then evaporated to yield purified caffeine. Separation of Excedrin yielded: a whitish-powder: aspirin
(155 mg, 62.0%); a white-crystal: acetaminophen (150 mg, 42.0%); and a whitish-yellow compound:
caffeine (20 mg, 30.8%). Aspirin 1H NMR (400 MHz, CDCl3) δ (ppm) 10.96 (singlet, 1H), 8.08-8.05
(doublet, 1 H), 7.35-7.18 (multiplet, 3H), 2.3 (singlet, 3H); Acetaminophen 1H NMR (400 MHz, CDCl3)
δ (ppm) 9.61-9.56 (singlet, 1H), 9.10 (singlet, 1H), 7.40-7.20 (doublet, 2H), 6.79-6.53 (doublet, 2H),
1.967 (singlet, 3H); Caffeine 1H NMR (400 MHz, CDCl3) δ (ppm) 7.26 (singlet, 1H), 4.00 (singlet, 3H),
3.59 (singlet, 3H), 3.42 (singlet, 3H).
Results and Discussion
In this experiment, aspirin, acetaminophen, and caffeine were all extracted from Excedrin using
column chromatography. Caffeine was further purified from acetaminophen utilizing liquid extraction.
The products were then characterized by 1H NMR analysis.
Before a column was constructed, the reaction mixture of the Excedrin tablet was analyzed using
TLC. Aspirin had the highest Rf value, while caffeine had the lowest Rf value. These Rf values
confirmed that even though aspirin is highly polar due to its carboxylic acid and ester, the aspirin is able
to hydrogen bond to itself, thus preventing it from interacting with the polar TLC plate. Acetaminophen
contains an amide and phenol, and had an Rf value between aspirin and caffeine. Caffeine had the
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lowest Rf values due to its six electronegative atoms. Caffeine contains two amides, as well as two
amines.
The column was slurry packed (50% ethyl acetate in hexanes) before loading the Excedrin. The
column was filled halfway with the stationary phase, and nitrogen was used to flash the column
throughout the experiment. Fractions of approximately 9.5 mL were taken to ensure that compounds
would not elute together. The first mobile phase (50% ethyl acetate in hexanes) was used to help elute
aspirin in twelve fractions. The mobile phase was then increased (25% ethyl acetate in hexanes) to elute
the acetaminophen. Twenty fractions of the second mobile phase were collected. The third mobile
phase (100% acetone) helped elute any caffeine or other compounds left the column.
Although aspirin and a large amount of acetaminophen eluted purely, the last eight fractions of
the second mobile phases yielded both acetaminophen and caffeine. The final mobile phase of acetone
fractions were also contaminated. As a result, liquid extraction was used to separate the caffeine from
the acetaminophen. After adding ether (20 mL) to the contaminated fractions, the acetaminophen was
extracted from the organic layer using KOH (1M 10 ml). The ether layer was then washed with sodium
chloride and anhydrous sodium sulfate. The aqueous layer was treated with HCl (6M 20 mL) in an
attempt to recover acetaminophen, but crystals never crashed out of the solution.
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H NMR analysis supported the purity of all three compounds. 155 mg of aspirin was recovered
(62.0%), while 105 mg of acetaminophen were recovered (42.0%). Only 20 mg of caffeine was
recovered (30.8%), but this was most likely due to the small amount of caffeine present in Excedrin (65
mg). These percent yields were well within the accepted range. For this experiment, chromatography
yields of aspirin are normally between 60-100%, while acetaminophen is normally 10-20%. Liquid
extraction of caffeine in this experiment was extremely successful since normal extraction yields are
extremely low (1-5%).6
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The 1H NMR analysis of aspirin showed 8 hydrogens. The acidic hydrogen of the carboxylic
acid was found at 10.96 ppm, while the methyl hydrogens were found at 2.34 ppm. The methyl’s slight
downfield shift is due to the ester’s polarity and inductive effect. The four aromatic hydrogens were
found between 8.08 ppm and 7.18 ppm. Three of the hydrogens attached to the aromatic ring were
found in a large multiplex (see Figure 3). Surprisingly, one of the aromatic hydrogens appeared as a
doublet that was distinct from the other three hydrogens. This is most likely due to the carboxylic acid’s
deshielding effect. Overall, the NMR characterization of aspirin confirmed the purity of the compound
that was seen on the TLC plates performed during the flash chromatography.
Acetaminophen’s characterization by 1H NMR showed a very strong phenol at 9.61 ppm (see
Figure 4). The hydrogen of the amine was found at 9.10 ppm as a strong singlet. Once again, a methyl
singlet containing three hydrogens was found at 1.9 ppm. However, since an amide does not have as
much of an inductive effect as an ester, the methyl shift for acetaminophen was not as extreme as the
methyl shift in aspirin (Compare functional groups in Figure 1). The aromatic hydrogens were found
between 7.40-7.20 ppm and 6.79-6.53 ppm. Both splitting patterns were doublets due to their
neighboring aromatic hydrogens. Most likely, the hydrogens closest to nitrogen represent the signals
that are farther downfield. Since both oxygen and nitrogen are donating groups, all aromatic hydrogens
are slightly shielded. However, nitrogen is less electronegative, so it will more easily donate the
elections. Small imprints of DMSO were found between 2-3.5 ppm, but there integrative values were
negligent. It is also possible, but unlikely, that these small impurities may be due to methyl groups in
caffeine. The lack of aspirin in the acetaminophen was observed since the there was no distinctive peak
around 11 ppm, which would have signified the hydrogen of the carboxylic acid.
Since only 20 mg of caffeine was purified through the extraction process, 1H NMR showed weak
signals. However, NMR characterization supported the purity of caffeine. There were three methyl
groups at 3.41 ppm, 3.59 ppm, and 4.00 ppm. “B” labeled hydrogens (see Figure 5) were the farthest
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downfield due to the large inductive effect of the two carbonyls. “D” labeled hydrogens were more
upfield due to the lack of strong inductive effects. The hydrogen attached to the sp2 carbon was found at
7.26 ppm. This downfield stretch was confirmed through known caffeine 1H NMR characterization that
showed an sp2 peak around 7.5 ppm.11
In conclusion, Excedrin was successfully separated into its three main compounds – aspirin,
acetaminophen, and caffeine. Column chromatography completely purified aspirin and a portion of
acetaminophen. However, liquid extraction was necessary to elute the caffeine from the remaining
acetaminophen. The overall yield of aspirin (62.0%), acetaminophen (40.0%), and caffeine (30.8%) all
indicate these separation techniques are very powerful in separating compounds of different polarities.
Using more Excedrin in future experiments may help increase the quantity of caffeine to help run
a better 1H NMR analysis. The percent yield in this experiment of caffeine was relatively high, yet it
was still difficult to gain an accurate 1H NMR reading. In addition, a longer column may fix the
problem of acetaminophen and caffeine eluting together. With a longer column, the compounds will
have a longer distance to travel, thus increasing the separation between the compounds. In the future, if
more time is allotted in lab, gravity column chromatography may yield a better separation of
acetaminophen and caffeine than flash chromatography. Finally, it would be useful to do this
experiment twice using two different methods. The separation could be performed by not only column
chromatography, but also completely by liquid extraction. Then, by comparing percent yields between
the two reactions, it would be possible to make conclusions about the effectiveness of column
chromatography compared to liquid extraction.
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References
(1) Huber, J.F.; Hulsman, A.R.; Analytica Chimica Acta. 1967. 38, 305-313.
(2) Soloshonok, V.A.; Angewandte Chemie International Edition. 2005, 45, 766-769.
(3) Peterson, E.A.; Sober, H.A.; Methods in Enzymology. 1962. 5, 3-27.
(4) Rummel, S.; Beiswenger, K.; Lab Guide for Chemistry 213: Introductory Organic Chemistry
(5) Laboratories. Haydn-McNeil: Plymouth, MI; 2015; 211-217.
(6) Revell, K.D.; J.Chem.Ed. 2011, 88, 1413-1415.
(7) Michael, J.; Bonny, B.; Cancer Prevent II, 2009, 181, 215-221.
(8) Crawley, B.; Saito, O.; Malkmus, S.; Fitzsimmons, B.; Hua, X.; Yaksh, T.; Neurosci Lett. 2008,
442, 50-53.
(9) Ricciotti, E.; Fitzgerald, G.A.; Arterioscler Thromb Vasc Biol. 2011, 31, 986-1000.
(10) Petzer, J.P.; Anel, P.; Current Medicinal Chemistry. 2015, 22, 975-988.
(11) Li, N.; Kaifa, T.; 2003, 15, 208-211
Supplemental Information:
Figure 1: The three common ingredients in Excedrin.
Figure 2: The extraction process used to extract caffeine from the acetaminophen-caffeine fractions
Figure 3: 40 MHz 1H NMR of aspirin
Figure 4: 40 MHz 1H NMR of acetaminophen
Figure 5: 40 MHz 1H NMR of caffeine.
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