Comparison of Base Extraction versus Column Chromatography for Separation of the

Components of Excedrin and the Synthesis of Acetaminophen

August Rothenberger

Department of Chemistry, The Pennsylvania State University, University Park, PA 16802

ajr6121@psu.edu

Abstract ..........................................................................................................................................

The techniques of base extraction and column chromatography were used to separate the components of
an Excedrin tablet into acetaminophen, aspirin, and caffeine. The components were identified by 1H NMR
spectroscopy and TLC chromatography. The column technique was determined to better separate the
components of the tablet due to higher percent recovery. Additionally, acetaminophen was synthesized
from p-aminophenol via an acyl substitution mechanism, then purified by recrystallization and identified
by IR and 1H NMR analyses.

Introduction ...................................................................................................................................

Medicines and pharmaceutical research was started in the late 19th century when Bayer performed the first
manufactured synthesis of an organic drug, the painkiller aspirin, which is found naturally in the willow
tree.1 Since then, chemists have explored the active ingredients in many natural remedies so that they
might discover new molecules to artificially synthesize and produce. Countless numbers of drugs have
been synthesized through the years, vastly improving the quality of modern medicine. Some of the
simpler drugs are sold as over-the-counter medications, like Advil® and Tylenol®, which contain
ibuprofen and acetaminophen, respectively.2 Many medications combine active ingredients to provide a
different effect to consumers. Extra-strength Excedrin® combines the analgesics acetaminophen and
aspirin with the stimulant caffeine, and is typically used for treatment of migranes.2 The active ingredients
are shown below in Figure 1.

Figure 1. Active Ingredients of Excedrin

The technique module of this report will describe the separation of an Excedrin tablet into its active
ingredients. The separation will be performed by base extraction and column chromatography. Base
extraction is used to separate a mixture of organic acids by their acidity. Starting with a weak base, the
strongest acid in solution can be deprotonated. The now charged molecule moves to an aqueous phase
where it can be removed.3 Progressively stronger bases can then be used to remove weaker acids until all
the mixture components are separated. If appropriate bases are used, the technique can showcase
extremely high precision in mixture separation.
Chromatography is a technique used to separate molecules by their polarity. Column chromatography and
thin liquid chromatography, TLC, will be used in this report. A typical column used for organic chemistry
is packed with silica, then a nonpolar solvent is used as a mobile phase. As the mixture flows through the
column, polar molecules will travel more slowly due to their attraction to the silica. Once separated, the
individual components can be collected as they exit the column.3 Typically, the column is pressurized to
elute the components more quickly, called flash chromatography. Column chromatography is used
extensively in organic synthesis to separate and purify product mixtures. TLC works by the same
principle as a column but operates on a much smaller scale, using drops of sample on a silica plate. It is
typically used to monitor the progress of chemical reactions and columns.3

The synthesis module will outline the formation of acetaminophen (1) from p-aminophenol (2) and acetic
anhydride. The key step in the reaction is a nucleophilic acyl substitution under acidic conditions to form
an amide linkage. Amides are extremely important in bioorganic chemistry as they are the linkage used in
peptides. Many amides can be synthesized by reacting ammonia or a primary/secondary amine with a
carboxylic acid derivative, typically under acidic conditions. A scheme for the synthesis of 1 as described
in this report is shown below (Scheme 1).

Scheme 1. Acyl Substitution of p-Aminophenol to Synthesize Acetaminophen

Results and Discussion..................................................................................................................

Base Extraction of Excedrin

Two Excedrin tablets were crushed into a fine, white powder, then dissolved in ether and filtered to
extract the organic active ingredients. In a separatory funnel, 1M K2HPO4 was added to remove aspirin,
which is fairly acidic with a pKa of 3.5 due to the carboxylic acid functionality.2 The aqueous layer was
removed, then 1M KOH was added to remove acetaminophen, which due to its phenol group has a pKa of
9.9.2 Acid was added to these two aqueous layers to neutralize the dissolved organics and force their
precipitation from solution. The solid product was then washed to procure 210.5 mg of aspirin and 12.6
mg acetaminophen. The ether was removed from the organic layer by evaporation to leave 28.0 mg
caffeine. One Excedrin tablet contains 250 mg acetaminophen, 250 mg aspirin, and 65 mg caffeine,4 so a
percent recovery can be calculated for the technique to be 42.1% aspirin, 2.52% acetaminophen, and
43.1% caffeine.
1
H NMR spectroscopy was performed to confirm the identity of these products. Data collection was
performed on a 400 MHz spectrometer in CDCl3 for all samples. The plots for aspirin, acetaminophen,
and caffeine are included and annotated as Supp. 1 – 3, respectively. The spectrometer was not shimmed
properly so the data are very noisy and splitting is non-ideal. For aspirin and acetaminophen, solvent
peaks are much larger than the sample’s, but it is still possible to confirm the structure identity. The
caffeine sample is essentially absent of product, but three peaks for the N-methyl groups are discernable
around 3.5 ppm indicating that it may be present in trace amount.
Column Chromatography Separation of Excedrin
Two Excedrin tablets were crushed and dissolved in ether. A column was packed with silica gel using a
mobile phase of 1 hexanes : 1 ethyl acetate. The sample was loaded and 30 mL of the same mobile phase
was eluted through the column and collected in equal volumes as fractions 1 and 2. The solvent was then
switched to 2 hexanes : 1 ethyl acetate to collect fractions 3 and 4. Finally, 30 mL acetone was eluted and
collected as fraction 5 and 6. TLC was performed on the fractions and the plates provided on page 18 in
the notebook pages. Fraction 2 contained two different products and fraction 6 no product, so they were
removed from the sample set. Fractions 3&4 contained the same product and were combined. Liquid was
removed from fractions 1, 3&4, and 5 by drying with nitrogen to procure solid residue of 244.0 mg, 22.4
mg, and 52.7 mg.

The products were identified by TLC and 1H NMR spectroscopy. TLC was performed on a sample of
Excedrin against standards of its components using 1 hexanes : 2 ethyl acetate as a mobile phase. The
plate is shown on notebook page 15. Comparing this plate to those of the column fractions, they can be
identified as 1: aspirin, 3&4: acetaminophen, and 5: caffeine. 1H NMR was ran on a 400 MHz
spectrometer for all products in CDCl3 and are included and annotated as Supp. 4 – 6. Again, the
spectrometer was not shimmed properly so the data experienced the same issues as the base extraction
samples, but crucial peaks are still identifiable. Fraction 1 contains a singlet at 11.82 which identifies the
carboxylic acid on aspirin, fractions 3&4 contains two downfield singlets at 8.12 and 7.63 for the phenol
and amide protons on acetaminophen, and fraction 5 contains three singlets integrating to about 3 for the
three N-methyl groups. With known products, the percent recovery for the technique can be calculated to
be 48.8% aspirin, 4.48% acetaminophen, and 81.1% caffeine.

Technique Comparison
Both the base extraction and column chromatography techniques proved to be an effective method of
separating the components of Excedrin. The base extraction gave decent yields for all products except
acetaminophen. The procedure was fairly straightforward and worked well, although the precipitation of
aspirin and acetaminophen from solution took a few days to occur. TLC could have been performed to
assess the purity of the products and strengthen the extraction results. Column chromatography produced
much better yields for all samples, but acetaminophen was still very low. The low recovery of
acetaminophen for both samples was likely due to its higher solubility in water compared to aspirin and
caffeine. The column procedure was straightforward, time efficient and ran very smoothly. Additionally,
the technique is more readily adapted to extracting a greater volume of sample than base extraction. For
these reasons, column chromatography should be considered the superior separation technique.

Synthesis of Acetaminophen
As shown in Scheme 1, acetaminophen can be synthesized via an acyl substitution of p-aminophenol
using acetic anhydride under acidic conditions. The mechanism for the reaction occurs by the activation
of the anhydride carbonyl followed by nucleophilic attack by the amine. A proton transfer occurs from the
amine to the carboxyl group, then reformation of the carbonyl removes acetic acid. Deprotonation of the
carbonyl yields the desired product. The reaction was carried out in a single step while heating, then
cooled to precipitate the product as a white powder. The crude product was purified by recrystallization in
20 mL water at 44.8% recovery. The final reaction yield was calculated by mass to be 15.5%. The product
recovery through precipitation likely led to some product loss, since some product must remain in
solution. The recrystallization yield would probably have been improved if it were carried out for a longer
period of time. The mechanism is technically reversible, but unlikely due to the high reactivity of acetic
anhydride, so recovery methods were likely major the main cause of product loss.

The pure product was characterized by IR and 1H NMR spectroscopy. The most important peaks in the IR
spectrum (Supp. 7) are the C=O stretch at 1651 cm-1 and C-N stretch at 1225 cm-1 signifying formation of
the conjugated amide. Other distinguishing peaks include the O-H stretch at 3322 cm-1 of the phenol, the
C=C aromatic stretching between 1609-1435 cm-1, and the out-of-plane bending at 836 cm-1 of para
substituted ring. An N-H stretch should also be present but may have been obscured by the phenol O-H
stretch. A 60 MHz NMR spectrometer was used to analyze the product (Supp. 8); some important peaks
include the singlet at 9.7 ppm of the amide proton, the singlet at 9.1 ppm of the phenol proton, the
doublets and 7.261 and 6.653 ppm for the protons on the para substituted aromatic ring, and the singlet at
1.963 ppm of the methyl. There exists a solvent peak for DMSO at 2.494 ppm and a water peak at 3.318
ppm which is likely due to failure to properly dry the NMR tube. The melting point of the compound was
about 171.5°C, which is consistent with literature, further showing the purity of the product.

Conclusions
The separation of Excedrin into aspirin, acetaminophen, and caffeine was successfully performed using
both base extraction and column chromatography techniques. 1H NMR was most useful in identifying the
components. TLC should have been performed for base extraction to assess the purity of the recovered
products. Column chromatography produced higher percent recoveries for all components and is thus
considered the superior separation technique. Acetaminophen was successfully synthesized at 15.5%
yield using a nucleophilic acyl substitution mechanism. The product was purified by recrystallization and
identified by IR and 1H NMR. The low percent yield is largely attributed to recovery methods.

Experimental .................................................................................................................................

General Methods
Reagents were purchased from Sigma-Aldrich and used without further purification. Reactions were
carried out in open atmosphere. 1H NMR was performed on a Bruker UltraShield™ 400 MHz and Varian
EM360A 60 MHz NMR spectrometer. Infrared spectroscopy was performed on a Thermo Electron
Corporation Nicolet 380 FT-IR.

Acetominophen (1) p-aminophenol (1.5 g, 13.7 mmol) was dissolved in DI water (25 mL) with conc.
phosphoric acid (85 drops, 4.25 mL). The solution was placed in a warm water bath and acetic anhydride
(2 mL, 21.2 mmol) was added. After 10 min, the solution was placed in an ice bath until crystals formed.
The crude product was filtered and washed, then recrystallized in water (20 mL) to recover the pure
product as a white powder (0.32 g, 15.5%). IR (neat) 3322, 1651, 1609, 1563, 1505, 1435, 1225, 836; 1H
NMR (DMSO, 60 Hz) δ 9.100 (s, 1H), 7.261 (d, 9.24 Hz, 2H), 6.653 (d, 9.24 Hz, 2H), 3.318 (s, 1H),
1.963 (s, 3H); MP (pure) 171.5°C.

References ......................................................................................................................................

1. Landau, Elizabeth. “From a tree, a ‘miracle’ called aspirin.” CNN Health. Published Dec 22, 2010.
Accessed Apr 7, 2018. < http://www.cnn.com/2010/HEALTH/12/22/aspirin.history/index.html>

2. Masters, K. “CHEM 213M: Medicines Chemistry Module.” Penn State Chemistry Dept. 2018.

3. Williamson, K. L. and K. M. Masters. Macroscale and Microscale Organic Experiments. Cengage

Learning, Boston: 2017.

4. “What is Excedrin (Acetaminophen, Aspirin, and Caffeine)?” Everyday Health. Accessed Apr 7, 2018.
<https://www.everydayhealth.com/drugs/excedrin>
Acknowledgements .......................................................................................................................

Reagents, materials, spectral instrumentation, and laboratory space were provided by the Penn State
Chemistry Department. Special thanks to Steven Taylor and Prof. Katherine Masters for assistance and
guidance in writing this report.