Li 1

Three Component Separation of Excedrin by Column Chromatography

Experiment 23: Three Component Separation of Excedrin by Column Chromatography
Joy Li
Valeria Alstadt
213W.004
4 April 2016

Introduction
Column Chromatography functions as a highly utilized purification technique in organic
chemistry. This can be attributed to the fact that it has the ability to purify nearly any mixture of solid or
liquid. The process uses concepts similar to those in TLC to separate and purify the components in a
mixture, based on their respective polarities. Unlike TLC, column chromatography provides the isolated
collection of the components in a mixture.
Excedrin is a painkiller composed of aspirin 1 (acetylsalicylic acid), acetaminophen 2 (4acetamidophenol), and caffeine 3 (1,3,7-trimethylxanthine) (Figure 1, Supplemental Information). These
active ingredients are each analgesics that function together to provide pain relief. Acetaminophen and
aspirin belong to a group of non-steroidal anti-inflammatory drugs (NSAIDS). These drugs function by
stopping prostaglandin production, preventing inflammation by inhibiting cyclooxygenase enzymes. 1
Acetaminophen has been shown to be a preventative measure for pain. 2 Aspirin has been identified as an
alleviator of pain, due to its efficiency in inhibiting inflammation. 3 Caffeine is included as a means to
amplify the abilities of aspirin and acetaminophen.4
The three components are separated via column chromatography in manner dependent on their
respective polarities. The polarities of the components in a mixture intended for column chromatography
are exploited to distinguish and separate the components. This is achieved by a specific "mobile" and
"stationary" phase within the column. The column is packed with silica gel which is considerably polar,
attracting other polar molecules. The polarities of the components, relative to one another, determine

Li 2
how attracted each component is to the silica gel, participating in attractive forces that allow the more
polar molecules to adhere more strongly to the silica, while the less polar molecules are pushed through
the column by the solvent. The solvent polarity is slowly increased throughout the procedure to compete
with the interactions of the silica with the more polar molecules, eventually pulling these polar
molecules out through the column. The elution of the components starts with the least polar molecule
and ends with the most polar molecule.5

Figure 1. Aspirin, Acetaminophen, and Caffeine structures.

Aspirin elutes from the column first, because it is composed of four electronegative oxygen
atoms: an ester, and a carboxylic acid group, plus a hydrogen-bond donor, making it the least polar of
the group. It has the highest TLC Rf value, traveling the farthest distance. Next, acetaminophen follows,
with three electronegative atoms: amide and phenol groups plus two hydrogen bond donors. It is
relatively more polar than aspirin, and is more attracted to the silica, eluting second. Finally, caffeine
contains six electronegative atoms: amide and amine groups, making it the most polar component of
Excedrin. Caffeine elutes last, with the most polar solvent, and the lowest TLC Rf value.
The goal of the experiment was to separate the three components of an Excedrin tablet through
column chromatography. The process was monitored by thin-layer chromatography, and column
fractions were isolated to produce aspirin, acetaminophen, and caffeine. The components were analyzed
and characterized by 60MHz 1H NMR.

Li 3
Experimental
Acetylsalicylic acid, 4-acetamidophenol, 1,3,7-trimethylxanthine. One Excedrin tablet, consisting of
acetylsalicylic acid (250 mg, 1.39 mmol), 4-acetamidophenol (250 mg, 1.65 mmol), and 1,3,7trimethylxanthine (65 mg, 0.335 mmol), was crushed and dissolved with dichloromethane (20 mL).
Silica gel (~1/2 mL) was added to the solution. The mixture was stirred over a warm water bath until the
dichloromethane evaporated. Slurry-packed silica gel column chromatography (50% hexanes/50% ethyl
acetate; 25% hexanes/75% ethyl acetate; 100% acetone) was used to separate the solid mixture, and the
column was monitored by TLC (25% hexanes/75% ethyl acetate). Similar fractions were combined into
three separate solutions, and each was evaporated with a stream of nitrogen, producing white crystal
acetylsalicylic acid (240.6 mg, 96.24%) 1H NMR (60 MHz, d-DMSO): (ppm) 7.931-7.851 (d, 1H),
7.679-7.498 (d, 1H), 7.646-7.052 (t, t, 2H), 2.250 (s, 3H); white powder 4-acetamidophenol (189.6 mg,
75.8%) 1H NMR (60 MHz, d-DMSO): (ppm) 9.640 (s, 1H), 9.157 (s, 1H), 7.463-7.224 (d, 2H), 6.7886.591 (d, 2H), 2.083 (s, 3H); and white powder 1,3,7-trimethylxanthine (49.1 mg, 75.5%) 1H NMR (60
MHz, CDCl3): (ppm) 7.521 (s, 1H), 3.999 (s, 3H), 3.586 (s, 3H), 3.409 (s, 3H).

Results, Discussion, and Conclusions

A tablet of Excedrin was separated into the major active ingredients aspirin, acetaminophen, and
caffeine. The separation was conducted through column chromatography of the pill with TLC
monitoring and the isolated components were analyzed by 1H NMR.
Column chromatography was utilized for the purpose of isolating and purifying the components
that make up Excedrin. Dichloromethane was used to dissolve the Excedrin tablet, and the remaining
mixture was separated by column chromatography with the use of three solvents over the course of the
procedure. The solvents, 50% hexanes and 50% ethyl acetate, 25% hexanes and 75% ethyl acetate, and
100% acetone, increased in polarity, and eluted acetylsalicylic acid, 4-acetamidophenol, and 1,3,7-

Li 4
trimethylxanthine, respectively. The elution of the three components into fractions was monitored by
TLC to identify the order of elution and the number of fractions to combine together for the collection of
each component separately. Column solvents were changed once TLC plates indicated the entirety of a
component had eluted and been collected. The components eluted in order of increasing polarity, with
aspirin first, acetaminophen second, and caffeine last.
Percent yields for aspirin, acetaminophen, and caffeine were found to be 96.24%, 75.8%, and
75.5%, respectively. Previously conducted experiments reported values of approximately 60-100% yield
for aspirin, approximately 10-20% yield for acetaminophen, and approximately 40% yield for caffeine. 5
The percent yield values produced were all significantly higher than the typical recoveries from the
previously conducted experiments. acetylsalicylic acid produced the highest yield, falling directly into
the 60-100% range. 4-acetamidophenol, and 1,3,7-trimethylxanthine yields were lower, both around
75%, which was still greater than the reported, typical yields.
The experiment was run one time; however, the use of nitrogen to flash the column due to time
constraints is likely to have caused the reported co-elution of acetaminophen and caffeine. One fraction
(10 mL worth) of solvent, acetaminophen, and caffeine together, was produced. Extraction was
considered to separate the two components out, but since the yields indicated sufficient product,
extraction was determined to be unnecessary. Previous to running the column, a TLC plate was prepared
with lanes for Excedrin mixture, acetaminophen standard, aspirin standard, and caffeine standard, and
was run in 25% hexanes/75% ethyl acetate. The presence of three spots in corresponding distances on
the TLC plate to the three standard spots confirmed that the Excedrin mixture was, in fact, composed of
aspirin, acetaminophen, and caffeine. The standards of aspirin, acetaminophen, and caffeine had R f
values of 0.64, 0.24, and 0.04, respectively. The Excedrin mixture lane displayed R f values of 0.6, 0.2,
and 0.08. This TLC plate in comparison to the fraction monitoring plates during the running of the

Li 5
column made clear what component had eluted from the column, and indicated how pure the fractions
were in terms of contaminations by other components.
Aspirin eluted entirely in fractions 1-4, in a total of 40 mL, with R f values of 0.6. Fractions 5-7
were empty of major components, and fractions 8-18 contained acetaminophen at R f values of 0.23.
Fraction 19 contained two spots, one of Rf value 0.2, and one with Rf value 0.08, indicating a co-elution
of acetaminophen and caffeine. Fractions 20 -25 contained caffeine, with spots of Rf value 0.08 and
0.12. All the Rf values measured during column chromatography were values considered consistent with
the three Rf values measured of the three standards and the three spots in the Excedrin mixture. Fractions
26-29 were free of spots, indicating all the components had eluted fully.
1

H NMR data of aspirin (Figure 2, Supplemental Information) supports the success of its

isolation. The peaks at 6.5-8.0 ppm indicate the four aromatic hydrogens, with doublet H f, at 7.8517.931 ppm, downfield of doublet Hc, at 7.410-7.679 ppm, which is downfield of triplets He,d, at 7.0527.372 ppm due to proximity of the ester group and the carboxylic acid group. H f and Hc had integration
values of 1H and He,d had an integration value of 2H. The lack of peaks in the 9-10 ppm region and the
3-4 ppm region indicates the absence of the alcohol and amine groups of acetaminophen and the amine
groups of caffeine, respectively. There is a peak missing in the 11-12 ppm range for the carboxylic acid
proton, however, this absence does not mean the product was not isolated. An additional peak at 1.174
ppm indicates contamination by water, but does not imply the presence of any of the other components.
Finally, a peak at 2.085 ppm with an integration value of 3H implicates the methyl group on the end of
the ester. Messy NMR data may have resulted from samples that were too dilute with weak signals.
The 1H NMR data of acetaminophen (Figure 3, Supplemental Information) indicates the
complete isolation of the product from the other two components. An amide singlet shift at 9.640 ppm,
with an integration value of 1H, and the phenol singlet shift at 9.157 ppm, with integration value of 1H,
supports the presence of acetaminophen. The doublets of the aromatic hydrogens at the 6.5-8.0 ppm

Li 6
range, each at 2H, plus the 3H peak at 2.083 ppm for the methyl group all agree with the classification
of acetaminophen as the second product eluted. A peak at 3.402 ppm indicates water contamination, but
not the presence of the other components.
The 1H NMR data of caffeine (Figure 4, Supplemental Information) features three singlet peaks
in the 3-4 ppm range, each at integration value 3H, indicative of the three methyl groups within the
molecule, next to electronegative atoms that cause a downfield shift. A singlet 1H peak at 7.521 ppm
indicates the single C-H bond present in caffeine, proving the presence of caffeine in the third and final
elution, and the absence of aspirin and acetaminophen. A peak at 2.171 ppm shows contamination by
acetone in the CDCl3 solvent, but not contamination by the other two components.
The separation of the three components: aspirin, acetaminophen, and caffeine, from a single
Excedrin tablet through column chromatography proved to be effective in the isolation and collection of
the three separate components. The TLC data confirms the separation of the three compounds based on
relative polarities and Rf values. It confirmed that three distinct components were eluted, and that each
eluted component corresponded to either aspirin, acetaminophen, and caffeine. 60 MHz 1H NMR data
demonstrated that none of the compound isolations had been contaminated by the presence of either of
the other two compounds. The products were each pure of the others, suggesting that an effective
separation was conducted. The yields of the three components were all either well within range of
typical yields, or significantly higher. As mentioned previously, the column was flashed during the
elution of acetaminophen due to time constraints. This may have contributed to the co-elution of
caffeine with acetaminophen, affecting yields and data. For future experiments, column flashing should
be avoided to help prevent the co-elution of the last two components with fairly similar R f values.
However, if time permits otherwise, flashing the column should be reserved only for the final elution of
caffeine in isolation, after all acetaminophen has eluted already. If flashing is further required, it should
only be utilized during aspirin elution, well before acetaminophen.

Li 7
References
1) Colebatch, A. N.; Marks, J. L.; and Edwards, C.J. Cochrane Db. Syst. Rev. 2011, 11, 872-878.
2) Doleman, B.; Read, D.; Lund, J. N.; and Williams, J. Region. Anesth. Pain M. 2015, 40, 706-712.
3) Kalathil, A. A.; Kumar, A.; Banik, B.; Ruiter, T. A.; Pathak, R. K.; Dhar, S. Chem. Commun. 2015,
52, 140-143.
4) Serefko, A.; Szopa, A.; Wla, A.; Woko S.; Wla, P.; and Poleszak, E. J. Neural Transm. 2015, 123,
463-472.
5) Revell, K. D. J. Chem. Ed. 2011, 88, 1413-1415.

Li 8
Supplemental Information
Figure 1. Structures of Aspirin, Acetaminophen and Caffeine
Figure 1. 60 MHz 1H NMR of acetylsalicylic acid.
Figure 2. 60 MHz 1H NMR of 4-acetamidophenol.
Figure 3. 60 MHz 1H NMR of 1,3,7-trimethylxanthine.