Ester Synthesis and Analysis:
Aspirin and Oil of Wintergreen
Christy Chan
October 23, 2014
Thursday Evening Lab Section
Partner: Deven Roberts
INTRODUCTION
The objectives of this experiment were to synthesize two organic esters, study the implications of
reactions, which do not go to completion, and use chromatographic techniques to evaluate the
purity of the products. A synthesis reaction is a reaction in which two or more reactants combine
to form a single product. Esters, which have characteristically fruity odors, are synthesized by
reacting an alcohol with an acid and is catalyzed by the addition of a strong acid and heat; this is
also known as esterification.
Carboxylic Acid + Alcohol
heat
à
Ester + Water
In this experiment, both methyl salicylate (oil of wintergreen) and acetylsalicylic acid (aspirin)
were synthesized with the use of salicylic acid. As an alcohol, salicylic acid reacts with acetic
anhydride to form acetylsalicylic acid, which is a white, powdery solid. As an acid, salicylic acid
reacts with methyl alcohol (methanol) to form methyl salicylate, which is a colorless and fragrant
oil.
Salicylic acid
Salicylic acid
+
+
Acetic anhydride
à
Methanol
à
Acetylsalicylic acid
Methyl salicylate
+
+
Water
Water
Acetylsalicylic acid (aspirin) is commonly used in the treatment of different medical conditions
such as pain, fever, and inflammatory diseases. Studies have also found that low doses of aspirin
consumed immediately after a heart attack can help reduce the chance of another heart attack or
of the death of cardiac tissue.1 However, the main side effects of aspirin include gastrointestinal
ulcers, stomach bleeding, and ringing in the ears; therefore, it is not recommended that children
under 16 years old consume aspirin for flu-like symptoms or viral illnesses.2
Methyl salicylate (oil of wintergreen) is naturally produced by many species of plants, most
likely to serve as an anti-herbivore defense. For example, when a plant is infected with
herbivorous insects, the production of oil of wintergreen attracts other insects that kill these
herbivorous insects.3 Oil of wintergreen can also be used by plants as a pheromone to warn other
plants of pathogens such as the tobacco mosaic virus.4
The final products of these syntheses were evaluated for purity with a chemical test for salicylic
acid and the use of two chromatographic techniques – thin layer chromatography and highpressure liquid chromatography. In aspirin synthesis, the chemical test was conducted by the
addition of an indicator (Fe3+), which changed color in the presence of salicylic acid. In thin
layer chromatography, compounds in mixtures are separated between the stationary phase (the
TLC plate) and the mobile phase (the solvent: a mixture of hexane and ethyl acetate) due to
differing polarities. With this test, ratios of fronts of the experimental product are calculated and
compared to controls to determine its purity. In high-pressure liquid chromatography, the
compounds in the mixture placed into the chromatograph separate based on polarity as well. The
retention times and peak areas of the different compounds in the mixture are compared to a
standard, which was created by putting pure compounds into the chromatograph. The data
obtained from a chromatography experiment are compared with controls to determine the
presence of impurities, where in this experiment is the presence of salicylic acid.
EXPERIMENTAL
During week one, to make oil of wintergreen, a 0.50-g sample of salicylic acid was weighed out
and transferred to a labeled test tube. Next, two mL of methanol was added to the test tube, along
with 15 drops of concentrated sulfuric acid. The test tube was swirled continuously and then
stopped with a cotton plug to prevent water from entering the reaction mixture. A boiling water
bath was made by filling a 400-mL beaker with water, and adding several boiling stones. The test
tube was added to the boiling water for 30 minutes. Next, two mL of cold distilled water was
added to the test tube before it was stopped with a cork. The test tube was shaken to mix the
contents. Using three mL of methylene chloride, the oil of wintergreen was extracted. The test
tube was stopped with a cork and inverted several times. The cork was removed to relive
pressure, and then placed back onto the test tube. The test tube was shaken for one minute, and
then allowed to stand to let the layers separate. Using a Pasteur pipet, the lower layer (methylene
chloride layer) was drawn up and transferred to a clean and dry test tube. The extraction was
repeated and the second layer of methylene chloride was placed in the same test tube as the first
layer. The methylene chloride layers were dried by the addition of a small amount of solid
anhydrous sodium sulfate. Next the layer was decanted, leaving the sodium sulfate behind, into a
clean and dry sample vial that was previously labeled and weighed. The vial was stopped loosely
with a cotton plug and stored so that the methylene chloride evaporates.
To make aspirin, a 0.50-g sample of salicylic acid was weighed out and transferred to a labeled
test tube. Approximately 25 drops was acetic anhydride was added to the test tube, followed by
two drops of concentrated phosphoric acid. The test tube was stopped loosely with a cotton plug
to prevent water from entering the reaction mixture. It was then placed in the water bath for ten
minutes before it was removed. While the mixture is still hot, two mL of cold distilled water was
added. Another three mL of cold distilled water was added and swirled. The test tube was placed
in an ice-water bath for five to ten minutes to allow for maximum crystallization of the product.
At the same time, 15 to 20 mL of distilled water is cooled to be used for the transfer and washing
of the crystals later. A side-arm flask and Büchner funnel was set up for vacuum filtration. The
contents of the test tube were carefully poured into the Büchner funnel and a rubber policeman
was used to scrape the solid into the tunnel. Small amounts of cold distilled water were used to
aid in the transfer of the solid. The solid was carefully transferred to a clean and dry sample vial
that was previously labeled and weighed. The vial was stopped loosely with a cotton plug and
stored for drying.
During week two, to determine percent yield, sample vials that were stored the previous week
were obtained and the cotton plugs were removed. The vials were weighed and the masses of the
products were determined by calculating the difference in weight of the vial before and after
product was added. Using theoretical yields, the percent yield for each compound was calculated.
To conduct a chemical test for salicylic acid, in a test tube, a small amount of aspirin was
dissolved in two mL of a 50:50 mixture of ethanol and water. This was repeated in a second test
tube with a small amount of salicylic acid starting material, and in a third test tube with a small
amount of pure acetylsalicylic acid. Next, ten drops of one molar iron chloride solution was
added to each test tube. The test tubes were compared to determine if the aspirin product
contained any salicylic acid.
To conduct thin layer chromatography, three test tubes were prepared (one with four drops of
experimental wintergreen, one with four drops of pure methyl salicylate, one with a small
amount of salicylic acid). Next, two mL of methanol was added to each test tube and swirled
gently to dissolve the sample. The TLC chamber was prepared by placing five mL of the TLC
solvent (50% hexane and 50% ethyl acetate) in a clean and dry 250-mL beaker. A piece of folded
filter paper was placed in the beaker and pushed against one side. The top of the beaker was
covered with aluminum foil and the mixture was swirled to saturate the beaker with its vapors.
Next, three evenly spaced spots were lightly made with a pencil on the TLC plate. Using a
different spotter for each substance, the samples were spotted onto the plate. The solvents
evaporated and then the TLC plate was placed on the opposite side of the filter paper in the
beaker. The beaker was covered. The TLC plate was removed when the solvent front was almost
at the top of the plate. When the TLC plate was thoroughly dried, it was placed under a UV lamp
and glowing shapes that appeared on the plate were traced lightly with a pencil. The color of the
spots, distance the spots traveled and the total distance the solvent front traveled were recorded.
The ratios of fronts were calculated for each spot on the TLC plate.
To conduct high-pressure liquid chromatography, a few grains of experimental aspirin were
dissolved in five to ten mL of methanol in a sample vial. This solution was injected onto the
chromatograph with the help of the professor. The resulting chromatogram was compared to that
of the standard sample provide by the professor. The retention times and areas for aspirin and
salicylic acid peaks were recorded. Using known concentrations and measured areas from the
standard chromatogram and the measured areas from the experimental chromatogram, the
concentrates of aspirin and salicylic acid in the sample were calculated. These concentrations
were then used to calculate the fraction of aspirin in the sample. This fraction was then used to
adjust the percent yield of the aspirin synthesis.
RESULTS
Table 1: Raw Data and Calculated Data of Oil of Wintergreen and Aspirin
Compounds
Oil of
wintergreen
(C8H8O3)
Aspirin
(C9H8O4)
Salicylic
Acid
Mass (g)
Sample
Vial Mass
(g)
Vial and
Product
Mass (g)
Product
Mass (g)
Theoretical
Yield (g)
Percent
Yield (%)
0.4999
12.9981
13.4524
0.4543
0.5507
82.50
0.5135
13.1250
13.5224
0.3974
0.6698
59.33
Sample Calculations of Oil of Wintergreen
Theoretical Yield of Oil of Wintergreen = 0.4999 g C7H6O3 x 1 mol C7H6O3 / 138.122 g C7H6O3
x 1 mol C8H8O3 / 1 mol C7H6O3 x 152.149 g C8H8O3 / 1 mol C8H8O3 = 0.5507 g C8H8O3
Percent Yield of Oil of Wintergreen = Experimental yield / Theoretical yield x 100 = 0.4543 g /
0.5507 g x 100 = 82.50%
Observations About the Physical Appearance of the Products
Oil of Wintergreen: clear, liquid, oil bubbles, peppermint and minty scent
Aspirin: white, wet, powder paste
Observations of the Iron Chloride Chemical Test for Salicylic Acid Performed on Aspirin
Aspirin with 50:50 mixture of ethanol and water + iron chloride: solution turned dark purple
Salicylic acid + iron chloride: solution turned golden
Pure acetylsalicylic acid + iron chloride: solution turned golden
Table 2: Thin Layer Chromatography Data and Observations
Sample
Dsolv (cm)
Dspot (cm)
Rf
3.72
0.8176
0.63
0.1385
Oil of
wintergreen
4.55
Salicylic acid
4.55
0.67
0.1473
Pure oil of
wintergreen
4.55
3.30
0.7253
Color
Spot was dark
purple
Spot was light
purple
Upper spots were
violet
Lower spots
were light purple
Spot was dark
purple
Sample Calculation of Rf
Rf (Spot B) = Distance traveled by the compound / Distance traveled by the solvent front = 0.67
cm / 4.55 cm = 0.1473
Solvent front
A: Sample of oil of wintergreen
B: Salicylic Acid
C: Pure oil of wintergreen
Table 3: High-Pressure Liquid Chromatography Data on Aspirin
Peak Identity
Aspirin
Salicylic Acid
Retention Time
(min)
7.50
9.15
Peak Area
(mAU)
1489386
201040
Concentration
(mg/mL)
0.0965
0.00603
Sample Calculation of Adjusted Yield of Aspirin
Concentration of Aspirin = Aspirin standard x Peak area of experimental aspirin / Peak area of
standard aspirin = 2.27 mg/mL x 1489386 mAU / 35039045 mAU = 0.0965 mg/mL
Concentration of Salicylic Acid = Salicylic acid standard x Peak area of experimental salicylic
acid / Peak area of standard salicylic acid = 1.51 mg/mL x 201040 mAU / 50308147 mAU =
0.00603 mg/mL
Adjusted Yield of Aspirin = Concentration of aspirin / (Concentration of aspirin + Concentration
of salicylic acid) x Percent yield of aspirin = 0.0965 mg/mL / (0.0965 mg/mL + 0.00603 mg/mL)
x 59.33% = 55.84%
Aspirin
Salicylic acid
Figure 1: Experimental High Pressure Liquid Chromatography Data on Aspirin
Figure 2: Standard High Pressure Liquid Chromatography Calibration Data on Aspirin
DISCUSSION
In order to study the implications of reactions that do not go to completion, two organic esters
were synthesized, and chromatographic techniques were used to evaluate the purity of the
products. First, the percent yields of aspirin and oil of wintergreen were calculated to determine
how much of the theoretical yields were produced. While a 100% yield is strived for, it is not
possible even with the best technique because the product could have been lost in certain areas of
the procedure. Also, in esterification reactions, no matter how much time is allowed, some of the
reactants remain. For example, in the synthesis of oil of wintergreen, when drawing up the
methylene chloride layer with the Pasteur pipet, it was very likely that not all of the methylene
chloride was transferred to the test tube. Next, when the methylene chloride was dried with
anhydrous sodium sulfate and then decanted, it was possible that not all of the liquid was
transferred and that some anhydrous sodium sulfate was transferred as well. Also, in the
synthesis of aspirin, when filtering the solution, which contained acetic anhydride, concentrated
phosphoric acid, and distilled water, the aspirin solid may not all have been transferred to the
Büchner funnel, or the filtration may have allowed some solid to filter through, losing the
product. Previous years demonstrate the percent yield to fall between 40-70%; in this
experiment, the oil of wintergreen had a 82.50% yield and the aspirin had a 59.33% yield (Table
1). However, the percent yield calculated was not an accurate reflection of purity because there
may have been a low percent yield, but the product could possibly contain only the ester
synthesized.
A chemical test was conducted to determine the purity of the aspirin qualitatively. The indicator,
Fe3+, turns violet when reacting with salicylic acid. A positive control was created by reacting
iron chloride with salicylic acid forming the violet color, and a negative control was created by
reacting iron chloride with pure acetylsalicylic acid forming a golden color. The experimental
aspirin was reacted with the iron chloride and its color was compared with the controls to
determine which compounds were present in the experimental product. With the chemical test,
the experimental product, when reacted with iron chloride, formed a golden color, identical to
that of the negative control. This suggested that the experimental product contained aspirin, and
possibly minute traces of salicylic acid.
A thin layer chromatography test was conducted to determine the purity of the oil of wintergreen
qualitatively. The TLC plate (a plastic plate covered with silica gel), which acts as the stationary
phase, was spotted with three different compounds dissolved in methanol – the experimental oil
of wintergreen, salicylic acid, and pure methyl salicylate, and then placed in a solvent (a mixture
of hexane and ethyl acetate), which acts as the mobile phase. When the solvent moved pass the
spots on the TLC plate through capillary action, molecules of the spotted materials separated
between the stationary phase and the mobile phase because of their differing polarities. Each
compound was characterized by its ratio of fronts, which is the distance that the compound
traveled divided by the distance traveled by the solvent front. The spot B was salicylic acid and
the spot C was pure oil of wintergreen, so these two spots served as controls for comparison with
spot A, which was the experimental oil of wintergreen. In this experiment, the Rf of salicylic acid
was 0.1473 and the Rf of pure oil of wintergreen was 0.7253 (Table 2). The Rf values of the two
spots found in the experimental oil of wintergreen were relatively similar to that of the controls
(0.1385 and 0.8176), suggesting that the oil of wintergreen was not really pure due to the
presence of salicylic acid in the final product.
A high-pressure liquid chromatography was conducted to determine the purity of aspirin. The
chromatograph is made up of a pump that pushes a solvent (the mobile phase) through a small
column (the stationary phase), which contains very small polymer beads that attract different
molecules based on their polarity. Different compounds pass through the column at different
rates, allowing for the separation of different compounds in a sample. Experimental aspirin was
dissolved in methanol and injected onto the chromatograph and the resulting chromatogram was
compared to that of the standard chromatogram, which showed the retention times and peak
areas of pure aspirin and pure salicylic acid. By comparing the experimental retention time and
peak area to that of the standard, purity can be determined. For example, the experimental peak
area of aspirin was 148936 mAU and the experimental peak area of salicylic acid was 201040
mAU, while the standard peak area for aspirin was 35039045 mAU and the standard peak area
for salicylic acid was 50308147 mAU; this indicated that the level of salicylic acid in the
experimental product was relatively low (Table 3). With these data, the percent yield was
adjusted to factor in purity of the aspirin. The experimental product had a 55.84% adjusted yield,
suggesting that the aspirin was not as pure as indicated with the chemical test of salicylic acid.
This may be due to fact that there may be salicylic acid in the product, but not even to trigger a
violet purple in the test.
CONCLUSION
The syntheses of oil of wintergreen and aspirin were successful, but the products were not pure.
REFERENCES
1. Julian, D.G.; Chamberlain, D.A.; Pocock, S.J. A comparison of aspirin and anticoagulation
following thrombolysis for myocardial infarction (the AFTER study): a multicentre unblinded
randomised clinical trial. BMJ 1996; 313:1429
2. Macdonald, S. Aspirin use to be banned in under 16 year olds. BMJ 2002; 325:988
3. James, D.G.; Price, T.S. Field-Testing of Methyl Salicylate for Recruitment and Retention of
Beneficial Insects in Grapes and Hops. Journal of Chemical Ecology 2004; 30:8
4. Shulaev, V.; Silverman, P.; Raskin, I. Airborne signalling by methyl salicylate in plant
pathogen resistance. Nature 1997; 385:6618