The Synthesis of Acetyl Salicylic Acid

Analytical Techniques

Acetylsalicylic acid is an effective analgesic (pain reliever), antipyretic (fever reducer), and anti-inflammatory agent derived from salicylic acid. It is one of the most used over-the-counter medications. Acetylation is important in the preparation of Acetylsalicylic acid because the phenol group in salicylic acid is associated with irritation to the lining of the mouth, esophagus, and stomach and can cause hemorrhaging of the stomach lining. The aim of this experiment was to synthesize acetylsalicylic and quantify the yield and purity using three analytical methods: melting point, absorbance, and IR spectra. Using the Aspirin lab protocol, 302SCI - The Synthesis and Analysis of Aspirin, aspirin was synthesized. The percentage yield was 75.94%, and the melting temperature was slightly lower than that of commercial aspirin at 132-134 °C. The purity of the compound was determined as 91.01% using spectrophotometric absorbance. In addition, the IR spectra resembled that of commercial aspirin. The narrow melting point range and its proximity to the literature value of 135-136 ^oC, along with the calculated 91.01% purity, confirm the successful synthesis of aspirin with a relatively low impurity content. The successful production of high-purity aspirin demonstrates the effectiveness of the acetylation process. However, the yield could be improved by using high-purity starting materials and optimizing recrystallization techniques to enhance the product's quality further.

Analytical Techniques

Objectives

The experiment aims to synthesize acetylsalicylic acid using acetylation of salicylic acid using acetic anhydride in the presence of phosphoric acid, determine the percentage yield and purity of the compound using three analytical methods, namely determination of the melting point, determination of absorbance, and using IR spectra.

Acetylsalicylic acid is an effective analgesic (pain reliever), antipyretic (fever reducer), and anti-inflammatory agent derived from salicylic acid. It is one of the most used over-the-counter medications. Aspirin derives its medicinal properties from its ability to inhibit the activity of cyclooxygenase (COX), leading to the formation of prostaglandins (PGs), which are associated with inflammation, swelling, pain, and fever, according to Vane and Botting (2003). PGs are signaling molecules that play a major role in several physiological processes, including the regulation of blood flow, inflammation, and pain sensation. There are two isoforms of the COX enzyme, namely COX-1 and COX-2.

From a strictly biochemical point of view, aspirin inhibits the activity of COX-1 and COX-2 through the following activities. First, by acetylating the enzymes where when aspirin is ingested, it undergoes a chemical reaction with serine 530, an amino acid residue in the active site of the COX enzyme (Giménez‐Bastida et al., 2018). This leads to an irreversible inactivation of the COX enzyme in a process known as acetylation. Second, aspirin inhibits the production of PGs. As mentioned above, aspirin acetylates COX, which usually converts arachidonic acid into prostaglandin H2 (PGH2), a precursor to various PGs (Holdgate, 2017). Thus, by ingesting aspirin, the production of PGs, mainly those involved in inflammation and pain sensation, is inhibited. By decreasing the levels of PGs, the inflammatory and pain signaling responses are reduced.

Given these activities, aspirin is used in the treatment of minor pain, including toothache, migraines, myalgia, and dysmenorrhea. Aspirin is also used in the treatment of acute and chronic inflammatory diseases such as rheumatoid arthritis and the symptomatic treatment of feverish states and flu or cold syndromes. It is important to note that due to its ability to inhibit COX irreversibly, aspirin has an edge over other non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX reversibly and temporarily. This is because it provides long-lasting pain relief from pain and inflammation.

However, its advantage comes with a caveat because aspirin’s irreversible effect on the COX enzyme carries a higher risk of gastrointestinal side effects and bleeding. This is based on the fact that COX-1 is critical for gastric mucosal protection through vasodilation, stimulation, and secretion of the gastroduodenal mucus (Crofford, 2017). Aspirin is marketed under different brand names, including Zorprin, Bayer Buffered Aspirin, and Durlaza, among others. It is marketed in the form of tablets, effervescent tablets, and suspension for oral use and in the form of injectable preparations. Aspirin is prepared through the process outlined below.

Literature Review

The term “Aspirin” was coined by Bayer, a drug and dye firm, in 1899 after two years of investigating acetylated organic compounds as possible new medicines. Bayer company modified the salicylic acid structure by replacing the phenol group with an ester group because the phenol group was associated with irritation to the lining of the mouth, esophagus, and stomach and can cause hemorrhaging of the stomach lining. Some background information is critical in understanding the evolution of aspirin to the medicine popular now. According to Miner (2007), in 1757, Reverend Edward Stone discovered the medicinal benefits of willow bark after discovering it reduces pain and fever. The active ingredient was later found to be salicylic acid. The compound was initially known as salicin because it was found to be very acidic when dissolved in water with a pH of 2.4. The compound was extracted by Johann A. Buchner in 1828, purified, and named salicin (Miner, 2007). Later, Henri Leroux, a French pharmacist, and Raffaele Piria, a Calabrian chemist in Paris, extracted it. The latter gave the compound its current name (salicylic acid).

The major breakthrough in the medicinal industry came after it was isolated from meadowsweet flowers (Spiraea ulmaria) by some German researchers, such as Karl Jakob Löwig, 1839. In 1860, Hermann Kolbe and his students at the University of Marburg managed to synthesize salicylic acid, then placed it on the market in 1874 at a price ten times lower than the acid extracted from salicin. In 1876, a group of German scientists, including Franz Stricker and Ludwig Riess, published in “The Lancet” the results of their therapies based on the administration of six grams of salicylates per day (Miner, 2007).

Based on existing research, taking acetylsalicylic acid daily significantly reduces the death rate from all forms of cancer by about a fifth of all genders. While it was difficult to quantify the impact on pancreatic, stomach, brain, breast, and ovary cancer, reduction was confirmed (Vaughan et al., 2016). This is because high concentrations of salicylate (sodium salt of acetylsalicylic acid) activate the AMPK (AMP-activated protein kinase) protein, which plays a vital role in the regulation of metabolism and cell growth (Hawley et al., 2012). Its activation has positive impacts on tumors and diabetes. Currently, researchers are focused on determining whether salsalate, an aspirin derivative, can prevent type 2 diabetes.

There are two analytical methods central in this report: spectrophotometric analysis and IR spectrometry. The purity of the compound was well quantified by the spectrophotometric absorbance of aspirin. The spectrophotometric principle is based on the fact that every chemical absorbs, transmits, and reflects light over a certain range of wavelengths (Libretexts, 2023). The amount of photons that pass through the cuvette and into the detector is dependent on the length of the cuvette and the concentration of the sample (Libretexts, 2023). As such, with a known intensity of light, as it passes through the cuvette, one can determine transmittance and, therefore, the absorbance, the number of photons absorbed. The unknown concentration is then determined using the Beer-Lambert equation. In contrast, IR spectrometers analyze a compound by passing IR radiation over a range of different radiations through a sample and measuring absorptions made by each type of bond (Notman, 2023). Since where different bonds absorb is known, one can identify a compound using an IR spectrum.

Method

Reagents and Apparatus

The apparatus included a 50ml conical flask, 5ml disposable pipette, 600ml beaker, Vacuum filtration apparatus, Spatula, Ice bath, Hotplate, Glass rod, Timer, Balance, Thermometer, Plastic pipette, Clamp and stand, measuring cylinder, Spectrophotometer, 50ml graduated cylinder, Centrifuge tubes, 100ml volumetric flask, Melt station, Glass capillary tubes, 10ml and 5ml bulb pipette, 250ml volumetric flask, IR spectrometer, 25ml beaker, and 25ml volumetric flask. The reagents included Salicylic acid, Phosphoric acid solution, Acetic anhydride, Distilled water, Ethanol, Salicylic acid, 0.025M iron (III) nitrate solution, and Water.

Procedure

1. Synthesis of Aspirin

Some water was placed in a 600 ml beaker and placed in the fume hood. The plate was put on the hottest setting, and a thermometer was used to maintain the temperature at 100°C. About 1g of salicylic acid was weighed and placed in a 50-conical flask, and the weight was recorded. 1.5 ml of acetic anhydride was pipetted and added to the flask in the fume chamber. Three drops of phosphoric acid were added and gently swirled. The conical flask was placed into the boiling water bath such that the level of the liquid in the conical flask was below the level of water in the boiling water bath. The contents of the flask were boiled at 100°C for 15 minutes. The contents were stirred using a glass rod to avoid vicious bubbling and to ensure the contents were dissolved. 2 ml of distilled water was added after ten minutes of heating. When vapor ceased, the flask was removed from the boiling water, and 20 ml of distilled water was added. The mixture was allowed to cool to near room temperature and then transferred to an ice bath for five minutes.

Washing the synthesized Aspirin

The mass of filter paper was taken to the nearest 0.001g before filtering the solid. The weighed filter paper was placed on the vacuum filtration apparatus and a small amount of distilled water squirted onto it to seal it to the funnel’s surface. The aspirator was turned on fully, and the contents of the conical flask poured through while swirling. After the liquid was drained, 10 ml of chilled water was poured on the sample twice to rinse it. The setup was left for a few minutes to dry the sample. The filter paper and product were then placed on a labeled petri dish and allowed to dry before being weighed.

1. Testing the IR Frequency

The sample was analyzed by an IR Spectrometer and a chromatogram of the synthesized aspirin compared to that of a commercial aspirin sample.

1. Melting Point

A small sample of the synthesized aspirin was obtained and ground with a mortar and pestle to make a powder. A capillary tube was packed with the sample to 3-4 mm (~1/8 inch) deep. The melting station was turned on, and the plateau temperature was set at 120°C. The capillary tube was inserted into the sample holder of the melt station, and the start key was pressed. When the plateau light and heating light illuminated, the start button was pressed again to heat the sample. The melting temperature range was recorded as when the first liquid drop appeared and when the last crystal disappeared. The stop button was then pressed, and the unit was allowed to cool between runs. This was used as an estimate, and the procedure was repeated to collect an accurate reading. The melting point was then determined.

1. Spectrophotometric Absorbance of Aspirin

250 ml of a 5.79 × 10-3 mol/L stock salicylic acid solution was prepared, and the mass of salicylic acid (0.2g) was recorded to the nearest 0.001 g. This step involved dissolving the salicylic acid in 10 mL of ethanol first, then adding distilled water. Five standard solutions of varying concentrations of salicylic acid were prepared as follows. First, 100.0 mL of the standard solution used in Trial 1 was prepared by quantitatively transferring 10.0 mL of the stock salicylic acid solution you prepared in Step 1 to a 100 mL volumetric flask. 0.025 M Fe(NO₃)₃ solution was added to the flask to the 100 mL mark. The second standard solution was prepared by adding 8 ml salicylic stock solution and 2 mL distilled water and topped to the 100 mL mark with 0.025 M Fe(NO₃)₃ solution. The third standard was prepared using 6 mL salicylic stock solution and 4 mL water, the fourth, 4 mL salicylic stock solution, and 6 mL water, while the last contained 2 mL salicylic stock solution and 8 mL water. All samples were topped up to the 100 mL mark using 0.025 M Fe(NO₃)₃ solution. The precise molar concentrations of the five standard solutions prepared were calculated and recorded.

A blank was prepared by filling a cuvette with 1ml of Fe(NO₃)₃ solution. The spectrophotometer was set to a wavelength of 530nm and calibrated by placing the cuvette in the holder, closing the lid, and pressing CAL. The data for the five standard solutions was collected, and the absorbance and concentration values were recorded. The synthesized aspirin was prepared for testing through the following steps. First, approximately 0.04g of synthesized aspirin was measured to the nearest 0.001g and transferred to a 25-beaker. 1ml of Ethanol was added to the beaker of aspirin and swirled to dissolve. 15ml of distilled water was added to the beaker. The solution was then mixed. The solution was transferred from the beaker into a 25ml volumetric flask; a beaker was rinsed with some more distilled water and poured into the volumetric flask. Distilled water was added, as needed, to fill the volumetric flask up to the 25ml mark. Mix the solution thoroughly. 5 ml of the synthesized aspirin solution was transferred from the 25-volumetric flask to a 100-volumetric flask. 0.025M Fe(NO₃)₃ solution was added to the flask to make precisely 100ml. 1 ml of this solution was taken and added to a cuvette, then measured, and the absorbance value of the treated aspirin sample was recorded.

Results

Masses (For Calculation of % Yield)

Table 1; Mass of Aspirin Synthesized

Since Salicylic acid is the limiting reagent and the mole ratio of salicylic acid to aspirin is 1:1, then the theoretical yield of aspirin is;

Thus, 0.0072 moles of aspirin would be produced. This is equivalent to;

Moles × Molar Mass = 0.0072 moles × 180.158 g/mol = 1.2971g = Mass

MELTING POINT EXPERIMENT (QUALITATIVE ANALYSIS- ASSESSMENT OF RELATIVE PURITY OF ASPIRIN)

Table 2; Melting Temperature of the Aspirin

Note:

Melting point of pure salicylic acid = 158-159 ^oC

IR ANALYSIS (CONFIRMATION OF PRODUCT IDENTITY)

Figure 1 Note. IR Spectra for the synthesized aspirin, comercial aspirin and Salicylic acid.

SPECTROPHOTOMETRIC ABSORBANCE OF ASPIRIN (QUANTITATIVE DETERMINATION OF UNREACTED SALICYLIC ACID)

0.001448moles

Mass=Molar mass × Moles=138.121 g/mol × 0.001448 moles = 0.2g

Preparation of the stock solution for the dilution standards:

Table 3; Mass of Salicylic Acid used in Stock

The dilution standards (‘trials’) are then prepared from the above stock solution as per below:

Preparation of the dilution standards (‘trials’) for the calibration graph:

Table 4; The Dilution Standards Respective Volumes

Standard 1;

Standard 2;

Standard 3;

Standard 4;

Standard 5;

Absorbance vs concentration of the dilution standards (‘trials’) for the straight-line calibration plot:

Table 5; Concentration and Absorbance of Standards

Figure 2 Absorbance vs Concentration Scatter Graph

Calculation of the relative % of unreacted salicylic acid within the synthesised aspirin sample using the equation of the straight-line calibration graph and the absorbance of the small aspirin sample:

Table 6; Synthesized Aspirin Percentage Purity

Thus,

Concentration of Salicylic acid in aspirin sample; in 100ml

In the 5ml,

Multiplying by dilution factor;

Mass = Moles×Molar Mass = 0.00002605mol × 138.121g/mol = 0.003598g

0.04g-0.003598g=0.0364g

(0.0364g/0.04)×100=91.01%

Discussion

In this experiment, acetylsalicylic acid was prepared from acetic anhydride and salicylic acid through acetylation, and three tests were done to determine the purity of the product. The yield obtained from the experiment was 0.985g, as shown in Table 1 above, which represents the 75.94% yield. The product melting temperatures were 132-134 °C, as shown in Table 2 above. The IR spectra produced for the synthesized aspirin resemble that of commercial aspirin for the most part, as shown in Figure 1 above. As shown in Table 6 above, the absorbance of a small sample of the synthesized aspirin is 0.15, which indicates that the mass of salicylic acid in the sample is 0.003598, and therefore, the percentage purity of the product is 91.01%.

The 75.94% percentage yield indicates that while the method used was relatively effective, some reactants were not converted into products. For this experiment, the percentage yield might have been affected by the process of crystallization, dissolving the reactants, or losing part of the product through filtration. The melting temperatures were determined as 132-134 °C. This is a slightly lower value than the literature value of 135-136 ^oC. Since the melting point of pure salicylic acid is 158-159 ^oC, the temperature recorded indicates that some of the impurities are either acetic acid or acetic anhydride, both of which have melting points lower than aspirin’s 135-136 ^oC.

The IR spectra attest to the purity of the product, with the spectra resembling that of commercial aspirin. The ester structure in aspirin explains the peak at around 1750 cm-1, while the peak at around 1680 cm-1 can be attributed to the carboxylic section. The strong stretch at about 2600 to 3200 can be attributed to OH stretching. The major determiner of the presence of salicylic acid in the product would be another band for the extra OH in salicylic acid, as seen at around 3250 in salicylic acid spectra. The produced aspirin does not seem to have such a band attesting to its purity.

The absorbance of the sample was 0.15, and using a scatter plot from standards of known concentration, the concentration of salicylic acid in the aspirin was calculated as 0.0000521 mol/L. Salicylic acid exclusively forms a colored complex with iron (III) nitrate, and its absorbance can be used to calculate the concentration based on Beer Lambert's law, which states that there is a linear relationship between the concentration and absorbance of a sample. This has been demonstrated in the form of a scatter graph in Figure 1 above. Based on the concentration, the moles and, consequently, the mass of salicylic acid were determined as 0.00002605mol and 0.003598g, respectively, indicating a 91.01% purity. This is a relatively high degree of purity, which explains why the salicylic acid was not identified from the IR spectra.

Conclusion

In conclusion, the aim of the experiment was met as aspirin was synthesized by acetylating salicylic acid in the presence of phosphoric acid. The percentage yield was 75.94%, and the melting temperature was slightly lower than that of commercial aspirin at 132-134 °C. The purity of the compound was determined as 91.01% using spectrophotometric absorbance. The purity of aspirin can be increased by using high-purity salicylic acid, enhancing recrystallization by offering more time, sublimation, and through column chromatography.