Experimental Procedures

Safety

• Wear lab safety glasses at all times
• HCl and H₂SO₄ are strong corrosive acids that are toxic and can cause burns.  Wear gloves to manipulate these reactants.  Wash your skin with large quantities of water in the case of an acid spill on your hands.
• Compounds used is this experiment are toxic and suspected carcinogens.  Work in the hood and wear gloves when manipulating chemical substances.

PART A: Synthesis of p-methylacetanilide ¹

1. In a 25 mL Erlenmeyer flask, dissolve 6.45 g of sodium acetate trihydrate (CH3COONa·3H2O) in 18 mL of distilled water (dH2O). Sodium acetate acts as a buffer during the reaction. The acetate ion also acts as a base in a deprotonation step in the mechanism. 
2. Stir vigorously until all the crystals have dissolved.
3. Place the solution aside for later use. 
4. Grind some p-toluidine with a mortar and pestle. Add 4.83 g of ground p-toluidine to a 500 mL Erlenmeyer flask. Add a magnetic stir bar and 120 mL of dH2O.
5. While stirring, add 3.9 mL of concentrated hydrochloric acid. 
6. Mix vigorously with the magnetic stir bar for two (2) minutes. Once all the p-toluidine has dissolved, verify the pH with a strip of pH indicator paper. If an insoluble brown impurity remains, remove it by gravity filtration.
7. While continuing to stir, add 6.3 mL of acetic anhydride. Quickly verify the pH.  Immediately add the sodium acetate trihydrate solution previously prepared. Verify the pH once again.
8. Stir vigorously to mix the reactants.
9. Maintain vigorous stirring while the solution is cooled in an ice bath and crystallization is occurring (15 min.). 
10. While cooling, prepare the TLC setup (developing chamber, mobile phase, stationary phase, standard solutions).
11. After cooling the product, collect it by vacuum filtration. Wash the crystals with small portions of ice-cold dH2O.
12. Verify the purity of the product by TLC only. It is not necessary to let the product dry before preparing the TLC solution. 
13. Allow product to dry during the week.
14. Weigh the product.
15. The rest of the product may be used in the next step without purification.

• Melting point: Determine the melting point of the dry product.
• TLC: Perform a small-scale analysis using a small silica gel chromatographic plate (4 cm wide x 10 cm high). Develop in a tall 200 mL beaker containing the mobile phase (10 mL ethyl acetate : 5 mL of hexanes). Which substances should you analyze?  Apply two (2) drops of 1 %  solutions in dichloromethane of the substances to be analyzed to the chromatographic plate.  The developed plate may be analyzed under a UV light.
• Infrared spectroscopy: Prepare KBr pellets with the product and with its standard (proportions of organic compound and KBr to be determined). Obtain the spectrum of each.

PART B: Synthesis of p-acetamidobenzoic acid ¹

1. Prepare a solution of potassium permanganate by dissolving 12.0 g of KMnO4 in 100 mL of hot water. Swirl to dissolve.
2. Place the solution aside, cover it with a watch glass and keep it hot on a hot plate for later use.
3. The p‑methylacetanilide left over from the first step, must be ground with a mortar and pestle before being weighed for this step.
4. To a 500 mL Erlenmeyer flask equipped with a magnetic stir bar, add 5.5 g of p‑methylacetanilide, 14.5 g of magnesium sulfate heptahydrate (MgSO4· 7H2O) and 300 mL of water. The magnesium sulfate acts as a basic buffer during this  reaction. 
5. Heat with stirring to 85°C on a hot plate.
6. While vigorously stirring the solution of p‑methylacetanilide, slowly add the warm solution of potassium permanganate with a plastique pipet (3mL). The addition should take approximately 30 minutes.  It is very important to add the permanganate solution slowly and uniformly to avoid local build-up of the oxidant.
7. After all the oxidant has been added, stir the solution vigorously for five (5) minutes.
8. Add 20 mL of ethanol to react with the excess oxidant.
9. Then, boil the solution until the dark mauve colour is dissipated. It is important to closely watch the boiling solution because it can very easily overflow.
10. To separate the brown manganese dioxide from the solution containing p‑acetamidobenzoic acid, place a 1 cm layer of diatomaceous earth (Celite) on the filter paper of a vacuum filtration setup. Do not forget to wet the filter paper with a small amount of dH2O before placing the layer of Celite on it. 
11. Wash the brown precipitate with hot water to dissolve adsorbed product.
12. If the filtrate is still violet-coloured, add 5 mL of ethanol, boil and refilter.
13. Cool the filtrate in an ice bath and acidify with 20% sulphuric acid until the pH is 3-4.
14.  Allow crystallization to occur for 20 min in the ice bath.
15. Collect the product by vacuum filtration, rinsing the crystals with small amounts of ice-cold dH2O.
16. Dry the crystals as much as possible by continuing suction.
17. Verify the purity of the product by TLC only.
18. The rest of the wet product may be used in the next step without purification.
    

• Melting point: Determine the melting point of the dry product. 
• TLC: Perform a small-scale analysis using a small silica gel chromatographic plate (4 cm wide x 10 cm high). Develop in a tall 200 mL beaker containing the mobile phase (1.5 mL glacial acetic acid : 4 mL of hexanes : 15 mL of dichoromethane).  Which substances should you analyze? Apply two (2) drops of 1 % ethanolic solutions of the substances to be analyzed to the chromatographic plate.  The developed plate may be analyzed under a UV light.

PART C: Synthesis of p-aminobenzoic acid ¹

1. In a 50 mL round-bottom flask, weigh 3 g of p-acetamidobenzoic acid.
2. Add a magnetic stir bar and 15 mL of 6 M HCl.
3. Set up a reflux apparatus as shown in Figure 6 below.
4. Begin stirring and heating slowly.  Reflux for 30 minutes.  Observe.
5. After the heating time is over, let the reaction mixture cool to room temperature. Continue to stir the reaction mixture and leave the condenser with circulating water in place. 
6. Add 7.5 mL of cold distilled water and mix before verifying the pH.
7. Then add 14 M aqueous ammonium hydroxide dropwise, until the pH is just slightly basic (pH = 7-8). To do so, add 50 drops of NH₄OH, mix and verify the pH. Repeat this two (2) more times for a total of 150 drops of NH₄OH.
8. Now add the NH₄OH more slowly, for example by portions of 10 drops before verifying the pH.  It will take 200 drops or more for the NH₄OH to neutralize the mixture.  Do not go past pH 8.
9. Add 1.5 mL of glacial acetic acid.
10. Stir vigorously and cool the solution in an ice bath.  It may be necessary to induce crystallization by scratching the side of the round-bottom flask with a glass rod.
11. Isolate the crude product by vacuum filtration using a small Büchner funnel. Wash the crystals with small portions of ice-cold dH2O.
12. Allow the crystals to dry and weigh them.  If performing a recrystallization immediately, weigh wet crystals.
13. Save about 10 mg of crude product for the TLC analysis.
14. Recrystallize the crude product from water on a hot plate.  If there are insoluble impurities, remove them with hot gravity filtration. If crystals are colored, decolorize using charcoal.
15. Analyze PABA using the four (4) analytical methods provided below.

• Melting point: Determine the melting of dry recrystallized PABA.
• TLC: Perform a small-scale analysis using a small silica gel chromatographic plate (4 cm wide x 10 cm high)  Develop in a tall 200 mL beaker in the mobile phase (1.5 mL glacial acetic acid : 4 mL of hexanes : 15 mL of dichoromethane).  Which substances should you analyze? Apply two (2) drops of 1 % ethanolic solutions of the substances to be analyzed.  The developed plate may be analyzed under a UV light.
  
• UV-Visible spectroscopy: Dissolve recrystallized PABA in an appropriate solvent and obtain the UV-Visible spectrum. Repeat with the PABA standard. Determine the wavelength of maximum absorption and calculate the molar absorptivity. 
• Infrared spectroscopy: Prepare KBr pellets with recrystallized PABA and PABA standard (masses of organic compound and KBr to be determined with Stephane). Obtain the spectrum of each.

ALWAYS make sure the hoses are securely attached to the condenser. A gentle flow of cooling water is all that is needed.  Check for leaks!   Organic chemists often need to heat a mixture for a long time and to be able to leave it relatively untended. The "reflux apparatus" shown allows such heating; it allows the reaction to be carried out at the boiling point of the solvent and yet it prevents loss of solvent or reagent due to evaporation. A condenser is attached to the boiling flask and is clamped in an upright position, the "reflux position," and cooling water is circulated to cause the vapours to condense as they rise up the condenser and thus prevent them from escaping. The upper level of the vapours in the condenser can often be seen as a reflux line. The direction of flow of the water should be such that the condenser will fill with the cooling water; water should enter the condenser at the bottom and leave from the top.  It is usually a good idea to ensure that the heating mantle can easily be removed by using a laboratory jack. Video

Figure 6. Reflux setup for the synthesis of p-aminobenzoic acid and video

Preparation of a sunscreen²

1. Choose your aqueous phase and your oil phase.  Note that the oil phase may be composed of only one oil (ex. 36 g of sunflower oil) or a combination of oil and butter (ex. 30.5 g of sunflower oil and 5.5 g of shea butter). Likewise, you may choose only one aqueous phase (ex. 100 g of hydrosol) or a combination of aqueous phases (ex. 50 g of hydrosol and 50 g of aloe Vera juice).
2. To a 400 mL beaker add 36 g of the oil phase, 5 g of stearic acid and 10 g of emulsifying wax, taring the beaker in between each addition.
3. To obtain a homogeneous mixture, heat gently to about 50^oC on a bain-marie until the emulsifier is melted, and the stearic acid has dissolved.  Do not swirl or stir the reactants during the melting process.
4. Meanwhile, in a 200 mL beaker, weigh 1.5 g of GeoGard Ultra.  Add 100 g of the aqueous phase and heat to about 35^oC.  Maintain at this temperature.
5. At this point, if you wish to prepare a sunscreen, add 24 g of zinc oxide (ZnO) to the oil phase (you may also add less ZnO, for example 12 g). Blend well with the mini-mixer equipped with the stir attachment. However, you may omit zinc oxide if you wish to just prepare a rich nourishing cream.
6. Once the zinc oxide has been completely mixed into the oil phase, work quickly to begin the addition of the aqueous phase. Slowly add the aqueous phase to the oil phase mixing vigorously with the mini-mixer equipped with the stir attachment.  
7. Once a smooth cream-like consistency is obtained, add 3 drops of vitamin E (antioxidant).  Incorporate well with the mini-mixer.
8. Next you may add an active ingredient from the list in Table 1 in the project handout document.
9. Place in sterilized labelled containers.
10. Let the cream cool completely before closing the containers to avoid contamination by water that may condense inside the sealed container.  Bacteria need water to proliferate!
11. Use the cream within 6 months.

References

1. Schoffstall AM, Gaddis BA, Druelinger ML.  Microscale and Miniscale Organic Chemistry Laboratory Experiments. 2e éd. Montréal: McGraw Hill Higher Education; 2004. Chapter 27, Amino Acids and Derivatives; p. 518-528
2. Fortin S. Cosmétiques non toxiques.  Montréal (QC), Canada : Les Éditions La Presse; 2016