Chemistry 261 Laboratory Experiment 5: Recrystallization & Hydrolysis of Acetanilide

Transcription

1 Chemistry 261 Laboratory Experiment 5: Recrystallization & Hydrolysis of Acetanilide Reading from Zubrick, 10 th Edition Safety, pages 1-10 Keeping a Notebook, pages Mining Your Own Data, pages The Melting Point Experiment, pages Recrystallization, pages Extraction & Washing, pages Reflux, pages Objectives Exploit differences in solubility as a function of temperature to purify a solid mixture, in this case impure acetanilide Utilize melting point determination to qualitatively assess product purity Investigate the reversibility of the formation of amides from acids (or acid derivatives) and amines Set up and run a reaction under reflux conditions Carry out an organic extraction, using changes in ph to effectively separate ionizable from non-ionizable compounds O HN Acetanilide 1

2 Introduction Acetanilide was the first aniline derivative found to possess analgesic 1 and antipyretic 2 properties. It entered the marketplace as Antifebrin in 1866, but was found to be too toxic and subsequently removed. It was not until 1948 that it was established acetanilide is a prodrug, being converted by the body to the para-hydroxy product acetaminophen. The para-hydroxy group allows for the rapid removal of acetaminophen (by linking it directly to sulfate or glucuronic acid) which lowers the toxicity and has resulted in one of the most widely consumed drugs over the last 70 years. As we will see later, functionalizing an aromatic ring to a phenol is not particularly easy and this is the case in the body as well. Thus, it is not surprising that the non-polar solvent benzene is toxic (causes bone marrow depression and soft tissue cancers) and has been largely replaced in the lab by other similarly structured molecules such as toluene (methyl benzene) or xylenes (dimethyl benzenes) which possess a more reactive benzylic position 3. Truly, it is remarkable that the body can carry out such a transformation at all, a feat made possible by the cytochrome p450 family of oxidative enzymes in the liver 4. Since acetanilide is an amide, it can be viewed as a condensation product between an amine and a carboxylic acid. However, reacting the 2 directly is difficult, as you may deduce given amines are weak bases and acids are, well, acidic. Thus, under anything but extreme temperature conditions ( o C) salt formation will occur. Instead, when trying to acetylate aniline, one would typically use acetic anhydride with an assist from pyridine. Instead of liberating water, the reaction would give acetic acid as the side product, and the pyridine would react with the acidic hydrogen of acetic acid to drive the reaction (if this seems mysterious, inquire either to me, or to available online sources). In fact, it has been found that at temperatures in the range of o C a number of carboxylic acids undergo dehydration to the corresponding anhydride, which makes it likely the actual mechanism of acid + amine forming an amide involves formation of the anhydride first before attack by the amine nucleophile and amidation can occur. 5 1 The difference between analgesia vs. anesthesia is a subtle one analgesia is specifically the removal of pain, while anesthesia is the removal of al sensation (think numbing agent). A local anesthetic removes not only pain, but the ability to perceive touch, heat, cold, pressure, etc. 2 Fever prevention or reduction 3 Unfortunately, benzene is still found at low levels in gasoline, with the EPA placing a 0.62 % limit in For a discussion of the mechanism of action by which cytochrome p450 oxidized hydrocarbons, including a brief discussion of kinetic isotope effects, see pages of Louden and Parise, 6 th ed. 5 D. Davidson and P. Newman, J. Am. Chem. Soc., 1952, 74,

3 The formation of an amide from an acid and amine is reversible, and water can be used to regenerate the initial amine and acid, given the appropriate temperature and ph environment 6 (or enzymatically in the body amide drug effects are often terminated by hydrolysis of the amide by amidases (e.g. lidocaine), and amide hydrolysis by proteases governs the breakdown of proteins into amino acids). We will undertake such a hydrolysis in this lab, first purifying acetanilide, then hydrolyzing the purified product. Since acetanilide shows limited solubility in cold water, we should be able to readily separate the products of hydrolysis from any unreacted acetanilide once the reaction mixture cools, since as a 2 carbon acid acetic acid is always 100 % water soluble, regardless of ionization status, and aniline can be trapped in H 2 O as the corresponding anilinium ion at sufficiently low ph Procedure Place 3.0 g impure acetanilide (which may contain sand and or brown sugar) in a 125 ml Erlenmeyer flask. Add 30 ml water and while stirring, bring the mixture to a boil on a hot plate. Add water in small (2-3 ml) increments, making certain to bring the mixture to a boil after each addition, just to the point where all of the acetanilide appears to have dissolved 7. No more than 50 ml water should be used 8 Cool the solution slightly and add approximately 100 mg decolorizing carbon 9. Reheat the mixture for 2 minutes, continuously stirring to prevent bumping Filter the hot mixture through a fluted filter paper. I recommend you expedite the process by using a light vacuum (side arm flask and rubber seat for your funnel). If crystals form inside the filter, dissolve them in a minimal amount of hot water dripped from a Pasteur pipette. Transfer to a beaker for ease of access to the crystals, and allow the hot filtrate to slowly cool to room temperature, undisturbed 6 Amides are stable in neutral, room temperature water 7 Regardless of how much boiling water you add, the sand is probably not going to dissolve 8 The aqueous solubility of acetanilide is 5.5 g/100 ml at 100 o C and 0.53 g/100 ml at 0 o C, thus if all of the sample was acetanilide, only 55 ml of boiling water would be needed to dissolve it 9 DO NOT ADD DECOLORIZING CARBON TO A BOILING LIQUID 3

4 Further cool the beaker by immersing in an ice-water bath for at least 10 minutes. Collect the crystals by suction filtration using a Büchner funnel and wash with a minimal amount of ice-cold water 10. Air dry the crystals under vacuum for 10 min, then transfer the crystals into a beaker and finish drying in a warm oven (60 o C) Weigh the crystals and determine the percent recovery from the crude mixture. Determine the melting point of the recrystallized mixture Hydrolysis of Purified Acetanilide CAUTION: Aniline is poisonous and a suspect carcinogenic agent. 2-naphthol is also toxic. Sulfuric acid, hydrochloric acid, and sodium hydroxide are corrosive. Work in the hood and handle materials carefully. Gloves are available. Like other amides, anilides (amides of aniline) may be hydrolyzed in either acidic or basic environments. However, the hydrolysis in base tends to be slow, and so we will carry out the hydrolysis in an acidic environment. A 70 % aqueous sulfuric acid solution works well, and is made by cautiously adding 40 ml of concentrated sulfuric acid (18 M) to 30 ml of water with cooling and stirring (or 20 ml H 2 SO 4 to 15 ml H 2 O depending on the number of groups) 11. Add 1 g of your recrystallized acetanilide 12 and 10 ml of the 70 % H 2 SO 4 to a 25 ml round bottomed flask (RBF) fitted with a reflux condenser and reflux 13 gently for 1 hour in the fume hood. At this time the reaction should smell strongly of acetic acid. Allow the mixture to cool, and then add 5 ml of water, and extract the aqueous layer once with 10 ml of toluene. This will remove any unreacted acetanilide. Do not discard this layer until you are certain you have the desired product. 10 A distilled water wash bottle placed in ice at the start of class would come in handy here 11 Remember to do what you otter and add acid to water 12 At the instructor s discretion, we may use stock acetanilide, so the purification and hydrolysis can be carried out simultanously 13 Refluxing may simply be thought of as boiling where the vapors formed are recondensed and returned to the reaction flask 4

5 Carefully, add 20 ml of saturated sodium carbonate via Pasteur pipette (dropwise initially) to begin neutralizing 14. Very Carefully add 6 M NaOH dropwise via Pasteur pipette until wide range ph paper gives a value > 8. This should require about 14 ml of the NaOH solution. In total you should have about 50 ml total volume. Transfer the mixture to a separatory funnel and extract with 3 x 20 ml with CH 2 CH 2 (methylene chloride, dichloromethane, DCM) 15 each time draining the DCM layer into the same beaker containing approximately mg of anhydrous Na 2 SO 4. Examine the ability of the drying agent to flow freely if it does dry the extract for min. If not, add another 100 mg and check again. Remove the organic layer from the drying agent and reduce the volume by about ¾ (to ca. 15 ml) using the Rotovap. Transfer the remaining aniline/dcm solution by pipette into a 50 ml beaker, making certain to record the mass of the beaker first, in case crystallization fails to occur. Place the beaker on ice and wait up to ½ hour to see if crystals form. If they do, retrieve them by vacuum filtration, dry them, and record the weight. If not, evaporate the remaining DCM under a stream of air while on a hot plate in the hood. You will likely be left with a viscous oil. In either case, report the mass of crude product and crude % yield. Please make certain to include physical properties of reagents and products, and I would like you to try your hand at making a purification flowchart, both for the initial purification of acetanilide, and the workup of the hydrolysis of acetanilide to aniline 16 We will conduct the classic diazotization test to see if in fact we isolated aniline (of course this test will not establish purity unless run against a spectrophotometric calibration curve). Really, what we will be doing is the kind of chemistry that gave rise to the German dye/chemical industry of the 1800 s (essentially acetanilide arose from this industry as the dye industry moved into pharmaceuticals). By coupling aniline to 2-naphthol, we will have generated a large conjugated resonance system and a lovely red dye. 14 Bear in mind you are generating H 2 CO 3 and thus CO 2 from a strongly acidic media the possibility of foam-over is very real 15 While most organic solvents float on water, it is again worth recalling DCM d 20 = 1.33 g/ml 16 Once I am convinced everyone has tried to make these purification flowcharts, I will provide my version to you 5

6 To about 0.2 g of the isolated crystals (or the remaining viscous oil) add 1 ml conc. HCl. Dilute with 3 ml H 2 O. Cool in ice and add several drops of saturated aqueous sodium nitrite. Slowly add this ice cold diazonium ion solution to an ice cold solution of 2-naphthol dissolved in excess 10 % NaOH (2-naphthol typically dissolves in about 3 parts aqueous media once ionized). If you isolated crystals, submit the remaining aniline to your instructor in a clean, clearly labeled vial, with the label including name, section, identity of contents and remaining mass. Bonus (2EC): Time permitting, you may also run an IR on your recrystallized acetanilide (cast film) and aniline (neat) and compare the 2. Pre-Lab Questions 1. If the solubility of acetanilide is 5.5 g/100 ml in boiling water and 0.53 g/100 ml at 0 o C, what is the best % recovery of acetanilide one can hope for when recrystallizing in on ice? 2. What is the effect of adding H 2 SO 4 to the reaction mixture in terms facilitating the hydrolysis reaction? You will have to look this up, but bear in mind if you have only modest nucleophiles like water about, you may have to crank up the electrophile strength 3. What is the effect of adding H 2 SO 4 to the reaction mixture in terms of the physical properties of the principal reactant and product? Are amides basic enough to be protonated completely under these conditions? 4. How do you rationalize protonation of the reactant for reaction vs. the lack of protonation with regard to physical properties (remember you will be removing unreacted acetanilide with toluene). Coming to grips with this question will truly unleash your capacities as an organic chemist, and remember, pk a s and the Henderson-Hasselbalch equation are your friend 6

7 Post-Lab Questions 1. Why is acetic anhydride a better choice for the synthesis of the amide than acetic acid? 2. How is it that mixing 40 ml of 18 M sulfuric acid with 30 ml of water gives a 70 % solution? 3. How many different naphthols are there? 4. What is the structure of the dye you formed (consult your text) 5. What are protecting groups? Would the formation of an acetamide derivative be a good way to protect the amine functionality? Why or why not. Bonus (1.0 EC): Using the curved arrow method, please show how 2 acid molecules can form an acid anhydride 7