Organic Chemistry Experiments (II)

Transcription

1 rganic Chemistry Experiments (II) Chem 316 Dr. Pr. Nejib MEKNI

2 List of Experiments List of Experiments N Experiment 1 Synthesis of Benzoin 2 2 Synthesis of Benzyl 5 3 Synthesis of Benzilic acid 7 4 Synthesis of Acetanilide 10 5 Synthesis of 4-bromoacetanilide 12 6 Synthesis of 4-bromoaniline 15 7 Condensation of Benzaldehyde with acetone 19 8 Synthesis of m-nitroacetophenone 24 9 Chemioselective Reduction of m-nitroacetophenone Synthesis of Fluorescein, a fluorescent dye 30 2

3 Introduction Introduction The experiments in second experimental workbook illustrate important concepts and principles in organic chemistry. In previous manual organic chemistry I, we have familiarized the students od the secondary year of the chemistry bachelor to the lab glassware, the balance the shot plate and to the general environment of the organic chemistry laboratory, in the third year of the bachelor, the students will be able to calculate the reactants and products amounts, totally synthesize and purify an organic reaction product. These intellectual and hand skills should be learned under the doctor guidance and control. The experimental workbook, contains different synthesis reactions types: direct and indirect reactions, in different reaction conditions: cold, middle and high temperature and the application of the major technical: dissolution, evaporation and separation and purification technical: extraction, crystallization and the product use as a reagent for the following reaction step as well as the life application. Herein we discover the concepts learned in the theoretical courses. The experiments are clearly written and provides enough information on experimental procedures, to be easily understand by the students, whose, will work with minimal supervision, and will be able to perform the experiments in the allowed laboratory period. The experiments are not only simple demonstrations, but also contain a sense of discovery. 1

4 Lecture 1, P1: Synthesis of Benzoin and Benzil Synthesis of Benzoin I. bjective: The synthesis and the purification of benzoin. II. Material and Products Apparatus: Quick fit condenser, 250 ml round-bottomed flask with Quick fit, Heater, Filter paper, Buchner funnel. Chemicals: Ethanol, Benzaldehyde, Sodium cyanide. Part A: Synthesis of Benzoin I. Background Treated with an alkali metal cyanide, in aqueous solution, aromatic aldehydes undergo condensation reaction yielding the α-hydroxyketone (benzoin). 2 Ar-CH + NaCN (aq) Ar-CH(0H)-C-Ar Using 1 mol of each one two different aldehydes, an unsymmetrical (mixed) benzoin is obtained. Ar 1 -CH + Ar 2 -CH NaCN or KCN (aq) Ar 1 -CH(H)-C-Ar 2 The reaction depends on the catalytic effect of the cyanide ion and the mechanism may be represented in the following way. II. Experimental 2Ph-CH + NaCN/EtH/H 2 Ph-CH(H)-C-Ph Place 32.5 ml of ethanol, mol (25 g, ml) of pure benzaldehyde and a solution of 2.5 g of sodium cyanide in 25 ml of water in a 250 ml round-bottomed flask. Attach a reflux condenser and boil the mixture gently for 30 min. Cool the contents of the flask (in an ice bath). Filter the benzoin crude and wash it with cold water, drain well and dry. 2

5 Lecture 1, P1: Synthesis of Benzoin and Benzil Crystallize the solid from about 175 ml of ethanol. After cooling, pure benzoin (a white, crystalline solid, m.p. 137 C) separates. Reserve the remainder product for the preparation benzil and benzilic acid. 3

6 Lecture 1, P1: Synthesis of Benzoin and Benzil Name:.... ID:.. Report Part A: 1. Why we used both ethanol and water as solvents? 2. Calculate the Molecular mass of benzaldehyde. 3. Calculate the number of moles of the benzaldehyde used to realize this reaction 4. Calculate the Molecular mass of benzoin. 5. Calculate the number of moles of the benzoin theoretical expected from the reaction. 6. Weigh the synthesized benzoin. 7. Calculate the yield of the reaction. 8. What is the role of the cyanide ion CN - in the reaction? 9. What are the particular precautions must be taken for the reaction. 10. Draw the scheme of the apparatus used in this experiment. 4

7 Lecture 1, P2: Synthesis of Benzoin and Benzil Synthesis of Benzil I. bjectives: The practical to the synthesis and the purification of benzyl. II. Material and Products Apparatus: Quick fit condenser, Quick fit 250 ml round-bottomed flask, Heater, Filter paper, Buchner funnel. Chemicals: Ethanol, Benzoin, Concentrated nitric acid. Part B: Synthesis of Benzil I. Background xidation of the α-hydroxyketone using a catalytic amounts of copper(ii) salts in acetic acid solution or concentrated nitric acid followed by ammonium nitrate regeneration, yields the corresponding diketone. Ar-CH(H)-C-Ar + HN 3 or Cu(Ac) 2 Ar-C-C-Ar HN 3 or Cu(Ac) 2 Benzoin Benzil II. Experimental In a 250 ml round-bottomed flask place 20 g (0.095 mol) of benzoin and 100 ml of concentrated nitric acid. Heat on a boiling water bath under the fume hood with occasional steering until the production of nitrogen oxides has ceased (about 1.5 hours). Pour the reaction mixture in a beaker containing ml of cold water, stir well until the oil crystallizes completely as a yellow solid. Filter the benzil crude, then wash it thoroughly with water to remove the nitric acid. Crystallize from ethanol. 5

8 Lecture 1, P2: Synthesis of Benzoin and Benzil Report Part B: 1. What is the role of the nitric acid in this reaction? 2. Can we use another acid such as HCl or H2S4 to realize this reaction? Why? 3. Why dose we heat the reaction mixture? 4. What are the organic functional groups existing in the benzil? 5. Calculate the molar mass of the benzil. 6

9 Lecture 2: Synthesis of Benzilic acid Synthesis of Benzilic acid I. bjectives: The practical synthesis and purification of benzilic acid. II. Material and Products Appartus: 50 ml Round bottom flask, Hot plate, Stand, Beaker, Funnel, Condenser, Water bath, stirrer etc. Chemicals: Benzil; Potassium hydroxide; Ethyl Alcohol; Hydrochloric Acid. I. Background Refluxed in aqueous-alcoholic potassium hydroxide α-diketones (Ar-C-C-Ar) undergo benzilic acid rearrangement to be converted into the corresponding benzilic acid salt. Ar-C-C-Ar + KH - (Ar) 2 -C(H)C0 2 -K + The mechanism of the reaction involves a nucleophilic attack of the hydroxide ion on a carbonyl carbon atom yielding the oxyanion (I) which undergoes a 1,2-nucleophilic shift of an aryl group as shown on the following scheme. Then, a proton transfer completes the reaction sequence. Direct conversion of a benzoin into the corresponding benzilic acid may be accomplished conveniently in good yield by action of an alkaline bromide solution at C. II. Experimental Place a solution of 3.5 g of potassium hydroxide in 7 ml of water, and 9 ml of ethanol 95%, then add 3.5 g (0.0167mol) of recrystallized benzyl in a 50 ml round-bottomed flask. The solution color change into deep bluish-black. Fit a reflux condenser to the flask and heat the mixture on a boiling water bath for min. Pour the contents into a beaker and allow to cool. The potassium benzyilic acid salt crystallizes out. Filter off the crystals under vacuum then wash with a little icecold alcohol. Dissolve the potassium salt in about 35 ml of water, and add slowly, under stirring 0.1 ml of concentrated hydrochloric acid from a burette. The precipitate thus produced is colored redbrown and is somewhat sticky. Filter off; the filtrate should be nearly colorless. Continue the addition of hydrochloric acid with stirring until the solution is acid to Congo red paper. Filter off the 7

10 Lecture 2: Synthesis of Benzilic acid benzilic acid under vacuum, wash thoroughly with cold water until free from chlorides, then allow to dry. The yield of the benzilic acid crude (light pink or yellow), is about 3 g (80 %). Crystallize the product from either hot benzene (about 6 ml/g) or hot water using a little amount of decolorising carbon ( g). The colored and sticky material obtained by the first precipitation may be recrystallized from hot water with addition of a little amount of decolorising carbon, and the pure benzilic acid is obtained (m.p C). 8

11 Lecture 2: Synthesis of Benzilic acid Report 1. Calculate the Molecular mass of benzil. 2. Calculate the number of moles of benzil used in this reaction 3. Calculate the molecular mass of benzilic acid. 4. Calculate the number of moles of the benzilic acid theoretically expected from the reaction. 5. Weigh the synthesized benzilic acid. 6. Calculate the yield of the reaction. 9

12 Lecture 3: Synthesis of Acetanilide Synthesis of Acetanilide I. bjectives Synthesize the acetanilide from the reaction of aniline with acetic anhydride. Purify the obtained acetanilide. II. Material and Products Apparatus: Hot plate, a 250 ml quick fit round-bottom flask, Quick fit condenser, beakers (150 ml, 250 ml), stirring rod, spatula, Büchner funnel, filter paper. Chemicals: Aniline, acetic anhydride. III. Background Acetanilide is a useful precursor to many pharmaceuticals such as acetaminophen and penicillin. This experiment involves four common functional groups in organic chemistry. The substrates (reactants) are liquids, whereas one of the products is solid. A solid product is often desirable since it may be easily crystallized. The reaction of aniline 1 with acetic anhydride 2 yields acetanilide 3 and acetic acid 4. So, all manipulations should be done under the fume hood. NH 2 HN CH 3 + H 3 C CH 3 + H CH Aniline Acetic anhydride Acetanilide Acetic acid IV. Experimental Scheme Suspend 18.6 g (0.2 mol) of aniline in 60 ml of water contained in a 250 ml quick fit roundbottom flask and add 24 ml (0.254 mol) of acetic anhydride. Fit a reflux condenser to the flask. Warm the mixture on a water bath under vigorously stirring (The solid dissolves). After 10 min, cool, filter off at the pump the obtained acetyl solid derivative and wash with a little cold water. Crystallize from hot water (about 150 ml) and dry. 10

13 Lecture 3: Synthesis of Acetanilide Report 1. Calculate the molar mass of aniline. 2. Calculate the number of moles of aniline used to realize this reaction. 3. Calculate the molar mass of the product (acetanilide). 4. Calculate the theoretical moles number of the acetanilide. 5. Calculate the theoretical mass of the acetanilide. 6. Weigh the produced acetanilide. 7. Calculate the percentage yield of this reaction. 8. For the reaction, which substrate is considered to be the nucleophile and which is the electrophile? Why? 9. Circle and give the name the functional groups in the structures of the following compounds: NH 2 + H 3 C CH 3 + H CH Aniline Acetic anhydride Acetanilide Acetic acid HN CH 3 11

14 Lecture 4, P1: Synthesis of 4-bromoacetanilide Synthesis of 4-bromoacetanilide I. bjectives: Practice the indirect synthesis of 4-bromoaniline and its purification. II. Material and Products Apparatus: Quick fit condenser, 250 ml Quick fit round-bottomed flask, Heater, Filter paper, Buchner funnel, Distillation column, distillation head. Chemicals: Ethanol, acetanilide, concentrated hydrochloric acid, Sodium thiosulfate. I. Background Since the amino group of aniline is a strong activator of aromatic ring, direct bromination is not selective (equation 1). The reaction conduces to a mixture of mono-, di- and tri- brominated compounds in both ortho and para positions. To synthesize p-bromoaniline, amino group need to be protected as acetamide, which slowed down the rate of the ortho bromination reaction vis-vice the para one (equation 2). So, the acetanilide can be para brominated (equation 3), then hydrolyzed back (disprotected) into to yield the p-bromoanline (equation 4). This strategy of protection/disprotection is an important tool in organic chemistry, especially for multi functional group substrates and multi-step synthesis. Equation 1: NH 2 2 NH 2 + NH 2 + NH 2 + NH 2 Equation 2: NH 2 HN CH 3 H 3 C CH 3 Aniline Acetic anhydride Acetanilide 12

15 Lecture 4, P1: Synthesis of 4-bromoacetanilide Equation 3: HN HN CH 3 2 CH 3 Acetanilide omine p-omoacetanilide Equation 4: HN CH 3 Acid Hydrolysis NH 2 p-bromoacetanilide p-bromoaniline Safety notes: Aniline is highly toxic. It is mutagen, sensitizer, and readily absorbed through the skin. Wear gloves when using bromine, a very corrosive substance. p-omoaniline is harmful if inhaled or absorbed through the skin. Part A: Synthesis of 4-bromoacetanilide II. Experimental omination of acetanilide: The preparation must be conducted in a fume hood. In a 250 ml quick fit round-bottomed flask, dissolve 13.5 g (0.1 mol) of acetanilide in 45 ml of glacial acetic acid. Dissolve in another small flask 17 g (5.3 ml, mol) of bromine in 25 ml of glacial acetic acid, then transfer the solution to a burette or a separatory funnel supported over the flask. Add slowly the bromine solution, with constant shaking to ensure through mixing: stand the flask in cold water. After adding all the bromine amount, the solution will have an orange colour due to the slight excess of bromine; a part of the reaction product may crystallize out. Allow the obtained mixture to stand at room temperature for 30 min with occasional shaking. Pour the reaction mixture into 400 ml of water; rinse the flask with about 100 ml of water. Stir the mixture well and if it is appreciably coloured, add just sufficient sodium metabisulphite (Na 2S 2 5) or sodium thiosulfate (Na 2S 2 3) solution, to remove the bromine excess (orange colour). Filter the crystalline precipitate on a Buchner funnel, wash thoroughly with cold water and press as dry as possible with a wide glass stopper. Crystallize from dilute methanol or ethanol. 13

16 Lecture 4, P1: Synthesis of 4-bromoacetanilide Report 1. Complete the following table: Structure of the compound Name Formula Molar Mass Molar umber 2. Theoretical yield calculation. 3. Percentage yield of product. 14

17 Lecture 4, P2: Synthesis of 4-bromoaniline Synthesis of 4-bromoaniline I. bjectives: The acidic hydrolysis disprotrction of the p-bromoacetanilide and the purification of the obtained p-bromoaniline. II. Material and Products Apparatus: Quick fit condenser, 250 ml Quick fit round-bottomed flask, Heater, Filter paper, Buchner funnel, Distillation column, distillation head. Chemicals: Ethanol, acetanilide, concentrated hydrochloric acid, Sodium thiosulfate. I. Background Herein we will practice the acidic hydrolyzed of the p-bromoacetanilide into the bromoanline (equation 5). Equation 1: NH 2 2 NH 2 + NH 2 + NH 2 + NH 2 Equation 2: NH 2 HN CH 3 H 3 C CH 3 Aniline Acetic anhydride Acetanilide Equation 3: 15

18 Lecture 4, P2: Synthesis of 4-bromoaniline HN HN CH 3 2 CH 3 Acetanilide omine p-omoacetanilide Equation 4: HN CH 3 NH 2 Acid Hydrolysis p-bromoacetanilide Equation 5: p-bromoaniline Safety notes: p-omoaniline is harmful if inhaled or absorbed through the skin. Part B: 4-omoaniline Experimental Hydrolysis of p-bromoacetanilide: Dissolve 18 g (0.084 mol) of p-bromoacetanilide in 36 ml of boiling ethanol contained in a 250 ml round-bottomed flask equipped with a reflux condenser. Add to the boiling solution in small portions, 22 ml of concentrated hydrochloric acid through a dropping funnel, then reflux for 40 min. Cool and dilute with 150 ml of water, and fit the flask with a condenser set for downward distillation. Distil the mixture and collect about 100 ml of the distillate constituted of water, ethanol and ethyl acetate. Pour the mixture into a 100 ml of ice-water, then, add 5 % sodium hydroxide solution under vigorous stirring until just alkaline. The p-bromoaniline oil separates, then crystallizes soon. Filter the crystals at the pump, wash with cold water and dry. Crystallize from dilute alcohol (m.p C). 16

19 Lecture 4, P2: Synthesis of 4-bromoaniline Report 1. Complete the following table: Structure of the compound Name Formula Molar Mass Molar umber 2. Calculate the theoretical yield. 3. Calculate the experimental percentage yield of the product. 17

20 Lecture 4, P2: Synthesis of 4-bromoaniline 4. Complete the following reactions. HN CH 3 2 HN CH 3 HN 3 H 2 S 4 5. Give an example of an activating group. 6. Give an example of a deactivating group. 7. Give an example of an ortho-para director. 8. Give an example of a meta director. 9. If 8.66 g of p-bromoacetanilide (C8H8N) is obtained from 6.25 g of acetanilide (C 8H 9N), what is the percentage yield? 18

21 Lecture 5: Condensation of Benzaldehyde with acetone Condensation of Benzaldehyde with acetone (The Calisen Schmidt reaction) I. bjectives: The synthesis and the purification of dibenzylideneacetone (1,5-Diphenylpenta-l,4-dien-3- one) II. Material and Products Apparatus: A 250 ml round-bottomed flask with Quick fit, Quick fit condenser, Heater, Buchner funnel, Glass funnel. Chemicals: Benzaldehyde, acetone, 95 % Ethanol, ethyl acetate. I. Background The retrosynthetic analysis of α,β-unsaturated ketones demonstrate that they can be obtained through various synthetic methods. These methods are applicable to aromatic aldehydes, which condense with aliphatic or mixed alkyl / aryl ketones using an aqueous alkali solution yield the α,β-unsaturated ketones (the Claisen-Schmidt reaction). The first step is an aldol species condensation, involving a nucleophilic addition of the carbanion derived from the methylated ketone on the carbonyl-carbon of the aromatic aldehyde. Hydroxyketone dehydrates spontaneously to form the conjugated, unsaturated carbonyl compound. 19

22 Lecture 5: Condensation of Benzaldehyde with acetone II. Experimental Synthesis of dibenzylideneacetone (1,5-Diphenylpenta-l,4-dien-3-one): Place a cold solution of 10 g of sodium hydroxide in 100 ml of water and 80 ml of ethanol, in a 250 ml round-bottomed flask, equipped with a stirrer and immerged in a water bath. Maintain the temperature of the solution at C, stir vigorously and add slowly a mixture of 2.9 g of acetone and 10.6 g of pure benzaldehyde. A flocculent precipitate forms few minutes. Continue the stirring for a further 30 min. Filte, and wash with cold water to eliminate the alkali as completely as possible. Dry the solid and crystallize from hot ethyl acetate (2.5 ml/g) or from hot ethanol 95 %. 20

23 Lecture 5: Condensation of Benzaldehyde with acetone Report 1. Why do we use both ethanol and water as solvents? 2. Calculate the Molecular mass of acetone and benzaldehyde. 3. What is the number of moles of the acetone and the benzaldehyde used to realize the reaction 4. Compare the used number of moles. 5. Calculate the Molecular mass of dibenzylideneacetone. 6. Calculate the number of moles of the dibenzylideneacetone theoretically expected from the reaction. 6. Weigh the synthesized dibenzylideneacetone. 7. Calculate the yield of the reaction. 8. What are the particular precautions must be taken in the reaction. 9. Draw the scheme of the apparatus used in this experiment. 21

24 Lecture 5: Condensation of Benzaldehyde with acetone Exercise g acetone reacted with g benzaldhyde yielding an actual mass of 1.00 g of dibenzalacetone. Calculate the theoretical and percent yield of the reaction. 2. Suggest the synthesis of the following compounds, using aldol or crossed aldol condensation. 3. What is the product of the aldol cyclization of hexanedioal in basic solution? 4. Give the structural form of the products of the condensation of the following compounds. a) acetone + benzophenone (1:1 ratio). b) cyclohexanone and cyclopentanone (1:1 ratio) 22

25 Lecture 5: Condensation of Benzaldehyde with acetone Exercise 2 1. If a student added two fold of acetone, it will react on itself. So, the product would be isolated as diacetone, alcohol or mesityl oxide. Write a complete reaction to support your result. 2. Draw the structure of the cis and trans isomers of the compound that you prepared. Why do you imagine that you obtained the trans isomer as the major, or the unique product? 3. Draw the complete mechanism of the product of the reaction between benzaldehyde and acetophenone in alkaline aqueous medium. 4. Complete the following reactions. 23

26 Lecture 6, P1: Synthesis of m-nitroacetophenone Synthesis of m-nitroacetophenone I. bjectives The synthesis of m-nitroacetophenone via the electrophilic nitration of acetophenone, using the mixture nitric & sulfuric acid. II. Material and Products Apparatus: A 250 ml Quick fit round-bottomed flask, Quick fit condenser, Heater, Buchner funnel, stir bar, Glass funnel. Chemicals: Sulfuric acid, Nitric acid, acetophenone, 95 % Ethanol, ethyl acetate. III. Background m-nitroacetophenone is a synthetic compound, used as intermediate in the dyes production and other organic compounds, having a fugitive emission during its production and use. If released to soil, m-nitroacetophenone displays high mobility. It is not volatile to the atmosphere from either moist or dry soil. Limited laboratory data indicate that the compound has a soil biodegradability potential under anaerobic conditions. m- Nitroacetophenone is not volatile from water, with a an estimated half-life for volatilization from a model river is 517 days. II. Experimental In a small Erlenmeyer flask, immerged in an ice bath, add slowly 8 ml of concentrated H 2S 4 to 5.3 ml of concentrated HN 3 to prepare a nitrating mixture. Cool this mixture to 0 C. In a 250 ml round-bottomed flask, place under swirling 20 ml of concentrated H2S4 and cool at 0 C. Add dropwise 8.0 ml of acetophenone to the cold H 2S 4 solution at such a rate that the temperature does not exceed 5 C. Cool this mixture to below 0 C in an 24

27 Lecture 6, P1: Synthesis of m-nitroacetophenone ice/salt bath: 1/3 (~ -5 0 C), then add dropwise (during about 15 min) the cold nitrating mixture, keeping the temperature at/or below 0 C. After the addition completion, maintain the temperature at 0 C for another min under occasional swirling. Pour the flask content into a beaker containing ~150 ml water and ~100 g of ice under vigorous stirring. When the excess ice has melted, filter the solid under vacuum and wash it well with cold water (the product might be sticky!). Add to the contents of the funnel 5 ml of ice-cold ethanol. Let it stand for about one minute, then complete the filtration, then, dry and crystallize from ethanol. If the solid do not appears, you need to extract the product from ethyl acetate. Dry the organic crude over Na2S4, then, vapor off the solvent. If the solid is not observed, you did not successfully perform the nitration! 25

28 Lecture 6, P1: Synthesis of m-nitroacetophenone Report 1. Give the nitration reaction scheme. 2. Calculate the Molecular mass of acetophenone. 3. The density of the acetophenone is 1.028, calculate the mass corresponding to 8 ml of acetophenone. 4. Calculate the number of moles of acetophenone. 5. Calculate the Molar mass of m-nitroacetophenone. 6. Calculate the number of moles of the m-nitroacetophenone theoretically expected from the reaction. 7. Weigh the synthesized m-nitroacetophenone. 8. Calculate the reaction yield percent. 9. What are the particular precautions must be taken during the reaction. 26

29 Lecture 6, P2: Chemioselective reduction of m-nitroacetophenone Chemoselective Reduction of m-nitroacetophenone I. bjectives: The study of the chemioselectivity via the reduction reaction of the m-nitroacetophenone. II. Chemicals and apparatus III. Background In a chemoselective reaction, a reagent that is capable of reacting with several functional groups would preferentially react with just one of the groups. If the reaction is not chemoselective, then the reagent will react with each of the functional groups. m-nitroacetophenone has two functional groups that are capable of being reduced a ketone and a nitro group. To further complicate matters, these two groups are both on an aromatic ring. Therefore the electron-withdrawing nature of these groups will have an effect on the reactivity of the other group, perhaps leading to greater or lesser reactivity than is expected otherwise. Each group will treat this compound with a reducing agent to determine whether the ketone, the nitro, or both are reduced in the reaction. Procedure In a 25 ml Erlenmeyer flask, equipped with a magnetic stirring bar, place 0.20 g of m- nitroacetophenone and 2.5 ml of absolute ethanol. Warm the flask on the lowest setting of a hotplate to dissolve the solid. Cool to room temperature for 2 minutes. Stir the mixture, and add a total of g of sodium borohydride in 3 portions, over a 2-3 min period. Add 2.0 ml of water and heat the mixture to boiling on a low setting of the hotplate. nce it has reached boiling, cool it slightly and observe carefully to see whether hydrogen gas is still being evolved (from the decomposition of sodium borohydride). If there are still bubbles forming, add 3 M HCl one drop at a time until the decomposition is complete. If there are NT any bubbles, then HCl does not need to be added. 27

30 Lecture 6, P2: Chemioselective reduction of m-nitroacetophenone Cool the reaction mixture in an ice-water bath for 10 minutes to produce a solid product. While it is cooling prepare a glass Pasteur pipette for the microscale extraction. Pour the reaction mixture into a test tube A. Rinse the flask with 2 ml of CH2Cl2 and add it also to the same test tube A. Use one pipette to mix the layers in test tube A, 4 or 5 times. After the phases separate, transfer (using the same pipette) the lower layer to a clean test tube B. Repeat extraction in test tube A, 2 more times with fresh 2 ml portions of CH2Cl2 (6 ml total transferred to test tube B). Add 2 ml of water to the 6 ml of extract in the test tube B. Mix layers and transfer the lower layer (6 ml) to a clean, dry small Erlenmeyer flask. Dry with anhydrous sodium sulfate. Decant the liquid away from the solid, or use a pipette to draw the liquid away. If you are careful, you should be able to do this without a cotton plug and leave all the solid drying material behind. Evaporate the solvent to reveal either a white solid or a yellow oil. Measure the mass, so that a percent yield may be calculated later. 28

31 Lecture 6, P2: Chemioselective reduction of m-nitroacetophenone Report 1. Give the reduction reaction scheme of the m-nitroacetophenone. 2. Calculate the Molecular mass of m-nitroacetophenone. 3. What mean chemioselectivity in this case? 4. Calculate the number of moles of m-nitroacetophenone. 5. Calculate the Molar mass of m-aminoacetophenone. 6. Calculate the number of moles of the m-aminoacetophenone theoretically expected from the reaction. 7. Weigh the synthesized m-aminoacetophenone. 8. Calculate the reaction percent yield. 9. What are the particular precautions must be taken during the reaction. 29

32 Lecture 7: Synthesis of Fluorescein, a fluorescent dye Synthesis of Fluorescein, a fluorescent dye bjectives The synthesis of fluorescein and the examination of its light properties. II. Material and Chemicals il or sand bath, large test tube (15x150 mm) or small Erlenmeyer flask, thermometer (200 0C, resorcinol, phthalic anhydride, H2S4, 0.1 M NaH solution, acetone, diethyl ether. Background Fluorescent detection technique has played a significant role on the advancement of some interesting living fields such as molecular biology and modern medicine and has achieved rapid development. Fluorescein is one of the widely used fluorescent probes in such applications. It was first synthesized by Bayer from the reaction of phthalic anhydride and resorcinol using zinc chloride as a catalyst. H H H + 2 ZnCl 2 or H 2 S 4 cc CH Phthalic anhydride Resorcinol Fluorescein Scheme 1. Synthesis of fluorescein from phthalic anhydride and resorcinol. This molecule have a very high fluorescence with an excitation and emission wavelengths of 494 nm and 521 nm respectively. Fluorescein is a weak acid with a pka value of 6.4. Its ionization equilibrium in water causes its ph-dependence in absorption and emission process over the range of 5 to 9. 30

33 Lecture 7: Synthesis of Fluorescein, a fluorescent dye Figure 1. Fluorescein under UV illumination. Experimental Synthesis of Fluorescein (a) Reaction Setup: Warm an oil or sand bath to a temperature of C. In a large test tube (15 X 150 mm) or a small Erlenmeyer flask place 0.2 g of powdered phthalic anhydride and 0.3 g of resorcinol, then, add 6 drops of 2M H2S4 (Do not add more than 6 drops). Stir the mixture briefly with a spatula. Place the test tube in the preheated bath. The reaction should run for 30 minutes at that temperature range. At the end of the reaction, remove the test tube from the bath and allow it to cool. (b) Reaction Workup Add 10 ml of acetone to the cold test tube and a stir bar. Use a ring stand and clamp, to place the test tube over a magnetic stirrer and stir the mixture for 5 to 10 minutes. The mixture should turn yellow as the crude fluorescein dissolves. If the entire product did not dissolve, add by portions of 5 ml acetone until the entire crude dissolves (do not excide 25 ml total). Poor the acetone layers in a 50 ml beaker. Evaporate the acetone to obtain orange residual crude. Dissolve the crude in 30 ml of diethyl ether and 1.5 ml of water. Place a stir bar in the solution and put the beaker over a magnetic stirrer for several minutes until dissolving all solids. Transfer this organic layer in a separatory funnel and wash with 15 ml of water. Discard the aqueous wash and wash with 10 ml of a saturated NaCl solution. Dry the organic layer over anhydrous sodium sulfate. Filter, then, place the dried solution in a beaker and emerge it in a water bath to evaporate the solvent, to obtain the product as an orange solid. 31

34 Lecture 7: Synthesis of Fluorescein, a fluorescent dye (c) Fluorescene observation: In 50 ml 0.1 M NaH solution, dissolve 5 milligrams (or spatula tip size) of the fluorescein sample. Place the solution in a vial and place it on a black non-reflective surface. Place a bright light source (sunny window works well) on the opposite side from the observer and note the appearance of the solution. The observed color is primarily due to absorbance of some wavelengths of visible light passing through the sample from the light source. Next observe the same solution at a position 90 degrees from the light source. The observed color from this angle is primarily due to fluorescence. Finally, in a darkened room, shine a long wave UV lamp to the sample vial. The vials should visibly glow from the fluoresced light. 32

35 Lecture 7: Synthesis of Fluorescein, a fluorescent dye Report 1. What is the used solvent for this reaction? 2. Calculate the Molecular mass of phthalic anhydride and resorcinol. 3. What is the number of moles of the phthalic anhydride and resorcinol used to realize the reaction? 4. Compare the used number of moles of both reactants. 5. Calculate the Molecular mass of fluorescein. 6. Calculate the number of moles of the fluorescein theoretically expected from the reaction. 7. What are the particular precautions that must be taken during the reaction? 33