‘LESSON 1: HOW TO SETUP AN EXPERIMENT 1. Lab diary In this course, we will record all the results in a lab diary. We take note of the experiment as detailed as possible and put in some discussion on the results. LAB DIARY EXAMPLE Employee: Experiment code: Date: 1. Reaction procedure: ............................................................................................. ..................................................................................................................................... 2. Reaction: (e.g.) Name Mol. weight Density Weight 0.99 1 µL (Volumn) Moles Equivalent 3. The actual experiment Entry Time Task 17/10 10:30 Stir at 60 °C 1 17/10 16:30 Quench in distilled water 2 18/10 10:30 Recrystallization with EtOH 3 Note: Describe the actually obtained phenomenon in detailed. Phenomenon Dissolved Precipitate Needle crystals 4. Results: Appearance: ..................................................................................................... Crude yield: ..................................................................................................... Isolated yield: .................................................................................................. Purity: .............................................................................................................. Rf: .................................................................................................................... H- NMR: ......................................................................................................... 5. Discussion: 2. Safety regulations 3. How to setup a reaction a. Procedure - Reaction procedure is the way we do the reaction from the start, the cooking, the working-up and the purification. - When we operate a procedure, we have an experiment with exact amount of each substance, particular obtained phenomenon, yield, result with reasonable deviation. - The more experiments we do, the more converging we have in the results. b. Reaction monitoring - To monitor a reaction, we must choose a method to visualize the most constituents in the reaction. In organic synthesis in liquid phase, monitoring methods could be crude NMR, GC, GC-MS, HPLC, LC-MS, TLC or even eye-test. - The most common method is using TLC, which will be performed in this course. Before performing a TLC, the sample from the reaction must be worked-up via a mini work-up to show up as many constituents as possible. - The reaction mixture will be compared to the starting materials. - Compounds on TLC could be visualized by visualization reagents (Dragendorff's reagent, KMnO4, the vanillin sulfuric acid reagent). c. Working-up Working-up a reaction is the procedure to stop the reaction immediately. Principle of this procedure is the isolating or decomposing one of the reactants included in catalyst or solvent. For example, a reaction in acidic environment can be worked-up by the neutralization using alkaline; a reaction using transition metal catalyst can be worked-up by using Celite to absorb the metal species or Na2S solution to precipitate the sulfide salts. d. Isolation and purification - After the reaction (confirmed by the monitoring step), the desired products will be isolated from the crude product by proper methods. - The isolation and purification methods could be precipitate-filter, liquid-liquid extraction (phase separation), distillation, crystallization, column chromatography or other modern chromatography methods. e. Column Chromatography Column chromatography is simple and the most popular separation and purification technique. Column chromatography consists of a stationary solid phase that adsorbs and separates the compounds passing through it with the help of a liquid mobile phase. Based on their chemical nature, compounds get adsorbed, and elution is based on differential adsorption of a substance by the adsorbent. In column chromatography, a cylindrical glass tube, which is plugged at the bottom by a piece of glass wool or porous disc, is filled with slurry (adsorbent) and a suitable solvent. Samples to be separated are mixed with silica and introduced at the top of the column and allowed to move with the solvent. With polarity differences, compounds are adsorbed at different regions and desorbed with suitable solvent polarity. The compound of higher adsorption ability will be adsorbed at the top and that with the lower one will be at the bottom. By adding the solvent at the top, compounds get desorbed and pass through the column and this process is called elution. A schematic diagram of column chromatography is shown in Fig. below. LESSON 2: Synthesis of benzimidazole derivatives 1. About benzimidazole - IUPAC name: Benzimidazole - Molecular structure: - CAS number: 51-17-2 - Mol. weight: 118.14 g/mol - Drug: Antitumor, antifungal, antiparasitic drugs - Appearance: colorless solid - Mol. formular: C7H6N2 2. Synthesis route 3. Procedure 2 sets of 0.54 g of o-phenylenediamine were dissolved in 1.5 mL of formic acid 90% solution and in 1.5 mL of glacial acetic acid respectively in 2 distinguished screw cap reaction vials. Set them in the heating block of a magnetic stirrer and let the reactions occurred at 100 °C for 2 h in fume hood. When the reactions are finished, quench them in water in a beaker. 10% sodium hydroxide solution was used to adjust the reaction mixtures to pH of 9-10 to precipitate the products. Crude products are filtered, washed with ice cold water. Dissolve the synthesized product in 15 mL of boiling water, add 0.1 g of activated carbon and digest for 15 min. Filter rapidly through a preheated funnel and a flask. Cool the filtrate to about 10 °C, filter off the crystals, wash with 10 ml of cold water and dry in the oven. 4. Reaction monitoring - TLC method: A sample was taken from the reaction into a test tube. The mixture was worked up and diluted by proper solvent and concentration. A pre-conditioning TLC of the solution was performed with o-phenylenediamine as the reference and using n- hexan/Ethylacetate/methanol 8:2:2 eluent. - NMR method: Compare the most special peaks of starting materials and products. 5. Identification of aspirin - Visualization: all o-phenylenediamine and bezimidazole derivatives can be stained by Dragendoff’s stain agents on the TLC. - Identification: Using GC-MS and NMR spectra of compounds. LESSON 3: THE SYNTHESIS OF ASPIRIN FROM WINTERGREEN OIL 1. About aspirin - IUPAC name: 2-Acetoxybenzoic acid - Molecular structure: - CAS: 50-78-2 - Mol. weight: 180.16 g/mol - Drug: Nonsteroidal anti-inflammatory drug - Appearance: white/pink crystal - Mol. formular: C9H8O4 2. Synthesis route 3. Procedure Step 1: 4 mL of wintergreen oil (methyl salicylate) and 40 mL of 6 M sodium hydroxide are added and then stirred in a round bottom flask. The mixture is gently boiled by heating for 30 mins. The mixture was acidified by H2SO4 22% solution and then cooled down in an ice bath. Precipitated solids are filtered and washed using a small amount of cold distilled water. The crude product is purified by recrystallization in boiled distilled water. The crystals was filtered, washed and dried in the oven. Step 2: Add 2.0 g of dried salicylic acid to a 50 mL round bottom flash containing 15 mL of acetic anhydride, magnetic bar, and condenser. Slowly add 2 drops of concentrated sulfuric acid (approximately 0.015 mL) to the mixture. The reaction mixture was stirred at 60 °C for 1 hour. When the reaction was finished, the product mixture was quenched in 150 mL of distilled water then stirred at 0 – 5 °C until aspirin needle crystals were formed. Crystal product was filtered on Buchner funnel. Dry and weight these crude crystals to calculate crude yield. The crude product is purified by recrystallization in hot EtOH 90º. The crystals was filtered, washed and dried in the oven. 4. Reaction monitoring - TLC method: A sample was taken from the reaction into a test tube. The mixture was worked-up and diluted by proper solvent and concentration. A TLC of the reaction mixture in the test tube was performed with wintergreen oil solution, salicylic acid solution as the references and using hexane:EtOAc eluent. - NMR method: Compare the most special peaks of starting materials and products. 5. Identification of aspirin - Visualization: Wintergreen oil and methyl salicylate can be visualized in 5% aqueous solution of FeCl3 but the aspirin is not. - Identification: Using GC-MS and NMR spectra of aspirin. LESSON 4: Synthesis of propranolol 1. About propranolol - IUPAC name: 1-(isopropylamino)-3-(1- - Molecular structure: naphthyloxy)propan-2-ol - CAS number: 525-66-6 - Mol. weight: 259.34 g/mol - Drug: beta-adrenergic receptor blocker - Mol. formular: C16H21NO2 - Appearance: White solid 2. Synthesis route 3. Procedure Step 1: Substitution reaction Add 1.25 g of 1-naphthol and 0.5 g of potassium hydroxide into 40 mL of ethanol 90° in a 100 mL 3-neck round bottom flask equipped with magnetic stir bar, dropwise funnel, temperature and condenser. The mixture was stirred for 10 minutes until the solid is well dispersed. Use dropwise funnel to add 4 mL epichlorhydrin in 5 minutes. The reaction mixture was heated reflux for 8 hours. Monitor the reaction by TLC with n-hexane/EtOAc 9:1 eluent. After the reaction, epichlorohydrin and solvent was removed under reduced pressure. The residue was dissolved in EtOAc then extracted by water 2 times. The organic phase was washed the last time by minimum amount of solution of NaOH 1M, dried over anhydride Na2SO4 then evaporated all the organic solvent to obtain an amber liquid (2). Step 2: Addition reaction Dissolve all the amber liquid (2) in 5 mL of MeOH in a 150 mL round bottom flask equipped with magnetic stir bar and condenser. 50 mL of isopropylamine was added then the reaction was refluxed for 4 hours. Monitor the reaction by TLC with DCM/MeOH 10:1 eluent. After the reaction, the solvent was removed under reduced pressure. Step 3: Isolation of the product Add 30 mL HCl 2M into the flask to dissolve the residue. The mixture was stirred overnight at room temperature. The mixture was washed with EtOAc on extract funnel. The water phase was neutralized by NaOH 6M until the pH value reaches 910. The mixture was cooled down to precipitate the amine product. Filter on Buchner funnel and wash with water to obtain desired product. LESSON 5: Synthesis of 9-fluorenone 1. About fluorenone - IUPAC name: 9H-Fluoren-9-one - Molecular structure: - CAS number: 486-25-9 - Mol. weight: 180.20 g/mol - Drug: It is used to make antimalaria drugs - Appearance: yellow solid - Mol. formular: C6H8N2O2S 2. Synthesis route 3. Procedure Step 1: Synthesis of 9-fluorenone 1 mmol of fluorene was dissolved in 2 mL of acetic acid in a 7 mL screw cap vial. 1 mmol of K2Cr2O7 was added slowly into the solution. The reaction was heated to 80 ºC for 30 minutes then the reaction was quenched in 15 mL of water. The precipitate was filtered and washed by water. The solid was dried properly before the purification using column chromatography. Step 2: Preparation of the column The column should be properly washed and completely dried before using. The bottom end of the column is packed with glass wool/cotton wool or an asbestos pad after which the stationary phase is packed. The slurry of silica in n-hexane is prepared along with the mobile phase and is poured into the column. Let the stationary phase stable before starting the chromatography. Step 3: Introduction of the sample - Wet method: The crude product was dissolved in the minimum amount of EtOAc then the solution was added onto the top portion of stationary phase in the column where it is absorbed. Afterward, a cotton pad is placed on the top to avoid the disturbance of the stationary phase during the introduction of the sample or mobile phase. - Dry method: The crude product was dissolved in EtOAc and a minimum amount of silica was added in a round bottom flask. The slurry mixture was dried on rotary evaporator to absorb all the compounds onto the silicagel. The dried silicagel was added carefully on the top of the stationary phase without disturbing this silicagel layer. Step 4: Run the column chromatography After introducing the sample, choose the proper eluent to let in run through the column. The eluent out was collected in tubes to distinguish fractions. All the fractions were monitored by TLC to isolate compounds out of the column. Pure fractions was collected in a round bottom flask and dried by rotary evaporator to recover the solvent. Notice: The disturbance in the stationary phase (adsorbent layer) leads to the irregular bands of separation.
