Experiment 17: NITRATION OF p-methylacetanilide H N O C H CH 3 + HNO3 p-methylacetanilide H2SO4 Acetic acid N O C CH 3 NO 2 Nitromethylacetanilide Objectives: To synthesize methylnitroacetanilide isomers using an electrophilic aromatic substitution. To purify product by recrystallization. To identify and determine purity of product by melting point, TLC and HPLC analysis. To characterize product by analysis of 1H-NMR and 13C-NMR spectra. Before coming to lab… Review these techniques: TLC analysis Recrystallization Suction filtration HPLC analysis Melting Point Analysis CHEMICAL EQUATION H N O C H CH 3 + HNO3 H2SO4 Acetic acid N O C CH 3 NO 2 H N O C CH 3 or NO 2 4-methylacetanilide C9H11NO 4-methyl-2-nitroacetanilide C9H10N2O3 MW: 149.19 MW: 194.19 o mp: 92-95oC mp:148-151 C INHALATION HAZARD, INGESTION HAZARD 4-methyl-3-nitroacetanilide C9H10N2O3 MW: 194.19 ELECTROPHILIC AROMATIC SUBSTITUTION E + E H +E + + H E E H + This reaction takes place in two steps: 1. Initial reaction of an electrophile, E+ with the aromatic ring. 2. Loss of H+ from the resonance stabilized carbocation intermediate to regenerate the aromatic ring. + H+ MECHANISM (Preliminary steps…) • Reaction of the aromatic compound with nitric acid alone is slow and potentially hazardous. • It is safer and faster to use a catalytic amount of sulfuric acid. •Sulfuric acid reacts with nitric acid to form the nitronium ion, a powerful electrophile. Sulfuric acid Nitric acid O H O N O + O H O S O H O • Sulfuric nitronium ion (STRONG electrophile) H O H O N O + HSO4- O N O + H2O acid protonates the hydroxyl group of nitric acid, allowing it to leave as water and form a nitronium ion. MECHANISM (Nitration of Aromatic Ring…) • Electrophilic aromatic substitution by the nitronium ion gives the nitro product. O O H H N CH 3 + + O N O O N CH 3 O+ N O H • Attack on the electrophile forms the sigma complex… N H O N + O + H3O .. + .. H3C O H H • …loss of proton to water… • …gives the aromatic nitro product. REACTIVITY OF AROMATIC RINGS The substituents ALREADY ON the aromatic ring affect the reactivity of the aromatic ring, or how FAST the substitution will occur. The substituent ALREADY ON the aromatic ring determines orientation of the substitution, or the position of the second (INCOMING) substituent. Both the methyl group (-CH3) and the acetamide group (-NHCOCH3) are o, p activators. The acetamide group is a STRONGER o, p director than the methyl group. Week 1: Synthesis Mix p-methylacetanilide and acetic acid in 50 mL Erlenmeyer flask. Prepare nitrating mixture in small test tube (CAUTION!). *IMPORTANT!* Cool this tube in ice water! Add nitrating mixture drop wise while swirling. Allow to react at room temp for 15 minutes. Add cold deionized water. Week 1: Crude Product Isolation Prepare suction filtration apparatus, seating filter with cold deionized water. Pour crude product slowly into center of funnel. Rinse with cold deionized water. Prepare CRUDE PRODUCT TLC and HPLC samples in small sample vials. Set crude solid aside to purify by recrystallization. Week 1: Preparation of Filtrate Samples Transfer 2 mL filtrate to a small test tube. Add 10% NaOH drop wise until neutral (check every 5 drops with pH Hydrion paper). Add 3 mL ethyl acetate. Cork tube and shake to mix. Allow layers to separate. Filtrate Prepare HPLC and TLC samples of FILTRATE using 5 drops of this solution for each. Remember to add appropriate sample solvent for each! Week 1: Purification Transfer CRUDE PRODUCT to 50 mL flask. Dissolve in hot 90:10 ethanol/water. Cool to room temp, then to 0oC. Suction filter. Seat filter paper w/90:10 ethanol/water. Prepare RECRYSTALLIZED PRODUCT TLC and HPLC samples. Week 1: Sample Submission Transfer small filter paper and product to a preweighed large filter paper and submit to instructor to dry until next lab period! Also, submit all TLC samples to instructor. At the end of lab, you should have submitted: To instructor… 3 TLC sample vials 1 packet of pure solid product For HPLC analysis… 3 auto sampler vials Table 17.1 Theoretical yield (g) Actual yield (g) Percent yield Experimental melting range (oC) Product appearance Week 1: Calculate this value based on how much reactant you ACTUALLY used, not just what the MANUAL SAYS to use. Week 2: Calculate this value after you have retrieved your dry product next lab! Week 2: Calculate this value after you have retrieved your dry product next lab! Week 2: Record this value as a Ti-Tf range Week 1: Record COLOR and PHYSICAL STATE of product. Table 17.2 Atom Economy (%) Week 1: Review Exp. 13 or Appendix I to review calculation! Experimental Atom Economy (%) Week 1: Be sure to calculate this value using actual weights/volumes that YOU used, not just what the MANUAL SAYS to use! Week 2: You need the PERCENT YIELD for this calculation! “Eproduct” Cost per Synthesis ($) Cost per Gram ($/g) Week 1: Be sure to calculate this value using actual weights/volumes that YOU used, not just what the MANUAL SAYS to use! Costs of stock bottles are given on p. 150. Week 2: You need the final ACTUAL YIELD value to calculate this! Week 2: Product Analysis TLC Analysis HPLC Analysis Prepare TLC plate with 6 lanes. Apply 3 provided standards along with samples of crude product, filtrate, and recrystallized product. Develop in 2:1 ethyl acetate/hexane. Visualize with UV lamp. Identify compounds and calculate Rf value. Compare sample chromatograms to provided standard to identify compounds in each sample submitted. Melting Point Analysis Obtain the experimental melting point of your purified product. Compare to the literature value to determine degree of purity. Table 17.3 TLC Rf values Compound Standards All Rf values are unitless! All Rf values are 2 decimal places ONLY! p-methylacetanilide Crude sample Filtrate sample If reactant present in this lane, it explains product loss! If major product present in this lane, it explains product loss! 4-methyl-2nitroacetanilide 4-methyl-3nitroacetanilide Recrystallized sample Table 17.4 Standards Compound Retention times (min) Crude Sample Retention times (min) Filtrate Sample Area % Retention times (min) Recrystallized Sample Area % Retention times (min) p-methylacetanilide 4-methyl2-nitroacetanilide 4-methyl3-nitroacetanilide • All samples are submitted for analysis during 1st lab period. Samples will be run, and chromatograms returned during 2nd lab period! Area % Product Analysis (NMR Spectroscopy) O H C N 8 CH3 9 1 H NO2 6 H 2 3 5 4 CH3 7 H Table 17.5 O H N C 8 N CH3 8 1 6 2 5 3 CH3 7 C 9 1 4 O H 6 CH3 9 NO 2 2 3 5 4 CH3 7 • Enter chemical shifts ONLY based on the spectra on pages 139 and 146. • Notice that the reactant is the product from Experiment 16. You should have already analyzed the NMR spectra! SAFETY CONCERNS Nitric acid and sulfuric acid are very corrosive! Use EXTREME caution when in use! Ethanol is flammable! Be careful when heating! WASTE MANAGEMENT Place all liquid waste from experiment into the bottle labeled “LIQUID WASTE”. Place solid waste in bottle labeled “SOLID ORGANIC WASTE”. Place all used TLC spotters and melting point capillaries in broken glass container, NOT trashcan! CLEANING Clean all glassware with soap, water, and brush if necessary. Rinse all glassware with wash acetone before returning to lab drawer. DO NOT return any glassware dirty or wet to lab drawer! All hotplates and MelTemps must be unplugged from outlets, with cords wrapped neatly.