MCMP 205 Laboratory Exercise 2 Exercise 2. Electrophilic Aromatic Substitution: Nitration of Phenol Required Reading: Chapter 16 in the lecture text. Pay special attention to sections 16.4 and 16.5. Objective: This experiment has been designed to understand the mechanism of electrophilic aromatic substitution. In this two week lab exercise, you will carry out nitration of phenol analyze the products using thin-layer chromatography during the first week. During the second week of this lab you will be carrying out <a href="/keyword/column-chromatography/" >column chromatography</a> to separate the various products formed in the reaction. This experiment will also demonstrate how thin-layer chromatography is used to monitor the progress of a column separation. Understanding the experiment Nitration of Phenol Aromatic compounds react with nitric acid, usually in the presence of sulfuric acid as a catalyst, to form nitroaromatic compounds. This reaction, called nitration, introduces a nitro group, --NO2, into the <a href="/keyword/aromatic-ring/" >aromatic ring</a> by electrophilic substitution. The electrophile in this reaction is the nitronium ion, NO2+, formed by the acid-catalyzed removal of the elements of water from HNO3. Phenol is a highly reactive aromatic compound that readily undergoes nitration at the ortho and para positions of the <a href="/keyword/aromatic-ring/" >aromatic ring</a> . The hydroxy group (--OH) donates electrons to the ring and activates it toward electrophilic substitution at ortho and para positions. Due to the highly activating nature of the hydroxyl group, phenol often undergoes multiple nitration to give 2,4-dinitrophenol (2,4,6-trinitrophenol at high temperature) along with the mononitro products. The highly activating --OH group of phenol necessitates the use of a mild nitrating reagent for the synthesis of mononitrated products. A convenient reagent for this purpose is nitrated <a href="/keyword/silica-gel/" >silica gel</a> , made by wetting a large excess of <a href="/keyword/silica-gel/" >silica gel</a> (SiO2) a drying agent, with a dilute solution of HNO3. The <a href="/keyword/silica-gel/" >silica gel</a> reacts with the HNO3 to form a salt between the nitric acid and the silicic acid in which the elements of water are eliminated. The resulting nitronium silicate, HSiO3NO2, present in the excess <a href="/keyword/silica-gel/" >silica gel</a> is a solid, mild nitrating agent that is easily dispensed. Furthermore, removal of any excess nitrating reagent is accomplished by a simple filtration. 1 MCMP 205 Laboratory Exercise 2 Monitoring the Reaction by Thin-layer Chromatography (TLC) One of the major applications of thin-layer chromatography is to monitor the progress of a reaction. It is usually possible to withdraw a small amount of reaction mixture and spot it on the TLC plate along with standard solutions of reactants (and products, if available). The TLC plate is then developed using a suitable solvent system. Appearance of new spots and disappearance of the spots corresponding to the reactants indicates that the reaction has gone to completion. Nitration of phenol can also be monitored by TLC. Just as you observed the separation of amino acids on paper chromatography in the last exercise, a mixture of nitrophenols can also be visualized on TLC. Introduction of the nitro group on the <a href="/keyword/aromatic-ring/" >aromatic ring</a> increases the number of hydrogen bonding sites. Therefore, various nitrophenols will be partitioned differentially between silica (stationary phase on TLC) and the solvent system. After the TLC is developed, it can be visualized by simply shining a UV lamp on the plate. The spots corresponding to the reactants and products in this reaction will appear dark on a fluorescent green background. (<a href="/keyword/silica-gel/" >silica gel</a> is not UV active by itself, but special UV-active TLC plates are manufactured by mixing some zinc sulfide with silica that makes it UVactive.) Separation of Nitrophenols by <a href="/keyword/column-chromatography/" >column chromatography</a> One of the most commonly used purification techniques used by synthetic chemists is <a href="/keyword/column-chromatography/" >column chromatography</a> . Whenever a reaction is performed, it is only seldom that it yields one product exclusively. Usually a mixture of products or a mixture of products with unreacted starting materials is obtained. In order to get the required product from that mixture, it is dissolved in a suitable solvent and loaded on top of a column containing a stationary phase (like silica or alumina). <a href="/keyword/column-chromatography/" >column chromatography</a> works on exactly the same principle as Thin-layer chromatography the differences being that column separation is carried out on a larger scale and the solvent is actually made to flow through the column under gravity (or by pressure). When using <a href="/keyword/column-chromatography/" >column chromatography</a> to separate a mixture of compounds, it is usually necessary to collect a large number of small fractions, analyze each fraction by TLC, and then combine fractions containing the same components (in our case, the same isomer). The principle of <a href="/keyword/column-chromatography/" >column chromatography</a> and analysis of fractions is well-illustrated in this exercise. When the column separation of the nitration reaction mixture was first carried out, twenty four fractions were collected and analyzed by TLC. The TLC profile of fractions and column progression is shown in Figure 2.1. The twenty-four fractions were spotted on a <a href="/keyword/silica-gel/" >silica gel</a> TLC plate (LANE 1 to LANE 24, respectively) along with standard solutions of 2,4-dinitrophenol, 2-nitrophenol, 4-nitrophenol and phenol (LANES A, B, C, and D, respectively) and the TLC plate was then eluted with an appropriate solvent system. 2 MCMP 205 Laboratory Exercise 2 As you can see in Figure 2.1, LANE 1 through LANE 10 contain only one spot having the same Rf as the spot in LANE B; this indicates that FRACTION 1 through FRACTION 10 contain pure 2-nitrophenol. LANE 11 contains two spots, one with an Rf the same as the spot in LANE A, and the other with an Rf identical to the spots in LANES 1 10; thus FRACTION 11 contains both 2-nitrophenol and unreacted phenol. LANE 12 and 13 contain two spots one with the same Rf as the spot in LANE A and the other same as the spot in LANE D. Thus, fractions 12 and 13 contain two products unreacted phenol and 4nitrophenol. In the same manner, LANES 15 to 22 contain a spot that has the same Rf value as the spot in LANE C. Therefore these fractions contain pure 2,4-dinitrophenol. This analysis helps us to identify the fractions that contain pure compounds. Figure 2.1. Analysis of the <a href="/keyword/column-chromatography/" >column chromatography</a> separation by TLC of the phenol nitration reaction products. LANE A is pure phenol; LANE B is pure 2-nitrophenol; LANE C is pure 2,4dinitrophenol; and LANE D is pure 4-nitrophenol. Procedure Transfer phenol (240mg, 2.55 mmoles) and diisopropyl ether (IPE, 2 mL) to a 5 mL conical vial containing a magnetic spin vane. Fill a 1 dram vial with diisopropyl ether to use throughout the reaction. Cover the conical vial with the screw cap containing septum and allow mixture to stir for 5 minutes at room temperature. Weigh out nitrated <a href="/keyword/silica-gel/" >silica gel</a> (1.0 g) into a 50 mL beaker then add slowly into the conical vial with a spatula over 15 minutes. The reaction of nitrated <a href="/keyword/silica-gel/" >silica gel</a> with the phenol is an exothermic reaction; the slow and even addition of the nitrated silica reduces the risk of raising the temperature and thus producing 2,4-dinitrophenol (undesired product). Once the addition of nitrated <a href="/keyword/silica-gel/" >silica gel</a> is complete, attach an air condenser and allow reaction mixture to stir for 30 minutes at room temperature. Set up the vacuum filtration apparatus using a Hirsch funnel and a filtration flask. Place the filter paper in the Hirsch funnel and wet it with 4 drops of diisopropyl ether. While agitating the mixture with a spatula, pour the reaction mixture with the spin vane into the Hirsch funnel. Wash the reaction conical vial by two 1-mL portions of acetone and transfer the washings into the funnel to rinse the <a href="/keyword/silica-gel/" >silica gel</a> . Transfer the solution in the filtration flask into a clean 5-mL conical vial using a Pasteur pipette. Wash the filtration flask with 1 mL of acetone and transfer the washing into the above 5-mL conical vial. Place the conical vial 3 MCMP 205 Laboratory Exercise 2 into a 50-mL beaker and leave it in your equipment box to allow the solvent to evaporate by next week. Analysis of Reaction by TLC Obtain a <a href="/keyword/silica-gel/" >silica gel</a> TLC plate (5 cm x 10 cm) and carefully draw a line with a soft pencil 1.5 cm from the bottom of the plate to mark the origin. Mark six separate spots by making six small, evenly spaced marks perpendicular to the origin line, number 1 through 6. Make sure to draw lines on the matte side of the plate not the glossy, and mark very gently as the silica can be scrapped off the plate. Dissolve your dried reaction mixture in 500 L of diisopropyl ether, and spot mixture only once on lane 1. Spot the standard solutions of phenol, 2nitrophenol and 2,4-dinitrophenol (3 times for each solution) in lanes 2, 3 and 4 respectively. Remember to keep your spots as small as possible (about the size of a small `o'). Develop the TLC plate with 8 mL of the eluent (diisopropyl ether:hexanes:acetone 32:65:3). Mark the distance traveled by the solvent front and allow the TLC plate to dry. Visualize the TLC plate by shining the UV lamp and outline the spots as you observe them. Calculate the Rf values for each spot as described in exercise 1. Separation of Reaction Mixture by <a href="/keyword/column-chromatography/" >column chromatography</a> (You are required to perform <a href="/keyword/column-chromatography/" >column chromatography</a> in groups of 3 students. Use only one reaction mixture for this operation. A pre-packed column will be given to you for this exercise. Please handle the column with care as it is expensive.) Collect 25 culture tubes and mark 2 mL mark on them by comparing with a reference test tube. Arrange these tubes in a test tube rack. Also make a 5 mL mark on a 25 mL vial by using acetone and a measuring cylinder. Clamp the column securely. Turn the stop cock on by turning it parallel to the column. Wet the column by adding 12 mL of hexane. It is important that the layer of sand covering the <a href="/keyword/silica-gel/" >silica gel</a> is not disturbed. Therefore, add hexane with a long Pasteur pipette by inserting it inside the column just above the layer of sand. When the hexane level just reaches the top of the sand layer, stop the column by turning the stopcock perpendicular to the column. To prevent air bubbles from forming in your column, the column should not run dry. Thus make sure that the solvent level remains above the level of the sand in the column at all times. Dissolve the dried reaction mixture in 500 L of isopropyl ether. Transfer this crude reaction mixture to the top of the column using a long Pasteur pipette. Wash the flask with 250 L diisopropyl ether, and transfer the wash to the top of the column. Turn the stop cock on until the solution of the crude reaction mixture reaches the top level of the sand and then stop the column again to add the eluent (diisopropyl ether:hexane:acetone, 40:52:8) to the top of the column. It will take about 50 mL of the eluent to complete the entire column chromatographic separation. Figure 2.2. Chromatography Column Start collecting the column eluent. First 5 mL should be collected in the marked 25 mL vial followed by collecting successive 2 mL fractions in marked culture tubes. Observe the 4 MCMP 205 Laboratory Exercise 2 different colors of fractions and on the column. After collecting 25 fractions obtain three TLC plates from your TA and mark the base line as mentioned in previous section. Spot every second fraction three times using fresh capillary tubes for each fraction. Develop the TLCs in three different developing chambers filled with the TLC developing solution and analyze the fractions. Observe the TLC plates to ascertain the success of column separation. On the basis of your observations, combine all the fractions that appear to be pure in a 25 mL vial. Aspirate the solvent from these fractions and determine their melting point. Record the IR spectrum of the isolated compounds by dissolving a very small amount in few drops of dichloromethane. Place a drop of this solution on the salt plate and let it dry. Repeat this procedure three times to accumulate enough material on the IR plate and record the IR spectrum. Observations and Results Your observations must contain your reaction TLC with outlined spots and calculated Rf values. Comment on the success of column purification on the basis of the TLC analysis of fractions. Record the IR spectra and melting points of the isolated compounds. Discussion 1. Why does the electrophile preferentially attack ortho and para positions of the <a href="/keyword/aromatic-ring/" >aromatic ring</a> in phenol? Draw the curved-arrow resonance structures of the carbocation intermediate to justify your answer. 2. Draw and discuss the mechanism of nitration of phenol. You must include the four important steps in your mechanism: a. Generation of electrophile; b. Attack of electrophile by the -electrons of the <a href="/keyword/aromatic-ring/" >aromatic ring</a> ; c. Stabilization of the carbocation intermediate; d. Loss of a proton to regain aromaticity. Pay close attention to the charges, arrows and lone pairs as you write the mechanism. 3. Why do ortho-nitrophenol and para-nitrophenol travel at different rates on TLC? Note that ortho-nitrophenol can form an internal (intramolecular) hydrogen bond. Show this hydrogen bond.. How would this hydrogen bond be expected to affect the retention of ortho-nitrophenol relative to para-nitrophenol on the column? (Note that <a href="/keyword/silica-gel/" >silica gel</a> is a potent hydrogen-bond acceptor. 4. What are the factors that affect the separation in <a href="/keyword/column-chromatography/" >column chromatography</a> ? You can compare gas chromatography and <a href="/keyword/column-chromatography/" >column chromatography</a> . What are the various variables that you would change to obtain a better separation? (Hint: think about the height-equivalent of a theoretical plate). 5