Lab 4: Separation of Organic Compounds Using Liquid-Liquid Extraction

Developed by Matthew Francis, MaryAnn Robak, and Kareem El Muslemany Introduction: Suppose you have just carried out the following reaction in the laboratory:

Figure 1. Oxidation of a primary alcohol. There are two different products that are expected (benzoic acid and benzaldehyde), which must be separated from one another to obtain pure materials. To make matters worse, most organic reactions do not go exactly according to plan. They commonly do not reach complete completion, producing a complex mixture of products, starting materials, salts, and other side products. To address these situations, chemists have devised various methods to separate organic molecules based on differences in their physical and chemical characteristics. Some of these methods include recrystallization, chromatography, and distillation. Liquid-liquid extraction is a complementary technique, and is one of the most common purification methods used to separate the various components of reaction product mixtures. The key idea is to partition the different compounds between an organic solvent (like ether or ethyl acetate) and a non-miscible aqueous layer that is either acidic or basic. When applied properly, this strategy can be used to extract one or more components selectively in order to isolate it or to simplify the reaction mixture that is left behind. In the example above, one could take advantage of the fact that benzoic acid is soluble in water at high pH (when it is deprotonated and therefore charged), but not under acidic conditions. Benzaldehyde is largely insoluble in water at both high and low pH. So, the reaction products would be shaken in a biphasic mixture of ethyl acetate and aqueous base. Using a piece of glassware called a separatory funnel (or sep funnel if you are in the know), the aqueous layer would then be isolated and transferred to a flask. Upon the addition of acid to this sample, the benzoic acid will be rendered insoluble, causing it to precipitate from the low pH water. The original ethyl acetate layer will contain the benzaldehyde, which can be recovered by evaporating the organic solvent. As this material will probably be contaminated with some of the starting alcohol (which is also mostly insoluble in water), a subsequent round of chromatography or distillation would likely be used to purify the material further. In laboratory research, this general strategy is adapted in various ways to meet the specific situation at hand. In this lab experiment, you will learn to use extraction techniques to separate a series of compounds based on their acid/base properties. Background Reading: Read the following sections in the Pedersen Lab Manual: Acids and Bases (4.1, 4.1.1) Extraction (7.6, 7.6.1) It may also be helpful to review the following sections: Solubility (6.1, 6.1.1) Evaporation (Column Chromatography Lab)

Pre-lab: In your pre-lab write-up, draw the structures and record boiling points of the extraction solvents. Also draw the structures and record the melting points of the compounds that will be extracted in Part 1. Also copy Table 1 (see below) into your lab notebook and fill in the structures and melting point ranges for the potential unknowns. Additionally, write out the reactions of benzoic acid with NaOH and of aniline with HCl. Finally, draw a flow chart of the separation technique you will use to separate your unknowns, similar to what is shown in Figure 2. Based on the volumes and concentrations of acidic and basic solutions given in the procedure, calculate the approximate volume of strong acid or strong base that will be needed to neutralize the weak acid or weak base solutions used in the extractions. Additionally, please answer the following questions prior to lab on a separate page in your lab notebook. Write out any necessary calculations. You will need the Evans pKa table (available on the bSpace site) to answer these questions. Since pKa values will be approximate, you need to state which pKa you used for your answer and list the compound you are using for comparison. Additionally, your pKa value should not have any decimal places since you are using approximate values. A student somewhere in the spiraling heights of Latimer Hall performs the following reaction:

Since the reactants and products are valuable, the student wants to recover all of the components of the reaction mixture. 1) Write out the acid-base reactions that each of these components (except water) would undergo. 2) For the carboxylic acid and the thiol, calculate the relative ratios of protonated-to-deprotonated compound at pH 2, pH 10, and pH 14. 3) Calculate the concentrations of NaOH and HCl that are required to generate aqueous solutions at pH 2 and pH 12. 4) Generate a flow chart similar to that shown in Figure 2 to indicate how you would separate and isolate each of these compounds. At your disposal is a basic solution (NaOH, aq) at pH=12, an acidic solution (HCl, aq) at pH=2, and a neutral solution (NaH2PO4) at pH=7. Use the precipitation method (similar to that used in Pt. I). General Work Guidelines: Your GSI will give a basic demonstration of how to use a separatory funnel, and then they will divide the laboratory section into groups of three. Divide the work so that each of you is able to practice each technique. Split up work so that everyone does at least one extraction or one precipitation in part I. Each person should do one extraction and one evaporation in part II. However, only one TLC needs to be run and recorded for part II. In part IV, each person should obtain at least one melting point. You will notice that in Part I, you use precipitation to isolate your compounds of interest. However, not all compounds respond equally well to precipitation. A simple solution to this problem is a second

extraction after neutralization, sometimes referred to as a back extraction. You will practice back extractions in Part IV. Please look at page 4 for further information. Safety Notes for this Laboratory Experiment: Vent the separatory funnels often while conducting the extractions, or excess pressure could develop. Your GSI will show you how to do this safely. Ensure that you are not pointing either end of the separatory funnels at the other lab students. The aqueous solutions used in this lab are strongly acidic and basic. They are hazardous if they come into contact with your skin. If they do, immediately wash the contact area with running water and alert your GSI. As with all labs, eye protection must be worn at all times. You will need to wear a pair of gloves while using the separatory funnel. These are available in the laboratory stockroom. Note that if you are allergic to latex, you should notify your GSI and the staff so that they can get you an alternative pair of gloves. Some of the compounds listed are harmful if they come in contact with the skin or are inhaled. Please immediately wash affected skin with soap and water and have a lab partner inform your GSI if any compound spills on you. Experimental Procedures: Part I. Separation of Known Compounds by Extraction. In the first section of the lab, you will be separating eosin Y, 9-fluorenone, and 4-bromo-N,Ndimethylaniline. You will use the general scheme shown in Figure 2 to separate these compounds based on their acid/base properties. The compounds will be precipitated and isolated by filtration.

Figure 2. Flow chart for the extraction of known compounds. First, obtain approximately 300 mg of a mixture of the carboxylic acid, the ketone, and the aniline from the fume hoods. Make sure that you note the exact weight used. The first student should add the

mixture to 30 mL of ethyl acetate in an Erlenmeyer flask. Swirl the Erlenmeyer flask until the compounds have dissolved, and then transfer the solution to a 125 mL separatory funnel. Add 15 mL of 0.1 M NaOH solution to the erlenmyer flask to rinse it, then add this to the separatory funnel as well. Cap the top of the separatory funnel and shake gently (while venting!) as instructed by your GSI. After the two layers have separated, remove the cap and drain the bottom aqueous layer into a labeled beaker. Repeat the extraction with an additional 15 mL of 0.1 M NaOH solution and drain the obtained aqueous layer into the same beaker. Student 2 can then assume control of the separatory funnel. Next, add 15 mL of 0.1 M HCl solution to the separatory funnel. After capping and shaking while venting, allow the layers to separate. Drain the aqueous layer into a new labeled beaker. Finally, add 30 mL of brine (saturated NaCl solution) to the separatory funnel. Cap and shake while venting, and then drain the aqueous layer into a third labeled beaker or flask. Finally, pour the organic layer into a fourth labeled Erlenmeyer flask. Add a few spatulas of anhydrous magnesium sulfate to the organic layer to remove any residual water while swirling the flask. Repeat until the white powder stops clumping and flows freely (it should look like a snow globe). While student 2 is performing the second extraction, student 3 can begin precipitating the dilute NaOH solution. Carefully add approximately 1 mL of 6 M HCl solution. Confirm that this has neutralized the NaOH by dipping the end of a glass rod into the solution and placing a suspended drop of the aqueous solution onto pH paper. Continue adding acid dropwise until the paper indicates pH<1. (Note the change in pH. How much strong acid/base does it take to change the pH to the desired values? Does this match the amount calculated in your prelab?) Place the container in an ice-water bath and wait for the precipitation to occur. Student 1 should repeat this procedure with the aqueous layer from the acid extraction, this time using 6 M NaOH solution to neutralize the acid (calculate how much 6M NaOH will be needed in your prelab). Again monitor with pH paper, adding additional NaOH solution until you reach pH>10. Place the container into an ice-water bath and wait for the precipitation to occur. Did the compound spontaneously precipitate, or did it require a drop in temperature? While you are waiting for the precipitations, gravity filter the ethyl acetate solution to remove the magnesium sulfate. Do so by placing fluted filter paper into a glass funnel. Slowly pour the organic solution through the filter paper in the funnel such that the solution drips into a dry labeled beaker. Since the separatory funnels will be reused in Part III, rinse them with acetone, then water. They can be reused, even if theyre still wet. Part II. Confirming Compound Separation by TLC. Each precipitate will be collected using a Hirsch funnel. Make sure that the plastic frit disc is firmly snapped in place in your funnel. Replacement frit discs are available in case it become clogged or otherwise damaged. Attach the funnel to a filter flask, using the neoprene adapter to ensure a good seal between the funnel and the flask. Attach the filter flask to your vacuum line and turn the vacuum on. Swirl the precipitate in solution and quickly pour the mixture onto the filter (while the solids are still suspended). Rinse the precipitate with a minimal amount of ice-cold water. Leave the vacuum on until the precipitate is dry (~5 min.), and take note of the mass. Place a small amount (~1-2 mg) of the precipitate into labeled small test tubes. Add 1-2 mL of ethyl acetate to each one and agitate the test tubes until the compounds have been partially dissolved in the ethyl acetate layer. Record your observations.

Prepare TLC plates for two different analyses. Spot samples of the products isolated from each of the aqueous layers onto the plate, and in the third lane spot the ethyl acetate solution that remained at the end of the experiment, and in the fourth lane spot a sample of the crude mixture. Develop the TLC plate with 35% ethyl acetate in hexanes, and visualize the compounds using both UV and I2. Spot authentic compound samples onto a second TLC plate and develop it using the same solvent mixture. Analyze the results of these TLC plates to gain information about both identity and purity of the compounds isolated from each layer of the extraction. Part III. Separation of Unknown Compounds by Extraction. Note: Student 3 should perform two of the extractions listed below, while the other two students should perform one extraction each. Divide up your work appropriately, ensuring that you record who is responsible for each activity. You will be given a mixture of an unknown aniline, a carboxylic acid, and a ketone. Record the code of your unknown mixture. Measure out and dissolve approximately half of the material in 30 mL of ethyl acetate. Repeat the extraction procedure listed in Part I to separate the compounds. NOTE: do NOT attempt to precipitate the compounds, as some will not readily precipitate. You will perform a back extraction to remove each compound from the aqueous layers. First, add the aqueous layer from your initial extraction to a beaker. Then, titrate the strong acid solution into the weakly basic aqueous layer. Continue until pH<1. Repeat this procedure for the weakly acidic aqueous layer, substituting the strongly acidic solution for a strongly basic one. Add this solution to a separatory funnel and repeat the extraction procedure; this time, after extracting the compound into 15 mL ethyl acetate, wash with brine. Then, remove the organic layer and place it in a beaker. Use magnesium sulfate to dry the solution. Place an aliquot (3-5 mL) of the solution obtained above in a 25-mL filter flask for the evaporation step. Each student should perform at least one evaporation. Ensure the cold trap is half-filled with acetone, and add dry ice slowly. Cap the filter flask with a cork, turn on the vacuum, and begin evaporating, using a room temperature water bath as needed. Spot samples of the crude mixture and each solution that was obtained, on a TLC plate, develop it, and analyze your results. Was complete separation of the three compounds achieved? Part IV: Identification of Unknown Compounds by Melting Point. Scrape the residue that remains after evaporation of the ethyl acetate solutions from part III and obtain a melting point for each compound. Once the precipitates from part I have dried, obtain the melting points of these compounds as well. Prepare 2 capillaries of each compound for measuring melting points. Do one quick ramp to determine the approximate melting range of each compound, and then a slower ramp to accurately measure the melting temperature. Using all of the information, discuss the results with your lab partner until you reach a conclusion as to which amine, carboxylic acid, and ketone you have. Confirm this result with your GSI. Clean-Up: Return all solid samples to your GSI. Pour all remaining solutions into the halogenated waste container, rinse the glassware with acetone into the waste container, then clean thoroughly using soap and water.

Table 1. Potential unknowns for this experiment. Name 4-Bromoaniline Benzocaine Phenanthrene 4-Acetyl-Biphenyl trans-Cinnamic acid p-Toluic acid -Methylcinnamic acid Discussion: Explain how your group identified the unknown compounds. Provide an explanation for why another extraction was used in Part III instead of precipitation. Did you observe any impurities in the TLC plates you developed? If so, suggest ways to improve the separation. If you encountered any other problems with this experiment, discuss those here as well. Include a concise conclusion (1-3 sentences). Followup Question: Upon the addition of acid to eosin Y, it becomes a yellow solid that is no longer soluble in water. This change can be explained by an intramolecular reaction that occurs when the compound becomes protonated. The color changes of pH indicators work in similar ways, yielding a noticeable change in appearance upon a change in pH. The reaction begins with protonation of the phenol oxygen (left). A second proton is consumed later in the reaction. Derive a mechanism using the arrow-pushing formalism that results in formation of the compound shown on the right. Structure Melting Point Soluble in acidic solution, basic solution, or neither?

Acknowledgments: This lab was adapted from an experiment developed by Profs. Steven Pedersen, Arlyn Myers, and Ahamindra Jain.