Lab D: Separation of Organic Compounds Using Liquid-Liquid Extraction Chem 112A Fall 2012
Introduction: In a liquid-liquid extraction, the goal is to partition the compounds to be separated between two immiscible solvent layers: an organic solvent layer (typically a nonpolar or moderately polar aprotic solvent such as hexanes, ether, dichloromethane, or ethyl acetate) and an aqueous (water) layer. The pH of the aqueous layer can be adjusted to cause compounds with acidic or basic functional groups to become either charged or neutral. Most neutral organic compounds (both nonpolar and moderately polar) tend to selectively partition into the organic layer, while ions or extremely polar neutral compounds tend to partition into the aqueous layer. Therefore, acid/base reactions with liquid-liquid extractions can be used to separate compounds from each other on the basis of differences pKa. To achieve efficient separation in an extraction, it is desirable to adjust the pH of the aqueous layer to a range which will allow at least a 100:1 ratio of the desired protonation state to the undesired state. The Henderson-Hasselbalch equation (pH = pKa + log([A-]/[HA])) provides a convenient method for predicting the equilibrium ratios of acid/conjugate base pairs based on the pH of the solution and the pKa of the acidic compound. Scenario/Example: 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 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. In the example presented in Figure 1, one could take advantage of the fact that under acidic conditions benzoic acid (pKa = 4.2) is neutral and will partition into the organic layer, but under basic conditions the conjugate base of benzoic acid (benzoate anion) is soluble in water. Benzaldehyde is neutral (and is neither acidic nor basic) so it would partition primarily into the organic layer at any pH. Therefore, the mixture of benzoic acid and benzaldehyde could be partitioned between ethyl acetate and a basic (high pH) aqueous layer. The aqueous and organic layers would then be placed in separate flasks. The organic layer would contain the
benzaldehyde, which could be recovered by evaporating the ethyl acetate (solvent). Upon the addition of acid to the aqueous layer, the benzoate ion would be protonated to form benzoic acid, causing it to precipitate due to low solubility in water. Filtration can be used to separate the solid benzoic acid from the aqueous solution. This purification strategy is depicted as a flowchart in Figure 2 (below). Technique Tip 1: Water is slightly soluble in most of the organic solvents used for extractions. Therefore, if the organic layer from an extraction contains a desired compound, extra steps are required to remove water (“dry” the organic layer) before evaporation of the solvent. Read about this in section 7.7 (p. 119-120) of the Pedersen lab text). Identify these extra steps on the flowchart in Figure 2. Technique Tip 2: Depending on the partition coefficient of a compound between the two layers in an extraction, a small but significant amount of the compound may stay dissolved in the opposite layer from the rest of the compound. To solve this problem, multiple extractions (with a portion of the solvent used each time) are often carried out at each stage of an extraction scheme. The multiple layers with the same solvent as each other are then combined before moving forward. Read about this in the example on page 113-114 of the Pedersen lab text). Identify the points in the flowchart in Figure 2 where this strategy might be employed. Technique Tip 3: You will be using a piece of glassware called a separatory funnel, which is designed to make it easy to separate two liquid layers from each other. During part I, your GSI will demonstrate the use of this glassware. On microscale, a pipette and test tube or cetrifuguge tube is often used instead of a separatory funnel. Read about these two methods on pages 115116 of the Pedersen lab text.
Figure 2. Flowchart for separation of benzoic acid from benzaldehyde. In laboratory research, this general strategy is adapted in various ways to meet the specific situation at hand. In this lab experiment, you will use extraction to separate a mixture of compounds based on their acid/base reactivity and then identify the components of the mixture by melting point analysis.
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 Questions: 1. Propose an explanation for the solubility trends described by this statement from the introduction: “Most neutral organic compounds (both nonpolar and moderately polar) tend to selectively partition into the organic layer, while ions or extremely polar neutral compounds tend to partition into the aqueous layer.” (hint: think about intermolecular interactions) 2. Calculate the pH of each of the following aqueous solutions: a. 1.0 M HCl (a strong acid) b. 1.0 M NaOH (a strong base) 3. What are the upper and lower pKa limits for functional groups that should be considered “acidic or basic” in the context of this lab technique? Base your answer on the values you calculated in question 2 and this statement from the introduction: “To achieve efficient separation in an extraction, it is desirable to adjust the pH of the aqueous layer to a range which will allow at least a 100:1 ratio of the desired protonation state to the undesired state.” 4. Based on your answer to question 3, should benzyl alcohol (the starting material in Figure 1) be considered acidic, basic, or neither? If the reaction pictured in figure 1 was incomplete, where would the remaining benzyl alcohol be at the end of the purification described in the introduction and depicted in Figure 2? 5. In the example provided in the introduction, precipitated benzoic acid was isolated from the acidic aqueous solution by filtration. If the benzoic acid did not precipitate upon acidification of the solution, how could it have been isolated instead? (Hint: more organic solvent will be required) 6. Draw curved-arrow mechanisms and calculate Keq for the following acid/base reactions: a. benzoic acid + NaOH b. aniline + HCl
Pre-lab Checklist: • • • •
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Draw the structures and record boiling points of the extraction solvents. Copy Table 1 (see below) into your lab notebook and fill in the blanks. Answer the prelab questions (above). Draw a flow chart (in the same format as Figure 2) depicting how your GSI will separate the mixture of 9-fluorenone, eosin Y and 4-bromo-N,N-dimethylaniline, based on the list of steps provided. Include predictions about the identities of each species present in each solution or precipitate. Don’t forget to include the strong acids, strong bases, and salts in your chart. Draw another flowchart (same format) showing how you will separate the unknown compounds from each other in part III. Make sure this flowchart accounts for what you will do if a precipitate does not form when expected (see prelab question 5).
Table 1. Potential unknowns for Part III. Name Structure 4-Bromoaniline Benzocaine Phenanthrene 4-Acetyl-Biphenyl trans-Cinnamic acid p-Toluic acid α-Methylcinnamic acid
Melting Point Acidic, basic, or neither?
Safety Notes: • • •
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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: You will work with a partner for this experiment. If necessary, the GSI will approve one group of three students. Part I. GSI Demonstration: Separation of Known Compounds by Extraction. In the first section of the lab, your GSI will demonstrate the use of a separatory funnel for extractions by carrying out the separation of 9-fluorenone, eosin Y and 4-bromo-N,Ndimethylaniline. The following steps will be used: 1. The mixture will be partitioned between ethyl acetate and a basic (NaOH) aqueous layer, then the layers will be separated. 2. HCl will be added to the aqueous layer from step 1 until a strongly acidic pH is obtained (a solid precipitate should result) 3. The organic layer from step 1 will be mixed with aqueous HCl, and then the layers separated. 4. NaOH will be added to the aqueous layer from step 3 until a strongly basic pH is achieved (a solid precipitate should result) 5. The organic layer from step 3 will be washed with brine, dried with sodium sulfate, and concentrated. 6. Each precipitate will be collected by filtration, rinsed with water, and allowed to dry on the filter with active air flow.
As the GSI carries out the above steps, record all of the observations you would typically make about your own work (steps taken, physical changes observed, etc.) and also modify the flowchart that you drew in your prelab as needed to make sure that it accurately reflects what happened and the fate of each of the compound in the mixture. Part II. Confirmation of Separation by TLC. With your partner, obtain a small sample (1-2 mg) of each of the three solid samples isolated by the GSI during the demo. Also obtain a sample of the starting mixture, and 1 mL of each of the aqueous filtrate layers from the samples that precipitated. Add 1 mL of ethyl acetate to each sample, mix well, and record your observations. Prepare TLC plate(s) containing spots of the 6 samples solutions you just prepared. For the biphasic samples, use the TLC capillary to selectively sample only the organic layer (not the aqueous one). Develop the TLC plate(s) with 35% ethyl acetate in hexanes, and visualize the compounds using both UV and I2.
Prepare and develop a second TLC plate containing samples of each authentic (pure) compound (provided by the stockroom) samples. Analyze the results of these two TLC plates to gain information about both identity and purity of the compounds isolated from each layer of the extraction. Record your observations and conclusions regarding these TLC plates in your notebook. Discuss any unexpected results with your classmates and GSI to make sure you understand them. Part III. Separation of Unknown Compounds by Extraction. 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 approximately half of the material (record the exact amount) and repeat the extraction procedure that your GSI used in Part I to separate the compounds from each other. (Save the other half of the unknown mixture in case you encounter a problem and need to start over.) Some of the unknown compounds do not precipitate readily from aqueous solution, even if they are neutral. However, it is possible to extract them from the aqueous layer into organic solvent. When you prepare your prelab, plan the additional steps that will be needed (don’t forget drying!) in the event your unknown compounds exhibit this behavior. Due to time restrictions, do not evaporate all of the solvent from each of your organic layers. Instead, concentrate only an aliquot of each solution in a filter flask, using the same procedure as you used for the column chromatography experiment. Your goal is to obtain enough of each solid to record a melting point. Spot samples of the crude mixture and each of the three purified compounds on a TLC plate, develop it, and analyze your results. Was complete separation of the three compounds achieved? If not, identify any impurities you can based on the TLC results. When you record your TLC results in your notebook, make sure that the lanes are clearly labeled to indicate which solution is which (a good way to do this is to give each solution a letter code in your flow chart, and then use the same letter code on your TLC plate.) Additionally, label which type of compound (acidic, basic, or neither) each spot on you TLC plate corresponds to. If you have a hypothesis about the actual identities of any of the unknowns, discuss your ideas with your lab partner and then record your hypothesis and reasons. Part IV: Identification of Unknown Compounds by Melting Point. Prepare melting point sample capillaries of each of the purified unknown compounds. Use a quick temperature ramp (10 degrees/min) to determine the approximate melting range of each compound using one set of samples. Then use a slower temperature ramp (2-3 degrees/min) to accurately measure each melting temperature with the second set of samples. Coordinate with other groups as needed to make sure the Mel-temp machines are being used efficiently (for example, if three different groups have all determined that one of each of their compounds melts in a similar temperature range to each other, all three samples can be run at once by one person).
Using all of the information you have collected, discuss the results with your lab partner until you reach a conclusion as to the identities of your unknown compounds. 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. Discussion: • •
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Explain how your group identified the unknown compounds. Did the unknown compounds in part III precipitate, or were further extraction steps necessary? Why might a precipitation be preferable to further extraction? Under what circumstances would extraction be desirable even if the compound does precipitate? What might cause compounds to not precipitate readily? 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 Questions: 1. Suppose that a researcher somewhere in the spiraling heights of Latimer Hall performs the reaction pictured below, but it does not proceed to completion (some starting material remains). The researcher wants to maximize the recovery of each of the valuable reaction mixture components (thioester, thiol, and carboxylic acid):
a. Identify each functional group in each compound as acidic, basic, or neither. Use table 4.1 on page 17 of the Pedersen lab text to estimate the pKa of each acidic or basic functional group, and write out the acid-base reactions that each of these components would undergo. b. Calculate the relative ratios of protonated-to-deprotonated compound at pH 2, pH 10, and pH 14. c. 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 buffer solution (NaH2PO4) at pH=7.
2. 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.
Acknowledgments: The following instructors contributed to the development of previous versions of this lab experiment: Kareem El Muslemany, Matthew Francis, Steven Pedersen, Arlyn Myers, and Ahamindra Jain.
