Introduction To A General Organic Work-Up

Often times the most difficult part of conducting an experiment is the work-up that follows. In this exercise you will be introduced to the fundamental techniques that all organic chemists must familiarize themselves with if they wish to be successful in the laboratory. Imagine this scenario. You are a graduate student working in the lab and you have just finished synthesizing phenylalanine, one of the 20 essential amino acids. You are going to use this molecule as starting material for additional experiments, but before you can do so you must first isolate phenylalanine. However, there’s an issue. The procedure you followed to make this molecule resulted in phenylalanine being dissolved in a solution of basic water. You could boil off all of the water using a rotary evaporator and collect your product, but using the rotavap to remove water will take too long, leave behind NaOH, and your time is precious. So now what do you do? Extractions work by taking a molecule that has limited solubility in one solvent and moving it into a different solvent. This second solvent is ALWAYS immiscible with the first solvent, and usually has a significantly lower boiling point. The fact of the matter is the only reason why phenylalanine is dissolved in water is due to the presence of the base, NaOH. You will learn later on that miscibility of reagents and solvents is dependent on polarity similarities (you may be familiar with the saying “like dissolves like”). Phenylalanine is not polar enough to dissolve in water, so this act is achieved by abstracting its acidic protons via the basic solution leaving phenylalanine as an anion. Anions dissolve more readily with water because they are polar. As a result, the only way you will remove phenylalanine from this basic solution is to give back the hydrogen ion that was taken from phenylalanine. To do this you have to introduce an H+

source that will change the pH of the water from basic to acidic. This is typically achieved via an HCl solution. The overall transformation is depicted below.

H2NOH

O

H2NO

O

H2NOH

O

NaOH HCl+ NaClNa

Phenylalanine Water-Soluble Anion Organo-Soluble

Scheme 1: Phenylalnine ion transformation Modifying a solution’s pH is a common method that organic chemists use to manipulate a substrate’s solubility. You can imagine it being more difficult for a neutral phenylalanine to dissolve in water rather than its anionic counterpart due to its large nonpolar side chain, the phenyl ring. As a result, this molecule is now ready to be brought into a solvent that has similar nonpolar properties. One of the more common extraction systems is water and ethyl acetate. These two solvents differ greatly not only in polarity,

but also by means of their intermolecular attractive forces. As a result, these two solvents are not miscible and a mixture of the two solvents will quickly separate into two distinct layers in a separatory funnel. In order to determine which solvent is which you simply have to look up the density values of the solvents. The more dense solvent will sink, in this instance water, while the lighter solvent, EtOAc, will float. These two layers are referred to as the aqueous and organic layers, respectively. To extract an acidic aqueous solution of phenylalanine into ethyl acetate, you will simply need to shake a separatory funnel containing both the aqueous solution and ethyl acetate for a minute and then collect the organic layer. This process is usually repeated two more times, to ensure that all of the organic substrates have been removed from the aqueous layer. Extractions are not bulletproof in terms of absolute partitioning between layers. It is possible that two solvents, while appearing to separate completely, may in actuality have very slight miscibility with one another. Hypothetically, lets say water exhibits 3% solubility in 1.0 L of EtOAc. This means that if you were to mix 1.0 L of water with 1.0 L of EtOAc, place them in a separatory funnel and partition the layers; approximately 30 mL of water will be dissolved in your EtOAc layer. On small-scale separations this is generally not too large of an issue, but will eventually become problematic should you want to evaporate your solvent with the intention of obtaining a yield. As a result, drying reagents are used to remove water from organic solvents. Magnesium sulfate and calcium chloride are granular substances that readily absorb water that has been unintentionally transferred to an organic solvent. When either of these substances is added to an organic solvent with unknown amounts of water, the materials will clump together as the water is absorbed while the organic solvents are left alone. Dissolved compounds (in our case, phenylalanine) are also left unmodified. Magnesium sulfate or calcium chloride should continue to be added to an organic solution until the granules are free flowing, it will almost resemble a snow globe. Once the solution has been “dried” using a reagent such as MgSO4, you are now charged with the task of removing the drying reagent from your solution. This can be achieved in one of two ways: gravity filtration or vacuum filtration. While gravity filtration is significantly easier in terms of set up and execution, it is slow, and is less efficient than vacuum filtering. As a result, you will be expected to carry out several vacuum filtrations throughout the semester. The set up for a vacuum filtration apparatus is depicted below.

Figure 1 – Vacuum Filtration Apparatus

The vacuum filtration method is an excellent means for removing drying reagents from solutions. The organic solution, termed the “mother liquor,” is allowed to pass freely through the Hirsch funnel while the undissolved, granular drying reagents are trapped above. While the drying reagent is trapped in the funnel, the vacuum filter pulls off any remaining organic solvent that may have clung to the outer coating of the granules while leaving the water absorbed by the drying reagents safely behind. It would be beneficial at this time to use a few extra milliliters of organic solvent to rinse out the glassware previously holding your solution and pour it through the filter containing your trapped drying reagent. This ensures as much product as possible has been removed from your original glassware. With the mother liquor now collected in your filter flask you are now ready to evaporate the organic solvent. Rotary evaporation, or “rotavaping,” is an organic chemist’s quickest means of removing solvent. It is safe and efficient and uses the basic PV = nRT principle by applying reduced pressure and elevated temperatures so that a solvent may boil off more readily. Upon completion of solvent evaporation, your compound can be left to dry overnight (usually under high vacuum), but for the purposes of obtaining a crude yield to continue on to the next step of a synthesis, a quick weight directly off the rotavap will be sufficient. Once you have the weight of your phenylalanine, calculate % recovery from the weight of your crude phenylalanine and the weight of your recovered phenylalanine. % Recovery = g. recovered compound x 100%

g. crude compound

Hirsch funnel w/adapter

Tubing to vacuum

125mL filter flask

Filter paper Clamp Ring Stand

Data Table: Have a copy of this in your notebook prior to the beginning of lab. Your data table should contain columns for starting weight of phenylalanine, weight of empty round bottom, weight of round bottom + phenylalanine, final weight of phenylalanine, and % recovery. PROCEDURE pH Optimization Charge a 25 mL Erlenmeyer flask with 250 mg of phenylalanine and dissolve it in 10 mL of 1.0 N NaOH solution. Acidify your solution to a pH of ~ 2. Do this by adding a 6 N HCl solution drop wise to your reaction flask. Swirl the contents of your flask gently for every 5 drops of HCl added (approximately). Test the pH of your solution by using a glass TLC capillary tube to place one drop of your solution on to pH paper. Once you have achieved a pH of ~ 2, transfer your solution to a separatory funnel. If crystals begin to form that is okay, simply rinse your Erlenmeyer flask with 10 mL of water and transfer this solution to your separatory funnel as well. Complete the rinsing step regardless of whether or not crystals have formed. Extraction Obtain 15 mL of EtOAc and place it in a beaker. Transfer 5.0 mL of EtOAc to your separatory funnel containing your acidified phenylalanine solution. Cap your separatory funnel with a glass stopper and shake the contents of your funnel vigorously for 5-10 seconds. With your hand on the cap, invert your separatory funnel so the stopcock is facing upwards and vent your funnel by turning the stopcock. A build up of pressure is normal when performing an extraction, but it does need to be vented. Remove the stopper and allow your two layers to separate. Remove the aqueous layer by turning the stopcock and draining it into a flask or beaker. Pour your organic layer into a clean Erlenmeyer flask. Pour your aqueous layer back into your separatory funnel and repeat the process 2 more times, combining your organic layers into the same flask after each extraction. Drying Your Organic Layer Depending on the amount of water transferred during your extraction, you may require more or less drying reagent than the person next to you. Start by adding a small amount of drying reagent to the flask containing your organic layer and swirling your flask (approximately 200 mg, you may eyeball this amount). If your drying reagent clumps together after the initial addition, then add a little more to your flask. What you should observe is a free flowing granular material similar to a shaken snow globe. Once this has been achieved, your solution has been adequately dried. Vacuum Filtration

Assemble your vacuum filtration apparatus according to the picture displayed above. You will need a vacuum flask, Hirsh funnel, filter adapter, and vacuum tubing. The vacuum tubing can be found in your fume hood. Attach the vacuum tubing to the stem protruding from the side of your flask and switch on your vacuum (found underneath your fume hood). Pour the flask containing your organic solution and drying reagent through the top of your Hirsh funnel and allow the mother liquor to collect in the flask below. Using an extra 5.0 mL of EtOAc, rinse out the flask that originally contained your solution and pour the remnants through the funnel. Transfer the mother liquor from your vacuum flask into a PREWEIGHED round bottom flask (weigh an empty, clean round bottom flask prior to mother liquor transfer). Again, use an additional 5.0 mL of EtOAc to rinse out the flask and transfer it into your round bottom. Rotary Evaporation See your TA when you are ready for this step. They will instruct you on how to rotavap as well as what components to inspect before you begin the process. Many experiments have been lost to the rotavap bath due to not pulling a vacuum; don’t let yours be one of them. It will cost you a large amount of points in the future. After your solvent has been completely removed, wipe down the outside of your round bottom to remove any water and weight your flask. Record this new weight and calculate a percent recovery. Taking an Infrared Spectrum (IR) Due to the fact that your samples are not completely dry, you will be taking an IR of phenylalanine from the stock sample that was provided earlier in the lab. The program that is used to do this is called EZ Omnic. This program will likely be opened when you use the IR, but the shortcut is located on the desktop of both IR computers should you need it. To use EZ Omnic:

1. Click the “Col Smp” button to start. 2. Enter your name when the message appears asking you to enter the spectrum title

and then click OK. 3. A message will appear telling you to prepare to collect the background spectrum.

BEFORE you click OK, place the white circular cover onto the sample area. This enables the IR to gather the background spectrum to use as a point of comparison against your sample.

4. Wait while the IR completes its scans. 5. When the scans have been taken, remove the white circular cover and add your

sample before you click OK. For Solid Samples:

Add a small amount of finely crushed solid sample onto the small crystal at the

center of the metal circle. Be sure to cover it completely but do not add so much

solid that there is a pile on the crystal. Turn the arm of the IR and twist the knob so that it firmly presses the solid sample into the eye. BE CAREFUL! Do not twist until the arm is in place and do not twist too much. 6. After clicking OK, wait for the scans to complete. 7. After the scans are complete, a message will ask if you want to add to the

window. Click Yes. 8. You want the spectrum to have transmittance on the y-axis. If it has absorbance

on the y-axis, click the button “% Trans” to change to transmittance. 9. At this time, click the “Find Pks” button. Adjust the bar on the left that has a

scale from 0 to 100 in order to allow the program to label some of the peaks with the wavenumbers. Do not allow it to label too many or you won’t be able to read any of the numbers. It is also ok if all the important peaks are not labeled by the program. Click “Replace” when you are finished.

10. Print from inside of the EZ Omnic program. 11. After you receive your printed spectrum, click the “Clear” button to remove your

spectrum for the next person. 12. Be sure to loosen and turn the arm to its starting position (for solid samples) or to

remove the white circular cover (for liquid samples). Carefully clean the crystal with a kimwipe and acetone.

Pre-lab Assignment Assuming you are working with an aqueous solution, propose two other solvents that may be beneficial for performing an extraction. In other words, name two solvents other than ethyl acetate that can be used for aqueous extractions. Provide the densities of each solvent and explain whether these solvents will be located above or below your water layer in the separatory funnel. Post-Lab Questions 1) Calculate your percent recovery from the exercise above. Show ALL mathematical steps. Include units and significant figures. Offer two reasons as to why your percent recovery may be above or below 100%. 2) Make a data table for your IR. Label the important peaks on your spectrum and in your table. Be sure to signify the functional group corresponding to each wavenumber.