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Identification of Unknowns Lab

In this experiment, your goal is to separate and identify two solid organic compounds - an alcohol and a ketone.When you come to lab, you will be given a vial which contains a 1:1 mix of a solid alcohol and a solid ketone. Column chromatography is the method that you will use to separate them. Before you can do the column, you need to determine which solvent system will give optimal column separation; this solvent system is determined by running a series of TLCs in different solvent systems. Once separated, each compound is identified by matching its melting point, IR, and NMR with that of known compounds.

This experiment hones your techniques in column and thin-layer chromatography: if you do not do these procedures properly, the compounds will not be separated. Once you have separated the compounds, identifying them is a puzzle for you to solve. The melting point, IR, NMR, and even appearance of each compound are clues about their identity. We provide you with a list of possible unknown compounds. (This situation reflects some real-world organic laboratory synthetic situations, since often a chemist is working in a scheme and has a pretty good idea of the identity of an isolated compound.) When you believe that you have properly identified each unknown compound, you will write supporting arguments for your compound assignments in a 1-2 page paper. Write the paper as if you are "convincing" someone else that the compounds are indeed the ones you state that they are.

Two lab periods are scheduled for this experiment. During the first lab period, run TLCs of the mixture to determine the ideal solvent for separation. During the second lab period, separate the compounds by microscale flash chromatography (this procedure is illustrated below; also review the section on column chromatography). If time allows during the second lab period, take melting points and run IRs of the separated compounds; if not, you can finish gathering the data during spare time in subsequent labs.

Thin Layer Chromatography (TLC) for ID Unknowns Lab

The first step is to run a series of TLCs of the mixture, each in a different-polarity solvent. The goal is to find a solvent system that gives an Rf of 0.25 - 0.35 for the faster-moving compound (the ketone). The rationale behind this is that an Rf in the above range indicates that the solvent is "ideal" for eluting that particular compound from a flash chromatography column (Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923-2925).

Four different solvent systems of varying polarity will be available. Each is a mix of hexanes and ethyl acetate; the least polar is 95:5 hexanes:ethyl acetate, and the most polar is 80:20 hexanes:ethyl acetate. The rule is if you want to have a compound run further up a plate (higher Rf), go to a more polar solvent.

Weigh out about 0.02 g of your unknown mixture and place it in a small vial. Label the vial "standard-unknown".
Add about 1 mL of acetone (estimate the volume knowing that a Pasteur pipet holds about 1.5 mL).
Stopper the vial. Mark the solvent level (in case the solvent evaporates). Use this solution for the TLC part of the experiment and save it (capped and labeled) for the next lab period. (You will use it as a standard when you run TLC's of the column fractions in the second part of this laboratory experiment.)
Prepare for TLC: Set up a developing chamber (a beaker, lined with filter paper , filled with eluting solvent to a depth of less than 0.5 cm, covered with a beaker) for each solvent that you plan to test (the photo shows solvents 1, 2, and 3). These chambers need to sit for awhile for the solvent vapors to saturate the atmosphere. In the meantime, cut several TLC plates, mark the origin, and mark each with the number of a TLC solvent that you plan to try.
Use the standard-unknown solution to spot the prepared TLC plates. (See the TLC page if you have forgotten how to spot/run TLC plates.)
Run (develop) each plate in a different solvent system - the solvent front will slowly move up the plate. This picture shows the solvent level after several seconds.
As soon as you take a plate out of the beaker, mark the solvent front - before the solvent dries. Visualize the developed plates under a UV lamp. Safety note: Wear your goggles whenever you use a UV lamp to protect your eyes from UV light.
While viewing under the UV lamp, circle all of the spots on the TLC plates. If you see more than two spots on your unknown-mixture TLCs, the darkest spots are always the ones that you want
Measure the Rf values. The Rf is equal to the distance traveled by the spot, divided by the distance traveled by the solvent front. You are looking for the system that gives an Rf of 0.25-0.35 for the faster-moving spot (this is the ideal system). Make sure that the slower-moving spot moves significantly slower in the same system. (Subtract the Rf of the slower-moving spot from that of the faster-moving spot and make use that the resulting number is at least 0.1.)

Microscale Flash Column Chromatography for ID Unknowns Lab

The second step in this experiment is to run a microscale flash chromatography column. Microscale flash chromatography was introduced in this course in the Electrophilic Aromatic Substitution experiment (the acetylation of ferrocene lab). In that experiment, the compounds you were separating were colored and the separation was relatively easy to follow. Most of the unknowns in this current experiment are not colored and the separation is monitored by taking multiple elution fractions and then running a TLC of each fraction. The TLC plates are then visualized under a UV lamp.

First, prepare the column as described in Steps 1-3 of the column chromatography procedure page. Pre-elute the column with hexanes. Use the dry method to add your compound to the column, after coating it onto silica.
Obtain 12 vials and label them 1-12, since you will be collecting at least 12 fractions from the column. Place vial #1 under the column. Fill the pipet with the solvent system you determined in the TLC part of this experiment.
Using a pipet bulb, force the solvent through the column until the solvent level is flush with the top of the column. When it gets to this point, remove the bulb. Again, be careful not to suck liquid or silica while removing the bulb. One way is to squeeze the bulb gently while removing it.
Again fill the pipet with solvent and force the solvent through the column. Repeat as necessary until the volume of the collected fraction is about 1 mL. (It might be helpful to measure 1 mL of a liquid into a vial to use as a reference.)
Replace vial #1 with vial #2, refill the column with solvent, and force the solvent through the column. Repeat until you have 6 fractions. Spot the fractions on a single TLC plate.
Please re-use the same microcap as long as possible: you need to rinse this microcap with acetone several times between each fraction. Fill the microcap with acetone, then touch the microcap to a paper towel until the solvent runs out; repeat 2 or 3 times.
Don't forget to spot a sample of the standard-unknown you prepared in the first part of the experiment on the same plate (if the solvent in the standard-unknown evaporated, add acetone up to the mark you made on the vial).
Develop the TLC plate with system #1, the most polar solvent. Remember to mark the solvent front as soon as you take the plate out of the beaker.
Observe the developed TLC plate under a UV lamp and decide whether to collect another 6 fractions using the same eluting solvent, or switch to the more polar solvent. In this example, fractions 1 and 2 do not have either the ketone or the alcohol. Fractions 3 and 4 have pure ketone. Fraction 5 has a lot of the ketone and a very small amount of the alcohol. Fraction 6 has a small amount of the ketone and some of the alcohol. All of the ketone is off the column and it is time to switch to a more polar solvent. (If this were not the case - meaning, if the ketone is showing up and the alcohol is not in fraction 6 - you would collect another 6 column fractions using the same eluting solvent and re-evaluate.)
Switch to the more polar solvent system (50:50 hexanes:ethyl acetate) and collect 6 fractions (fractions #7-12).
Spot fractions 7-12, spot the standard-unknown, and then run the TLC plate in solvent 1. Observe the developed TLC plate under a UV lamp. If the slower-moving compound is completely off the plate, you are finished. If not, collect another 6 fractions and re-evaluate. In this example, fractions 7, 8, and 9 have only the slower-moving spot, the alcohol. Fractions 10-12 have nothing. Both compounds are off the column, so no further elution of the column is needed.
Circle the spots and calculate the Rf values for your lab report.
Decide which fractions contain pure samples of unknowns. In this example you could probably combine fractions 3, 4, and 5 and have pure ketone. If you want to be safe, combine only fractions 3 and 4, although you may or may not have enough sample for IR and melting point analysis. Combine fractions 7-9 for pure alcohol. Place each combined sample in a separate side-arm flask and find a rubber stopper that fits the flask (make sure the stopper is not too little to be pushed into the flask).
Remove the solvent by connecting the stoppered side-arm to the vacuum pump apparatus in your labroom. A dish of warm water under the flask expediates the process. (See also solvent removal.)
If your sample still looks wet or gummy, put the container in an ice bath and scrape it with a spatula.
Once your samples are fully dried, scrape them out of the flask and into vials for storage. The next step is to identify them by melting point, IR and NMR.

Melting points, IRs, and NMRs

For each separated unknown: