Identification of 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.

Note: To see a larger view of each image, click on it and it will open in a new window.

(2) Pre-elute the column with hexanes.

Add hexanes to the top of the column.

The hexanes will move slowly down the column by gravity. To speed up the process, see the next step.

Use a pipet bulb to force the hexanes through the column...

...but stop applying pressure when the hexanes-level is just at the top of the silica gel as shown in the photo to the right.

Remove bulb -- be careful not to suck liquid into the bulb.

Add more hexanes and force the solvent through again, but do not let the hexanes level fall below the top of the silica gel column. Do this enough times so that all of the silica gel is wet and it begins dripping out the bottom.

If you are not ready to load your sample onto the column, it is okay to leave the column at this point. Just make sure that it does not go dry -- keep the top solvent level above the top of the silica by adding solvent as necessary.

(3) Prepare the sample to load onto the column.

The sample is loaded by the dry method as explained below. (Reference: the generic microscale flash chromatography procedure)

Weigh out 100-120 mg of your unknown mixture. Put it in a small side-arm flask and add about 75 mg of silica gel

Add acetone dropwise, about 1-2 mL, until the unknown begins to dissolve (the silica gel will not dissolve).

Swirl to dissolve the unknown mixture. It will look like a slurry because of the silica gel. You can help it dissolve by warming it in a beaker of hot water.

Connect the side-arm flask to the vacuum outlet and remove the solvent under reduced pressure. Or if you are not in a hurry, you can let the solvent evaporate at room temperature/pressure, or you can hold it in your hand or in hot water (but not a steam bath!) to expedite the evaporation of the acetone.

When dry, scrape the mixture with a spatula until it is a fine powder like flour.

(4) Load the sample onto the silica gel column.

(5) Elute the column.

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.

Force the solvent through the column until the solvent level is flush with the top of the column.

Here is the proper level again. 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 the 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 6 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 solvent in the standard-unknown evaporated, add acetone 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 our example, it is time to switch to the more polar solvent. (For details on this decision, click on the picture.)

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 our example, both compounds are off the column and it is time to combine pure fractions. (For details on this decision, click on the picture.)

(6) Combine pure fractions.

Decide which fractions contain pure samples of unknowns. The picture at the right shows all 12 fractions under the UV lamp. Fractions 3-5 will be combined for the ketone, and fractions 7-9 will be combined for the alcohol.

Circle the spots and calculate the R_f values for your lab report.

Combine fractions so that you have pure samples of each unknown.

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 remove the solvent under reduced pressure.)

The picture at the right shows the dried samples, still in the vacuum flasks.

If your "dried" sample still looks wet, put the container in an ice bath and scrape it with a spatula.

Here are the two separated unknowns (transferred to small vials), shown next to the original mixture. The next step is to take melting points and run IRs and maybe NMRs. (Back to the ID Unknown main page.)

Plug a Pasteur pipet with a very small amount of cotton. Use a wooden applicator stick to tap it down snugly but do not compact it too much.
Fill the Pasteur pipet with silica gel to about half a centimeter below the indentation in the glass pipet. This is one way to fill the column with silica: turn it upside down and press the top into a jar of silica.
Another way to fill the column is to pour the silica gel in using a small beaker.
Either method you choose, tamp it down on the benchtop and add more silica gel until it is almost to the indent in the pipet.
The picture to the right shows the proper level of the silica in the pipet. Clamp the column to a ring stand using a small 3-pronged clamp and place a collection flask under it.

Add hexanes to the top of the column.
The hexanes will move slowly down the column by gravity. To speed up the process, see the next step.
Use a pipet bulb to force the hexanes through the column...
...but stop applying pressure when the hexanes-level is just at the top of the silica gel as shown in the photo to the right.
Remove bulb -- be careful not to suck liquid into the bulb.
Add more hexanes and force the solvent through again, but do not let the hexanes level fall below the top of the silica gel column. Do this enough times so that all of the silica gel is wet and it begins dripping out the bottom.
If you are not ready to load your sample onto the column, it is okay to leave the column at this point. Just make sure that it does not go dry -- keep the top solvent level above the top of the silica by adding solvent as necessary.

Weigh out 100-120 mg of your unknown mixture. Put it in a small side-arm flask and add about 75 mg of silica gel
Add acetone dropwise, about 1-2 mL, until the unknown begins to dissolve (the silica gel will not dissolve).
Swirl to dissolve the unknown mixture. It will look like a slurry because of the silica gel. You can help it dissolve by warming it in a beaker of hot water.
Connect the side-arm flask to the vacuum outlet and remove the solvent under reduced pressure. Or if you are not in a hurry, you can let the solvent evaporate at room temperature/pressure, or you can hold it in your hand or in hot water (but not a steam bath!) to expedite the evaporation of the acetone.
When dry, scrape the mixture with a spatula until it is a fine powder like flour.

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.
Force the solvent through the column until the solvent level is flush with the top of the column.
Here is the proper level again. 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 the 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 6 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 solvent in the standard-unknown evaporated, add acetone 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 our example, it is time to switch to the more polar solvent. (For details on this decision, click on the picture.)
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 our example, both compounds are off the column and it is time to combine pure fractions. (For details on this decision, click on the picture.)

Decide which fractions contain pure samples of unknowns. The picture at the right shows all 12 fractions under the UV lamp. Fractions 3-5 will be combined for the ketone, and fractions 7-9 will be combined for the alcohol.
Circle the spots and calculate the R_f values for your lab report.
Combine fractions so that you have pure samples of each unknown.
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 remove the solvent under reduced pressure.)
The picture at the right shows the dried samples, still in the vacuum flasks.
If your "dried" sample still looks wet, put the container in an ice bath and scrape it with a spatula.
Here are the two separated unknowns (transferred to small vials), shown next to the original mixture. The next step is to take melting points and run IRs and maybe NMRs. (Back to the ID Unknown main page.)

Identification of Unknowns Lab: Microscale Flash Column Chromatography