1. Silica Gel Slurry

1. Pour the silica gel into an Erlenmeyer flask. The weight of the packing material should be roughly 50x that of the sample being separated. If the compounds being separated have very similar R_f values, then it may require using a larger amount of silica per sample, which is the case in this example.
   1. Place 10 g of silica in the Erlenmeyer flask, since 50 mg of sample (45 mg of fluorenone and 5 mg of tetraphenylporphyrin) are being isolated.
2. Add the solvent system (hexane/dichloromethane, 70%: 30%) to the Erlenmeyer flask containing the silica gel. Add enough solvent to ensure that all of the silica gel is well solvated. The silica will not dissolve, but the mixture will be visually noticeable when solvated. Once the solvent has been added swirl the Erlenmeyer flask to ensure that all of the silica is well solvated.

2. Preparation of the Column

1. Select the appropriately sized column. Typically the column should be filled about half way with silica gel slurry. The larger the sample being purified, the larger the column required.
2. Plug the bottom of the column with a piece of glass wool. Using a long rod, make sure the wool is firmly lodged in the bottom of the column just above the stopcock.
3. Once the wool is firmly in place, apply a thin layer of sand over the glass wool.
   Note: If the column is equipped with a glass frit above the stopcock, this step should be omitted.
4. Clamp the column in the vertical position to a ring stand.
5. Using a funnel, gently pour the prepared slurry of silica gel into the column. You may need to add additional solvent to transfer the slurry from the Erlenmeyer flask to the column. Using a pipette, wash down any silica gel that sticks to the sides of the column.
   1. As the silica gel is settling in the column, gently tap the sides of the column to ensure that the silica gel packs tightly and excludes any air bubbles.
6. Open the stopcock and allow solvent to drain into a clean Erlenmeyer flask until just before the silica gel and the solvent front meet. The silica gel should never go dry until the procedure is complete.
7. Place a thin layer of sand on top of the silica gel (Figure 1). Using a pipette, wash down any sand that may have stuck to the sides of the column.
8. Drain any additional solvent until the sand is dry, but not down to the silica gel layer.

Figure 1. The proper setup for a column chromatography experiment prior to the addition of the sample.

3. Adding the Sample to the Column

1. Dissolve the sample in the smallest amount of solvent possible (using the same solvent that was used to make the silica gel slurry).
2. Using a pipette, gently add the sample to the top of the column.
3. Once the sample has been applied to the top of the column, open the stopcock and allow the solvent to drain through the sand layer but not the silica gel layer. Use a very small amount of solvent to wash down any sample that may have clung to the sides of the column. Drain this additional solvent through the sand layer as well.

4. Eluting the Sample through the Column

1. Using a pipette, very gently add 4–5 mL of solvent in such a manner that does not disturb the sand layer.
2. Place a funnel at the top of the column and very slowly and gently fill the remainder of the column with solvent.
3. Open the stopcock and allow the solvent to drain through the column.
4. Begin collecting the mobile phase as it drains from the column into test tubes.
   1. Test tubes should be placed in a test tube rack in a sequential manner.
5. Add additional solvent to the top of the column as needed until all the desired compounds have eluted from the column.

5. Recovering the Constituents

1. If the compounds are colored, then they can be visually identified. However, if the compounds are colorless, they will have to be identified using ulta-visible (UV) light (if the compounds contain conjugation) or with the appropriate stain. The purity of the compounds can be verified using thin layer chromatography.
2. Identify the test tubes that contain the desired compound(s).
3. Merge all of the fractions that contain the desired isolated compound(s) into a pre-weighed round-bottom (RB) flask. Do this for each compound being isolated.
4. Evaporate the solvent by placing the RB flask on the rotary evaporator.
5. Once all of the solvent has been removed, weigh the RB with the dried product and subtract the initial weight of the RB to obtain a yield.