2.14: Column Chromatography

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.

Column chromatography is a versatile purification method used to separate compounds in a solution. A solution mixture is carried by a solvent through a column containing an adsorbent solid, called the stationary phase. The combined solvent and sample mixture is called the mobile phase.

Molecules in the mobile phase travel through the column at different rates based on their chemical properties?and their affinity for the stationary phase. Thus, each compound exits the column at a different time. Once the compounds have been separated and purified?they can be further processed?or are ready for distribution. This video will introduce the basics of column chromatography, then demonstrate the technique with the purification of organic compounds.

In column chromatography, molecules reversibly adsorb to the stationary phase as they flow through the column, thereby slowing their progress. Compounds that interact weakly with the stationary phase are faster to exit the column, or elute. Compounds that interact strongly with the stationary phase are slower to elute. The stationary phase is an adsorbent powder or gel such as silica gel or alumina. Silica gel and alumina are highly polar?so they interact strongly with polar compounds and solvents, and weakly with nonpolar molecules. The stationary phase is loaded into the column as a slurry with the solvent?and is then packed by flowing solvent through the stationary phase. When properly packed, the stationary phase is homogeneous from top to bottom and contains no air bubbles or dry patches, as uneven flow caused by these irregularities interferes with the separation of compounds. The solvent,?or eluent, is typically an organic solvent supplied from a reservoir. In general, nonpolar solvents only elute nonpolar compounds, whereas polar solvents elute both polar and nonpolar compounds. If a mixture contains compounds of significantly different polarities, a series of increasingly polar solvents may be used to elute all of the compounds of interest. The mobile phase flow rate is usually controlled by a stopcock at the bottom of the column. Pauses in flow are kept to a minimum to avoid diffusion of the compounds. The mobile phase leaving the column, called the eluate, is collected in fractions to preserve the separation of compounds. Now that you understand the principles of column chromatography, let's go through a procedure for the purification of a mixture of organic compounds.

To begin the procedure, obtain the equipment as noted in the text. Weigh a round-bottomed flask for each compound to be isolated and record the mass. Next, weigh the sample?and dissolve it in the minimum volume of solvent needed. The appropriate solvent should be predetermined using thin layer chromatography. The Rf value should be between 0.2?0.3. Then, determine the amount of silica gel required for the stationary phase based on the dry weight of the sample and the difference in migration distance of the compounds of interest based on the TLC pre-screening. Pour the appropriate amount of silica gel into an Erlenmeyer flask. Add the solvent to the silica gel until the slurry is translucent and moves freely when the flask is swirled. Next, select a column large enough that the silica gel will fill it halfway. If the column does not have a glass frit, place glass wool into the column and firmly press it to the bottom with a long rod. Cover the glass wool with about 2 cm of sand to prevent silica from passing through the glass wool. In a fume hood?clamp the column to a ring stand, allowing sufficient space below to accommodate the test tubes.

Place a funnel into the column and ensure that the stopcock is closed. Pour the slurry into the column, gently tapping the sides as the slurry settles to exclude air bubbles. Rinse the funnel, flask, and walls of the column with solvent to transfer all of the gel into the column.

Place an Erlenmeyer flask under the column. Open the stopcock and allow the solvent to drain into the flask until the solvent level is just above the silica gel, and then close the stopcock. Pour about 2 cm of sand onto the gel. Gently rinse down any sand stuck to the sides of the column with solvent. Drain the solvent as needed so the sand is mostly dry, but the silica remains completely covered.

To start the separation, add the sample to the column without disturbing the sand. Use small portions of solvent to rinse down any sample adhering to the column walls and to rinse out the sample container. Carefully drain the solvent until the level is just above the silica. Then, with a pipette, gently add 4?5 mL of solvent without disturbing the sand layer. Place a funnel into the column and slowly fill with solvent. Remove the flask and replace with a labeled test tube. With the first test tube in place, open the stopcock and collect the eluate until the test tube is nearly full.

Continue collecting fractions until all desired compounds have been eluted, proceeding sequentially through the labeled test tubes. When finished, close the stopcock.

For each compound isolated,?combine the pure fractions in a pre-weighed round-bottomed flask. Remove the solvent from the flask on a rotary evaporator and then weigh the round-bottomed flask containing the dry compound. For more information, see this collection's video on rotary evaporation.

This sample contained a mixture of tetraphenylporphyrin, or TPP, and fluorenone. The dark reddish-purple TPP was eluted first, followed by the yellow fluorenone. The purity of each isolated compound was confirmed by NMR spectroscopy.

Column chromatography is used in purification and analysis in a variety of scientific fields.

High performance liquid chromatography, or HPLC, is a form of column chromatography that provides excellent separation between compounds and can incorporate specialized detectors such as a radiation detector for radiolabeled molecules. Using HPLC, a radiolabeled phospholipid can easily be isolated from a mixture of many others even if it makes up a small percent of the mixture. This information can help elucidate the production, regulation, and functions of many important biomolecules.

Flash chromatography is a variant of column chromatography in which the mobile phase moves through the column under air or gas pressure rather than by gravity flow alone.

This creates a faster flow rate, minimizing diffusion for better separation. The desired compound is collected in a few pure, concentrated fractions, as shown with thin layer chromatography, resulting in excellent post-purification yield and purity.

The usual column apparatus is not appropriate for separating small volumes, but some mixtures are not compatible with specialized techniques such as HPLC. Small-scale purification is performed with glass pipette columns, with a pipette bulb used for small-scale flash chromatography. This is particularly useful when preparing a sample for specialized purification techniques or as a final step following large-scale purification.

You've just watched JoVE's introduction to column chromatography. You should now be familiar with the principles of column chromatography, a procedure for silica gel column chromatography, and some applications of the technique.

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