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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.

Thanks for watching!