In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.

The mixture to be analyzed by column chromatrography is applied to the top of the column. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as the solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, it is called flash chromatography, a "state of the art" method currently used in organic chemistry research laboratories.

The Adsorbent

Silica gel (SiO₂) and alumina (Al₂O₃) are two adsorbents commonly used by the organic chemist for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles.

Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400 mesh for flash columns.

Results were less than acceptable when large 60-200 mesh material was used, but remarkably improved when a  200-400 mesh material was in the column. Equally important: particle sizes less than 40 microns offered no significant improvement in resolution in this system.  

The Solvent

The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the successful application of column chromatography as a separation technique.

Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent is first used to elute a less-polar compound. Once the less-polar compound is off the column, a more-polar solvent is added to the column to elute the more-polar compound.

Column Chromatography Procedure

Packing a (silica gel) column:

1. Use a piece of wire to add a plug of cotton to the bottom of the column. There should be just enough cotton that the sand and silica will not fall out of the column.
2. Clamp the column to a ring stand and add enough sand to fill the curved portion of the column.
3. Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3 full with the initial eluent.
4. Prepare a slurry of silica in the initial eluent by pouring dry silica into a beaker of eluent. (Add a volume of silica gel, such as 20 mL, to approximately double the volume of eluent, 40 mL.) CAUTION: keep the dry silica in your hood and be careful not to inhale the lightweight substance.
5. Quickly but carefully pour the slurry into the column. Stir and pour immediately to maximize the amount of silica that goes into the column instead of remaining behind in the beaker. You may find a clean spatula or glass rod helpful in transferring the silica.
6. Remove the pinch clamp to allow solvent to drip into a clean flask. Tap on the side of the column with a rubber stopper or tubing to help the silica settle uniformly.
7. Use a Pasteur pipet to rinse any silica that is sticking to the sides of the column. Allow the silica to settle while eluent continues to drip into the flask.
8. Once the silica has settled, carefully add sand to the top of the column. Sand is heavier than silica. If the silica has not settled, the sand may sink into the silica instead of forming a layer on top of it. (You may need to rinse down sand that sticks to the side of the column.

Loading a sample onto the column:

9. Drain eluent from the column until no solvent remains above the surface of the sand.
10. Using a long Pasteur pipet, carefully add your sample to the column.
11. Drain eluent from the column until no sample remains above the surface of the sand.
12. Use ~ 1 mL of eluent to rinse your container and pipet. Add this milliliter of sample to the sand. Drain eluent from the column until no liquid remains above the surface of the sand.
13. Repeat step 12 two or three times to completely transfer your sample onto the silica gel. If you do not do and repeat step 12, your sample will remain in the sand instead of on the silica. Sample remaining in the sand will dissolve in the eluent that you add in step 14, ruining the possibility of good separation of components.

Eluting the sample:

14. Once you have rinsed your sample onto the silica, carefully add eluent to the top of the column. To avoid disturbing the top of the column, it's a good idea to carefully pipet an inch or two of solvent onto the column instead of pouring solvent directly onto the sand.
15. Add more eluent as necessary. The eluent collected prior to the elution of sample can be recycled. The composition of the eluent can be changed as the column progresses. If the eluent composition is to be changed, ALWAYS start with least polar solvent/mixture and change to the more polar solvent/mixture.

Analyzing the fractions:

16. Analyze the fractions by thin-layer chromatography to determine a) if the fraction contains more than one component and b) if fractions can be combined without affecting the purity of those fractions.

Other Tips:

• The success of your separation will be dependant on how well you pack and load the column. It is important to have level sand and silica. It is also important to carefully and evenly add your sample to the packed column.
• Do not allow the silica to dry out as the column progresses. Cracks will form within the silica column if it dries, and compounds can fall down the cracks instead of partitioning between mobile and stationary phases.
• Compounds pass through sand quickly and do not stick to it. Sand is used at the bottom of the column to help ensure a level silica gel line. The bottom of the column is typically cone shaped. If no sand were present at the bottom of the column, molecules traveling down the center of the column would encounter less silica gel than molecules traveling down the edge, closer to the glass. As a result, a particular component would elute as a broader band which is undesirable.
• Sand is used at the top of the column to aid even loading of the sample. Sample diffuses evenly through the sand. Once the pinch clamp is removed from the bottom of the column, sample loads evenly onto the silica. Without sand, the sample would be added directly to the silica and would stick wherever it is added, not evenly across the surface of the silica.