Chromatography column

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A chromatography column is the main device used in chromatography for the separation of chemical compounds.

General

A chromatography column consists of a glass tube with a glass or PTFE stopcock at one end. Some columns also have a fritted glass above the stopcock, to help when creating the stationary phase. Some chromatography columns have a ground glass joint at the top, where a gas inlet adapter can be placed to create positive pressure inside the column.

How to use a chromatography column

Materials needed

  • Chromatography column (Duh!) preferably with fritted glass
  • Stationary phase materials: fine silica gel (crystalline is often used), alumina, cellulose, clean fine sand
  • Mobile phase (or eluent): Ideally you need a solvent system in which the desired compound has an Rf of around 0.3;
  • Test tubes and test tube rack
  • Gas inlet adapter and keck clip (optional)

Packing the column

  • Make sure the chromatography column is dry and the stopcock works;
  • Place the chromatography column in a clamp, with the stopcock facing down, like a burette;
  • Close the stopcock;
  • If your column lacks a fritted glass, use a glass wool to act as a replacement, to prevent the stationary phase from leaking through the stopcock;
  • After you placed the glass wool, add sand or some other coarse granular material until you make an even level above the curvature of the column tube;
  • Add a bit of solvent to the sand and make sure the solvent level is high enough, so that when you add the slurry of stationary phase, the sand level remains undisturbed;
  • If your chromatography column has a fritted glass disc, the previous 3 steps are not required;
  • Create a slurry of stationary phase by mixing the stationary phase with your chosen solvent;
  • Proceed to carefully fill the column with stationary phase material;
  • Allow the stationary phase to settle;
  • Make sure the level of the stationary phase is even and there are no air bubbles or other defects;
  • Drain the mobile phase until it reaches the level of the stationary phase;

Loading the column

  • Make sure the mobile phase is drained to the level of the top of the stationary phase;
  • Using a pipette, gently add a narrow band of concentrated sample to the top of the silica gel column (stationary phase), by placing the tip of the pipette as close as possible to the top of the stationary phase;
  • Drain the mobile phase to bring the sample into the silica gel column, and make sure the level of the phase matches the level of the stationary phase;
  • Using a pipette, gently add a more mobile phase to the top of the stationary phase, by placing the tip of the pipette as close as possible to the top of the stationary phase;
  • The column is ready to use;

Running the column

  • The column is loaded as previously described;
  • Open the stopcock to let the mobile phase drain;
  • Place a dropping or separatory funnel with the mobile phase above the column and adjust the stopcock to constantly replenish the column, to keep the level of the mobile phase above the level of the stationary phase;
  • If you need to use air pressure, this setup cannot be used, though a dropping funnel may work, you'll just have to connect the gas inlet adapter to the dropping funnel, and your system becomes overly complicated;
  • As the band of your sample moves down, it should separate into several narrow bands;
  • Mark the test tubes to identify the collected phases;
  • Collect each band into a different test tube, as well as intermediary phases;
  • Continue collecting until no more phase can be collected;

Emptying the column

  • Wash the column one more time with mobile phase;
  • Turn the column upside down and place a dish or beaker below to collect the falling silica gel, as it dries;
  • Remove the column from the clamp;
  • Scrub any leftover stationary phase then wash the column to remove any traces of it;
  • Wash the column using cleaning baths or an oxidizing mixture;
  • Rinse it with water and let it dry;

Availability

Chromatography columns can be bought online or from lab suppliers. Price depends on the type and brand.

DIY chromatography column

You can use a wide diameter burette as a makeshift chromatography column, though you won't be able to separate a significant amount of compound.

References

Relevant Sciencemadness threads