Column Chromatography Notes

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Media for Separation

• Silica: Slightly acidic medium. Best for ordinary compounds, good separation is achieved.
• Florisil: Mild, neutral medium. 200 mesh can be effective for easy separations. Less than 200 mesh best for purification by filtration. Some compounds stick on florisil, test first.
• Alumina: Basic or neutral medium. Can be effective for easy separations, and purification of amines.
• Reverse phase silica: The most polar compounds elute fastest, the most nonpolar slowest. See "Preparative Reverse-Phase Flash Chromatography, A Convenient Method for the Workup of Reaction Mixtures." Kuhler, T. C.; Lindsten, G. R. J. Org. Chem. 1983, 48 (20), 3589-3591 and "Reverse Phase Flash Chromatography: A Convenient Method for the Large Scale Separation of Polar Compounds." O'Neil, I. A. Synlett 1991, 661-662.

  Flash Column Chromatography

  "Rapid Chromatographic Techniques for Preparative Separation with Moderate Resolution."
  Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43 (14), 2923-5.
  How To Run a Flash Column

  Solvent Systems

  Flash column chromatography is usually carried out with a mixture of two solvents, with a polar and a nonpolar component. Occasionally, just one solvent can be used.
  
  The only appropriate one-component solvent systems (listed from the least polar to the most polar):
  1. hydrocarbons: pentane, petroleum ether, hexanes
  2. ether and dichloromethane (very similar polarity)
  3. ethyl acetate
  The most common two-component solvent systems (listed from the least polar to the most polar):
  1. Ether/Petroleum Ether, Ether/Hexane, Ether/Pentane: Choice of hydrocarbon component depends upon availability and requirements for boiling range. Pentane is expensive and low-boiling, petroleum ether can be low-boiling, hexane is readily available.
  2. Ethyl Acetate/Hexane: The standard, good for ordinary compounds and best for difficult separations.
  3. Methanol/Dichloromethane: For polar compounds.
  4. 10 percent Ammonia in Methanol Solution/Dichloromethane: Sometimes moves stubborn amines off the baseline.

  Rules of Thumb:

  • A compound with an Rf of 0.5 in 10 percent ethyl acetate/hexane will have an Rf of 0.5 in 20 percent ether/hexane. This conversion factor is general.
  • Methanol can be used as polar solvent, but only up to 10 percent of the mixture. More than 10 percent methanol can dissolve the silica gel.

  Tips:

  • Dichloromethane can dissolve compounds better, but it will take longer to run through the silica.
  • Benzene is sometimes useful as the non-polar component, but is usually avoided because of toxicity.
  • If your compound is sensitive to acid, put 1-3 percent triethylamine in your solvent system to neutralize acid in the silica gel. The Rf of your compound may increase a little bit, check first.

  Column Chromatography on a Small Scale

  To purify <25 mg of a compound:
  1. Use a 5 inch disposable glass pipette as your column.
  2. Choose a solvent such that the Rf of the desired compound will be lower than usual- around 0.2.
  3. Place a cotton plug at point where the pipette narrows, and pack with sand/silica just as you would a normal glass column, leaving an inch or two of silica-free space at the top.
  4. Apply your compound, and elute as usual, using either a pipette bulb or tygon tubing hooked to a compressed air source to flash the solvent through.

  You'll have to refill often, and experiment with fraction size, depending on how difficult the separation is. Because it is easy to slowly increase polarity on such a small scale (aka a solvent system "gradient"), it is possible to separate components of very similar Rf this way.

  For 25-100 mg of compound, you might consider preparatory thin layer chromatography.

  See also:Troubleshooting Column Chromatography, Thin Layer Chromatography Notes,TLC How to, Extensive Discussion on Column Chromatography