Purification technique-Column Chromatography

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>> Wednesday, August 04, 2010

Sorry guys, it has been quite busy days so couldn't connect with you often. Today's post will be on column chromatography (demonstration on how to make column). As a medicinal chemist, I can't imagine my work without column chromatography at least single run a day. 
Actually, this post is especially for the freshmen who still are not sure on how to make column. At the same time, I have great expectation from expertise to share the information which they think is missing in this post.

Column chromatography, technique for purification or separation of single compound from mixture of compounds, contains two phases i.e. stationary and mobile phase. Stationary phase are solid materials, commonly silica and alumina, through which compounds are allowed to run down by the help of appropriate solvents called as mobile phase. Usually mixture of solvents is used as mobile phase. The most common solvent combination is ethyl acetate/ hexane and Methylene chloride/ methanol.
Process of making column 
First put small piece of cotton or glass wool into the column (to outlet). Support the column with clamps. Little sea sand is poured into the column (0.5 to 1.0 cm in height). Cotton wool and sand are used to prevent silica falling from the column. Solvent, usually less polar, is poured into the column (for e.g if you are using solvent mixture of Ethyl acetate and hexane as mobile phase, it's better to make column in less polar solvent i.e. hexane). Silica slurry is prepared in appropriate solvent and poured into the column while stirring. The valve is opened to allow the dripping of solvent. The adsorbed silica is rinsed down the column using pipette. Silica is allowed to settle down for a while. Thereafter valve is closed, silica level is marked and gently tapped with soft materials like cork rings or hand. Slight pressure can be applied (but be sure to open the valve). Small amount of sea sand is poured (In case of wet loading, I put sea sand first and then sample but in case of dry loading I put sample first and then sea sand on the top of sample). That’s it; your column is ready. Dry loading is demonstrated in the video embedded below.

Things to remember while making column,
1.     Make sure the column is dry (keep the column in oven prior to use).
2.     Appropriate amount of cotton wool or sand should be used. Excess amount makes the opening tight resulting slow rate of solvent dripping.
3.     Silica slurry should be stirred enough to remove any air bubble. It is advisable to make the column one night before using so that silica will be well settled and avoids trapping of any air bubbles.
4.     While making silica slurry, be careful to avoid inhalation of light silica particles (Always put on the mask and do your work in fume hood).
5.     Make sure to remove any air bubble trapped in the column by tapping or applying slight pressure.
6.     Be careful while applying pressure. Pressure should be released slowly to prevent formation of cracks in the column.
7.     Be sure that silica is settled properly before adding sand. Otherwise the sand, being heavier will sink into the silica.
8.     While loading sample (wet loading), make sure the level of solvent is just in middle of sand layer.
9.     You can add your point. Leave it in comment section and it will be updated here.


  1. Really nice article. Keep posting. I also would like to suggest you a link about analytical chemistry at http://www.adichemadi.com/common/htmlfiles/analytical.html.

    You can check your knowledge at this link.


  2. @aditya thanks.I will try my best to come up with more good posts.

  3. can u suggest me the method of removal of small amount of activated carbon(charcoal) from a solvent mixture having material in it!!!!

    1. Thanks for stopping by!
      Regarding the removal of charcoal, I would suggest you to filter through celite and wash with organic solvent like EtOAc (if your compound dissolves in it).

  4. Can you post some pictures about how the column should NOT look like? For instance a column filled with voids and channels.

  5. 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.


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