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Column Chromatography Theory


Chromatography (from Greek χρώμα:chroma, color and γραφειν:graphein to write) is the collective term for a set of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.

Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.

What is column chromatography?

Column Chromatography is a preparative technique used to purify compounds depending on their polarity or hydrophobicity. In column chromatography, a mixture of molecules is separated based on their differentials partitioning between a mobile phase and a stationary phase. The different size column could be used for this technique.

Setting up a microcolumn (used for samples less than 200mg).

A pasteur pipette is used as the column and usually 1g of adsorbent (ie silica/alumina) is used to separate the mixture.

    pasteur pipette
  1. Obtain a Pasteur pipette and ~1g of silica or alumina oxide.
  2. Setup the Pasteur pipette in such a way that it is straight and secure it with a clamp. A small amount of glass wool should be put into the base of the pipette using the other Pasteur pipette or wire. This is to prevent any of the fine particles from going through. Silica was then added to the pipette via the short stem funnel. The top of the silica must be flat/level. If the silica is not flat, then gently tap the pipette on a bench until it levels (this technique calls dry loading).
  3. You can use column with a small clamp to make a stopcock to control the flow rate (not necessary in our case).
  4. Small amount of sand could be added at the top of the column (about 2 mm high) to help the sample loading (it will help sample to go smoothly through the column). In this case the sand layer at the top must be flat as well.
  5. Wet the column using appropriate solvent/solvents (hexanes in the video case). It helps to compact the column (wet loading). The column cannot be dry at this point (you should continually add solvent to the column and keep its surface wet until you finish experiment).
  6. Collect the fractions using labelled test tubes or vials.
  7. Put the column in a container labelled PASTEROUS PIPETTES WASTE.