last updated Thursday, January 14, 2016

Thin layer chromatography is used to monitor reactions, check for purity of compounds and to optimize solvent mixtures for column chromatography. It is a micro-technique that requires very small quantities of compound. On the other side, column chromatography is one of the most widely used methods to purify compounds from undesired side products.

Below is a simple setup of chromatography column. It is a glass cylinder and a tapered joint with stopcock at the bottom. Using the same principles as TLC, the chromatography column can, with the utilization of the proper amounts of stationary phase (i.e., silica, alumina, cellulose), size of column (diameter and length), and type of solvent system, separate various components based on their respective polarities. With the aid of air pressure from the top (hence the name flash chromatography), one may collect solvent fractions from the column, which contain isolated components (purified).

Setup (dry packing):

A 25 mL burette is clamped securely in the hood. A small cotton ball is then placed on the bottom. Next, 0.5 cm of sand are layered on top of it. The sand is leveled and then ~25 cm of silica gel are added. The powder is leveled again and then another ~0.5 cm of sand are placed on top. About ~150 mL of the eluent mixture are required for your experiment. The column is carefully filled with the eluent to approximately 1 cm from the top. The stopcock at the bottom of the column is opened and a big beaker is placed underneath. A thermometer adapter (or rubber stopper) with a glass tube of appropriate diameter on the top is attached to the top. Then the regulator valve (at the copper line) is opened slowly while holding on to the tubing. This will generate a pressure that pushes down the solvent and will increase the speed of elution. When the solvent front is ~1 cm away from the sand at the head of the column, the adapter is taken off and more solvent added. This procedure is repeated until the entire column is wetted. After this happened, the solvent level is allowed to reach the sand level. (For the epoxidation step the column has to be pre-treated with a 1% NEt3 solution in hexane to neutralize the acidic residues!)

Running the column:

The product is dissolved into 2-3 mL of your eluent solvent. The solution is then carefully added to the top of the column. The solution is allowed to absorb on the column. The container used is rinsed with 2-3 mL of eluent and also loaded onto the column. Then the column is carefully filled with eluent. The air pressure is used to force the solvent through at a rate of ~10 mL per minute. The eluent leaving the column is collected in clean test tubes.

Upon completion, every other fraction of the eluent is checked by TLC (starting with the 3rd test tube) to identify the fractions containing the product. Since the concentration will be fairly low, heavy spotting will be necessary at this point. The product fractions containing the product are combined and the solvent removed by evaporation. keeping in mind that the products are liquids in this experiment (epoxidation).

Important pointers

1. The column can also be filled using a slurry of the stationary phase in a non-polar solvent or the solvent that will later be used for the elution. This technique avoids the presence of fine powders and often time reduces the formation of bubbles.

2. If the is packed dry (see above), the solvent has to be added slowly, because often times the adsorbent will react with the solvent and liberate heat. The resulting vapors will cause cracks and bubbles in the column.

3. Using either technique, any cracks and air bubbles in the column should be avoided. Both will deteriorate the quality of separation due to the lack of interaction or the broadening of the bands.

4. The sand on top of the stationary phase serves as a buffer. It permits the person running the column to refill the column with solvent without disturbing the stationary phase itself.

5. Generally, a very concentrated solution of the mixture in a non-polar solvent should be used. The solution should be allowed to run down at the side of the column container. Then the solvent (eluent) is added very carefully on top.

6. If the solvent (eluent) evaporates before it drips in the collection flask and a solid residue forms, some of the solvent is used to wash the solid at the tip down.

7. Microscale column chromatography is usually performed in a Pasteur pipette. It works the same like the macroscale column chromatography. Which type of column is needed depends on the ease of separation and the quantity to separate. Typically, about 25-50 times the amount of mixture to separate are used as stationary phase to get a good separation.