The sample to be separated is loaded in solution in a suitable solvent, preferably as concentrated as possible. Dilute samples tend to separate less efficiently.
1. When preparing the column as described above, also prepare a series of small test tubes for collecting the different fractions that will come out of the column as the separation proceeds. Also have masking tape and a marker to label the tubes, and of course a test tube rack.
2. After preparing the column as described above, allow the solvent to flow until its level comes very close to the top of the solid support, but not below it.
3. Load the sample solution in small amounts, allowing the solvent to flow after each load until it comes very close to the top of the solid support, but not below it. By doing this you never have large amounts of solvent present above the solid support.
4. After the sample is loaded, add the first eluting solvent (usually a low polarity one) in small amounts, just as you did with the sample. Once again, do not allow the solvent level to fall below the top of the solid support. By doing this you allow for complete adsorption of the sample onto the solid support.
5. Now you’re ready to proceed with the separation. Fill the top of the column with eluting solvent, and allow it to flow. If your sample is colored, as it is in experiment 15, you will see the sample move and separate into “bands” as the components of the mixture begin to separate.
6. As the solvent level reaches the top of the solid support, add more solvent to keep it from falling below the top of the solid.
7. As the solvent is flowing and you see the bands move down the column, be ready to collect different bands into separate test tubes. These collections are called fractions. If it is not clear where the band starts, play it safe by starting the collection before the band reaches the bottom of the pipette. It’s better to have an excess of solvent in your fraction than to lose material.
8. The less polar components of the mixture will come out first. The more polar components might lag far behind, moving very slowly. Once the less polar fractions come out, you can gradually increase the polarity of the eluting solvent to speed up the movement of the more polar bands. This is done by gradually increasing the percent of a more polar solvent from zero to whatever it takes to effect the separation. For example if your initial solvent was hexane, you can increase the polarity of the solvent by switching to 20% acetone / 80% hexane.
9. Continue this process until you’re satisfied that all the components of the mixture have come out with good separation. Keep in mind that abrupt changes in solvent polarity might cause poor separations. If there is a technique where patience is a premium asset, chromatography is definitely the one.