4.2.2. Sample preparation

Crude hexane honey bee extracts are normally already suitable for subsequent analysis without further purification. In some cases CHC are separated for the purpose of behavioural bioassays; in particular, HC can be separated from oxygenated compounds using column chromatography on silica gel.

To do so the following method can be used:

  1. Prepare a column, this is done by packing a Pasteur pipette (clogged at its tip with glass wool to prevent the gel running down the pipette) with 100-500 mg of silica gel (200–400 mesh, 60 Å).
  2. Condition the column by passing hexane through it 2-3 times.
  3. Add the sample to the column using a small volume of solvent.
  4. Elute the column with 1-5 ml of hexane to collect the HC.
  5. Several elutions can be done to ensure collection of all HCs.
  6. Elute the column with 1-5 ml of ether or acetone if interested in more polar compounds.
    To further fractionate CHCs, saturated and unsaturated components in the apolar fraction can be separated.
  7. Prepare a column packed with 100-500 mg of 10% silver nitrate on silica gel (200–400 mesh, 60 Å).
  8. Elute the column with 1-5 ml of hexane to collect saturated hydrocarbons.
  9. Elute the column with 1-5 ml of ether to collect unsaturated HC.
    In order to remove silver ions,
  10. Reduce the ether fraction under nitrogen.
  11. Pass the eluate through an identical silica column.
  12. Elute with hexane.