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:
- 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).
- Condition the column by passing hexane through it 2-3 times.
- Add the sample to the column using a small volume of solvent.
- Elute the column with 1-5 ml of hexane to collect the HC.
- Several elutions can be done to ensure collection of all HCs.
- 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.
- Prepare a column packed with 100-500 mg of 10% silver nitrate on silica gel (200–400 mesh, 60 Å).
- Elute the column with 1-5 ml of hexane to collect saturated hydrocarbons.
- Elute the column with 1-5 ml of ether to collect
In order to remove silver ions,
- Reduce the ether fraction under nitrogen.
- Pass the eluate through an identical silica column.
- Elute with hexane.