Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure.

Because floral fragrance is critical for attracting pollinators, this method could be used to better understand or even enhance pollination of native flower species. With this method, fragrance volatiles are sampled using a non-destructive method with an easily-transported machine. This methodology uses rapid sampling, cutting sampling time from two to three hours to 10 minutes.

These methods provide a way to quickly sample volatiles produced by flowers and could be used to sample volatiles from other specimens as well, such as insect pheromones. To begin, select a flower that is initially unopened, and mark the flower with flagging tape to ensure repeated sampling of the same flower. Boil oven bags in water for 30 minutes to remove residual plastic compounds.

To dry, bake in an oven at 175 degrees Celsius for 60 minutes. Once the bags have dried, add a polypropylene bulkhead union to each corner of the closed end of the oven bags. Rinse all bags and tubes with 75%ethanol and let them air dry.

Then, bake the bags and the tubes in an oven at low heat, between 74 to 85 degrees Celsius for 30 minutes. Depending on the duration of the blooming time, if possible wait at least 24 hours after blooming to collect the sample. Cover the selected flower with a baked oven bag.

Cinch the bag together tightly with a plastic zip tie below the flower to prevent unwanted air flow into the bag. Wearing sterile neoprene gloves, attach a tube from the air outlet of the collection equipment and connect it to one of the bulkhead unions on the oven bag. On the other bulkhead union, attach a glass filter cartridge containing porous polymer adsorbent.

Attach a second tube to the collection equipment on the vacuum input. Connect the end of the second tube onto the glass volatile collection filter cartridge. Turn on both the air pump and the vacuum at the same time and set at 0.05 liters per minute.

The head space around the flower is filled with air but not overinflated. The system pulls air from the bag through the filter, trapping the floral volatiles. Allow the machine to run for 10 minutes.

And then turn off both the air pump and vacuum. Disassemble the tubes and the glass filter cartridge. Place the filter into a glass vial and screw on a cap.

Seal the vial with PTFE pipe thread tape. Store the sample in a freezer until GCMS analysis. Repeat this process with an empty oven bag to collect a blank air sample as a control.

For consistent replications of the same volatiles, repeat sample collection at the same time each day, as some flowers produce varying fragrance levels over the course of the day. First, remove the glass filter cartridge from the freezer and place it into a GCMS in the injector port. To release head space volatiles collected on porous polymer adsorbent from the adsorbent, heat the thermal collection trap to 220 degrees Celsius for eight minutes within a flow of helium gas at 1.2 milliliters per minute.

Compare chromatograms of collected volatiles to identify common reoccurring peaks. After identifying the peak volatiles, use Pherobase to determine if they have been previously described in floral fragrances. In this study, representative data from the GCMS are shown as a chromatogram.

Peaks between 10 and 15 minutes are floral volatiles. The collected volatiles were referenced in Pherobase to determine if they have been previously described in a floral fragrance. For example, compound number 21, with a retention time of 10.311 has been identified as benzaldehyde.

Benzaldehyde is searchable on Pherobase with a list of all flower species, organized by plant family, from which that fragrance compound has been identified. The orchid species are shown here, from which benzaldehyde has been determined to be present in the floral fragrance. Previous methods took two to four hours per sample.

The use of a solvent to remove the sample from the filter would result in a solvent peak on the GCMS. This would hide the compounds from zero to 3.5 minutes. This method allows for all compounds to be seen.