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Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure.
Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important step to conservation of flowers that are threatened or endangered. Because floral fragrance is critical for attracting pollinators, this method could be used to better understand or even enhance pollination. We present a protocol using a portable charcoal air filter and vacuum to collect floral fragrance volatiles, which are then analyzed by a GC-MS. By using this method, fragrance volatiles can be sampled using a non-destructive method with a machine that is easily transported. This methodology uses a rapid sampling procedure, cutting sampling time down from 2-3 hours to approximately 10 minutes. Using GC-MS, the fragrance compounds can be identified individually, based on authentic standards. The steps used for collecting fragrance and control data are presented, from material setup to collecting the data output.
Flowers typically produce a fragrance used to attract pollinators. These fragrances are made up of many chemical compounds all acting together as a floral blend1,2,3. Without these fragrances, flowers would be less likely to pass on their genetic information using pollinators. Floral fragrance has been documented in many flowering plant families, with Orchidaceae being one of the more common families studied4. To understand the role of floral fragrance in pollination, it is important to nondestructively collect and analyze the chemical compounds being emitted from the flowers at different times of the day and during the several days to weeks the flower blooms are open, as fragrance can vary over time5.
An early protocol for this kind of sampling was developed by Heath and Manukian6. The goal of their sampling methods was to reduce stress on the specimen (e.g., plants, insects) being studied. Earlier papers documented that destructive procedures to the plant were required, such as removing blooming flowers in order to collect the fragrance. More recent floral fragrance publications by Cancino and Damon7,8 used similar methods. This study put the flowers in glass chambers and passed purified air over them; then fragrance compounds from the chamber were absorbed onto porous polymer adsorbents in clear Pasteur pipettes. The fragrances were collected for at least two hours during this study. Sadler et al.9 carried out floral fragrance studies on an epiphytic orchid in south Florida, much like the original study10. Again, this study required the flowers to be sampled for over two hours to collect the fragrance volatiles, with fragrance collected onto the porous polymer adsorbent. The paper here presents a non-destructive method that allows for much quicker sampling, lasting only 10 minutes. Also, instead of using a glass chamber oven baking bags are used, which allow for more flexible movement of the chamber and reduce the chances of damage to the flowers. These bags come in several sizes allowing the option to select the size of bag that will easily fit individual samples without damaging the sample or the surrounding material. The adsorbent used in this study was Tenax Porous Polymer Adsorbent. This differs from Porapak, because the sample can be thermally desorbed onto the GC-MS column for analysis, eliminating the use of a chemical solvent.
The methods in this study provide a way to quickly sample fragrance volatiles produced by flowers and could be used to sample volatiles from other specimens as well, such as insect pheromones, or mushroom volatiles. The reduced time for sampling means there is less stress on the sample and the ability to collect many samples in a short period of time. For example, in Sadler et al.9, the flower was only fragrant at night, so only two or three samples could be collected each night. With the method here, samples could be taken all night at 15-20-minute intervals from the same flower. Additionally, by using bags instead of glass chambers, the headspace can be suspended easier for sampling in the field for in situ collection on endangered or threatened plant species. Using the method presented here, we were able to sample flowers 1.5 to 2 meters above ground. These methods are incredibly useful for fragrance collection in the laboratory and the field, and provides researchers with a sampling technique that is fast and non-destructive to the sample.
NOTE: Perfumes or scented lotions and products must not be worn during any of these procedures.
1. Flower selection
NOTE: Flowers used can be either naturally growing in the environment or kept under artificial environmental conditions. Temperature, humidity, and light level during collection can vary based on the specific flower species used, and what type of data is being collected. For example, data has been collected during the day and at night for the same flower to determine if fragrance varies over time of day, and collected from both in situ and greenhouse flowers.
2. Material preparation
3. Volatile collection
NOTE: Sterile neoprene gloves need to be worn throughout this process, as contacting the bag or filter cartridges can contaminate the samples.
4. GC-MS
5. Data analysis
Representative data from the GC-MS are shown as a chromatogram in Figure 1. In addition to the chromatogram, a data file of results is also provided (Supplementary File 1). This data file provides the retention time for each peak (RT), and an identification of what compound that peak is (Library/ID). Peaks between 10:00 and 15:00 minutes are floral volatiles, due to the molecular weight of the compounds10. The numbers above the peaks signify the reten...
Though this technique is incredibly valuable for its sampling speed and portability, one limitation is using it for epiphytic species, or those growing on trees and not from the ground. In the original study10, one of the flowers sampled was epiphytic. Because the machine is too heavy to hang freely, a stable, elevated base must be made for sampling. Additionally, the machine can either be plugged in to an electrical outlet or battery powered, so if there is prolonged field sampling, there must be...
The authors declare no conflicts of interest.
USDA-ARS Research Project number 6036-22000-028-00D. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agriculture Research Service of any product or service to the exclusion of others that may be suitable. Additionally, the University of Florida Biology Department-Lewis and Varina Vaughn Fellowship in Orchid Biology (2017), and a University of Florida Graduate Research Fellowship (2014-2018) provided funding as well. We also thank Cindy Bennington from Stetson University for the orchid plant used during filming of this video.
Name | Company | Catalog Number | Comments |
Bulkhead Union | Cole-Palmer | UX-06390-10 | |
FEP tubing | Cole-Palmer | UX-06407-60 | |
Gas Chromatography | Hewlett Packard | 6890 | |
Glass Wool, Silanized | Sigma-Aldrich | 20411 | |
Inlet liner | Agilent | 5062-3587 | |
Mass Spectrometer | Hewlett Packard | 5973 | |
Reynolds oven bag | Reynolds Consumer Products | Turkey size | |
Tenax Porous Polymer Adsorbent | Sigma-Aldrich | 11982 |
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