Porapak q

Volatile compounds from whole maize plants, with and without stemborer eggs, were collected by headspace sampling^{7 (link)}. Volatiles were collected from at least 4 plants with and 4 plants without eggs per genotype. Prior to volatile collection, 4-week old maize seedlings were placed inside oviposition cages ( $80\times 40\times 40$  cm) into which six gravid female stemborer moths were introduced and kept overnight for oviposition. Concurrently, control plants were kept inside similar cages, but without stemborer moths. Volatiles were collected the following day, starting from the last 2 h of photophase, for 24 h. Leaves of plants with or without eggs were enclosed in polyethyleneterephthalate (PET) bags (volume 3.2 L, $\simeq$ 12.5 mm thickness) heated to 150 °C before use and fitted with Swagelock inlet and outlet ports. Charcoal-filtered air was pumped (500 mL min⁻¹) through the inlet port. Volatiles were collected on Porapak Q (0.05 g, 60/80 mesh; Supelco, Bellefonte, PA, USA) filters inserted in the outlet port through which air was drawn at 300 mL min⁻¹. After entrainment, volatiles were eluted with 0.5 mL dichloromethane (Sigma Aldrich) for use in subsequent bioassays. Volatiles were collected from 1,168 plants representing 146 genotypes.

Cassava plants were maintained in cages without whitefly infestation. VOCs were collected using sterile plastic cooking bags (Qualitá cooking bags composed of polyester [poly(ethylene terephthalate) or PET], 27 × 41 cm, max 200 °C [RMBPACK Machines and Packaging Ltd., Colombo, PR, Brazil]). After cooking, identical precleaned polyester bags were opened and baked in an oven (100 °C) for 2 h. The bags were inflated with clean air and subsequently deflated (thrice) to remove any residual contamination, and the VOCs were collected using a push–pull system. Six cassava plants in six identical precleaned plastic bags were used for each extraction. The VOCs were trapped on a Porapak Q (50/80 mesh, 0.05 g, Supelco Inc., Bellefonte, PA 16823, USA). The absorbent was placed in a glass tube and inserted at the top of the bag. Air was filtered through activated charcoal and then pumped into the plastic bag at a flow rate of 600 mL/min per plant before being collected at a flow rate of 400 mL/min. After the collection was complete, the trapped VOCs were desorbed with 500 µL of double-distilled hexane (HPLC grade), and the samples were stored at −20 °C until analysis and bioassay. The samples were collected in the laboratory for 60 h.

Volatiles were collected from virgin male and virgin female C. brassicola by placing single-sex groups into a silanized glass chamber with a glass frit at the upwind end (12 cm × 4 cm; Hamilton Laboratory Glass, Margate, Kent, UK). Insects were anesthetized briefly with CO₂ to facilitate transfer from rearing vials to the glass containers. Air was drawn into the glass chambers at 200 ml/min through an activated charcoal filter (20 cm × 2 cm, 10–18 mesh; Fisher Scientific, Loughborough, UK) using a vacuum pump (DA7C; Charles Austen, West Byfleet, UK). Air flowed out of each chamber via a Pasteur pipette (4 mm i.d.) containing Porapak Q (200 mg, 50–80 mesh; Supelco, Gillingham, Dorset, UK) held between plugs of silanized glass wool. Porapak was cleaned through Soxhlet extraction with chloroform for 8 h and washed with dichloromethane prior to use. Dead midges were removed daily and replaced with newly-emerged individuals of the same sex. Most of the work was done with a collection from 68 females and one from 34 males made over 3 d, as these contained most material. Volatiles trapped on Porapak were eluted with 3 × 0.5 ml dichloromethane (Pesticide-Residue Grade) and stored at 4 °C until use.

The tomato floral scent was collected using a dynamic headspace extraction device [40 (link)]. Prior to flowering, individual buds were isolated in fabric mesh bags (mesh diameter: 2 mm) to prevent visits by bees. Once the flowers opened in the next morning, they were placed in a polyethylene plastic cup (upper diameter: 490 mm, bottom diameter: 370 mm, height: 35 mm, volume: 35 mL) to collect scent. The flowers in the cups opened normally and were not jostled. Each cup contained an inlet and an outlet to let air in and out. The inlet was made in the middle of the lid of the cup. A vacuum pump (QC-1B, Beijing Institute of Labor Instrument, Beijing, China) was utilized to pull fragrant headspace air through sorbent traps at a flow rate of 1.5 mL/min. Air purified via an activated charcoal column (100 mm length × 5 mm diameter) was pumped through the cup, and volatiles were forced into a glass trap (120 mm length × 8 mm diameter) containing 50 mg Porapak Q (80/100 mesh, Supelco, Bellefonte, PA, USA). All of the connections were made with Teflon tape. The collection of volatiles was conducted over 6 h from 09:00 to 15:00. Then, the glass traps were eluted twice with a total of 300 μL of HPLC grade hexane (Sigma-Aldrich, Oakville, ON, Canada), and the volatile compounds were stored in sample vials (2 mL; Agilent Technologies, California, USA) at −80 °C until use.

The Ethane50 culture was equally distributed in six 150-ml serum flasks using 20 ml inoculum and 80 ml medium. Three replicate cultures were amended with 0.05-MPa ethane in 0.1-MPa N₂-CO₂, while 3 negative controls were amended with 0.15-MPa N₂-CO₂. Both treatments were incubated at 50°C. Weekly, 0.5-ml headspace gas samples were analyzed for ethane content using an Agilent 6890 gas chromatograph in splitless mode equipped with a packed column (Supelco Porapak Q, 6 ft by 1/8 ft by 2.1-mm stainless steel column, oven temperature 80°C). The carrier gas was helium (20 ml per minute), and hydrocarbons were detected by flame ionization detection. Each sample was analyzed in triplicates and quantified against ethane standards of 5, 10, and 100%. Derived concentrations were converted into molar amounts by taking the headspace size, pressure, and temperature into account. Results were corrected for sampled volumes. Sulfide concentrations were measured as described above. To determine sulfate concentrations, 1 ml of sample was fixed in 0.5 ml zinc acetate. Samples were diluted 1:50 with deionized water (MilliQ grade; >18.5 MΩ), and samples were measured using nonsuppressed ion chromatography (Metrohm 930 Compact IC Metrosep A PCC HC/4.0 preconcentration and Metrosep A Supp 5-150/4.0 chromatography column).

We used a headspace collection system to collect headspace volatiles from plants. Living test plants were placed in a clean roasting bag. The bag was sealed around the plant stem with a self-sealing strip about 20 cm above soil-height^{34 (link)}. Humidified, charcoal-filtered air was pulled through the bag with a pump (Beijing Institute of Labour Instruments, China) at 300 ml·min⁻¹ and passed over an adsorbent cartridge. The adsorbent cartridge was a 0.5 × 10 cm glass column containing 50 mg of adsorbent (80/100 mesh, Supelco, Bellefonte, PA, USA). The Porapak Q (50 mg, 80–100 mesh, Supelco, Bellefonte, PA, USA) was held between plugs of glass wool. Each sample was aerated for 8 h. Volatiles were eluted from the adsorbent cartridge with 500 μl redistilled hexane at room temperature. An internal standard of 0.5 μg of benzaldehyde (99%, Fluka Production) was added to the extract for chemical quantification^{43 (link)}. The final extracts were reduced to 50 μl using a slow stream of nitrogen and then subjected to gas chromatography mass spectrometry (GC-MS) and gas chromatography-electroantennographic detection (GC-EAD). If not used immediately, extracts were stored in glass vials at −18 °C until use.