TITLE: Solvent Extraction of Polydimethylsiloxane (PDMS) Tubing to capture Volatile Organic Compounds from the Headspace and the Rhizosphere
AUTHORS: Gareth Thomas, Anusha Mohan-Kumar, Alexander N. Borg, John Caulfield, Michael A. Birkett, József Vuts

[DESCRIPTION]
This protocol describes a step-by-step guide for sampling Volatile Organic Compounds (VOCs) from the headspace/rhizosphere for synthetic blends and biological samples using polydimethylsiloxane (PDMS) silicon tubing, based on methods reported in Thomas et al. (2024) and Vuts et al. (2020). The advantages of using solvent elution when sampling headspace with PDMS tubing are that a liquid sample is generated, which can be used for multiple chemical (e.g. Gas Chromatography/coupled GC-Mass Spectrometry/Nuclear Mass Resonance Spectroscopy), behavioural (e.g. olfactometry) and electrophysiological (e.g. GC-electroantennography) tests, enabling a potential link to be established between VOC content and biological activity.

 

 


[STEPS]
SECTION: Conditioning PDMS tubes prior to headspace collection
1. Cut PDMS tubing (1 mm i.d. × 0.4 mm wall thickness) to desired length. The length of tubing can be variable depending on the headspace or rhizosphere set-up. In our previous work, the following lengths of PDMS tubing were used:

For headspace Volatile Organic Compound (VOC) collections: 2.5 cm-7.5 cm
For synthetic VOC blend collection from the rhizosphere: 30 cm 
For root VOC collection from the rhizosphere: 130 cm 

 

SECTION: Conditioning PDMS tubes prior to headspace collection
3. After 24 hours, place PDMS tubing into a glass vessel. 

 



SECTION: Conditioning PDMS tubes prior to headspace collection
4. Attach glass vessel to supply a constant flow of purified nitrogen, and turn on nitrogen supply. The PDMS tubes should be facing in the direction of the flow, enabling nitrogen to flow through the tubing. 

 

SECTION: Conditioning PDMS tubes prior to headspace collection
5. Place glass vessel containing PDMS tubes into a modified heating oven (170˚C) for a minimum of 1.5 hours.
SECTION: Conditioning PDMS tubes prior to headspace collection
6. After 1.5 hours, use heat-resistant gloves to remove the glass vessel from the oven and allow to cool. Keep nitrogen flow on whilst the vessel is cooling.
SECTION: Conditioning glass chamber/metal plates prior to headspace collections
7. Wash glass chamber (6 cm height × 12 cm diam.) using Teepol detergent and distilled water.
 

SECTION: Conditioning glass chamber/metal plates prior to headspace collections
8. Rinse Teepol detergent off glass chamber using distilled water.
SECTION: Conditioning glass chamber/metal plates prior to headspace collections
9. Rinse glass chamber with 100% acetone.
SECTION: Conditioning glass chamber/metal plates prior to headspace collections
11. Place the glass chamber and metal plates in a modified heating oven (150˚C) for a minimum of 1.5 hours.
SECTION: Conditioning glass chamber/metal plates prior to headspace collections
12. Remove the glass chamber and metal plates from the oven using heat-resistant gloves and allow to cool.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
13. Place a filter paper disc (12.5 cm) onto a metal plate (12 cm diam.).
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
14. Place a Petri dish (5 cm diam.) on top of the filter paper.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
15. Place a small filter paper disc (0.6 cm diam.) onto the Petri dish.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
16. Using a small square of tin foil, cover the outlet at the top of the glass chamber.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
18. Using forceps, insert a small hole through the top of the tin foil to place the PDMS tubing through.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
19. Using cotton gloves, carefully place the PDMS tubing through the hole in the foil, so it is suspended above the headspace. Where possible, avoid skin contact with conditioned PDMS tubing, using cotton gloves/forceps, to reduce the chance of contamination of PDMS tubing with skin VOCs.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
21. Apply 10 µL of a synthetic VOC blend of compounds onto the filter paper disc, and immediately place the glass chamber over the Petri dish.

SECTION: Application of synthetic VOC blend for sampling from aerial headspace
22. Attach the glass chamber to the metal plate using bulldog clips.
 

SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
30. Place the glass chamber (10 cm height × 12 cm diam.) on top of the metal plate, confining the orange within the headspace.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
25. Place filter paper (12.5 cm) on top of the metal plate.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
26. Place orange fruit onto filter paper.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
27. Using a small square of tin foil, cover the outlet of the glass chamber.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
31. Using cotton gloves, carefully place the PDMS tubing through the hole in the foil, so it is suspended above the headspace. 
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
32. Attach the glass chamber to the metal plate using bulldog clips.
SECTION: Conditioning PDMS tubes prior to headspace collection
2. Soak cut pieces of PDMS tubing in 100% methanol for a minimum of 24 hours.
SECTION: Eluting sorbed VOCs from the PDMS tubing
35. Remove the PDMS tubing from the headspace, and attach the tubing to the end of a glass Pasteur pipette.
SECTION: Eluting sorbed VOCs from the PDMS tubing
36. In a fume hood, clamp the glass Pasteur pipette and PDMS tube so they are suspended.
 



SECTION: Eluting sorbed VOCs from the PDMS tubing
37. Place a 1.1 mL pointed GC vial underneath the PDMS tubing.
SECTION: Eluting sorbed VOCs from the PDMS tubing
38. Measure 1 mL of redistilled solvent (diethyl ether/hexane) into an LC-MS vial.
SECTION: Eluting sorbed VOCs from the PDMS tubing
39. Take up 1 mL of redistilled solvent (diethyl ether/hexane) and pass through the clamped glass Pasteur pipette, which will pass through the PDMS tube and into the vial. 
SECTION: Eluting sorbed VOCs from the PDMS tubing
41. Solvent will pass through the pipette and PDMS tubing into the pointed glass vial, eluting the VOCs sorbed to the PDMS tubing, to generate a liquid extract. This can be analysed multiple times by gas chromatography (GC), coupled GC-mass spectrometry (GC-MS), GC-electroantennography (GC-EAG) and behavioural assays.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
23. After one hour, remove the PDMS tubing from the headspace using cotton gloves, and refer to 'Eluting sorbed compounds from the PDMS tubing'. 

N.B. certain parameters (e.g. dose of synthetic VOC blend/length of tubing/length of sampling time) can significantly influence compound recovery (Thomas et al., 2024; Vuts et al., 2020).
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
54. Grow maize (cv. Delprim) in soil for 16 days.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
56. Sterilize sand (0.25–0.71 mm grain size) in the oven for 48 hours at 80˚C. 
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
57. Mix the sand with distilled water (v/v) to obtain a substrate with 15% moisture content. 
SECTION: Eluting sorbed VOCs from the PDMS tubing
40. To remove the remaining solvent in the PDMS tube, attach pipette bulb teat to the Pasteur pipette and pass a bolus of air.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
60. Place sand into the beaker to achieve a ca. 1 cm depth of substrate at the bottom of the beaker, and place the PDMS probe on top of the sand. Both ends of the tube should extend 1 cm beyond the rim of the beaker. 
 

SECTION: Application of synthetic VOC blend for sampling from aerial headspace
24. To determine background VOCs, the carrier solvent alone (diethyl ether/hexane) should be applied to the filter paper as a blank control, which should be performed each time the experiment is set up.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
43. Heat-sterilise sand at 80˚C for 48 h.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
63. Release five second instar larvae of western corn rootworm (Diabrotica v. virgifera) onto the surface of the sand, and allow them to feed on the maize roots for 5 days.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
64. After 5 days, place a 1.1 mL screw-cap GC vial on one side of the tubing, insert a Pasteur pipette into the other end and pass 2 mL of redistilled solvent (diethyl ether/hexane) through the tubing. Solvent will pass through the pipette and PDMS tubing into the pointed glass vial, eluting the VOCs sorbed to the PDMS tubing, to generate a liquid extract. This can be analysed multiple times by gas chromatography (GC), coupled GC-mass spectrometry (GC-MS), GC-electroantennography (GC-EAG) and behavioural assays.


 


SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
45. Mix sand with distilled water to achieve a 15% moisture content (v/v).
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
46. Add sand to 250 mL beaker to achieve a ca. 1 cm depth of sand at the bottom of the beaker.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
48. Fill up beaker with sand medium and compact with a metal spoon.

N.B. soil can also be used as a medium in a similar way.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
49. Prepare synthetic VOC blend in redistilled solvent (diethyl ether/hexane), such that 100 µL of the blend will yield a 1 mg dose (100 µg/µL blend).

N.B. this dose is based on the methods from Vuts et al. (2020).
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
50. Make a 0.5 cm diam. hole in the medium using a hollow glass rod.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
51. Insert a 100 µL glass micropipette through the glass rod down the hole to mid-depth of medium, and inject 100 µL of the synthetic VOC blend. Cover the hole with sand.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
47. Place the PDMS tube into the beaker, ensuring that the tubing runs along the opposite sides of the walls of the beaker, and both ends of tubing reach over the beaker.
 
 Set-up for extraction of synthetic VOCs from the rhizosphere, using PDMS tubing.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
61. Transfer one 16-day-old plant to the beaker containing the sand. 
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
58. Bend a 19 cm long glass tube (0.5 cm diam.) into a U shape (height: 8 cm and base: 3 cm), using a Bunsen burner.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
59. Coil PDMS tube (130 cm) around the glass tube ensuring that the hollow inside of the PDMS tube is not obstructed anywhere, to allow for a flow of solvent during elution. Fasten the PDMS tubing at the top of the U shape with PTFE tape. This is a PDMS probe.

 


SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
55. Wash 250 mL glass beakers with Teepol detergent and then rinse with distilled water and acetone; oven-dry at 150˚C for a minimum of 2 hours.  
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
52. After 1 h, place a 1.1 mL screw-cap GC vial on one side of the tubing, insert a Pasteur pipette into the other end and pass 1 mL of redistilled solvent (diethyl ether/hexane) through the tubing. Solvent will pass through the pipette and PDMS tubing into the pointed glass vial, eluting the VOCs sorbed to the PDMS tubing, to generate a liquid extract. This can be analysed multiple times by gas chromatography (GC), coupled GC-mass spectrometry (GC-MS), GC-electroantennography (GC-EAG) and behavioural assays.


 


SECTION: Conditioning glass chamber/metal plates prior to headspace collections
10. Repeat steps 7-9 for the metal plates.
SECTION: Eluting sorbed VOCs from the PDMS tubing
42. In the case of GC analysis via thermal desorption, directly place the PDMS tubing after step 33 into an empty Tenax tube with a glass wool plug. Seal Tenax tubes with PDMS into glass ampoules under a stream of nitrogen and store at -20˚C until ready for GC/GC-MS analysis. 
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
33. After one hour, remove the PDMS tubing from the headspace using cotton gloves, and refer to 'Eluting sorbed compounds from the PDMS tubing'.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
62. Fill the remainder of the beaker with sand.
SECTION: Sampling VOCs from the plant rhizosphere, e.g. maize (Zea mays)
65. To remove the remaining solvent in the PDMS tube, attach pipette bulb teat to the Pasteur pipette and pass a bolus of air.
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
20. Prepare a synthetic VOC blend of compounds in redistilled solvent (diethyl ether/hexane), such that 10 µL of the blend will yield a 100 µg dose (10 µg/µL blend).

N.B. this dose is based on the methods from Thomas et al. (2024).
SECTION: Application of synthetic VOC blend for sampling from aerial headspace
17. Attach the septum and red screw cap to the inlet at the side of the glass chamber.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
34. To determine background VOCs, an empty glass chamber should be used as a blank control, which should be set up exactly the same way as the treatment containing the orange fruit, and should be performed each time the experiment is set up.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
28. Attach the septum and red screw cap to the inlet at the side of the glass chamber.
SECTION: Sampling VOCs from headspace of biological sample, e.g. orange (Citrus sinensis)
29. Using forceps, insert a small hole through the top of the tin foil to place the PDMS tubing through.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
53. To remove the remaining solvent in the PDMS tube, attach pipette bulb teat to the Pasteur pipette and pass a bolus of air.
SECTION: Application of synthetic VOC blend for sampling from the rhizosphere
44. Wash 250 mL glass beakers with Teepol detergent and then rinse with distilled water and acetone; oven-dry at 150˚C for a minimum of 2 hours.