Protocol

Overview

This protocol describes how to measure volatiles released from plant root using gas chromatography coupled to mass spectrometry.

Background

This method aims at analyzing plant root volatiles using SPME (Solid Phase MicroExtraction). This technique allows a semi quantitative evaluation of volatiles which are produced by roots.

Materials/Equipment

• Aluminium foil
• Liquid nitrogen
• Mortars and pestles
• Precision balance
• 20mL glass vials with a septum in the lid (Supelco, 20mL)
• 100 μm PDMS solid phase microextraction fibre (SPME; Supelco c/o Sigma-Aldrich Chemie GmbH Buchs, Switzerland)
• Gas Chromatograph (__NOTE__: this protocol is designed for Agilent 6890 series GC system G1530A)
• DB1-MS Apolar column (DB1-MS, 30m, 0.25mm internal diameter, 0.25 μm film thickness)
• Mass Spectrometer (__NOTE__: this protocol is designed for Agilent 5973; transfer line 230^∘C, source 230^∘C, ionization potential 70 eV)
• Enhanced ChemStation and Productivity Tools MSD ChemStation software, E.02.00.493, Agilent Technologies, Inc.

Procedure

Collecting root samples

• Cautiously wash the root system with tap water
• Cut off the root system from the aboveground parts
• Immediately wrap the roots in aluminium foil
• Freeze the samples in liquid nitrogen.
• Samples can be stored at -80^∘C until use

Preparing the samples

• Grind the roots in liquid nitrogen to a fine powder using a mortar and a pestle
• Place 0.3g of powder in a glass vial (Supelco, 20mL) with a septum-lid
• Samples can be stored at -80^∘C until use

Operating the GC-MS

On the GC: Load the 100 μm PDMS solid phase micro extraction fibre

On the computer:

• Using the Enhanced ChemStation software, create/load the following method (from “File” menu):
  • Exposure of the fiber to the headspace in the vial:
    • 20 min at 35^∘C
  • Injector:
    • Automatic insertion of the fiber into the injector port of the gas chromatograph. Manual injection can also be performed. In this case, select “manual insertion”.
    • Temperature: 250^∘C
  • Inlet:
    • Fiber exposure: 2 min
    • Mode: Splitless
    • Gas flow: Helium, pressure: constant, 50.6kPa
  • Column (DB1-MS, 30m, 0.25mm internal diameter, 0.25 μm film thickness):
    • Gas flow: Helium,
    • pressure: constant, 50.6kPa
    • Flow: 0.9 mL/min
    • Average velocity: 35cm/sec
    • Select: MSD detector
  • Oven
    • Temperature: 60^∘C for 1 minute before ramping to 220^∘C at a rate of 10^∘C per minute followed by a post run of 5 minutes at 250^∘C
  • Mass Spectrometer (MSD)
    • Acquisition mode: Scan 20-220 atomic mass unit
• Save the method
• Load the method (from “File” menu)
• Edit the sequence of samples and select the file (from “Sequence” menu). Indicate a datapath to save the data in one folder
• Run the sequence

Data analysis

• Analysis of data can be realized using Productivity Tools, MSD ChemStation software.
• The peaks can be analysed by comparing retention times and mass spectra with those of the NIST05 Mass Spectra Library, provided with the MSD ChemStation software. Different libraries can be built by running pure compounds on the GC-MS to obtain their retention times and mass spectra. To perform the analysis:
  • Select libraries from the “spectrum” menu, “select library”. If several libraries are used, enter the quality threshold of each of them.
  • From “Chromatogramm” menu, select “MS signal Integration parameters” to enter integration parameter.
  • Select a sample to analyse
  • Integrate all peaks and export peak areas and compound identification results to excel (“Export Results” menu).

See example chromatogram attached at base of page.

Links to resources and suppliers

http://www.home.agilent.com/agilent/home.jspx cc=US&lc=eng

Literature references

Rasmann S, Kollner TG, Degenhardt J, Hiltpold I, Toepfer S, Kuhlmann U, Gershenzon J, and Turlings TCJ (2005). Recruitment of entomopathogenic nematodes by insect-damaged maize roots. ”Nature” 434: 732-737.

Health, safety & hazardous waste disposal considerations

Liquid nitrogen is a dangerous hazard. One cubic foot of liquid nitrogen will expand to 696 cubic feet of 100% gaseous nitrogen at 70°F, and can lead to asphyxiation. Liquid nitrogen should then be stored and used in well-ventilated spaces, and safety equipment such as cryogenic gloves, eye and face protections should be worn by workers.