Team:Valencia UPV/Project/modules/methodology/sample preparation

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Methodology

SAMPLE PREPARATION- HEADSPACE-SOLID-PHASE MICROEXTRACTION



Headspace solid-phase microextraction is one of the most convenient techniques for analysing Volatile Organic Compounds (VOCs). It is a very sensitive, inexpensive, robust and easy-to-use technique since it does not require the use of solvents and is able to detect metabolites up to ppt (parts per trillion) with a low sample quantity.



In our case, we chose this technique coupled to a Gas Chromatography-Mass Spectrometry detection (see GC-MS) to analyse the presence of pheromones ((Z)-11 hexadec-1-ol, (Z)-11 hexadecenal, (Z)-11 hexadecenyl acetate)[see biosynthesis] in our samples, Nicotiana benthamiana leaves [see agroinfiltration].



This technique is based on the detection of volatiles present in the headspace of a vial. The volatiles diffuse from the sample (Solid phase) to the headspace (Gas phase) and are captured by an absorbent, a polymer-coated fiber with high affinity for them. After desorption from the fiber, volatiles are analysed by GC-MS.



solid_phase_extraction

Source: Metabolomics: Methods and Protocols. Humana press, Totowa, New Jersey.


In order to perform an accurate analysis of the sample, this technique must follow 3 simple steps:



  • Sample preparation: it is vital for a proper analysis that samples are unaltered during the whole process.
  • Therefore the biological material, leaves in this case, must be immediately kept on liquid Nitrogen after the removal from the plant.
  • Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap headspace vial.
  • Afterwards, EDTA and saturated solution of CaCl2 are added to inhibit enzymatic activity and stabilize the sample.
  • Finally, they are sonicated to induce the release of volatiles from the solid phase to the gas phase in the headspace of the vial.
  • Trapping of volatiles: volatiles present in the gas phase must be extracted for further analysis. Therefore a fiber coated with an adsorbent polymer is introduced in the headspace, and ‘traps’ the volatiles from the sample.
  • Desorption: release of the volatiles from the fiber by thermal desorption which takes place in the insertion port of the Gas chromatograph.


Now the sample is ready to be analysed by GC-MS. (see GC-MS)



For further information check the detailed protocol (see protocol)



References


  1. Wolfram Weckwerth., 2007. Metabolomics: Methods and Protocols. Humana press, Totowa, New Jersey.

  2. Zhouyao Zhang , Janusz Pawliszyn (1993). Headspace solid-phase microextraction. Anal. Chem., 1993, 65 (14), pp 1843–1852.




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