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Team:Valencia UPV/Project/modules/methodology/sample preparation
From 2014.igem.org
Project > Modules > Methodology > Sample Preparation HSPME
The Idea
Headspace solid-phase microextraction (HS-SPME) is one of the most convenient sample extraction methods for analysing Volatile Organic Compounds (VOCs). It is a very sensitive, inexpensive, robust and easy-to-use technique that does not require the use of solvents, being able to detect metabolites at the parts per trillion (ppt) level.
How It Works
HS-SPME is based on the extraction of volatiles present in the headspace of a vial by an adsorption/desorption process. The volatiles diffuse from the sample to the headspace where are captured by an adsorbent, a polymer-coated fiber with high affinity for the target compounds. The adsorption process is completed when equilibrium between the sample, the headspace and the fiber is reached. The fiber is then transferred to the injection port of, most commonly, a gas chromatograph where the captured compounds are desorbed for analysis.
Figure 1. Schematic representation of the headspace solid-phase microextraction adsorption/desorption. In the adsorption step the fiber is exposed in the headspace of a vial containing biological material, where volatile organic compounds (shown in red) are adsorbed by the fiber. In the desorption step, the fiber is introduced in the Gas Chromatograph injection port where the volatile compounds are released by thermal desorption.
We have used HS-SPME coupled to Gas Chromatography-Mass Spectrometry detection to analyse the presence of pheromones ((Z)-11-hexadecen-1-ol, (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate) in our samples, Nicotiana benthamiana leaves.
Below is a brief outline of the different steps of the extraction process:
- Sample preparation:
- Leaves must be immediately frozen in liquid nitrogen after removal from the plant to avoid any changes in their volatile profile.
- Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap vial, avoiding defreezing.
- EDTA and a saturated solution of CaCl2 are then added to the sample in order to inhibit enzymatic activity and nonenzymatic oxidation, thus stabilizing the sample.
- Finally, samples are sonicated to favour the release of volatiles from the plant material.
- Trapping of volatiles: the SPME fiber is introduced through the septum into the headspace of the vial in order to ‘trap’ the volatiles from the sample. During the adsorption process the sample is heated under continuous agitation to favour the diffusion of volatiles from the sample.
- Desorption: release of the volatiles from the fiber by thermal desorption in the injection port of the Gas chromatograph.
Now the sample is ready to be analysed by GC-MS.
For further information check the detailed protocol.
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References
- Wolfram Weckwerth., 2007. Metabolomics: Methods and Protocols. Humana press, Totowa, New Jersey.
- Zhouyao Zhang , Janusz Pawliszyn (1993). Headspace solid-phase microextraction. Anal. Chem., 1993, 65 (14), pp 1843–1852.
