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

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<div align="center"><img class="img-title" alt="Methodology" src="https://static.igem.org/mediawiki/2014/1/10/VUPVMethodology-title.png"></img></div><br/>
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<p><h3 class="hook" align="left"><a>Project</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules">Modules</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology">Methodology</a> > <a>Sample Preparation HSPME</a></h3></p><br/></br>
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<p class="subpart">SAMPLE PREPARATION- HEADSPACE-SOLID-PHASE MICROEXTRACTION</p><br/><br/>
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<div align="center"><span class="coda"><roja>S</roja>ample <roja>P</roja>reparation: <roja>H</roja>eadspace <roja>S</roja>PME</span> </div><br/><br/>
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<p>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.</p><br/><br/>
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<p class="subpart">The Idea</p>
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<p>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].</p><br/><br/>
 
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<p>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.</p><br/><br/>
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<p>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.</p><br/><br/>
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<p class="subpart">How It Works</p>
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<p>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.</p><br/><br/><br/>
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<div align="center"><img width="600px" src="https://static.igem.org/mediawiki/2014/8/88/VUPVSample_preparation_1.jpg" alt="solid_phase_extraction" title="Solid Phase Microextraction"></img></div><br/>
 
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<div align="center"><p style="font-size: 0.8em; width: 70%;">Source: Metabolomics: Methods and Protocols. Humana press, Totowa, New Jersey.</p></div><br/>
 
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<div align="center"><img width="550px" src="https://static.igem.org/mediawiki/2014/a/a7/VUPVSample_prep1.png" alt="solid_phase_extraction" title="Solid Phase Microextraction"></img></div><br/>
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<div align="center"><p style="text-align: justify; font-size: 0.8em; width: 670px;"><b>Figure 1</b>. 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.</p></div><br/>
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<p>In order to perform an accurate analysis of the sample, this technique must follow 3 simple steps:</p><br/><br/>
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<p>We have used HS-SPME coupled to Gas Chromatography-Mass Spectrometry detection to analyse the presence of pheromones (<span class="red-bold">(Z)-11-hexadecen-1-ol</span>, <span class="green-bold">(Z)-11-hexadecenal</span>, <span class="blue-bold">(Z)-11-hexadecenyl acetate</span>) in our samples, <i>Nicotiana benthamiana leaves</i>.</p><br/><br/>
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<p>Below is a brief outline of the different steps of the extraction process:</p><br/><br/>
<ul class="method">
<ul class="method">
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<li><a class="black-bold">Sample preparation</a>: it is vital for a proper analysis that samples are unaltered during the whole process.
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<li><a class="black-bold">Sample preparation:</a>
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     <li>Therefore the biological material, leaves in this case, must be immediately kept on liquid Nitrogen after the removal from the plant.</li>
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<ul>
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     <li>Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap headspace vial.</li>
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     <li>Leaves must be immediately frozen in liquid nitrogen after removal from the plant to avoid any changes in their volatile profile.</li>
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     <li>Afterwards, EDTA and saturated solution of CaCl2 are added to inhibit enzymatic activity and stabilize the sample.</li>
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     <li>Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap vial, avoiding defreezing.</li>
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     <li>Finally, they are sonicated to induce the release of volatiles from the solid phase to the gas phase in the headspace of the vial.</li>
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     <li>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.</li>
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     <li>Finally, samples are sonicated to favour the release of volatiles from the plant material.</li>
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</ul>
</li>
</li>
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<li><a class="black-bold">Trapping of volatiles</a>: 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.</li>
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<li><a class="black-bold">Trapping of volatiles</a>: 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.</li>
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<li><a class="black-bold">Desorption</a>: release of the volatiles from the fiber by thermal desorption which takes place in the insertion port of the Gas chromatograph.</li>
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<li><a class="black-bold">Desorption</a>: release of the volatiles from the fiber by thermal desorption in the injection port of the Gas chromatograph.</li>
</ul><br/><br/>
</ul><br/><br/>
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<p>Now the sample is ready to be analysed by GC-MS. (<a class="blue-bold">see GC-MS</a>)</p><br/><br/>
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<p>Now the sample is ready to be analysed by <span class="blue-bold">GC-MS</span>.</p><br/><br/>
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<p>For further information check the detailed protocol (<a class="blue-bold">see protocol</a>)</p><br/><br/>
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<p>For further information <a class="blue-bold">check the detailed protocol</a>.</p><br/><br/>
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Latest revision as of 01:38, 18 October 2014

Project > Modules > Methodology > Sample Preparation HSPME



Sample Preparation: Headspace SPME


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.




solid_phase_extraction

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.







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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