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

From 2014.igem.org

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<p>When it comes to analysing volatile compounds Gas Chromatography (GC) coupled to Mass spectrometry (MS) is unequivocally the first choice. The combination of both techniques allows the separation and identification of each single volatile molecule present in the sample.</p><br/><br/>
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<p>When it comes to analysing volatile compounds Gas Chromatography (GC) coupled to Mass spectrometry (MS) is unequivocally the first choice. The combination of the separation resolution provided by chromatography with the structural information provided by mass spectrometry allows the quantification and identification of each single volatile molecule present in the sample.</p><br/><br/>
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<p>First of all, the sample must be prepared and volatilized (in case the sample is not in gas state). This is not our situation, since we are analysing volatiles extracted by HS-SPME (<a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_preparation" class="normal-link-page">see Sample preparation - HS-SPME</a>). Once the sample is in gas state it can be introduced in the gas Chromatograph.</p><br/><br/>
 
<p class="subpart">GAS CROMATOGRAPHY</p><br/>
<p class="subpart">GAS CROMATOGRAPHY</p><br/>
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<p>As every chromatography technique, it is based on the separation of the components of a mixture, which is called the mobile phase or eluent, when it is flowing through a stationary phase.</p><br/><br/>
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<p>As every chromatography technique, gas chromatography is based on differential partitioning of the components of a sample between a mobile phase that acts as sample carrier (a pure gas such as N2, He or H2 in the case of gas chromatography) and the stationary phase coating the chromatography column.</p><br/><br/>
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<p>The molecules of the mixture are separated by selective retention, it is to say, if they have more affinity for the mobile phase, they will flow faster while if they have higher affinity for the stationary phase, they will be more retained by it. Therefore molecules will last different times flowing through the stationary phase, it is called retention time.</p><br/><br/>
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<p>The molecules in the mixture are separated as they flow through the column by selective retention.  Molecules with higher affinity for the mobile phase will flow faster and elute the column first, whereas those with higher affinity for the stationary phase will take longer to pass through the system. The retention time of a particular substance (the time it takes to pass through the column) depends on the type of column used, its length, and set temperature. </p><br/><br/>
<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/4/47/VUPVGas_chromatography_1.jpg" alt="analytes"></img></div><br/>
<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/4/47/VUPVGas_chromatography_1.jpg" alt="analytes"></img></div><br/>
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<p>In the case of Gas chromatography, the mobile phase is composed of pure gases that act as carriers, N2, He or H. The stationary phase is a capillary column with a inner hollow where the mobile phase flows through. The inner surface of the column is coated with the stationary material. The column is inside an oven where the temperature is raised in order to increase the volatility of the analytes, decreasing the analysis time without losing resolution.</p><br/><br/>
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<p>The last part of the chromatograph is the detector where a signal is registered as the compounds elute from the column. In GC-MS the mass spectrometer acts as detector.</p><br/><br/>
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<p>The retention characteristics of the column depend on its length, material and the temperature of the oven.</p><br/><br/>
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<p>As the analytes flow through the column and become separated, they arrive at the detector where they will be identified.</p><br/><br/>
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<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/2/22/VUPVGas_chromatography_2.jpg" alt="sample_injector"></img></div><br/>
<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/2/22/VUPVGas_chromatography_2.jpg" alt="sample_injector"></img></div><br/>
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<div align="center"><p style="font-size: 0.8em; width: 70%;"><span class="black-bold">Figure 1. </span>.Gas Chromatography diagram</p></div><br/>
<div align="center"><p style="font-size: 0.8em; width: 70%;">K. Murray/ Wikimedia Commons / CC-BY-SA-3.0.</p></div><br/>
<div align="center"><p style="font-size: 0.8em; width: 70%;">K. Murray/ Wikimedia Commons / CC-BY-SA-3.0.</p></div><br/>
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<p>Example of a chromatogram obtained by GC: each peak corresponds to a different molecule.</p><br/><br/>
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<p>Example of different molecules separated by GC:</p><br/><br/>
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<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/b/bf/Gas_chromatography_3_2.jpg" alt="molecules_gc"></img></div><br/>
<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/b/bf/Gas_chromatography_3_2.jpg" alt="molecules_gc"></img></div><br/>
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<div align="center"><p style="font-size: 0.8em; width: 70%;"><span class="black-bold">Figure 2. </span>.Chromatogram obtained by Gas chromatography</p></div><br/><br/>
<br/><p class="subpart">MASS SPECTROMETRY</p><br/><br/>
<br/><p class="subpart">MASS SPECTROMETRY</p><br/><br/>
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<p>Once the analytes arrive at the detector, they can be identified by mass spectrometry. This technique works by ionizing the analytes coming from the GC and measuring the abundance of the formed ions.</p><br/><br/>
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<p>Mass spectrometry is an analytical technique capable of separating charged ions according to their mass-to-charge ratio (m/z) and measuring their abundance.</p><br/><br/>
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<p>The three basic components of a mass spectrometer are:</p>
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<ul class="method">
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<p>The first step is to ionize the analytes. There are many methods but the one we used was Electron Ionization (EI). This basically works by bombing the molecules with high-energy electrons (70eV), which break the molecules into charged fragments of a range of different masses, which are characteristic for each compound.</p><br/><br/>
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<li>The ion source, where ionization takes place. </li>
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<li>The mass analyser.</li>
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<li>The detector. </li>
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<p>Then, the resulting fragments can be analysed according to their mass/charge (m/z) ratio. There are many different mass analysers; the quadrupole analyser is the chosen one in this case. It consists of four cylindrical rods, two of them having positive electric potential while the other two are negatively charged. A radio frequency voltage is applied between the rod pairs creating an oscillating electric field. Only the ions with a given m/z will maintain its trajectory and cross the quadrupole to reach the detector, while the rest will be deflected.
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</ul>
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<p>MS systems differ in the methods used to generate and separate the ions. Our MS system employs electron ionization (EI), a quadrupole mass analyser, and an electron multiplier detector.
</p><br/><br/>
</p><br/><br/>
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<p>Finally, a detector can define the amount of ions with a given m/z. There are also many types of detectors. In this case, we used an electron multiplier. These detectors can amplify the signal of a given ion into an electronic current, like a cascade. The more quantity of ions that arrive, the greater the electron current produced. Therefore, the system is capable of quantifying the arriving ions by measuring the produced electric signal.</p><br/><br/>
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<p><b>How it works</b>
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</p><br/>
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<ul class="method">
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<li><b>Ionization:</b>
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The different compounds in the sample mixture enter the ionization source as they elute form the column. There, they are bombarded with high-energy electrons (70eV), which break the molecules into charged fragments of a range of different masses, which are characteristic for each compound.
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</li><br/>
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<li><b>Analysis:</b>
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The resulting fragments are separated according to their m/z ratio in the quadrupole analyser. A quadrupole consists of four cylindrical rods, two of them having positive electric potential while the other two are negatively charged. A radio frequency voltage is applied between the rod pairs creating an oscillating electric field. Only the ions with a given m/z will maintain its trajectory and cross the quadrupole to reach the detector, while the rest will be deflected. The voltage applied can be continuously changed (full scan) to monitor a range of m/z values, or it can be set to monitor only specific m/z ions (single ion monitoring mode, SIM)
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</li><br/>
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<li><b>Detection:</b>
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The ions that cross the analyzer reached the detector which converts ions to electric currency. The more quantity of ions that arrive, the greater the electron current produced. Therefore, the system is capable of quantifying the arriving ions by measuring the produced electric signal.
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</li><br/>
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</ul>
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<p>As a result a mass spectrum for each compound is obtained, i.e., the pattern of the ion fragments in which that compound breaks down, characterised by their m/z ratio and their relative abundance. This mass spectrum is characteristic for each substance and therefore a very valuable tool for compound identification.</p><br/><br/>
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<div align="center"><img width="600px" src="http://2014.igem.org/wiki/images/9/9e/VUPV_mass.png"></img></div><br/>
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<div align="center"><p style="font-size: 0.8em; width: 70%;"><span class="black-bold">Figure 3. </span>(Z)-11-Hexadecn-1-ol mass spectrum</p></div>
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<div align="center"><p style="font-size: 0.8em; width: 70%;"><span class="black-bold">Source</span>. NIST Chemistry Webbook</p></div><br/>
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<p>After the entire analysis process, the pheromone analysis results were obtained (<a href="http://2014.igem.org/Team:Valencia_UPV/Project/results/pheromone_analysis" class="normal-link-page">see Results: pheromone analysis</a>)</p><br/><br/>
 
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<p>You can find the results of the GC-MS analysis <a hrf=http://2014.igem.org/Team:Valencia_UPV/Project/results/pheromone_analysis> here.</a></a></p><br/>
<p>To see more details about GC-MS conditions <a class="blue-bold">see Protocol</a>.</p><br/><br/>
<p>To see more details about GC-MS conditions <a class="blue-bold">see Protocol</a>.</p><br/><br/>
<div align="center">
<div align="center">

Revision as of 00:43, 18 October 2014


Project > Modules > Methodology > Sample Analysis GC-MS



Sample Analysis. GC-MS

The Idea


When it comes to analysing volatile compounds Gas Chromatography (GC) coupled to Mass spectrometry (MS) is unequivocally the first choice. The combination of the separation resolution provided by chromatography with the structural information provided by mass spectrometry allows the quantification and identification of each single volatile molecule present in the sample.



GAS CROMATOGRAPHY


As every chromatography technique, gas chromatography is based on differential partitioning of the components of a sample between a mobile phase that acts as sample carrier (a pure gas such as N2, He or H2 in the case of gas chromatography) and the stationary phase coating the chromatography column.



The molecules in the mixture are separated as they flow through the column by selective retention. Molecules with higher affinity for the mobile phase will flow faster and elute the column first, whereas those with higher affinity for the stationary phase will take longer to pass through the system. The retention time of a particular substance (the time it takes to pass through the column) depends on the type of column used, its length, and set temperature.



analytes

The last part of the chromatograph is the detector where a signal is registered as the compounds elute from the column. In GC-MS the mass spectrometer acts as detector.



sample_injector

Figure 1. .Gas Chromatography diagram


K. Murray/ Wikimedia Commons / CC-BY-SA-3.0.


Example of a chromatogram obtained by GC: each peak corresponds to a different molecule.



molecules_gc

Figure 2. .Chromatogram obtained by Gas chromatography




MASS SPECTROMETRY



Mass spectrometry is an analytical technique capable of separating charged ions according to their mass-to-charge ratio (m/z) and measuring their abundance.



The three basic components of a mass spectrometer are:

  • The ion source, where ionization takes place.
  • The mass analyser.
  • The detector.

MS systems differ in the methods used to generate and separate the ions. Our MS system employs electron ionization (EI), a quadrupole mass analyser, and an electron multiplier detector.



How it works


  • Ionization: The different compounds in the sample mixture enter the ionization source as they elute form the column. There, they are bombarded with high-energy electrons (70eV), which break the molecules into charged fragments of a range of different masses, which are characteristic for each compound.

  • Analysis: The resulting fragments are separated according to their m/z ratio in the quadrupole analyser. A quadrupole consists of four cylindrical rods, two of them having positive electric potential while the other two are negatively charged. A radio frequency voltage is applied between the rod pairs creating an oscillating electric field. Only the ions with a given m/z will maintain its trajectory and cross the quadrupole to reach the detector, while the rest will be deflected. The voltage applied can be continuously changed (full scan) to monitor a range of m/z values, or it can be set to monitor only specific m/z ions (single ion monitoring mode, SIM)

  • Detection: The ions that cross the analyzer reached the detector which converts ions to electric currency. The more quantity of ions that arrive, the greater the electron current produced. Therefore, the system is capable of quantifying the arriving ions by measuring the produced electric signal.

As a result a mass spectrum for each compound is obtained, i.e., the pattern of the ion fragments in which that compound breaks down, characterised by their m/z ratio and their relative abundance. This mass spectrum is characteristic for each substance and therefore a very valuable tool for compound identification.




Figure 3. (Z)-11-Hexadecn-1-ol mass spectrum

Source. NIST Chemistry Webbook


You can find the results of the GC-MS analysis here.


To see more details about GC-MS conditions see Protocol.