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Team:Valencia UPV/prueba pag
From 2014.igem.org
Project > Modules > Methodology > Dynamic Headspace ST
The Idea
In order to analyse the amount of pheromones released by our Sexy plant into the environment (see Release), we performed a dynamic headspace sampling technique. This technique allows analysing the volatile organic compounds (VOCs) from the plant, including insect pheromones (see Biosynthesis).
This technique consists of a container where the plant is introduced (Figure 1 [1]). This container is sealed except for two conducts. One from which a continuous pure air stream (Figure 1 [2]) flows through the container acting as a carrier in order to facilitate the release of volatiles from the plant. This pure air comes from a pump (Figure 1 [3]). The other conduct allows the purge of air from the headspace of the container, and leads directly to an absorbent matrix capable of trapping the VOCs, including our pheromones (Figure 1.[4]). We used Tenax ® TA adsorbent resin adsorbent, which has high affinity for organic volatiles.
Once all VOCs have been trapped and enriched on the adsorbent matrix, they are ready to be analysed by GC-MS.
No conclusive results were obtained in this analysis.
Figure 1. Volatile Organic Compounds from Nicotiana benthamiana being analysed by dynamic headspace sampling technique.
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.
The retention characteristics of the column depend on its length, material and the temperature of the oven.
As the analytes flow through the column and become separated, they arrive at the detector where they will be identified.
K. Murray/ Wikimedia Commons / CC-BY-SA-3.0.
Example of different molecules separated by GC:
MASS SPECTROMETRY
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.
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.
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.
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.
After the entire analysis process, the pheromone analysis results were obtained (see Results: pheromone analysis)
To see more details about GC-MS conditions see Protocol.
