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Team:Valencia UPV/Project/modules/methodology/sample analysis
From 2014.igem.org
SAMPLE ANALYSIS - GAS CHROMATOGRAPHY-MASS SPECTROMETRY
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.
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 (see Sample preparation - HS-SPME). Once the sample is in gas state it can be introduced in the gas Chromatograph.
GAS CROMATOGRAPHY
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.
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.
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 gas molecules arrive at the detector, they can be identified by mass spectrometry. This technique works by ionizing the gas molecules coming from the GC and measuring the relative abundance of the formed ions.
The first step is to ionize the gas molecules. There are many methods but the one used was Electron Ionization (EI). This basically works by bombing the molecules with high-energy electrons until they break into fragments of different charge and mass.
Then, the resulting fragments must 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 be detected, the rest will be deflected.
Finally, a detector can define the amount of ions with a given m/r. There are also many types of detectors. In this case, an electron multiplier was used. 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. Therefore, the system is capable of quantifying the arriving ions by measuring the produced electric signal.
After all the analysis project, the pheromone analysis results were obtained (see Results: pheromone analysis)
To see more details about GC-MS conditions see Protocol.
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