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Team:Valencia UPV/Project/modules/methodology/sample analysis
From 2014.igem.org
Project > Modules > Methodology > 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.
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.
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.
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.
