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Team:TU Darmstadt/Protocols/GC-MS
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For our measurement approach we choose the following [https://2012.igem.org/Team:TU_Darmstadt/Protocols/GC-MS/GC-Protocol GC-Parameter] | For our measurement approach we choose the following [https://2012.igem.org/Team:TU_Darmstadt/Protocols/GC-MS/GC-Protocol GC-Parameter] | ||
| - | [[File:SIM_Scan_optimal_method.jpg |900px| GC-MS spectra | + | [[File:SIM_Scan_optimal_method.jpg |900px| GC-MS spectra |center ]] |
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We came down on the following approach (see protocol below) where we extract and purified our substances out of the supernatant. Therefore, we have to transform TPA into the corresponding di-methyl-ester : | We came down on the following approach (see protocol below) where we extract and purified our substances out of the supernatant. Therefore, we have to transform TPA into the corresponding di-methyl-ester : | ||
| - | [[File: Estermechanismus.png | 700px| Ester mechanism| | + | [[File: Estermechanismus.png | 700px| Ester mechanism| center]] |
Here we illustrate the reaction-mechanism of the ester reaction that we observed and thus used for our sample preparation. | Here we illustrate the reaction-mechanism of the ester reaction that we observed and thus used for our sample preparation. | ||
Revision as of 20:45, 25 September 2012
Contents |
Theory
Gas chromatography mass spectrometry (GC-MS)
To validate that our designed bacteria is able to digest PET and convert it into chemicals which can be used by the organism, we decided to use the "gold standard" for substance identification gas chromatography-mass spectrometry. The gas chromatograph (GC) separates all substances via a purification column, afterwards the exact mass of each purified substance is measured by mass spectrometer. This gives a clearly proof of the exact amount of the substance which are produced by our designed organism. The difficulty in achieving good measurements of terephthalic acid (TPA), the main component of PET, is given by itŽs physical properties. The aromatic body carries hydrophobic properties, while the two acid groups in para position are hydrophylic, this ambivalent character makes it a challenge to purify TPA by conventional purification columns. Since we searched various houres, That's why we first had to develop a new experimental setup to prepare our probes for the analysis.
GC-MS Purge and Trap
The purge and trap method is used to get an anaylt out of a water sample. Therfore, we purge our sample with an inert gas. Moreover, we trap our analyt with an hydrophobic trap as well as water. While gasing out the whole sample with the inert gas, the anaylt accumumlate on the trap. After that, we release the analyt from the trap with an temperature shift. Now, our analyt is ready to pass the column for the analysis.
Development of a brand new method
To measure TPA we have to embed it into a an appropriate matrix. One requirement to a matrix is that it has to be free from water. Hence we tested aceton. methanol and cycl. hexan. Unfortunately, TPA was only soluble in methanol (0,1 g/l). In our case, a matrix like cycl. hexan would be easier for the extraction of TPA out of our water samples. For our measurement approach we choose the following GC-Parameter
We came down on the following approach (see protocol below) where we extract and purified our substances out of the supernatant. Therefore, we have to transform TPA into the corresponding di-methyl-ester :
Here we illustrate the reaction-mechanism of the ester reaction that we observed and thus used for our sample preparation.
