Hi Ghosthunter100,
That is a really great question – it shows that you’ve been thinking about this in depth, well done! 🙂
I don’t actually isolate the chemicals that they release (their pheromone) as such, but I do analyse them. There is only one major compound that forms their emissions, and it is very volatile (that means that it is has a very low boiling point) so exists in the air as a gas (so that it travels and can reach other flies to attract them).
Let me backtrack and just go briefly over what I do as a whole. What I actually do is get the flies to eat compounds that I have made which contain a different isotope of hydrogen (a heavier form of hydrogen) called deuterium (as well as regular hydrogen). The idea is that the flies P450 enzymes that make the pheromone might be able to use some of these these deuterium-labelled compounds as substrates to make the pheromone out of. If they do use them, then some of the pheromone that they produce will contain deuterium. As I said, deuterium is a heavier form of hydrogen. Hydrogen weighs 1 mass unit, and deuterium weighs 2 mass units. So the deuterium-labelled pheromone will be a bit heavier than normal pheromone, and we can detect these mass differences using a technique called mass spectrometry (MS).
Let me now get back to your question. The flies do release very small amounts of pheromone, so the way I analyse it is by using two techniques together – one is called solid-phase microextraction (SPME), which is the way I collect some pheromone to analyse, and the other is gas chromatography-mass spectrometry (GC/MS), which is what I use to analyse the ‘collected’ pheromone. I do these feeding experiments inside glass flasks with rubber seals, so the pheromone remains trapped inside the flasks, and are in the air inside the flasks because they are so volatile. I stick the SPME needle through the rubber seal into the flask, and then the fibre on the end of the needle is exposed to the air and pheromone inside the flask. Some of the pheromone gets ‘adsorbed’ onto the fibre (which means sticks to the outside of), then I can pull it out of the flask to analyse it. If you would like to see what an SPME apparatus looks like, have a look at the diagram here: http://cool.conservation-us.org/jaic/articles/jaic44-01-002.html (scroll down a little to the picture). SPME is a really good technique for analysing very small amounts of chemical.
GC/MS is actually two techniques rolled in one. Gas chromatography (GC) is a way of separating out different chemicals based on how volatile they are (how easily they become gas/vapour) using high temperatures. MS involves chemicals being bombarded with energy and then the chemicals fragment (break into) smaller pieces. The mass spectrometer measures the masses of these pieces/fragments. So a GC/MS instrument has a GC part that you inject your sample into, and then your sample is vaporised and carried through a really long column by helium gas. The different chemicals in your sample separate out and the computer the instrument is attached to gives you a graph that shows you peaks for each different chemical (this graph is called a ‘gas chromatogram’). After the GC part in the instrument is a MS detector. Each separate chemical/peak then gets to this detector and is fragmented, and the computer this time gives you a bar graph showing you the masses of different fragments/pieces (this graph is called a ‘mass spectrum’). Different chemicals break differently to give you fragments with different weights.
So, once I’m done with the SPME sampling of the emitted pheromone, I stick the SPME fibre into the GC part of the GC/MS instrument (this part of the instrument is called the ‘injection port’) and the pheromone is vaporised and removed off the fibre, and then goes through the GC column and then gets to the MS detector. The gas chromatogram and mass spectrum that I get then shows me if their is deuterium in the emitted pheromone or not, which tells me if the eaten compounds are being used to make pheromone or not!
Hi Ghosthunter100,
That is a really great question – it shows that you’ve been thinking about this in depth, well done! 🙂
I don’t actually isolate the chemicals that they release (their pheromone) as such, but I do analyse them. There is only one major compound that forms their emissions, and it is very volatile (that means that it is has a very low boiling point) so exists in the air as a gas (so that it travels and can reach other flies to attract them).
Let me backtrack and just go briefly over what I do as a whole. What I actually do is get the flies to eat compounds that I have made which contain a different isotope of hydrogen (a heavier form of hydrogen) called deuterium (as well as regular hydrogen). The idea is that the flies P450 enzymes that make the pheromone might be able to use some of these these deuterium-labelled compounds as substrates to make the pheromone out of. If they do use them, then some of the pheromone that they produce will contain deuterium. As I said, deuterium is a heavier form of hydrogen. Hydrogen weighs 1 mass unit, and deuterium weighs 2 mass units. So the deuterium-labelled pheromone will be a bit heavier than normal pheromone, and we can detect these mass differences using a technique called mass spectrometry (MS).
Let me now get back to your question. The flies do release very small amounts of pheromone, so the way I analyse it is by using two techniques together – one is called solid-phase microextraction (SPME), which is the way I collect some pheromone to analyse, and the other is gas chromatography-mass spectrometry (GC/MS), which is what I use to analyse the ‘collected’ pheromone. I do these feeding experiments inside glass flasks with rubber seals, so the pheromone remains trapped inside the flasks, and are in the air inside the flasks because they are so volatile. I stick the SPME needle through the rubber seal into the flask, and then the fibre on the end of the needle is exposed to the air and pheromone inside the flask. Some of the pheromone gets ‘adsorbed’ onto the fibre (which means sticks to the outside of), then I can pull it out of the flask to analyse it. If you would like to see what an SPME apparatus looks like, have a look at the diagram here: http://cool.conservation-us.org/jaic/articles/jaic44-01-002.html (scroll down a little to the picture). SPME is a really good technique for analysing very small amounts of chemical.
GC/MS is actually two techniques rolled in one. Gas chromatography (GC) is a way of separating out different chemicals based on how volatile they are (how easily they become gas/vapour) using high temperatures. MS involves chemicals being bombarded with energy and then the chemicals fragment (break into) smaller pieces. The mass spectrometer measures the masses of these pieces/fragments. So a GC/MS instrument has a GC part that you inject your sample into, and then your sample is vaporised and carried through a really long column by helium gas. The different chemicals in your sample separate out and the computer the instrument is attached to gives you a graph that shows you peaks for each different chemical (this graph is called a ‘gas chromatogram’). After the GC part in the instrument is a MS detector. Each separate chemical/peak then gets to this detector and is fragmented, and the computer this time gives you a bar graph showing you the masses of different fragments/pieces (this graph is called a ‘mass spectrum’). Different chemicals break differently to give you fragments with different weights.
So, once I’m done with the SPME sampling of the emitted pheromone, I stick the SPME fibre into the GC part of the GC/MS instrument (this part of the instrument is called the ‘injection port’) and the pheromone is vaporised and removed off the fibre, and then goes through the GC column and then gets to the MS detector. The gas chromatogram and mass spectrum that I get then shows me if their is deuterium in the emitted pheromone or not, which tells me if the eaten compounds are being used to make pheromone or not!
There’s the long-winded explanation of it! Sorry if my descriptions of the various techniques are not very clear – it difficult to explain things without being able to actually show you the instruments and point at things. If you would like to see some pictures and read more about GC/MS, then please check out these sites: http://en.wikipedia.org/wiki/Gas_chromatography, http://en.wikipedia.org/wiki/Mass_spectrometry, http://en.wikipedia.org/wiki/Gas_chromatography%E2%80%93mass_spectrometry.
If you watch NCIS, you might notice that Abby uses GC/MS and has an instrument near the door in her lab! 🙂
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