Hello everyone,
I want to ask one thing, should I add the internal standard in the reaction mixture after completion of reaction and then starting material, product and the internal standard with ether or do I first extract everything with ether and then add internal standard ?
Which method can give me good results and which is the standard method to use internal standard?
You need to provide more details what kind of experiments you are doing. What are you extracting from which source, DNA, RNA, protein, any other ingredients...?! What's your internal standard you want to add to determine what?
Actually I am doing organic reaction oxidation coupling of primary amine to imiine and i want to check its conversion via GC. so after reaction i extract all the starting material with ether so when should I add the standard after reaction before extraction or after completely extract with ether and then add what is the standard method
If the intended use of internal standard is to monitor the extraction recover, it should be added to the initial sample before extraction; in other words the IS must be submitted to same steps that can cause a loss of sample during extraction.
An internal standard's purpose generally is to guide you only as an identified peak in your chromatograph peaks while the surrogate standard is the standards you use to allow you to check for recovery. Surrogate standards are spiked into samples before you do any experimental work on your sample. In short, internal standard should be spiked at the end of your experimental work just before GC analysis.
So it depends on your purpose of using a standard, if you want to check it's recovery you should use a surrogate standard and if you want to use it as a peak identification then you should us an internal standard. Either way it's a different standard that you have to use. Meaning the compounds in a standard should not overlap with another standard orelse it defeats the purpose of using a standard.
If you add the internal standard before extraction, it is actually a surrogate compound. The best way is to add a surrogate before extraction which will allow you estimate the extraction efficiency, provided that you add an internal standard, just before GC analysis. The internal standard allows to correct any instrumental variations.
An internal standard should be added after all other sample preparation is complete, immediately before GC analysis.
It is used to correct the analyte peak area for instrument response, i.e. if the instrument response drops, the internal standard peak area will reduce to the same proportion as the analyte peak area.
There are two possibility if you also want to assess the extraction efficiency or extraction method (to compare the losses during extraction) then you can add internal standard after completion of reaction or after stopping the reaction. Otherwise for it will be also not a problem to extract everything and then add internal standard in extract and analyse it.
Both ways are acceptable. It depends what exactly you are checking. If you know about extraction method how does it go then you add internal standard otherwise add after all stopping the reaction.
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