During sample preparation for GC-MS, Where i followed the given protocol:
10 mg of dried biomass + 1.7 ml methanol + 2 ml chlorofoam + 0.3 ml H2SO4 ----kept in water bath (100 degrees) for 140 min. Later, after cooling the reaction mixture, distilled water was added.
I saw only one sample giving proper phase separation (between organic phase and aqueous phase) ---see Pic 1
whilst others did not gave any phase separation, even though these samples were prepared by the same protocol---see Pic 2
Please note that the sample giving phase separation was that of the external standard (i.e. PHB; Polyhydroxybutyrate)
Ensure minimum 20% of distilled water to the total volume of organic solvent.
Allow it for more time to get both the layers to be separated.
Check the preparation of solvents.
https://www.youtube.com/watch?v=rnFjiwu87Zc
https://www.spectroscopyonline.com/view/rapid-sample-preparation-and-fast-gc-ms-ms-analysis-pesticides-and-environmental-contaminants-fish-1
https://www.rsc.org/events/detail/40312/hands-on-sample-preparation-for-gc-and-gc-ms
https://ives-openscience.eu/4673/
DOI: 10.5772/intechopen.84259
Good luck.
be sure the derivatization vial has a Teflon cap or septum, and I recommend making a mark on the bottle to be sure there is no missing solvent during the derivatization reaction. On the other hand, put 200 ul benzene before adding the water
You are significantly above the boiling point of your solvent. Are you refluxing? If not, you're just boiling away your solvent and making a mess with your biomass and the acid/methanol.
Secondly, are you SURE you're supposed to add the solvent while heating? Check your method - perhaps you should be heating with just the biomass and the H2SO4 and methanol (methylating), and then adding the solvent to extract? Even so, you're well above the boiling point of the methanol, so you're going to lose your derivatizing agent unless you seal the vial or reflux.
The fact that you are using chloroform tells me that you have found a very old method. You should look for a newer method - newer methods are written without the assumption that you are a skilled chemist and are much easier for neophytes.
I think the chloroform must be in the reaction (methylation vial)
the role of chloroform as an aggressive extraction solvent.
So, you can not make derivitization (methylation) without extracting your sample
about the boiling point of methanol and chloroform, So, I was advised you to use a thermostable Teflon seal with cap and make a line mark on the bottle to confirm your sealing is good and there is no missing solvent and derivatization reagent during the reaction
and the 200 ul benzene used to avoid the emulsification and perform good separation layers (as makeup solvent)
like the inserted image
Best regards
https://www.researchgate.net/deref/https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DrnFjiwu87Zc
An amount of 5 grams of biochar add by a 15 ml mix of n-hexane: acetone (ratio 4:1) and Shake up for 5 hours to homogenize the extract. Extract the precipitate for 24 hours and filter by using filter paper to separate liquid and solid.
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