In my proposal ,My supervisor asked me why l didn't do isolation and l was stuck how to answer this question , can someone help me please how l can answer this , assuming you were in my position.
Dear, if you are asked for only extraction, then you might go for simple extraction through different extraction protocols like maceration. By this method, you can only get the whole extract of your sample plants, not specific molecules or compounds.
For isolation of compounds, you must need to separate your extract in different solvents depends on the polarity level (petroleum ether, chloroform, ethyl acetate, ethanol, methanol etc.), then dry those separated fractions. Check the separation of compounds by TLC to confirm the solvent system for each fraction. Then finally you can go for column chromatography and separate; or run through HPLC for separation sub-fractions which have more than one spot (compound) on TLC plate and less than 4-5. NMR, Mass spectra is also very important for proper isolation and structure modification of compounds.
Best of luck for your experiment. Regards
Phytochemistry study doing the following steps:
1- collection 2- washing 3- drying 4- grinding 5- extraction with different solvents, start with non-polar ( hexane or petroleum ether), ethyl acetate, 70%methanol.
6- take TLC for each fraction ( extract) to know number of components in each fraction. 7- ISOLATION of single component using different chromatography techniques ( best on is MPLC), if it is not available using column or preparative chromatography 8- identify the single pure components using IR, 1H NMR, 13C NMR, MS, element analysis,....etc 9- Application of single components according to different fields.
Hello,
generally there are two ways to isolate single compounds:
1. Bioassay-guided fractionation
Using this approach you start to test the whole extract in your biological assay and if there is a certain acitivity (e.g. antimicrobial activity, antioxidant activity etc or whatever you are interested in or which activity is characteristic for your plant material) you start with the extraction of this extract using solvents with increasing polarity, i.e. starting with lipophilic solvents (e.g. petroleum ether) and coming finally to most polar solvents (e.g. n-butanol). With these respective extracts (petroleum ether extract etc. which have been reduced to dryness and redissolved in solvents) you can test your activity again. If there is an activity you can do a further fractionation by column chromatography and/or HPLC. After testing the activity again, you can proceed by further fractionation. Finally, you will end up at a single compound.
2. Systematic fractionation
This usually needs more time. You start with the whole extract which is extracted by solvents with increasing polarity, i.e. starting with lipophilic solvents (e.g. petroleum ether) to most polar solvents (e.g. n-butanol). The respective extracts (petroleum ether extract) etc. are further fractionated by means for column chromatography and/or HPLC. These subfractions are then again further fractionated by CC or HPLC. At the end you will get single compounds.
Depending on your plant material and your project you have to select one of these two methods, i.e. it is important to differentiate, whether you are looking for a certain biological activity and the responsible chemical substance or whether you want to characterize a plant material in an analytical (chemical) way by the isolation of single compounds. Both approaches will take some time as, apart from the separation/ fractionation process, the isolated compounds need to be elucidated, using NMR, Mass spectra, IR, UV-spectra etc.!
Kind regards and much luck,
Klaus Peter
Dear, you need to make enough litterature to know exactly which solvent or mixture of solvent used. The choose of solvent depend of metabolites researched
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