I can not separate any comounds. I have isolate some but they were not pure. The one spot become more than 2 spot. I do not know how can I resolve them. They are very nonpolar which does not let me to use even reverse and Size exclusion chromatography. Can anybody help me in this topic.
u can use PTLC and CC by changing ur solvent ratio. u can also wash ur isolate with other less polar solvent like Chloroform, ethyl acetate or acetone,thus u wil get more clear fractions in these solvents , which can resolve further easily.
Hi Mahdi,
Relax... hexane extract usually is the easiest one to process phytochemistry job.
By now, I think you have a TLC profile of the crude extract and some idea about the nature of the compounds (fatty acids, triterpenes, steroids, diterpenes, etc.). If you know what you want, try to find at literature a specific technique to isolate it.
But if you want to process a classic or bioguided phytochemistry study you can divide the crude extract in fractions of diferent polarity, using fast filtration in silica gel. The advantage of this process is you will have around 5 fractions only, containing classes of compounds with similar polarity. So TLC or any other analysis can be less complicated.
After solvent elimination, CC of each fraction or those most interesting (by TLC profile or biological effect), also will give best results.
I have got the 20 fraction from the hexane extract by eluting with increseang amout of hexane ethylacetate ratio. I tried to re chromatographed of some of the fraction. I just could isolate 3 pure compounds which are known. This plant has a lot of secondry metabolite and I could not isolate them. I have used any kind of solvent but there is no result. I want to work on the other extract. :(
Mahdi - what kind of isolation techniques do you have available? Is it mainly preparative TLC, or can you use preparative LC as well? If it is very lipophilic, normal pahse might be your best chance. There are also "intermediate" phases, e.g. a diol stationary phase which may be worth trying. One thought: if your single spot on a TLC turns out to be three spots, it can be co-eluting compounds, but it could also point to a stability issue (the compound is degrading during the run). You can find stability problems by "turning" the developed (but no spray-reagents for detection!!!) TLC by 90 degrees and -re-run it. If you end up with several spots per lane again, you have a stability problem.
Mahdi,
I agree with damaris silveris's suggestion.you can divide the crude extract in fractions of diferent polarity, using fast filtration in silica gel. The advantage of this process is you will have around 5 fractions only. then columchromatogropy can be followed with silicsgel -230-400mesh size.Effective separation of pure compounds is possible.but elution will be very slow.(refer: the book -Separation Chemistry by R.P.Budhiraja, new age international publications)
Mr.Mahdi
Its tidious to separate. you have to use the column chromatography for the same,increase the polarity of solvent gradualy and take very small fractions collect them and perform the TLC by using the different mobile phase and with probable active compounds.It will will give idea that particular fraction have some active compound ,then againn you perform the same process for exptraction with bit changes in the extraction solvent system with respect to probability of compound as nature,type etc and repeat for several time .
Dear try to use column packed with sephadex LH 200 and methanol: dichloromethane 1:4 as a mobile phase
Hi Mahdi,
Did you try "wash" those fractions? If not, try it in already dried (without solvent) fractions, starting from the less polar of them. And remember: it is a"try and err" mission and requires huge patience:
drop enough amount of solvent on material. The idea is find one able to solve part of fraction; the insoluble part usually is simplier to solve than full fraction. Sometimes you can isolate compounds (e.g. sterols, like sitosterol) using this trick.
I suggest you try first ethyl eter, because if it does not work, you can easily evaporate it and starall the process again. Try also: acetone (usually good results), methanol; mixture ethyl eter: ethanol (1:1). The last one is my favourite :-).
I hope it works!
hi
the non-polar compounds in the extract can either isolated totally using silica gel column or neutral aluminium oxide for column, for me i like the second one because it’s more effective ,with both we use system (benzene - ethyl acetate 86-14) either all together or gradual elution , at the end if you got mixture , you need to run re-column with narrower diameter 0.5 cm with 50 cm length, this helps a lot, or you can use multiple developing preparative TLC
Also you can use different method from the beginning , run saponification to the pet ether extract using 10% alcoholic pot. Hydroxide and separate the sap. from unnap. And follow the same previous procedures , this is more effective
Hi stern,
there are some qualitative test s available to confirm the presence allakoids, terpenoids , steroids, sapanin, tannins etc. Before we go for isolation, these test can be done with the crude extract, so that isolation will be carried out accordingly.
Hi stern,
there are some qualitative test s available to confirm the presence allakoids, terpenoids , steroids, sapanin, tannins etc. Before we go for isolation, these test can be done with the crude extract, so that isolation will be carried out accordingly.
Hi Mahdi,
I agree with Stefan Gafner,
Before continuing the separation, you have sorted from the good system of eluent for TLC and also tried the suggestion of TLC by 90 degrees and -re-run it, if you a stability problem, a buffer medium used or added a drop of acid or base
good luck
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