I run a column of pet. ether extract and collected 25 fractions from pet.ether: ethyl acetate (80:20) mobile phase. One fraction showed single spot on TLC in same solvent system but difficult to get it as in crystal form. when i concentrate it in reduced pressure it forms green color sticky mass. Can anyone suggest how I can separate it as pure and crystallize form?
At first, you have to know that not all compounds can be crystallized.
I think you can try to solublize the compound in the least amount of ethyl acetate then put some methanol and watch the solution. If you notice one crystal put the solution in the refrigerator till complete crystallization.
At first, you have to know that not all compounds can be crystallized.
I think you can try to solublize the compound in the least amount of ethyl acetate then put some methanol and watch the solution. If you notice one crystal put the solution in the refrigerator till complete crystallization.
thanks Dr.Ahmed for your valuable suggestion. I know that not all compounds can be crystallized. If the fraction of which i m talking about not crystallize in that case what i do to next. Its a green semisolid sticky mass how we purify it. Can we convert it into powder form by giving some treatment if yes please guide me.
Dear Dr. Pandya also thanks to you. the isolated fraction is soluble in ethyl acetate or slightly soluble in pet ether..............
Dear dr Mishara
Try to use sephadex LH 20 to purify your fraction with eluent CHCl3 to MeOH 5 to 5.
I think it may oily because of its nature
It may be a mixture of unsaturated fatty acids or something like that.
Your isolated mass seems to be fatty material which should be soluble in non polar solvents like hexane or petroleum ether. I would suggest you to put a little methanol in your isolate and keep it in the fridge overnight. If there is no change, it cannot be crystallized because it is surely fatty material which will be sticky in consistency.
If you get a single spot in TLC and you are confirm about the absence of any other compound in the isolated fraction, go for the identification process using FTIR first. You will get the peaks of fatty acids as a confirmation. Then proceed for NMR and Mass analysis for structural elucidation. I hope you will reach to a conclusion in this way.
Good Luck and regards
In general there are twoor three ways. If the solvent is volatile, you can wait and carefully watch an open container while the solvent is evaporating. Frequently small crystals are formed in a ring at the top of the solution on the container once the solution becomes oversaturated. By removing the remaining solution from the bottom of the container carefully with e.g. a pasteur pipette, the crystallized compound can be collected by dissolving in an appropriate solvent.
It is also possible to make use of the fact that generally compounds are less soluble at lower temperatures. If your solution is heated until it is nearly saturated (If volume too low, it leads to mass of insoluble stuff; add some hot solvent until everything is again dissolved) you can allow it to cool SLOWLY. Crystals may appear.
Another way is to mix the solution with a miscible solvent with a different polarity in which the solute would be less soluble or insoluble. Acetone would be a good candidate for a petroleum ether solution. Add to hot solution until turbidity is just seen, heat to dissolve and then cool slowly. Good luck, crytallization may be more of an art than a science.
Petroleum ether extracts usually contain Fatty acids, sterols.You dissolve the fraction in ethyl acetate(minimum qty.) and then go on adding Hexane till you see slight precipitation.Warm to dissolve and then keep in cool condition for crystallisation.
Respected Jacobus Nicolaas Eloff Sir, I applied your suggestion to crystallize compound from pet ether fraction. When i add warm sol. of Acetone to the fraction, get turbid solution then keep it to cool. After 48 hrs i saw the fraction then i found there is white color precipitate with some impurities at the bottom of the soln. Can you suggest me again what i do next? Is that precipitate may be a compound. Could i do any test for confirming that.?????
The next step is to filter the precipitate from the mother liquor, dissolve a known mass in a known volume of a a volatile solvent. Then place about 20-50 microgram on a TLC plate and separate the components. If only one compound is seen with a general spray reagent (unlikely, unless you were very careful) you may have a pure compound.
There is another way as well, if your fraction containing only a few compounds is in a glass container and the solvent is allowed to evaporate, there may be small crystals on the side of the test tube or container where the solvent has evaporated. If you remove the rest of the solution with a pasteur pipette, you may be able to dissolve the crystals that could be relatively pure with an appropriate solvent. Good luck.
Usually to determine the structure by NMR MS 90-95% purity should be sufficient.
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