My Natural product, Methanol extract when filtered some solid compound remains and it is only soluble in water not in any other solvents. It contains a mixture of compounds. How can I separate the compounds present in crude extract by column chromatography.
If we are using water and methanol ratio as a mobile phase it will be little bit difficult to distill the solvent and to separate compound from water again.@ J-Y Jo
Yes Mr.Mariusz u are right, the problem is still active and it will majorly occurs in the areas like this natural products
Can u suggest any thing...
Ok. Then which type of compounds you want separate? Polar from nonpolar? Or maybe you want make some chromatography open column only polar and semi polar compounds?
Sir actually while extraction of plant compounds itself I had extracted separately from Non polar to polar solvents like Pet ether or hexane, ethyl acetate, methanol or ethanol and water extracts. I am trying to separate compounds present in crude Methanol extract by Column chromatography. I have started column with hexane and increased the ratio with ethyl acetate and after getting 100% ethyl acetate, i have run the same column with increasing ratio of Methanol. I have added methanol to ethyl acetate and running the same column. Now the problem comes....the compound comming out when distilling it, it is sticking to Round bottom flask itself...It is not getting dissolving in Methanol and it is slightly dissolving in DMSO and completely dissolving in Water... Now the problem is the compound is having 2 to 3 spots in TLC and how can i separate it and how can i proceed it further???
1 THING - TRY USED DRY LOAD SYSTEM - MEAN -
1. if you have something on the bottom of flask - weight, and then dissolv in:
1.A - first small volume of water and add some volume of methanol - you must check how many methanol you can add.!!
1.B - then add 1 g silica gel - (its depend from how many mg you have for inject 1g sample for 100 g silica in column plus add 1 g silica for sample - something like that) - and evaporte under vacume in rotary, and then you could liophilized. - you must recive dry silica gel with yours sample inside them.
2. Prepare column (must be wet and calibrate with mobile phase) IMPORTANT - level the mobile phase has to bee this same what adsorbent, very careful apliicate your dry silica gel with sample on the top, press gentle and start :))))))
3. first delicate for wetting this silica and the go on.......
SECOND - try C18!
maybe yours coumpounds have to few differences between polars activity, so you try -
1. put 1 g C18 in the syringe 6 ml reservouir - with frit on the bottom and top - press.
2. make activations with pure methanol - 5 ml to the waste, then water - 5 ml to the waste, and then mobile phase - 5 ml to the waste - i SUGGEST GRADIENT CHLOROFORM:METHANOL FROM 100:0, 90:10, ETC TO 0:100,
2 TIMES VOLUME OF COLUMNS MEAN APROX. 2 ML IS.
3. COLLECT EACH FRACTIONS AND CHECK WHAT YOU HAVE.
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