I am working on natural products and I am facing too many problems while separating compounds from crude plant extract. I tried all the ways, but it was no use. Now the ultimate step is to separate compounds by preparative HPLC only. But we lack that facility in our lab. So, I need your help in this regard. If anyone has this facility, kindly provide me an opportunity to do this work. And my mentor is ready for collaborative study also.
Use XADs, HP-20 resins followed by C-18, sepahadex LH-20 packing material for separation of natural product compounds.
I ve gone through ur question. I am also facing the same problems at last i find it. But it cost upto 25,000 per sample for preparatory. If yu want to separate it send samples with standards.
Use XADs, HP-20 resins followed by C-18, sepahadex LH-20 packing material for separation of natural product compounds.
I agree with the answer of Dr. Robin. Did you try to isolate the compounds using other than conventional chromatography stationary phase? e.g silica gel for column chromatography or alumina? Well, Prep.HPLC is only tool, but you have to decide before that which stationary phase you really need. Best Luck.
@Robin, dude I did the thing what you said in our convesation before. For another extract am not getting suitable solvent system for TLC optimization. What to do for that?
if u could have mention what type of extract eg. methanolic , water extract etc it could be easy to tell TLC mobile phase.
Most of the preperative work for natural extract purification is done on preperative layer TLC plates.95% of the work is done on silica surface.Pl. revert.
Let's try a simple way first, by using selective solvents for extraction. Firstly Hexane and then DCM, finally MeOH. Which one we want to continue, Non polar fraction (in Hexane), semipolar fraction (in DCM) or polar fraction (in MeOH) ? later on we can seperate the choosen fraction by VLC methode (Vacuum Liquid Chromatography) as a simple and flash method.
You said you tried all the ways. What were they? Perhaps that information can help us to help you. Also, is the extract polar or non-polar in nature?
Another cheap and efficient way to assist separation is to derivatize the extract via a chemical reaction (eg. acetylation, etc) which can be easily removed later.
You can also try on prepTLC. Separate your compounds on prepTLC plates and isolate as said by Sanjay. Because prpTLC is simple and cheap method. if you will get prep HPLC, there are so many problems with that-like standarisation of mobile phase, separation, resolution, collection etc. So you should first try with prepTLC then go with prepHPLC.
Gudluck
:)
Like Dr. Andrew Lamm said, we really need more information to help. The plant species would be helpful since certain groups of plants tend to make certain compounds.
Flash chromatography is an alternative to prep HPLC, especially if you can get an automated system. Glass columns will work but care is required to pack the columns and it will take more time.
Here's a link to a poster where I purified flavanoids entirely with flash chromatography:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
Depends on the compound types, before prep HPLC separation, you can try a similar way using reversed phase column chromatography with C-18 silica gel as the sorbent. RP-TLC will help you to determine the suitable solvent system.
I will add to above Achmad_Hafid/ comment, using solvent partitions to your crude extracts before doing any column chromatography separations
Isolation of phytochemicals can be quite cumbersome. But I dont agree with you that you have "tried all the ways." It would have been helpful if you had told us those methods you tried. However, I suggest you consider the following, if you have not yet done so. Do SUCCESSIVE partitioning of your extract into different solvents of different polarities e.g hexane, ethyl acetate, butanol and methanol. Or use acid-base extractions as explained by J.B. Harbone (Phytochemical methods). You can now run prep TLC on each fraction after ascertaining a suitable solvent system using normal TLC. This method gives you an idea of the polarity property of components in each of the fractions. For instance, it is expected that the hexane fraction will contain non polar components etc. This will increase your chances of isolating the components. Another approach is to isolate components by using methods selective to them. For instance, there are general methods for isolating flavonoids, saponins, alkaloids etc which are available in literature ( I suggest you consult J. B . Harbone's book). But whichever approach you follow, isolation of plant components is a tedious task. If you are lucky, you get quick results, otherwise, you have to "sweat it out." I wish you the best.
What type of extracts you are working on? I mean which solvent extract. If you are working on low to medium polar solvent extracts it will be easy for you to separate compounds from these extracts by normal column chromatography. If you are using high polar solvent extracts then you can go for HPLC/Preparative HPLC
I have found using a succession of different techniques works well. Preparative HPLC is good as one of those techniques but I have not found it works on its own, as each HPLC peak of a crude extract may still contain several components. I have used a combination of LH20 columns (to separate by size/shape), normal phase silica columns and reverse phase columns to be effective. However, one of these has always been a high performance system (e.g. HPLC). One high performance method I have used with success is HPTLC - high performace silica TLC plates from Merck. They give much better separation than ordinary plates. They are good as a last step as they will only take 10mg, and I'm afraid they are not cheap, but they do work well.
you could use pre TLC plates or column chromatography , eluting the column and collecting fractions which you can check by TLC
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