what is the best method to reduce sample loss during purification process?I did column by using silica gel to remove unwanted compound. but, the yield of the compound is about 30% of the starting material.
is there any suggestion for the purification process rather than by using silica gel column chromatography (solid phase extraction) to reduce the loss of target compound.
my compound is extracted from the natural product.
a) Recrystallization (using two solvent), but it depends on solubility of your key compound and impurities
b) Chromatography on silica gel plates (you can make plates yourself), but/ of course, it limits amount of unpurified compound (but you can use a few plates)
In a case of unsuccessful separation you can washed you compound from plate with appropriate solvent
And HPLS (if you have such equipment)
Minimum loss purification technique is column chromatography. Use 200-400 or 400-700 mesh size silica depending upon your spots difference and resolution.
Others techniques are selective precipitation and slurrying.
There isn't quite enough information to answer the question properly.
Natural product extracts are often tars which contain a significant volume of extraction solvent. It is possible that your sample contains this solvent? After purification, the resolved peaks allow the solvent to evaporate more easily than the original tar.
What solvent system are you running? It is possible that the solvent isn't strong enough to elute the more polar compounds. I use a technique that I call "wide polarity range chromatography" . I essentially chain solvent gradients together so that I elute nearly everything and purify the desired compound in the correct solvent polarity- in natural products, one often doesn't know which compound is active. The technique can be used with silica gel. One can run hexane->ethyl acetate -> methanol -> water. The links describing wide polarity range chromatography are below.
Another possibility is that some compounds are volatile and are lost during evaporation- how did you extract the mixture? Such compounds are often found in a hexane extract.
Here is the link to wide polarity range chromatography:
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Application%20Notes/Wide%20Polarity%20Range%20Chromatography%20App%20Note.pdf#search=wide%20polarity
I've extended the technique to silica as well and use the technique as part of a column screening program:
http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20Poster.pdf#search=wide%20polarity
Note that good silica gel (made from high purity silica) doesn't dissolve in water or methanol unless the solution is above pH 7.5. Keep the mobile phase neutral or acidic!
what the polarity of your compound of interest? Depending on the polarity and of the grade of purity you can use liquid partition and/or cromatograph in fase reverse.
regards
Having specific details of the compound is critical for any useful suggestion. If not interested to share in public you can reach me in private at [email protected]. I have expertise in the purification various compounds from natural products.
Cheers
Jagan
It is immanent to any separation technique that the mass of your isolated target compound is smaller than the mass of the starting material. There would be no purification otherwise. If the recovery is much smaller than expected, you should consider that the analytical methods that you used for sample characterization had not been able to recognize a major part of the impurities. For example residual sovents as stated by Jack Silver, or sugars if you use HPLC-UV.
Many thanks for your comments and help. really appreciate it. will try any possible method that has been recommended.
I think LC is the best for purification of compounds but it depend on various factors and u must have a concrete background in chromatography. Other techniques for purification of compounds are Distillation, Re-crystallization, Sublimation etc. But for your purpose, first do extraction and then go for LC.
Thanks
Preparative HPLC is there for purifying the compounds. Small Quantity of compound can be purified using this Technique.
Re-crystallization is a method other than column chromatography to purify compound. For this, You have to remember the solvent in which your compound should be soluble not impurity.
Purification method will depend upon the nature of sample and the possible matrices. Samples with dirty and complex matrices are best cleaned up by column chromatographic methods. Today there are many techniques available for this purpose like SPE, GPC etc.
try polyamide column i think it doen't maintain mass but generally it used for separate flavonoid
Or you may try to precipitated your compound in organic solvant like acetone or chloroform. .. based on similar study of your compound
Good luck
check for literature,if your compound crystallizes in any solvent like acetone,methanol or IPA...better do re crystallization process for pure compound
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