I've used methanol and water (100% methanol to 100% water) for the isolation and the column was packed using silica gel (60-120 mesh size) and methanol. But, the purity of the active fraction was as low as 70-80%. Importantly, no peak (signal) was observed in 13C NMR. Thank you in advance.
Did you use RP silica gel to run the column? If not, then you might have eluted all the binder out of normal phase.
If you had used RP silica for isolation and gone for 13C NMR, I think concentration is too less or number of scans are not sufficient.
I would suggest you to use gradient elution for isolation of compound. that would be more suitable to collect more components. I would suggest you read the articles and books suggested by Dr. Rao above.
Actually, if you're not familiar with the isolation job, I suggest you to co-work with an isolation specialist. Compound isolation requires much time, efford and experience. Doing it yourself would not be efficient, I guess.
Isolation from plant products is not so easy as many people suggested.
As Rajeswara Rao read some basic principle in isolation from natural resources, generally people used to extract in different solvents like chloroform, methanol, toluene. Based on the polarity difference, compound will be in extracted in respective solvents concentrating the solvent and followed by purification by gravity column.
Final purification generally made in HPLC as well as to confirm the purity, later they used to proceed for characterization.
Hope this would provide an idea to you, but i strongly recommend to follow Rajeswaran Rao advice
It appears you are running aqueous normal phase (also called HILIC), which is a useful technique, and may be entirely appropriate for water soluble compounds.
Ways to improve the purification on silica include:
- Dry load the sample; this often improves the resolution greatly
- Pack your column in 1-2% water; this forms a hydration layer which improves the chromatography
- Other organic solvents are useful for HILIC such as acetonitrile and acetone, which are miscible with water and may cause selectivity changes on silica
- If you feel that you need to add a solvent modifier, use an acid (such as acetic acid, formic acid, or trufluoroacetic acid), NOT a base (like ammonium hydroxide). Silica dissolves above pH 7.5-8 when using polar solvents.
You can also consider other columns such as alumina, diol, and amine columns. Also consider ion exchange columns as well.
Here are some references:
Silica flash HILIC:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN84_RediSep_Rf_Gold_Silica_and_Highly_Polar_Solvents.pdf
Other column types:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN78_HILIC_Purification_Strategies_for_Flash_Chromatography.pdf
Poster, with another silica example:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Strategies_for_the_Flash_Purification_of_Highly_Polar_Compounds.pdf
Ion exchange applications:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN87_RediSep%20Rf_SAX_Column_Applications.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN40_Purification_of_Alkaloids_with_RediSep_Rf_SCX_Columns.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN98_Hints%20for%20Strong%20Ion%20Exchange%20Resins.pdf
you can also use rotavapour for the isolation of compounds like alkaloids etc.
Check this link for several publications:
http://www.slideshare.net/rahulbs89/extraction-of-plant-contituents
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Pharmaceutical Botany
- Plant Extracts