I am trying to develop a solvent system for HPLC analysis of my extracts.
What are you looking for?
There is really no single solvent that can resolve everything in a methanol extract. I've isolated everything from compounds with a steroid group (non-polar, lipophilic, TLC with hexane/ethyl acetate on silica) through alkaloids (dichloromethane: methanol TLC system on silica). Please note I'm assuming you mean a silica TLC system.
actually it is crude methanol extract. I want to find suitable solvent system for further column and HPLC. My extract amount is small. I want to analyze the crude in HPLC. Is it Possible? But usually i am looking for phenolics,flavonoids.
I suspect it will need some cleaning since there will be alkaloids and maybe some other things likely in the mixture. You can use hexane/ethyl acetate on silica and many phenolics and flavanoids will move well. Some phenolics (anthocyanidins, tannins) may need a stronger solvent system DCM/methanol. Methanol water could be used as a solvent system, but not on TLC.
I've had good luck extracting phenolics on an SAX (strong ion exchange) column followed by C18 pourification. I also found an LH-20 step also helped clean the mixture. Here a poster on how I purified these compounds:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
You can also try polyamide resin (several gradients: water to 100% methanol followed by methanol to 100% acetone; finish with acetone to 100% water containing 5% ammonia.)
Thank you very much for your suggestions. I am very new in natural product research. things r little bit difficult to me. I hope it will help me. thank you again.
The binder in TLC plates is generally gypsum (calcium sulfate) or silicic acid. The gypsum binder often falls apart when exposed to water, in my experience.
Solvent less polar than methanol at diffrent proportion...chloroform...Dichloromethane....ethyl acetate
Here's a collection of flavanoids and how they were purified:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN85_Purification_Strategies_for_Flavones_and_Related_Compounds.pdf
What about methanol with a combination of other less polar solvents?
Depending on the solvents (like hexanes) you may have miscibility problems. Try ethanol instead. Isopropanol works but it's viscous (higher pabk pressure or slower flow rates). Solvents that work with methanol are listed in Dr. Brajesh Kumar's post.
Thanks for your continuous suggestions which are very helpful for me as a beginner. Sometimes people use filter paper in the TLC tank during TLC for saturation. What is the benefit of saturation actually?
You need the TLC tank saturated with vapor for 2 reasons. First, the plate runs faster if solvent isn't evaporating from the plate. More importantly, it helps maintain the solvent mixture as the plate develops. If one solvent evaporates faster than the other, you'll get a retention factor different than one where the TLC chamber was saturated. With some solvents, it can take some time to saturate the chamber. Minimize the time the cover is removed from the chamber when inserting or removing plates.
so we should saturate first and then insert the TLC plate, right?
Dr. Huq- yes, you should. The filter paper helps that to happen faster. I know some people that keep ~10 (or more!!) TLC tanks filled with their favorite solvent systems (they do a lot of TLC/flash chromatography) and refresh them once or twice a week. They do a lot of this work so they save time because the solvents are mixed and the tanks are saturated.
thank you very much. I am using FeCl3 as visualization reagent for flavonoids. but the color I see it is also seen after usinf anysaldehyde and heating. but anysaldehyde shows more spots(probably presence of terpenoids). but what color should show after using FeCl3 and heat for flavonoids/phenolics?
Could be several colors- purple, green, blue. Enols will react too. I saw a reference mentioning that steroids react to make red spots with acidic ferric chloride.
Can you refer any book on visualization reagents and colour changes for TLC?
You can refer the following books which are considered milestones in Chromatography, this will definitely end up ur search regarding the querry you have now:
1. Stahl E (1967). Text Book of Chromatography, 2nd Ed., Springer-Verlag, Berlin.
2. Wagner H, Baldt S. (1996). Plants Drug Analysis, Springer Verlag, Berlin Heidelberg,
3. WHO (1989). The Validation of Analytical Procedures used in the Examination of Pharmaceutical Materials, WHO/Pharm. 89541, rev 2 Genf,.
4. Harborne JB (1973). Phytochemical Screening Methods In: A Guide to Modern Techniques of Plant Analysis. 1st Edition, Chapmann and Hall, London.1973
Regards and gud luck
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