How can I Separate the Alkaloids, glycosides, Triterpenoids, Seponins, Flavonoids from the crude extract without loosing any of the compound by single extraction procedure.
Another, simple, method to extract the alkaloids is to partition them between dichloromethane and aqueous acetic acid (pH 4). After extraction with acid, take the aqueous layer and make it basic with ammonium hydroxide and partition again with dichloromethane. The free-base alkaloids will be in the organic layer after the second basic extraction.
Non-polar compounds will remain in the first dichloromethane extract, while polar compounds will be in the aqueous layer. The remaining salt in the aqueous layer is ammonium acetate which is volatile and can be removed with the water. Formic acid would work too, and also makes a volatile salt with ammonium hydroxide.
for alkaloids use this method
Total alkaloid extraction
The dried powder of each plant leave (50 g) was mixed with 20%
alcoholic granulated and dried at room temperature before the
extraction of total alkaloids. The granulated powders were extracted
with benzene for 6 h. The extracts were shaken with three
successive portions of 25 cm
3
of 5% sulphuric acid and decolorized
by heating with activated charcoal. The hot solution was then
filtered using Whatmann No.1 filter paper. The combined filtrate
was acidified with ammonium solution (pH 8.5), transferred into a
separatory funnel and extracted with three successive portions of
20 cm
3
chloroform and the extracts combined. The chloroform was
distilled off to give the total alkaloids extract (Kokate et al., 2002).
Total alkaloids extracted from the leaves were tested against the
targeted microorganism using disc diffusion method. Stock solution
of the extract was prepared by dissolving 20 mg of each extract in 5
cm
3
of chloroform to obtained 4000 µg/ml. Using a micropipette, 0.3
ml, 0.2 ml and 0.15 ml were separately drawn into vials and the
volume adjusted to 2 ml to give approximate concentration of 6x10
,
4x10
2
and 2x10
2
, respectively. A control was similarly set up using
only chloroform.
regards
please refer this book phytochemical methods by Harborane
also pharmacognosy by Tris and Evans
also refer research paper regarding phytochemicals related with your plant
Alkaloids and flavanoids can be captured by ion exchange chromatography. The neutral compounds can elute off silica. Alternatively, I run something I call "Wide Polarity Range" chromatography; essentially a series of chained gradients.
Alkaloid ion exchange purification:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Isolation_and_Purification_of_Alkaloids_with_SCX_Columns.pdf
Alkaloid purification:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Alkaloid_Purification_Strategies_Using_Flash_Chromatography.pdf
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN59_Purine_Purification_Strategies.pdf
Flavanoids:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Purification_of_Phenolic_Flavonoids.pdf
Flavanoid ion exchange:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN87_RediSep%20Rf_SAX_Column_Applications.pdf
Wide Polarity Range:
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Wide%20Polarity%20Range%20Flash%20Purifications.pdf
IF you wan pure compounds go for open column chromatography silica gel thats the only way you can isolate in pure form for identification of the compounds through spectra or crystallography and use Libberman and other reagents to identify the class.
Dear Mr. Jack,
Thank you very much for sharing informative knowledge with me.
Dear Ms. Sujata,
if you throw some lights on separation of separation of total alkaloid from crude extract same way saponins, coumarins, terpenes, etc. from same extract without loosing any of the biomolecules.
If you dont want to loose quantitatively I think you have to isolate first open column chromatography and then do the various test and the structure elucidation will tell which group of compounds it belongs to. I am just sharing my work .
Dear Ms. Sujata,
Thank you very much for sharing your knowledge.
With regards
you are welcome just try to get crystal I think it will make things easier
Another, simple, method to extract the alkaloids is to partition them between dichloromethane and aqueous acetic acid (pH 4). After extraction with acid, take the aqueous layer and make it basic with ammonium hydroxide and partition again with dichloromethane. The free-base alkaloids will be in the organic layer after the second basic extraction.
Non-polar compounds will remain in the first dichloromethane extract, while polar compounds will be in the aqueous layer. The remaining salt in the aqueous layer is ammonium acetate which is volatile and can be removed with the water. Formic acid would work too, and also makes a volatile salt with ammonium hydroxide.
But if you want to isolate in the form which was in the plant ,addition of bases might alter the structure as even in extraction method compounds break down .If it is for estimation may be it will be ok.
For triterpenoids you can use chloroform/methanol increasing the polarity from non polar to polar
Dear Devendra, each of the classes of compounds you listed has well known specific methods of isolation. It will probably be difficult giving you details on each of them through this medium. I therefore suggest you consult the book "Phytochemical Methods, A Guide to Modern Techniques of Plant Analysis' by J. B. Harborne. You will find answers to your questions in this excellent book. Good luck!
Start with hexane. Extract your plant material with hexane. You will get non-polar compounds viz. fatty acids etc.
Then Extract with chloroform for alkaloids followed by column chromatography for purification of chloroform fraction.
Use butanol (sat. butanol) for triterpenoid saponins followed by column chromatography
Remaining plant material with 70% methanol for phenolics.
Gudluck
:)
Hello Robin..whatever you suggested is simple. I like it. It will be more beneficial as you give details with references. Thank you.
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