try isolation with nonpolar solvent ,like nhexane or petroleum ether. you can even try preliminary analysis by cold extraction with methanol overnight and then run itsTLC with toluence:methanol (8:1)and checking it on uv as well as after devertatsing it with 10 %methanolic suphuric acid result it may come pink or blue. Also refer some paper for further perliminary tests.
Detection of phytosterols:
Salkowski’s test: The extracts were treated with chloroform and filtered separately. The filtrates were treated with few drops of concentrated sulphuric acid, shaken and allowed to stand. If the lower layer turns red, sterols are present. If lower layer turns golden yellow triterpenes are present.
Libermann Burchard test: The extracts were treated with chloroform solution and few drops of conc. 1 ml of acetic anhydride solution followed by sulphuric acid. A blue green colour shows the presence of phytosterols.
I think you have to try with Lieberman Burchard test to be sure you have steroids in your material. If you have it, they can be as aglycons or glycosides, the first one are less polar you can extract them using dichloromethane , the others are more polar and you can use methanol to extract them.
Try extraction with nonpolar solvent follwoed by unsaponfication of extract. Then perform TLC to pin point your steroids using Anisaldehyde Sulphuric acid reagent. If you are intrested in isolation then go for column using combination of hexane and ethylacetate as eluent as per your experimental condition
To isolate:
Most steroid (non-glycosidic steroids) will come out with less polar solvent extraction (hexane, dichloromethane). Separation could be done with traditional column chromatography using hexane:ethyl acetate. Just let the tubes (fractions) dry in your fume hood, relax and come back in the next day, most steroids will give white solids around inside tube's wall (i.e tiny needle). You can always re-purify with another column chromatography or prep-TLC using high grade solvent (HPLC grade). This will remove grease contaminant (i.e from lower grade solvent used in the first column).
Cheers.
Procedure for extraction of steroid is, that powder plant material is extracted with Ethanol , filtered , dried the filtrate in china dish on water bath .The dried material present in the dish is dissolved in chloroform , filtered and used for steroid identification test . Both tests proposed by Mr. Sandeep sachan are the best for steroids identification but I prefer the Libermann Burchard test: The extract is treated with chloroform solution and few drops of conc. 1 ml of acetic anhydride solution followed by sulphuric acid is added .A blue green/green color shows the presence of steroids or phytosterols.
Test for Steroids (Salkowski test): Five ml of chloroform extract was mixed concentrated H2S04 (3 ml) and shaken. If the reaction is positive it gives red coloration on standing. Liebermann-Burchard and Molish test also good to detect steroids.
The procedure for extraction of sterols is: extraction with 80% ethanol and fractionation with pet ether, chloroform, ethyl acetate and n-butanol. the most of steroids go to chloroform fraction. subjecting the CHCI3 part in SG column with eluthing the gradient solvent with petroleum ether/EtOAc (20:1-0:1) to yield steroids.
Liebermann -Burchard and Salkowskii tests are the best for detecting the presence of steroids in plant extract,while column chromatography is best with N-hexane and N-hexane; Ethylacetate mixtures as eluting solvents. Most steroids will be eluted from 95:5 to 80:20
The brief steps are:
First of all you isolate the pure fraction from plant root extracts using following techniques. First run TLC to know the type of fractions. After that Column Chromatography with HPLC, after isolated pure fraction run FT-IR, LC-MS, HNMR, CNMR for structure elucidation. For phytochemical tests you can read my papers too.
Dear Jitendriya Panigrahi
The attached paper may be useful to you for planning your research now and in future.
Good luck.
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