After extraction (in Soxhlet) of Prunus spinosa L. fruits, I want to find content of phytosterols (beta sitosterol) in extract using the HPLC.ED. Any suggestions?
In TLC system Hexane/Acetone (3:1) b-sitosterol has a retention factor of 0.5 approx. and it worked for me. In fact, such phytosterol was precipitated by adding acetone to the fraction.
Methanol: water (80:20) has worked in many cases. Otherwise Acetonitrile:water (90:10) is also found useful in phytosterol HPLC analysis
Gud Luck
Regards
For TLC analysis, the best solvent system for Phytosterols is Chloroform:methanol (8:2). You can try the variants in dilution in this case with these two solvents. Further addition of toluene will also help to get a better resolution.
Its my own experience with phytosterols and I got very good results with this solvent system.
Hopefully it helps you too.
Again gud luck
Best Regards
GC would be jettera khan HPLC for phytosterol separation either in native form up to 300 degre or derivatized ( BSTFA ). Anyway HPLC or TLC could be perfor med with poor resolution between the different phytosterols (ODS with ACN /water 70% to 90% gradient for HPLC with detection : MS or LSD or UV 250 nm).
Hopefully it can help you .
Before going to TLC or HPLC you need to do a simple identification test for your Phytosterols in plants . The test is that , extract your Phytosterols containing extract with chloroform.Take 1ml of this chloroform extract in a test tube and add 0.5ml of acetic anhydride and then add 1-2 of conc H2SO4 and observe the changes. You will observe a brown or red color ring in the test tube and a greenish or bluish coloration over the ring, which indicates the presence of Phytosterols.
First of all, you should perform the Liebermann- Burchard test for phytosterols and then proceed for chromatography. It mainly depends on which type of extract you are using. If you are using a hexane or petroleum ether extract, then Hexane:Ethyl acetate (8:2) is the best and if you are using an ethanolic extract, then chloroform:methanol is undoubtably the best.
Phytosterols are also spotted in ethanolic extract very often. So choose the solvent as per your conditions.
Gud Luck
If you aim to isolate single sterols or conjugates, generally, solvent extractions with hexane, methylene chloride, or chloroform are used.
THIN-LAYER CHROMATOGRAPHY. A Laboratory Handbook. Second Edition, Revised and Expanded. Edited by Egon Stahl; This book is useful for tLC studies
Try with Chloroform: Methanol (V/V) if its not working then try with Chloroform: Methanol: Water V/V
yes for TLC and HPTLC of stigmasitosterol
Toluene:Methanol:Formic Acid (7:27:0.3) and Absoption at 530
TLC and HPTLC of Betasitosterol
Toluene:Ethyl Acetate: metahnol: Glacial Acetic acid (8:1:0.5:0.3)
For both sterols you have to use RP-HPTLC plates for good resolution.
for more details refer Stahl.
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