As it was suggested by Jack Silver and Amr El Demerdash, TLC plates prepared with Silica gel coated with silver nitrate (Si gel is treated with silver nitrate dissolved in methanol as follows: stir 100 g Silica gel G with 10 g silver nitrate dissolved in methanol (100 mL) to get an homogeneus slurry and then evaporate the solvent in a rotavap. Use the argentic silica gel thus obtained to prepare the plates). Use hexane-acetate 83:17 to run the plate (two developments).

The best method to separate a mixture of sitosterol-stigmasterol is by preparative HPLC using an ODS column (C18 column; 5 u particle size; 10 mm inner diameter x 200 mm) and absolute methanol at 2 mL/min as eluting solvent. Use an injector with a 2 mL loop. Inject 1.0-1.5 mL of the sterol mixture dissolved in absolute methanol (solubility ca. 2 mg per mL) and collect the stigmasterol and sitosterol peaks separately. Retention time (Rt) stigmasterol ca. 52 minutes; Rt sitosterol ca. 60 min.

Try the separation in Preparative HPLC system with appropriate column,solvent system and detector. you can estimate the quantity exactly and separate the compounds too

I'm assuming silica TLC?

I can't access the paper to see if it is applicable, but the synthesis of beta-sitosterol from stigmasterol is described in the citation below. Perhaps they have a purification technique that would work for you.

McCarthy, F. O.; Chopra, J.; Ford, A.; Hogan, S. A.; Kerry, J. P.; O'Brien, N. M.; Ryan, E.; Maguire, A. R. Synthesis, Isolation and Characterization of β-Sitosterol and β-Sitosterol Oxide Derivatives. Org. Biomol. Chem. 2005, 3, 3059-3065

@ Jack Silver...Yes.. it was silica gel G TLC. Glass plates coated with silica gel G were used.

The only difference between them is a bouble bond- maybe try argurated TLC plates (TLC plates treated with silver nitrate). The silver nitrate forms a complex with double bonds so the stigmaterol, having two alkenes, may elute later than the sitosterol which has a single alkene.

Here's a link that purports to having resolved these compounds, although it looks like they ran many TLC plates:

http://scialert.net/fulltext/?doi=pjbs.2007.4174.4176

May you can use TLC grafted with AgNo3 to separat them

It is difficult to separat beta-sitosterol and stigmasterol.However you can try to use short column, and elute using pet. ether, then increase the polarity by ethyl acetate. collect the fractions with small volume. At least you will get two fractions with pure compounds, one in the first collecctions and other in latest fractions. Also you can check in GC-Ms the major compound.

As it was suggested by Jack Silver and Amr El Demerdash, TLC plates prepared with Silica gel coated with silver nitrate (Si gel is treated with silver nitrate dissolved in methanol as follows: stir 100 g Silica gel G with 10 g silver nitrate dissolved in methanol (100 mL) to get an homogeneus slurry and then evaporate the solvent in a rotavap. Use the argentic silica gel thus obtained to prepare the plates). Use hexane-acetate 83:17 to run the plate (two developments).

The best method to separate a mixture of sitosterol-stigmasterol is by preparative HPLC using an ODS column (C18 column; 5 u particle size; 10 mm inner diameter x 200 mm) and absolute methanol at 2 mL/min as eluting solvent. Use an injector with a 2 mL loop. Inject 1.0-1.5 mL of the sterol mixture dissolved in absolute methanol (solubility ca. 2 mg per mL) and collect the stigmasterol and sitosterol peaks separately. Retention time (Rt) stigmasterol ca. 52 minutes; Rt sitosterol ca. 60 min.

Acetone-hexane or pet ether is best

@Cesar Catalan. Indeed a very good method suggested by you. In first method what did you mean to say by '(two developments)'. Shall I run the same plate twice. I can't get you.

Secondly another problem is that I don't have a Preparative HPLC instrument for carrying out the method suggested by you. Is it possible to have some conventional technique to do the same like TLC (which I am trying repeatedly), normal HPLC or HPTLC?

Regards

Try two D TLC using two different solvent systems and running the plate in two direction.

Dear all

I would like to add the photograph of the TLC plate and the behavior of both the standards on the same TLC plate. I hope this will help all to understand my problem more effectively. This is the photograph of standard compounds and their Rf values can be easily seen to be very close. When they are present in the same extract, I should say they literally merge with each other posing problems in their separation. In the figure, B represents beta-sitosterol and S represents stigmasterol.

Thanks

Dear Dr. Alok Nahata,

it is not [possible to saparate the both of them by any convensional method. You ahve to opt for the Prep.HPLC. Even i have came across with the same problm. The HPTL (prep) is the only solution.

regards

Pl. refer paper ,hope will meet your requirement

@ Sanjay Poman. I could not find any paper attached with your post. Please attach the paper.

Thanks and regards

Sorry i missed my answer in previous posting.

Try Acetone : Hexane ( 1:3 ) solvent system on MerckMillipore TLC plate Silica 60 20X20 format for initial work up. You can spot 1000 ppm solution in pet ether on prep. plate of the same material.Pl. revert.

Thank you sir I will try...

Were you able to solve the problem of separation of sitosterol from other sterols? If so what methods did you use?

It is very difficult to separate both componds. A column chromatography using silver nitrate impragnated silica gel would be a good attempt.

GC-MS is best technique to identify Beta-sitosterol and stigmasterol in any plant extract sample. It is single quadrupole technique. The source use is Electron Ionization (EI).

@sanjay Poman. Could you please share that reference that you mentioned? Please do add the citation if you are unable to attach it. Thanks.

In some plant extracts, sitosterol, stigmasterol and campasterol appear as ternary mixture which is inseparable in normal phase silica gel TLC in various solvent systems. However, derivatization (like acetylation) may improve the situation. Trimethylsilyl derivative of the mixture followed y HPLC analysis will help in quantification of the compound.

Solvent system= Chloroform: Methanol (8:0.6), then spray with H2SO4 (10 % in ethanol) and heat in oven at 110 degree Celcius for 5-10 minutes. Pink spot of beta-sitosterol at Rf 0.34 and stigmasterol at Rf 0.85

spot the plate and place it in iodine chamber before u run into mobile phase. The iodine will saturate the double bond of stigmasterole and thats make the structure more different than beta sitosterol. Run the plate to 2 cm. Dry it in oven and run again upto solvent front (~8 cm).

Dear ketan variya sir , what mobile phase should be used ?

You can not separate beta-sitosterol and stigmasterol on same TLC plate. They are too similar; you should use HPLC.

K. Naumoska and I. Vovk, Analysis of triterpenoids and phytosterols in vegetables by thin-layer chromatography coupled to tandem mass spectrometry, J. Chromatogr. A 1381, 2015, 229–238.

Dear Alok Nahata

As suggested by J.  Anyam,  it would be very difficult to separate beta-sitosterol and stigmasterol on the same silica gel TLC plate. These two compounds are too similar and  too close each other on TLC. HPLC should be use.

I will suggest that these can be separated by HPLCanalysis as suggested by Cesar Catalan.

Kindly refer to this publication  Rapid purification of gram quantities of β-sitosterol from a commercial phytosterol mixture

Narayanan Srividya · Deanna Brooke Heidorn · Bernd Markus Lange

BMC Research Notes 2014, 7:182

For larger quantities its easier to purify using column chromatography with other methods in tandem.