Chromatography Forum

I am looking for a method for separating brassicasterol, campesterol, stigmasterol, and B-sitosterol. I did find a partial method that uses a C18 column, but the stigmasterol and the campesterol co-eluted (if i remember correctly). Any help would be a great help!

Doug

Did you already try a C8 column? Campesterol has one carbon less the sitosterol and stigmasterol has a double bond more then sitosterol. rom my experience these compounds will most likely elute together on a C18 column.

How are you detecting these sterols? Their UV response is not very good. Perhaps you do not require a sensitive method?

Sterols like this can be separated very easily by GC with much higher efficiency than HPLC; they work better if you make the trimethylsilyl derivatives. Maybe you don't want to go down this road though.

I plan on using a ELSD for detection. I have already separated them by GC, but I need a small amount of one of the sterols and was hoping to be able to separate it out of a mixture.

I have been fighting with sterol separations for quite a while...
My problem is related to phytosterol esters (fatty acid esters) in combination with fish oil included in the matrix.

I did have one try where I hydrolized my phytoesters before analysis which simplified my chromatogram, however it didn't help because these components co-eluted with fish oil components. To cut to the chase however, I got good separation of the free esters from each other with these conditions.

M.P.
A = 5%ACN/95%IPA
B = ACN
60%A:40%B Isocratic
Column oven @45C
UV detection @204nm
Phenomonex Gemini C18 5u 4.6X250

The K' of most of these peaks is proportional to the amount of ACN you have, you can increase-decrease resolution fairly easily by adjusting the %B component.

Also If I remember correctly there is an application in one of the back issues of LC-GC's application notebook for separating free sterols... Unfortunately I don't recall which issue.