Chromatography Forum

Hi all,

i wish to know what mobile phase can be used in reverse phase HPLC for separation of phytosterols? i am new to HPLC and need genuine suggestions. Please help out!!!! its URGENT

A Google search on the words "phytosterol" and "HPLC" returned 32,500 hits.

ya i know that.....thanks for this expert advice.

i would like to seek some genuine advice. Please help

That *is* expert advice. The first thing you need to do is to read some of those references to get some idea of the columns and mobile phases that have been used. Then you can come back with more specific questions to help you make an informed decision.

Try this: ES Industries cn-hs column, 2.5% isopropyl alc in heptane, 1 ml/min, 40 deg C. ELSD detection. Isocratically separates many phytosterols such as those in rice bran fatty acid distillates and related cmpds. You can play around with a shallow ipa gradient if you need to but those conds are a good place to start. Also not all CN columns are created equal it didnt work on a CN column for RP, eg the Zorbax SB-CN. CALL ES Inds and they can help you out too. Good luck!

I know you asked for RP but trust me NP works great for these cmpds.

should i continue analysis of phytosterols with HPLC or switch over to GC? as the problem i am facing is detection via UV detector in HPLC

@ljc
Initially i also assumed dat Np will work best with these lipids but afte going through literatures, i found that RP is better option as phytosterols are polar lipids. what do you suggest? and why?

The phytosterols don't give good UV response, if any, we used evap light scattering, and the NP eluent works really well with that detector 'cause they're so volatile. NP chromatography is kindof a "lost art" but modern bonded phase NP columns are very reproducible and re-equilibrate quickly. The problem is that method dv is tricky as just small amonts of polar modifiers lead to big swings in your selectivity and RTs. Also the waste handling issues, etc. But if you found a good reference for RP go for it. I'm still not certain as to the UV response of these compounds, to my recollection they were poor chromophores.

Only as a suggestion. The UE has published a Reglament 2568/91 over Olive oil that includes the analytical procedures for this Oil.
It includes the dfferent sterols separation (TLC) and determination by Capillary GC as a way to look for possible adulterations.
Perhaps you can profit this method