Chromatography Forum

Friends,

I have had some frustrating experiences trying to develop a good HPLC procedure for sterol analyses (Sitosterol, stigmasterol, etc). These compound have poor UV absorption, they are difficult to dissolve in aqueous solvents (even when you initially start with 100% organic).

The basic problem, besides the poor UV absorption, is that they have low solubilities in aqueous solvents (H2O+ MeOH, ACN, Acetone, iPrOH, etc) and sometimes require down to 30-50% organic content in the mobile phases in order to be adequately retained in RP columns. This makes MPs and sample solvents not very compatible and results in poor peak shapes.I have not tried normal phase yet.


Can anyone recommend RP columns that could have more retention for these compounds and therefore require higher % organic mobile phases?. This could make the MPs and sample solvents more alike. I have tried Zorbax SB-C18 and Luna C18 (2) columns with some success. Has anyone tried C30 or Hypercarb columns?, Is there anyone with experience using silica columns and Normal Phases MPs?

thanks,

josebenjamin

Is there any special reason why you like to use HPLC. Sterols are easily analysed by GC (MS), often as acetates.
If you go for HPLC, try normal phase

I agree with Alex-GC is a much better method. You can get much higher efficiencies by GC, detection is simple with a flame ionisation detector, and you can use good solvents for sterols like hexane and dichloromethane. There was discussion of this in the "cholesterol in eggs" thread a week or so ago.

Friends,

It is true that GC sounds more attractive in several ways. However, I am in a hurry to get this done and I do not have the right GC column and equipment, and reagents. That means, short column with thin film and high temperature phase, and on column injection. Do you know how well this can be done without derivativatization?

If you see the literature on this particular subject you will find that most references report LC analyses, but they rarely show a chromatogram. Those few that I have found on GC analysis use derivativatization and on column injection.

If you have specific information on results of underivatized sterol analyses I would like to see it.

Thnaks,

josebenjamin

Derivatisation to the acatates is easy: just add 1 ml acetic acid anhydride and 1 ml of pyridine, leave it over night and evaporate to dryness (o.k. evaporating can take some minutes)

Alex

You can try analysing sterols by GC without derivatising them. I think you will get some peaks. How quantitative the analysis is though, I do not know. It is possible that you will get problems due to the adsorption of the compounds on active surfaces. Probably this will depend on the inertness of your column and any dirt which has accumulated in the front end from previous use. The performance may deteriorate as the number of samples analysed increases.

Victor, Alex,

Thanks for your comments. Looking at all the options I have now, it seems like Normal Phase HPLC is the best one. The solubility problems here will be less and the solvents and mobile phase compatibilities will be much better.

I have found a reference that describes argentation chromatography as a good technique for esterols. This is fine, but the kind of selectivity it provides is not really necessary for the samples I have.

The GC option is still open, but I have to configure an instrument to do on-column injection and buy a special column as well.

josebenjamin

I have seen compounds VERY similar to these analyzed on an Alltech Platinum EPS C18, with an aq. 0.1% phosphoric acid/acetonitrile gradient, with very good resolution of isomers.

Although these sterols are nominally covalent, the presence of 0.1% H3PO4 does sharpen the peaks.

Good Luck !!!

JMB