Chromatography Forum

Has anyone else dealt with phytosterol esters analysis by HPLC?

I have used GC for this type of analysis before, however it was a mess, derivitization using MSTFA in pyridine. Columns tended to die rather quickly because of the high temps required to get a separation. This is not a usable method for me anymore because I am now testing for phyto-esters in a matrix that is mostly fish oil.

Now I have been working with HPLC, UV detection at 204nm, using isocratic ACN:IPA.

The problem is this: To get a good separation from fish oils and phyto-esters I need selectivity that will separate the fatty acids from the phyto-esters. With column selection I can get a separation of oil from phyto esters, but without selectivity between related pairs of phyto esters that have varied fatty acid ester groups.

The best column I have found so far gives me a separation of phyto esters from fish oil components but leaves my phyto-esters in a series of partially resolved groups that I suspect only vary by their ester group.
This is Phenomonex's new Gemini column 5u C18 250X4.6. Has anyone else done any work with a separation that requires two different types of selectivity?

Thanks
Rick

Rick,

If possible, try normal phase. Just a diol column, Heptane(or hexane):IPA. if selectivity is not o.k., you can add some ACN (then mix externally - there is a solubility gap) .Backpressure is rather low, so 2 or even three 25cm columns with 3µm silica gel is no problem.

Thanks, this gives me some ideas to work with,

However I have run into the same problem as several others on this board with solvents like hexane, acetone or methylene chloride giving me huge pump pressure variations.

I will start digging and see what columns we have around here that are normal phase.

I will check to see what kind of % of very volitile solvents I can get away with using this pump system.

Thanks
Rick

I ran across some literature about improving selectivity of varying saturation of fatty acids by adding Ag+ Ions to either the mobile phase or using silver impregnated packing. Apparently they claim that there is an interaction between the unsaturated fatty acid bonds and the silver ions in solution.

I am however hesitant to add silver nitrate to my mobile phase, I was curious if anyone had any information on this kind of separation. I am unsure if silver ions would be safe to use with my column. Has anyone else worked with silver ions as a mobile phase additive?

Thanks

Rick

I've heard of adding silver salts to GC phases during the coating process to improve selectivity between hydrocarbons of varying unsaturaion. I imagine that the same things would occur in LC, predominantly in the mobile phase (where the silver ions are). I don’t know if the silver ions would become impregnated in the stationary phase or not. Something to seriously consider is the solubility of the silver salt in organic/water solutions, especially if doing gradients.

What must be going on is a electron donor : acceptor interaction. If these are beneficial to the separation, one might be better off with diphenyl stationary phase, or a PS-DVB that is highly crosslinked (Dionex claims that their NS-1 is tolerant of all organic solutions without swelling, as well as acid and base). Jordi also makes a column like this (available in Alltech catalog). These columns should also be capable of electron donor : acceptor interactions. As to whether or not these columns can achieve your separation or not….

The addition of silver ions to the stationary phase is use in normal phase chromatography under the name of "argentation chromatography". I have not seen anything on this in a long time. Here is an article that allows you to follow up on the technique: D. Cagniant, "Argentation Chromatography: Application to the Determination of Olefins, Lipids, and Heteroatomic Compounds" in Chromatographi Science Series Vol 57, Dekker, 1992.

I don't recall any reversedphase applications, and I do not think that it would work in RP.

Thanks for the info on silver ions, I appreciate your help.

Rick