Chromatography Forum

Hello,
I am starting a new project that I need a bit of help with. I have not been able to find any information on HPLC methods for determining Cholesterol and Fats in Eggs.
I am very new to the world of HPLC and any help with the methods would be greatly appreciated.
Thank you in advance,

Ann Lucas
NC A&T
Greensboro, NC

Ann- Gas Chromatography is a far better method for determining cholesterol than HPLC. I guess you might not have a sensitivity problem for eggs (there is only 1 isolated double bond in cholesterol if you have a UV detector on your HPLC) but the column efficiency in GC is much higher than in HPLC.

Victor,
Thank you for your reply. As I said, I am very new to this and I would like to have as much information as possible. Could you send me the method for GC? I do have a UV detector on my HPLC, but it is always better to have a plan B if needed. If you do not want to post that information here, let me know and I'll send you my email address directly. Still looking for HPLC method.
Ann Lucas

We also use GC for chlolesterol, with cholestane as internal standard. We updated the published procedure years ago (for example, to use capillary columns), but unfortunately the procedure is deemed proprietary.

Ann - I do not have a recipe for this method that you can follow blindly, but you should be able to find one in the literature if you want such a thing.

Basically, you can analyse cholesterol on a non-polar column (e.g. OV1) or a slightly polar column (e.g. SE-54, or anything with about 5% phenyl groups). It will also work on a polar column, but these are generally less temperature stable, so why bother?

You could use split injection probably for your samples. Maybe a temperature program up to 300 degrees centrigrade. People ususally make the trimethylsilyl derivatives of cholesterol to get rid of the -OH group although it will still work with the underivatised compound. You can make the TMS derivatives with the reagent BSA/TMCS. 5 alpha chosestane is an alkane which will do as an internal standard especially to get round any vagaries of split injection.

Sorry-that's 5 alpha cholestane. I'm talking about capillary colums too-choose one with film thickness 0.15-0.25um otherwise the temperature required to elute cholesterol may be too high. A standard FID will give you much better sensitivity than HPLC-UV, even though you might not need this with eggs......