Chromatography Forum

We're currently using a Restek C8 Ultra II, 5um 150x4.6mm HPLC column and to date have been happy with its performance.

I have however a one off request for an analysis of L-Carnitine - the method I'd like to use as a base for its development requires an acetonitrile-phosphate buffer mix with sodium 1-octanesulphonate.

The phosphate buffer is supposed to be at pH 2.0 and the specs of the column have a pH range of 2.5 - 8.0 - I don't believe it should be an issue - but in should it be okay to use (it is a one off analysis after all), or would we be better off with a more rugged column at low pH? I note that there are a number of competing manufacturers with a range of 2.0 - 8.0.

Thanks in advance.

You may try Zorbax SB Columns which can run at pH from 2 to 8 or even lower.

While Restek C8 might be adequate for this application, Zorbax SB C8 is definitely more stable because of the steric hindered side group adjacent to the anchor point.
Here is an alternative approach for this application using mixed mode chromatography. The following links lead to two carnitine related applications done on a 3x50-mm Acclaim Trinity P1 column. This column provides cation-exchange, anion-exchange and reversed-phase retention at the same time. In these two examples, both carnitine and counterion were retained and resolved easily. If carnitine is the only thing of interest, a simple isocratic method with phosphate buffer can do the job, and no ion-paring agent is needed. If a MS compatible buffer is desired, simply replacing the phosphate buffer with ammonium acetate buffer with similar buffer concentration will do.

http://www.dionex.com/en-us/images/pdf- ... rinity.pdf
http://www.dionex.com/en-us/images/pdf- ... nityP1.pdf

Thanks for the replies people.

Interesting links re: the mixed mode chromatography, if we get much more in the way of requests for zwitterionic species I'll certainly keep these in mind.

This is only a one off request (at least to date) so I'm not sure if a purchase of a new column (be it Zorbax or Trinity P1) can be justified. I saw the pH range of the Zorbax columns in my literature research prior to posting so will recommend to my colleagues to get one of these in the future.

Carnitine is the only analyte of interest however it needs to be resolved from Crotonylbetaine (L-Carnitine Impurity A) which apparently is an issue. A colleague who kept her mobile phase at pH 2.5 has had problems, and, assuming the pKa value of 3.8 for Carnitine is correct, the pH she's operating at could be why.

I'm impatient to roll up the sleeves and onto the machine to see how these analytes behave; so if you think the column won't have lasting problems after a one off development and quant and I'll have a crack tomorrow...

I am interested in knowing the outcoming. Meanwhile, I will try your application with the Trinity P1 column, and will let you know if there is anything interesting.

I would not advise running the Restek column at pH of 2.0. It may not show harmful effects immediately, but will eventually start removing C8 phase from the silica and expose active silanol sites.