Chromatography Forum

N-hydroxyl group interacts with silica gel stationary phase and therefore, causes problem for the direct assay. A pre-derivatization in terms of methylation of N-hydroxyl group was used in a few publications. I am wondering if there is any good mixed mode column that can retain N-hydroxyl group containing compounds without the need of pre-derivatization and makes the direct assay possible. Any suggestions and references will be appreciated.

WW

May I ask what the reported problem is with silica-based columns?

Two problems were reported. One was irreversible absorption of small amounts of compounds onto the packing material resulting non linear responses and the other was tailing peak. Being more specific, the cpd contains N-hydroxypyridone group that has chelating function and this chelating function was the reason for the problems, according the the reports.

Thanks,
WW

Hi WW -

For chelating compounds, you will need a well endcapped column.
All of our columns utilize a novel endcapping system - so our silica - based columns work well for compounds that have chelating sites.

You may want to try an amino column for your application. We have one, Unison UK-Amino. If you email me, I can forward you some data.

You may have a better chance with some of the modern columns, potentially best with hybrid columns. However, if the authors were correct and the problem is due to chelation with metal, even the highest purity packing is not going to help you, since you are working with a stainless steel system with stainless steel columns and stainless steel frits. If a problem remains, you need to consider a complexing agent in the mobile phase, which is no good for MS. Alternatively, a regular wash of the column with EDTA should be OK.

Thank you, Uwe and Bryan, for your help. I am wondering if there is any volatile chelating agent that is compatible with Mass system. Has anybody used it in LC/MS assay?

WW

Bryan, Can you please give your e-mail address? I would like to learn more about your well endcapped amino column. Uwe, What concentration of EDTA is good for the regular column wash and how often? I would like to try using endcapped amino column with regular EDTA column wash and will inform you if it works. Thanks.

Hi WW -

It's bevans@silvertonesciences.com