Chromatography Forum

Hi everyone!

I am really new to HPLC but I am currently trying to develop a method to detect and quantify acetamide and butyramide in groundwater. We have a Jasco HPLC system with fluorescence detection. We are planning on purchasing a Inertsil ODS-4 C18 column (250mm x 4.6mm, 5um) for the detection of other compouds.

My initial thoughts were to use a mobile phase of 5% acetonitrile with 95% phosphoric acid solution at 35C and 1mL/min on this column.

Has anyone doen separation of acetamide and butyramide by HPLC? or Does anyone have any advice on adjusting this method (column, mobile phase, flow rate, temp, etc)?

Thanks!

At least the acetamide is a very polar compound, and you may need a column that is compatible with a higher amount of water.

The analytes are not ionic in nature, so you do not need phosphoric acid or a buffer (unless of course other components of your sample require this.

You should use a low detection wavelength - 210 nm.

If you have reversed phase column and only a fluorescent detector then I would suggest you go with ortho Phthalaldehye (opa) derivatization. It is essentially instantaneous at room temperature and will provide you with a fluorescent derivative. Another great reason to do this is that your amide will also be nice and greasy and so amenable to reversed phase.

You would need to order some OPA and two other things. Sodium tetraborate and N acetyl cysteine are what I used I think. Lots of recipes are out there.

emission 436-475nm (max 455nm) with excitation at 330-390nm (max.340nm)

OPA for amides???

I used OPA for a chiral butyramide and it worked great. I could separate the diasteomers in a 3 minute method.

I used a chirobiotic T with high ACN for a HILIC method on my Waters Premier for a non-chiral quick screen.

Or maybe my memory is getting bad. I was studying catalytic conversion of the amine to the amide. Maybe I did OPA of the amine and ran LCMS on the amide? Let me think about this some more.