Chromatography Forum

Hi,

I have done a quick search and the boards and have read the threads about this, but I am not familar with HPLC and some of the posts I don't really understand. I have been asked to develop a HPLC method for nucleotides as my project with the possibility of coupling to ESI-MS. I have been doing some reading and am looking at trying this method.

Mobile phase A: Ammonium acetate (15mM) adjusted to pH 5.8 with acetic acid
Mobile phase B: MeOH

I am using a mix of 4 dNTP's, is this a good place to start?

Thanks

I should probably say I am going to use a discovery C16 Amide column.

The other method I was thinking of is using is:

A = 20 mM Tris-HCl, pH 9
B = 20 mM Tris + 0.3 M Citrate + 0.1 M NH4Cl, pH 9

You need to bear in mind that nucleotides are rather polar molecules. In the past, I have analysed nucleotides and I used to do it with an isocratic method. Try this:

Iscratic Mobile Phase: 0.1M Potassium Dihydrogen Phosphate adjusted to pH 7.0 with Dipotassium Hydrogen Phsophate.

Column: A HILIC column. Maybe an SB-Aq column (from Agilent) or ODS-Aq (from YMC).

Thanks for the reply - I have no budget for a new column unfortunately.

Your Tris and Nh4Cl buffer is not volatile and thus no compatible with ESI-MS

Thanks the citrate tris buffer I am using with LC-ICP-MS