I need to separate two compounds with pKa 5,19 and 10,96. Which buffer should I use and which pH is more suitable?
It depends on your molecule, sample matrix, and the column. A C18 column is very robust at an acidic pH, so I would suggest;
1. pH 3.0 phosphate buffer if using a PDA/DAD detector
2. 0.1% formic acid if using an MS detector
If you have a mixture of acid and basic compounds, I would try ion-pair method. You can do it two ways. 1) you pair the acid analyte with basic reagent such as TBAOH at high pH. Your acid compound will be neutral and stick to the C18 while your basic compound is not ionized and also stick to the C18 column. 2) you can pair the basic analyte with Hexanesulfonic acid to make it neutral at low pH while your acid in not ionized (or being ion-suppressed) and stick to the C-18 column.
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