Hydrophobic compounds with a strong acidic group such as phosphate, sulfate, and a-substituted carboxilic acids are preferably analyzed on a cation-exclusion Primesep 500 column. Anion-exchange columns (Primesep D, B2) provide very strong, sometimes irreversible retention due to both RP and IE combined interactions. At a neutral pH, the retention decreases significantly because of repulsion phenomena of the negatively charged column surface and negative charge of the analyte itself. Good peak symmetry and high efficiency is typical for most acidic compounds.
Hydrophobic strong acid compounds have a very strong interaction with the stationary phase by reverse phase mechanism. A high concentration of organic modifier is usually required to get reasonable retention time. To reduce interaction of such hydrophobic molecules, the column with the same surface charge as the analytes is a preferred option. Primesep 500 is a column which has strong negative charge at a pH above 5, which can be easily obtained by using ammonium formate (AmFm) or ammonium acetate (AmAc) salt as ionic modifier. Typical conditions include an MeCN gradient and 10-50 mM AmAc or AmFm.