Rationalization of retention and overloading behavior of basic compounds in reversed-phase HPLC using low ionic strength buffers suitable for mass spectrometric detection.

The retention and overloading behavior of some basic (and acidic) compounds has been studied on different RP-HPLC columns in buffers of varying ionic strength. Anomalous retention patterns of acids and bases were found on one phase in low-pH, volatile buffers such as formic acid, favored for mass spectrometric analysis. Unusual retention compared to that in higher ionic strength phosphate buffers is attributed to the presence of positively charged sites existing on this phase at low pH. Overloading of bases as well as acids is shown to be a function of mobile-phase ionic strength. This result is a logical consequence of previous suggestions that mutual repulsion of ions held on the hydrophobic surface of the stationary phase, rather than overload of silanols, is largely responsible for overloading on pure silica RP columns. Thus, overloading occurs much more readily in low ionic strength formic acid buffers. Appreciable loss of efficiency can occur in such buffers when only 50 ng of some bases is analyzed on a standard-sized column.