Study of overloading of basic drugs and peptides in reversed-phase high-performance liquid chromatography using pH adjustment of weak acid mobile phases suitable for mass spectrometry.

Serious losses in column efficiency for ionised basic drugs and peptides occur due to overloading of C18 phases when weak acid mobile phases of low ionic strength suitable for mass spectrometric detection are used. Measurable changes in retention time and efficiency can be observed even for levels around 0.01 microg of basic drugs on 0.46 cm I.D. columns; the overloading process is a continuum, rather than an event which takes place only once a certain threshold value has been reached. Overloading can be reduced by increasing the mobile phase pH, e.g to the pKa of the weak acid, which increases the ionic strength, allowing a greater degree of ion pairing and perhaps also physical screening of the adsorbed solute ions by buffer ions. Buffer capacity is also optimum at its pKa. Silanol ionisation and kinetic tailing effects were mostly absent at aqueous pH up to 4.75 on the hybrid inorganic-organic C18 phase employed in this study, except when analysing the most highly charged peptides. The exact cause of overloading of these ionic species is unclear.