Acceptance of capillary isoelectric focusing (cIEF) as a routine analy
tical method has been limited by variable migration times, irreproduci
ble patterns, and sample precipitation. Poor separation reproducibilit
y can be traced to a number of factors. In this paper we explore the p
ossibility of eliminating reagents without buffering capacity, and the
use of internal markers. An often overlooked optimization stage of cI
EF is mobilization. We demonstrate that hydraulic mobilization can be
finely tuned by manipulating the pressure applied, and that ion additi
on mobilization can be greatly improved by properly selecting the type
of ion used and its concentration. By limiting the pH range of the am
pholytes to 8.5 or less, hundreds of runs on an individual capillary a
re possible, which is a necessity for routine analysis.