Abstract
Low repeatability of migration time, peak area, and linearity (p__I vs__. mobilization time) is a problem often encountered in capillary IEF (cIEF) and is mainly caused by protein precipitation and protein–wall interactions. In order to study the influence of these phenomena, the effect of different classes of additives on repeatability of migration time, peak area and linearity of a mixture of seven model proteins has been investigated. Moreover, the influence of these additives on protein signal suppression in MALDI‐TOF MS has been studied. The optimal ampholyte blend (stabilizes pH gradient) to be used depends on the selected UV detection wavelength. All tested ampholyte blends show a significant and comparable signal suppression in MS. The best detergent (to prevent precipitation and wall interaction) should be determined for each sample individually, but generally polyethylene oxide and zwitterionic detergents show good repeatability for migration time (RSD <4.5%) and peak area (majority <10%). The RSD of R^2^ is <1.3% for the hydrophilic protein mixture. However, these components cause severe signal suppression in MS. Therefore glucoside detergents should preferably be used for MS coupling. Viscosity‐increasing agents (for hydrodynamic wall coating and to minimize diffusion) in particular cellulose derivatives, give good repeatability for migration times (RSD <4.5% at lower concentrations), peak area (except for high concentration methylcellulose and hydroxyethylcellulose all within 7.5%), and correlation (p__I vs__. migration time), but severe signal suppression is observed in MALDI‐TOF MS. Overall, cIEF repeatability and linearity can significantly be improved by adding the appropriate components. However, when the system is coupled to a MALDI‐TOF MS, compromises have to be made between high repeatability and linearity on one hand and MS signal intensity on the other.