Capillary Isoelectric Focusing of Proteins with Carrier Ampholyte pH Gradient and Immobilized pH Gradient

Abstract

Capillary isoelectric focusing (CIEF) of proteins, which involves the separation of proteins on the basis of differences in their pI values has been investigated. To achieve efficient protein separations in carrier ampholyte pH gradient, different coating methods were studied to reduce the electroosmotic flow (EOF) and proteins interactions with capillary inner surface. It could be concluded that, Glycidoxypropyltrimethoxysilane-hydroxypropyl cellulose (HPC) coated capillary column worked better with pressure mobilization while surfactant-HPC coated column yielded superior separations with chemical mobilization. Another alternative to achieve CIEF is by immobilized pH gradient (IpG), which is a relatively new technology and it offers several advantages over carrier ampholyte pH gradient. This part of study involved the design of monolithic capillary columns with reactive functional groups that were required to immobilize the ampholyte pH gradient.On the average, CIEF with IpG monolithic columns provided convenient approach to achieve similar or improved CIEF separation than with carrier ampholyte pH gradient.