Journal of Chromatography A, 979 (2002) 155–161 www.elsevier.com / locate / chroma Preparative-scale isoelectric trapping separations using a modified Gradiflow unit a a a a b b David Ogle , Agnes Ho , Toby Gibson , Dennis Rylatt , Evan Shave , Peniel Lim , b, * Gyula Vigh a Gradipore, French’ s Forest, NSW 2086, Australia b Chemistry Department, Texas A&M University, College Station, TX 77842, USA Abstract The Gradiflow BF200 preparative electrophoretic unit (Gradipore), which has been developed for size-based and charge-sign-based protein separations and in which the hydraulic flow path of the recirculating sample stream in the separation cartridge is orthogonal to the electric field, has been modified to carry out binary protein separations using the principles of isoelectric trapping. The disposable separation cartridge contained three isoelectric membranes which, along with the cartridge holder, formed the anode and cathode compartments and the anodic and cathodic separation compartments. The utility of the modified instrument was demonstrated by effecting a binary separation of chicken egg white across an isoelectric point 5.5 isoelectric membrane. The desalting and subsequent binary separation steps proved to be remarkably rapid, due to the favorable combination of short electrophoretic path, high electric field strength and large effective isoelectric membrane surface area.  2002 Elsevier Science B.V. All rights reserved. Keywords: Isoelectric focusing; Preparative electrophoresis; Isoelectric trapping; Membranes; Gradiflow; Proteins 1. Introduction rates, respectively. This requires means to effectively dissipate Joule-heat and keep convective mixing to a As indicated by the annual recurrence of multiple minimum. reviews, interest in all forms of preparative-scale, When the target compounds are ampholytic, such non-gel, electrophoretic separations has grown as proteins, isoelectric focusing (IEF) in carrier steadily over the last 7 years [1–12]. Success of a ampholytes or low conductivity Bier’s buffers [13] is proposed preparative electrophoretic separation often favored over zone electrophoresis, because the method depends not only on its ability to create focusing mechanism at least partially offsets the adequate separation selectivity (by zone electropho- undesirable effects of diffusion and convective mix- resis, isotachophoresis or isoelectric focusing), but ing [3–6]. Isoelectric trapping (IET), an alternative also on its ability to balance the opposing require- to recirculating IEF [3] or free-flow IEF [4,5], ments that favor high resolution and high production achieves separation of proteins by trapping them in pure, isoelectric form [7,8] in compartments formed by immobilized isoelectric gel segments [14–17] or *Corresponding author. Tel.: 11-979-845-2456; fax: 11-979- isoelectric membranes [18–34] whose isoelectric 845-4719. E-mail address: vigh@mail.chem.tamu.edu (G. Vigh). points (pI values) bracket the pI values of the 0021-9673 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0021-9673(02)01601-1