Sanjiv Lalwani Evan Shave Helen C. Fleisher Kingsley Nzeadibe M. Brent Busby Gyula Vigh Department of Chemistry, Texas A&M University, College Station, TX, USA Alkali-stable high-pI isoelectric membranes for isoelectric trapping separations Alkali-stable, high-pI isoelectric membranes have been synthesized from quaternary ammonium derivatives of cyclodextrins and poly(vinyl alcohol), and bifunctional cross-linkers, such as glycerol-1,3-diglycidyl ether. The new, high-pI isoelectric mem- branes were successfully applied as cathodic membranes in isoelectric trapping separations in place of the hydrolytically more labile, polyacrylamide-based cathodic isoelectric membranes, and permitted the use of catholytes as alkaline as 1 M NaOH. The new high-pI isoelectric membranes have shown excellent mechanical stability, low electric resistance and long life times, even when subjected to electrophoresis with current densities as high as 80 mA/cm 2 . Keywords: High-pI ampholytes / Isoelectric focusing / Isoelectric membranes/ Isoelectric trap- ping / Poly(vinyl alcohol) / Quaternary ammonium cyclodextrins DOI 10.1002/elps.200305944 1 Introduction Over a decade ago, Faupel et al. [1] introduced isoelec- tric trapping (IET) in multicompartmental electrolyzers (MCEs) for the preparative-scale isoelectric focusing separation of proteins (for a review see, e.g., [2]) as an alternative to recirculating isoelectric focusing (IEF) (for a recent review see, e.g., [3]) and continuous free-flow IEF (for a recent review see, e.g., [4]). In the very suc- cessful MCE system, immobilized isoelectric gel seg- ments [5] or isoelectric membranes [6] were used to iso- late the separation compartments of the MCE from each other. The isoelectric points (pI values) of the membranes increased monotonously from the anode to the cathode. In an electric field sufficiently strong to counter the effects of cross-membrane diffusion, the proteins were trapped, in pure isoelectric form, in the respective com- partments that were formed by membranes whose pI values bracketed the pI values of the proteins of interest [1]. In the IsoPrime, the first commercial version of the MCE, the isoelectric membranes were formed on glass fiber filter disks from polyacrylamide-based isoelectric hydrogels [7]. In an attempt to make the IsoPrime system more rugged, Cretich et al. [8] replaced the polyacryl- amide-based isoelectric membranes with polyacryl- amide-based isoelectric beads. Recently, MCEs became important in proteomics [9–12] by providing a tool for the efficient prefractionation of complex samples (for a recent review see, e.g., [13]). In one of the commercially available MCE prefactionators, the IsoelectrIQ 2 [11], the polyacrylamide-based hydrogels are supported by polysulfone membranes. In the other commercially available MCE prefactionator, the Zoom unit [12], the polyacrylamide-based isoelectric hydrogels are supported on porous polyethylene discs. In an even further miniaturized MCE, the polyacrylamide-based iso- electric hydrogels are formed in situ in the short glass tubes that connect the adjacent wells in the rows of a 96- well titer plate [14]. Recently, a four-compartment, membrane-based elec- trophoretic unit, the BF200IET [15] that offers processing rates as high as 6 mg/min [16] was introduced for the preparative-scale, binary IET separation of ampholytic substances. The thin (approximately 150 mm) mem- branes of the BF200IET contain a poly(ethylene ter- ephthalate) fabric that supports the polyacrylamide- based isoelectric hydrogels. Thus, one can conclude that irrespectively of their mechanical design, the cur- rently known IET systems rely on the use of polyacryl- amide-based isoelectric hydrogels, membranes or beads that are formed by copolymerizing Immobiline chemicals, acrylamide, and bis-acrylamide [7, 8, 17, 18]. Correspondence: Dr. Gyula Vigh, Department of Chemistry, Texas A&M University, College Station, TX, 778452-3012, USA E-mail: vigh@mail.chem.tamu.edu Fax: 1979-845-4719 Abbreviations: â-ALA, b-alanine; BSH, benzenesulfonic acid; BzTMAOH, benzyltrimethylammonium hydroxide; CAR, carno- sine; CDP , mono(6-deoxy-6-pyridinium)-b-cyclodextrin; CDQ, quaternary ammonium derivative of cyclodextrin; DMPG, N,N- dimethyl-N-(3-phenoxypropyl)-D-glucaminium bromide; DS, average degree of substitution; GDGE, glycerol-1,3-diglycidyl ether; GLU, glutamic acid; GTMA, glycidyl trimethylammonium chloride; HIS, histidine; HMMB, 4-hydroxy-3-(morpholino- methyl)benzoic acid; IDA, iminodiacetic acid; IET , isoelectric trapping; LYS, lysine; MABA, m-aminobenzoic acid; 3PPA, 3-pyridine-propionic acid; PreMCE, pressure-mediated capil- lary electrophoresis; PVA, poly(vinyl alcohol); TYRA, tyramine 2128 Electrophoresis 2004, 25, 2128–2138  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim