Helen C. Fleisher Gyula Vigh Department of Chemistry, Texas A&M University, College Station, TX, USA Hydrolytically stable, diaminocarboxylic acid-based membranes buffering in the pH range from 6 to 8.5 for isoelectric trapping separations Diaminocarboxylic acid carrier ampholytes, such as L-histidine, 2,3-diaminopropionic acid, L-ornithine, and L-lysine, were reacted with glycerol-1,3-diglycidyl ether (GDGE) and poly(vinyl alcohol) (PVA) in the presence of sodium hydroxide to produce hydro- lytically and mechanically stable hydrogels, supported on a PVA substrate, for use as buffering membranes in isoelectric trapping (IET) separations. The pH values of the DACAPVA membranes were determined with the help of small-molecule pI markers and proteins and were found to be in the 6 , pH , 8.5 range. The membranes were successfully used to isoelectrically trap small ampholytes, desalt ampholyte solutions in IET mode, and effect the binary separation of chicken egg white proteins. Keywords: Buffering membranes / Diaminocarboxylic acids / Immobilized carrier ampholytes / Isoelectric focusing / Isoelectric membranes / Isoelectric trapping / Poly(vinyl alcohol) hydro- gels DOI 10.1002/elps.200500198 1 Introduction Isoelectric trapping (IET) protein separations in multi- compartmental electrolyzers, pioneered by Martin et al. [1], Faupel et al. [2, 3], and Righetti et al. [4, 5], became important alternatives to chromatographic methods of protein purification [6]. Earlier achievements of this pow- erful preparative separation technology and instrumenta- tion [7] in the general area of laboratory-scale preparative protein purification have been reviewed extensively, such as in [8, 9]. Recently, IET has been applied as a pre- fractionation method in the field of proteomics [10, 11], and the first results from this exciting new field have been reviewed in [12, 13]. The quintessential feature of classical IET [1–5] is that the purified proteins are isolated in the form of isoionic solu- tions in compartments that are formed by buffering membranes [1] (also known as isoelectric membranes [2– 5, 14–16] and zwitterionic membranes [17, 18]), whose pH (pI) values bracket the pI of the target protein. Typically, the solutions containing the proteins are recirculated through the separation compartments. The flow direction is tangential to the membranes, while the direction of the electric field is orthogonal to the membranes. This arrangement moves the proteins through the buffering membranes into the appropriate separation compart- ments [1–18]. Historically, the buffering membranes have been made by copolymerizing acrylamide, N,N’-methylenebisacryl- amide, and Immobiline chemicals (acrylamido weak acid and acrylamido weak base derivatives) [5, 14–18] over a substrate that acts as a mechanical support (such as a glass fiber filter [5, 14–18], a porous polyethylene disk [11, 19], a polysulfone membrane [10, 12, 20], a woven poly- (ethylene terephthalate) fabric [21] or a capillary [22]). The pH value of the buffering membrane is adjusted by selecting the appropriate Immobilines (their pK a values) and their concentrations (according to the Henderson- Hasselbalch relationship) [23–27]. As IET technology has matured and possible partial hydrolysis of the gel mem- branes during long separations became a concern, Chiari Correspondence: Professor Gyula Vigh, Department of Chemis- try, Texas A&M University, College Station,TX 77842–3012, USA E-mail: vigh@mail.chem.tamu.edu Fax: 1979-845-4719 Abbreviations: CAR, L-carnosine; DACA, diaminocarboxylic acid; DACAPVA, mid-pH membrane prepared from a diamino- carboxylic acid, glycerol-1,3-diglycidyl ether, and poly(vinyl alco- hol); DAPA, 2,3-diaminopropionic acid; DAPAPVA, mid-pH membrane prepared from 2,3-diaminopropionic acid, glycerol- 1,3-diglycidyl ether, and poly(vinyl alcohol); GDGE, glycerol-1,3- diglycidyl ether; HIS, L-histidine; HISPVA, mid-pH membrane prepared from L-histidine, glycerol-1,3-diglycidyl ether, and poly(vinyl alcohol); HMMB, 4-hydroxy-3-(morpholinomethyl)- benzoic acid; IDA, iminodiacetic acid; IDAPVA, low-pH mem- brane prepared from iminodiacetic acid, glycerol-1,3-diglycidyl ether, and poly(vinyl alcohol); IET , isoelectric trapping; LAB, labe- talol; LYS, L-lysine; LYSPVA, mid-pH membrane prepared from L-lysine, glycerol-1,3-diglycidyl ether, and poly(vinyl alcohol); MABA, m-aminobenzoic acid; ORN, L-ornithine; ORNPVA, mid- pH membrane prepared from L-ornithine, glycerol-1,3-diglycidyl ether, and poly(vinyl alcohol); PVA, poly(vinyl alcohol); QPVA, high-pH membrane prepared from glycidyltrimethylammonium chloride, glycerol-1,3-diglycidyl ether, and poly(vinyl alcohol) Electrophoresis 2005, 26, 2511–2519 2511  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim General