Available online at www.sciencedirect.com Journal of Chromatography A, 1182 (2008) 226–232 Integration of various stacking processes in carrier ampholyte-based capillary electrophoresis Jean-Marc Busnel a,c,∗ , Thomas Le Saux b,d , St´ ephanie Descroix a , Hubert H. Girault c , Marie-Claire Hennion a , Shigeru Terabe b , Gabriel Peltre a a Laboratoire Environnement et Chimie Analytique, UMR CNRS 7121, ESPCI, 10 rue Vauquelin, 75005 Paris, France b Graduate School of Material Science, University of Hyogo, Kamigori, Hyogo 678-1297, Japan c Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique F´ ed´ erale de Lausanne, EPFL-SB-ISIC-LEPA, Station 6, CH1015 Lausanne, Switzerland d D´ epartement de Chimie, UPMC-CNRS-ENS, ´ Ecole Normale Sup´ erieure, 24 rue Lhomond, 75231 Paris Cedex 05, France Received 1 October 2007; received in revised form 10 December 2007; accepted 27 December 2007 Available online 6 January 2008 Abstract Field-enhanced sample stacking, field-enhanced sample injection as well as electrokinetic supercharging have been successfully integrated in carrier ampholyte-based capillary electrophoresis. Through the analysis of different test sample mixtures, it has been shown that the carrier ampholyte-based background electrolytes, in spite of their very low conductivity, allow efficient online preconcentration of analytes by field- amplified techniques. Sensitivity enhancement factors of the same magnitude as those usually encountered with classical conductive background electrolytes have been obtained in such carrier ampholyte-based buffers. Depending on the online preconcentration method that has been integrated, sensitivity enhancement factors between 50 and several thousands have been reached. © 2008 Elsevier B.V. All rights reserved. Keywords: Carrier ampholytes; CABCE; FESS; FESI; EKS 1. Introduction The use of isoelectric background electrolytes (BGEs) for capillary zone electrophoresis (CZE) has been introduced by Hjerten et al. in 1995 [1]. The use of such BGE in CZE allows the application of high electric field strength without inducing any significant Joule heating in the system because of their low conductivity. So far, different kinds of compounds have been used as low conductivity buffers in CZE. Through different studies, amino acid-based buffers have been proven to be a valuable alterna- tive to classical BGEs. As an example, we can mention that the tryptic peptide map of bovine -casein was performed in less than 10 min in an aspartic acid-based buffer while 80 min were necessary in an 80 mM classical phosphate buffer [2]. Cysteic ∗ Corresponding author at: Laboratoire d’Electrochimie Physique et Ana- lytique, Ecole Polytechnique F´ ed´ erale de Lausanne, EPFL-SB-ISIC-LEPA, Station 6, CH1015 Lausanne, Switzerland. Fax: +41 21 693 36 67. E-mail address: jean-marc.busnel@epfl.ch (J.-M. Busnel). acid [3], histidine or the His-Gly dipeptide [4] and iminodiacetic acid [5–7] have also been used as low conductivity buffers in CZE. The main drawback of amino acids is that only few of them exhibit a sufficient buffering capacity to be used as BGE in CZE [8]. In this context, we have been interested in the develop- ment of isoelectric buffers composed of narrow pH cut of carrier ampholytes (CAs). CAs are usually used as a very heteroge- neous mixture in isoelectric focusing (IEF). In such a mixture, covering, for example a pH 3–10 range, a very high number of compounds can be found [9–11]. To obtain solutions containing only CAs with close isoelectric points (pI), we fractionated a wide pH range mixture of CAs by preparative IEF in a gran- ulated gel [12]. With this fractionation technique, between 25 and 30 narrow pH cut fractions of CAs have been obtained. As the CAs are the sole buffering species in each narrow pH cut, the fraction pH should be rather close to the pI of the con- tained CAs. As a consequence, each fraction should present a low conductivity value. This has been confirmed by the physico- chemical characterization of the 25 narrow pH cuts obtained 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.12.079