Sang-Hee Shim Asif Riaz Kihwan Choi Doo Soo Chung School of Chemistry, Seoul National University, Seoul, Korea Dual stacking of unbuffered saline samples, transient isotachophoresis plus induced pH junction focusing A dual stacking mechanism based on transient isotachophoresis (TITP) and induced pH junction focusing is demonstrated as a means to increase the concentration sensi- tivity in capillary electrophoresis of highly saline samples. When stacking was carried out with an unbuffered saline sample of fluorescein between two zones of low mobility background electrolyte at high pH under an electric field of reverse polarity, two tran- sient peaks at both boundaries of the sample zone were observed. One peak at the rear boundary could be inferred as a transient isotachophoretic stacked zone. Through computer simulations of an unbuffered sample with a high concentration of sodium chloride, we showed that the fast moving zones of sodium and chloride ions induced pH changes at both boundaries to satisfy the electroneutrality condition and that the peak at the front boundary was due to the induced pH junction. To verify the pH changes, an indicator, thymol blue, was added to an NaCl solution and the color changes under an electric field were observed. The proposed mechanism was sup- ported by observing the dual stacking procedure for an unbuffered sample of 4-nitro- phenol and measuring additional sensitivity enhancements by dual stacking for ten weakly acidic compounds. For the ten analytes including nucleoside phosphates, every dual stacking of an unbuffered sample exhibited an additional enhancement up to 86% larger than that of usual transient isotachophoresis of the corresponding buffered sample without loss of separation efficiency and reproducibility. Therefore, it would be useful to skip over buffering in sample preparation for TITP, contrary to the general recommendation. Keywords: Capillary electrophoresis / pH junction / Simulation / Stacking / Transient isotacho- phoresis DOI 10.1002/elps.200305411 1 Introduction The pH of the background electrolyte (BGE) can have a significant influence on capillary electrophoresis (CE) and thus buffer solutions have been used for stable separa- tions. When preparing a sample, a buffer solution with a similar composition to the BGE is usually used. However, when the electrolyte system is composed of different seg- ments within a capillary in a proper condition, solutes can be focused into sharp zones since the migration velocity is governed by local properties such as the local electric field, viscosity, pH, and additive concentrations. Utilizing this fact, several on-line concentration techniques have been developed for improving the concentration sensitiv- ity in CE [1, 2]. Sample stacking is one of the most com- mon approaches [3, 4]. When the sample is prepared in a lower conductivity matrix than the BGE, a higher electric field is established across the sample zone and solutes are accelerated and stacked at the boundary of the BGE zone, where their migration velocities become signifi- cantly lower. Various sample stacking formats including large-volume sample stacking have been developed [1, 2, 5–8]. However, real samples such as physiological and environmental ones usually contain a high concentration of salts that may hamper stacking. In order to enhance the detection sensitivity for highly saline samples, sev- eral schemes have been developed including isotacho- phoresis which employs a discontinuous electrolyte sys- tem consisting of leading and/or terminating electrolytes [9–11]. The pH discontinuity has also been used for several focusing techniques that can be applied to highly saline samples. pH-mediated stacking has been reported to occur through titration of the sample with acid or base Correspondence: Prof. Doo Soo Chung, School of Chemistry, Seoul National University, Seoul 151–747, Korea E-mail: dschung@snu.ac.kr Fax: +82-2-877-3025 Abbreviations: TAPS, N-tris(hydroxymethyl)methyl-3-aminopro- panesulfonic acid; TEA, triethylamine; TITP, transient isotacho- phoresis Electrophoresis 2003, 24, 1603–1611 1603  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/1005–1603 $17.501.50/0 CE and CEC