Research Article Investigation of the pH gradient formation and cathodic drift in microchip isoelectric focusing with imaged UV detection This paper reports the protein analysis by using microchip IEF carried on an automated chip system. We herein focused on two important topics of microchip IEF, the pH gradient and cathodic drift. The computer simulation clarified that the EOF could delay the establishment of pH gradient and move the carrier ampholytes (CAs) to cathode, which probably caused a cathodic drift to happen. After focusing, the peak positions of components in a calibration kit with broad pI were plotted against their pI values to know the actual pH gradient in a microchannel varying time. It was found that the formed pH gradient was stable, not decayed after readily steady state, and migrated to cathode at a rate of 10.0 mm/s that determined by the experimental conditions such as chip material, internal surface coating and field strength. The theoretical pH gradient was parallel with the actual pH gradient, which was demonstrated in two types of microchip with different channel lengths. No compression of pH gradient was observed when 2% w/v hydroxy- propyl methyl cellulose was added in sample and electrolytes. The effect of CAs concentration on current and cathodic drift was also explored. With the current auto- matic chip system, the calculated peak capacity was 23–48, and the minimal pI difference was 0.20–0.42 for the used single channel microchip with the effective length of 40.5 mm. The LOD for the analysis of CA-I and CA-II was around 0.32 mg/mL by using normal imaged UV detection, the detected amount is ca. 0.07 ng. Keywords: Cathodic drift / Microchip isoelectric focusing / pH gradient DOI 10.1002/elps.201000395 1 Introduction As a popular and powerful separation tool, IEF has been adopted in proteomic communities for a long history. The principle is that the amphoteric compounds (e.g. proteins and peptides) are separated according to their isoelectric points (pI) in a pH gradient generally formed by carrier ampholytes (CAs) when an electric potential is applied [1]. As well known, the pH gradient is conventionally created by using amphoteric electrolytes. Besides such normal way, a couple of reports exist about the thermally generated pH gradient on the basis of most buffers exhibiting a noticeable dependence of pK a on temperature [2], or pH gradient created based on the electrolysis of water without CAs [3]. In the field of bioanalysis, IEF in a slab gel is a high- throughput and routine tool, but which is a time-consuming procedure and lack of quantitative capability. Hjerte ´n first pioneered capillary IEF (cIEF) in a free solution, and studied the pumping or electrophoretic elution to mobilize the focused protein zone to a single-point monitor [4]. The cIEF owns advantages of automation, rapid analysis, high concentration factor and good quantitative capability; never- theless, the traditional gel-based IEF is still often used and popular because the results are robust in biologists’ eyes [5]. In comparison with cIEF, IEF in a gel media not only affords stable pH gradients but also avoids EOF interference occurred in a fused silica capillary. Otherwise, the sample detection in cIEF needs to transport the focused bands past a single-point detector [4, 6–8], so the diffusion and band broadening are unavoidable. In order to obtain wide applications of protein analysis, much work remains to be done to improve and setup reliable cIEF method with high accuracy and reproducibility. MCE has gained great progress over past few decades. Miniaturization means low sample and chemical consump- tion, high-speed and potential integration of multiprocess in a chip, so MCE is always active in electrophoretic area from Zhongqi Xu 1,2 Noboru Okabe 1 Akihiro Arai 3 Takeshi Hirokawa 1 1 Applied Chemistry, Department of Chemistry and Chemical Engineering, Graduate School of Engineering, Hiroshima University, Japan 2 College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, P. R. China 3 Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Co., Nishinokyou, Nakagyou-ku, Kyoto, Japan Received June 9, 2010 Revised July 27, 2010 Accepted August 18, 2010 Abbreviations: CAs, carrier ampholytes; HPMC, hydroxy- propyl methyl cellulose; LPA, linear polyacrylamide; WCI, whole column imaging Correspondence: Professor Takeshi Hirokawa, Applied Chemis- try, Department of Chemistry and Chemical Engineering, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan E-mail: hiro77@hiroshima-u.ac.jp Fax: 181-824-22-7192 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2010, 31, 3558–3565 3558