ELSEVIER Journal of Chromatography A, 767 (1997) 231-239 JOURNAL OF CHROMATOGRAPHY A Capillary isoelectric focusing with anionic coated capillaries Donna M. Whynot a, Richard A. Hartwick b, Susan Bane a'* aDepartment of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902-6016, USA bpharmAssist Analytical Laboratory, Inc., RD1 Box 248A, Whitestore Road, South New Berlin, NY 13843, USA Received 23 September 1996; revised 12 November 1996; accepted 11 December 1996 Abstract Capillary isoelectric focusing (clEF) is a powerful analytical technique for the separation of proteins, cIEF can be pertormed in capillaries with reduced electroosmotic flow, or in capillaries with controlled electroosmotic flow. This work introduces an alternative method for clEF with controlled electroosmotic flow through the use of an anionic coated capillary. The method is shown to be applicable to basic, neutral and acidic proteins, with some peak broadening for acidic proteins. It also proves to have good reproducibility with R.S.D.s around 3%, a linear relationship between migration time and isoelectric point and quantitative capability. The total analysis time is shorter and sample preparation is simplified over other clEF methods. Keywords: Isoelectric focusing; Coated capillaries; Proteins 1. Introduction Isoelectric focusing (IEF) is a powerful technique used for the separation of protein mixtures based on differences in isoelectric points (pI). In this method, the proteins to be analyzed migrate through a pH gradient until they reach the pH at which they have zero net charge, i.e., the isoelectric point. Historical- ly, 1EF was most commonly performed in a gel media, either in a tube or in a slab format, These methods are limited by a number of factors such as Joule heating, difficulty with quantitation, and the time and labor required for analysis. Hjert6n and coworkers introduced IEF in the capillary format (clEF) in an attempt to overcome the problems with the conventional method [1-5]. Higher field strengths can be used in cIEF since the small diameter of the capillaries, 50-100 Ixm, can *Corresponding author. dissipate the Joule heating efficiently. These high field strengths can lead to higher resolution and faster analysis. Hjertfn speculated that cIEF should be performed in capillaries in which the electro- osmotic flow (EOF) is completely eliminated in order to maintain stable focused protein zones [1]. Coatings such as linear polyacrylamide and methyl- cellulose were used to eliminate the EOF [2]. The coated capillaries were then filled with a mixture of the ampholytes to be used to create the pH gradient and the proteins to be analyzed. An acidic buffer was placed at the anode and a basic buffer at the cathode. When the electric field was applied, the pH gradient was formed with the most acidic ampholyte at the anode and the most basic ampholyte at the cathode. The proteins then moved to the position in the gradient where the pH is equal to its pI. As the focusing process occurred, the current decreased to a minimum when the focusing was complete [6]. Detection of the protein zones can be achieved by 0021-9673/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S0021-9673(96)01101-6