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In this report, an on-line coupling of capillary isoelectric focusing (CIEF) to capillary electrochromatography (CEC) is developed via a nanoinjector valve for performing two-dimensional (2D) proteomics separation. CIEF constitutes the first separation dimension, while CEC operates as the second separation dimension. Besides the orthogonal migration mechanisms of the two capillary-based separation modes, which lead to a 2D system whose overall peak capacity is the product of the peak capacity of the individual modes, the solvent of the CIEF mode is a weak eluent for the reversed-phase CEC (RP-CEC) mode, thus, allowing the transferring of focused fractions from CIEF to CEC without inducing band broadening, and instead zone sharpening would result. In fact, the transferred focused protein fraction from the CIEF column to the CEC column will stay tightly adsorbed to the inlet top of the CEC column until it will be eluted and separated into its protein components with a hydro-organic mobile phase. The theoretical peak capacity of the CIEF-CEC 2D platform is estimated at n(CIEF) (= 560) x n(CEC) (= 97) = 54 320. This peak capacity is more than needed for proteomics profiling. Also, only a fraction of this peak capacity is needed when looking at heart cuts for performing subproteomics. The 2D platform described here offers the convenience to generate the needed peak capacity to solve a given proteomic separation problem. This is facilitated by the RP-CEC dimension, which ensures rapid isocratic separation of proteins and peptides and rapid solvent change and column equilibration and avoids lengthy gradient elution. The RP-CEC column is based on neutral C17 monolith, which offers high separation efficiency and relatively high column permeability. To the best of our knowledge, the proposed 2D platform combining CIEF and CEC is reported for the first time for proteins and proteomics.

A novel silica-based stationary phase was developed for use in capillary electrochromatography (CEC) at relatively high electroosmotic flow (EOF). The silica was first bonded with a relatively hydrophilic layer bearing strong sulfonic acid groups. To this charged polar sublayer, octadecyl functions were covalently attached to yield the nonpolar top layer. This novel stationary phase, referred to as octadecylsulfonated silica (ODSS), was packed in bare fused-silica capillaries or in capillaries with the same coating as the sublayer on the silica-based stationary phase. The resulting packed columns were evaluated in CEC using alkylbenzenes as the test model solutes. Good separations can be achieved in less than 8 min, much faster than when using a regular octadecyl silica capillary column. Due to the permanent negative charge provided by the sulfonated sublayer on both the capillary walls and the silica particles, the magnitude of the EOF remained more or less constant over a wide range of pH, and its magnitude can be conveniently varied by the applied voltage.

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