Isoelectric Focusing in Capillaries and Channels Packed with Sub-Micron Silica Particles

Abstract

Isoelectric focusing (IEF) is demonstrated in capillaries and channels packed with sub-micron particles for the first time. Packings of silica particles were chemically stabilized by a mixture of methyltrichlorosilane and (chloromethyl)phenylethyl-trichlorosilane at a ratio of 1:20 (v/v). The silica surface was modified with polyacrylamide prepared by surface-initiated atom transfer radical polymerization to minimize protein adsorption. It was shown that the silica surface was coated with a dense polyacrylamide layer after 4 hr polymerization with a surface coverage of acrylamide of 84 μmol/m2. Both irreversible and reversible adsorption of proteins was negligible when the proteins migrated through capillaries coated with polyacrylamide prepared by 4 hr polymerization. Capillaries packed with 900 nm silica particles eliminate the problem of unwanted hydrodynamic flow between reservoirs during IEF. A mixture of three proteins including trypsin inhibitor, carbonic anhydrase II, and myoglobin was successfully separated by IEF. The time required for focusing in the packed capillaries was increased by only a factor of 2 compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing using packed capillaries. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H = σ²/L = 0.64 μm. This limits the resolution to 0.1 pH units for the 2 cm capillary having a pH range of 3−10, giving a theoretical peak capacity of 47. IEF in the channels packed with 680 nm silica particles was also studied. The packings in the channels are stable after being chemically modified with a brush layer of polyacrylamide. IEF in the packed channels obtained a resolution of 0.023 pH units at 400 V/cm. The focusing was typically completed in less than 30 min. Increasing the electric field up to 1000 V/cm can continually improve the resolution of IEF in packed channels. Diffusion of proteins after electric field is removed slows down with decreasing particle size. Therefore, for the detections where remobilization is not required, the resolution can be maintained by packing the channels with smaller particles to slow down the diffusion.