Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization.

This review presents the need for replacing gels in 2D separations for proteomics, where speed, high-throughput, and the ability to characterize trace level proteins or small samples are the current desires. The theme of the review is isoelectric focusing, which is a valuable tool because it pre-concentrates proteins in addition to separating with high peak capacity. The review traces the technological progress from gel IEF to cIEF to packed capillaries with immobilized gradients for cIEF. Multiple capillary techniques are progressing toward meeting the current desires, providing extremely high sensitivity with regard to concentration and to small samples, integrated automation, and high peak capacity from multiple dimensions of separation. Capillaries with immobilized pH gradients for cIEF are emerging, which will alleviate interference from ampholytes and improve reproducibility in separation times when this valuable technique can be used as one of the dimensions.

Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing by using packed capillaries. The capillary dimensions are 100 μm i.d. and 2 cm in length, and the packing is made of 0.9 μm nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of two 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. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D=6.1(±0.3)×10-8 cm2/s for the packed capillary vs. D=28.8(±0.3)×10-8 cm2/s for the open capillary. 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 unit for the 2-cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.

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