Department of Chemistry, University of Arizona, Tucson, Arizona 85721, United States, and Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.
Anal Chem. 2010 Nov 1;82(21):8910-5. doi: 10.1021/ac101680z. Epub 2010 Oct 8.
Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing 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 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. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D = 6.1(±0.3) × 10(-8) cm(2)/s for the packed capillary vs D = 28.8(±0.3) × 10(-8) cm(2)/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 = σ(2)/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.
在使用填充毛细管的毛细管等电聚焦中，无需外加电场即可实现压力驱动的再迁移。毛细管尺寸为 100μm ID 和 2cm 长，填充材料为 0.9μm 无孔硅胶颗粒，表面经聚丙烯酰胺刷层化学修饰。结果表明，可逆和不可逆吸附均可忽略不计。填充毛细管消除了储液器之间不需要的流体动力流动的问题。聚焦了三种蛋白质：胰蛋白酶抑制剂、碳酸酐酶 II 和肌红蛋白。与开放毛细管相比，填充毛细管聚焦所需的时间仅增加了 2 倍，在 200V/cm 时，不到 15 分钟即可完全聚焦。填充毛细管允许使用更高的电场，分辨率不断提高，至少可达 1500V/cm。填充后，即使关闭电场，扩散也会受到阻碍：对于胰蛋白酶抑制剂，填充毛细管的 D = 6.1(±0.3)×10(-8)cm(2)/s，而开放毛细管的 D = 28.8(±0.3)×10(-8)cm(2)/s。在再迁移过程中，由填充引起的扩散展宽来自涡流扩散，其由板高 H 描述，H 是单位长度的方差：H = σ(2)/L = 0.64μm。这限制了分辨率为 0.1 pH 单位，对于具有 3-10pH 范围的 2cm 毛细管，理论峰容量为 47。
