Plate Heights below 50 nm for Protein Electrochromatography Using Silica Colloidal Crystals

• Published in: Analytical chemistry (Washington) Vol. 82; no. 24; pp. 10216 - 10221
• Main Authors: Wei, Bingchuan, Malkin, Douglas S, Wirth, Mary J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-12-2010
• Subjects: Analytical chemistry; Animals; Capillary Electrochromatography - methods; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Colloids - chemistry; Crystals; Cytochromes c; Electric fields; Enzymes; Exact sciences and technology; Humans; Muramidase; Other chromatographic methods; Proteins; Proteins - isolation & purification; Ribonuclease, Pancreatic; Silica; Silicon Dioxide; Water; Enzyme; Capillary electrophoresis; Device; Esterases; System on a chip; Silica; Colloid; Protein; Plate; Glycosylases; Cytochrome c; Electric field; Efficiency; Glycosidases; Formic acid; Acetonitrile; Electrochromatography; Hydrolases; Height equivalent to a theoretical plate; Limit; Diffusion; Pancreatic ribonuclease; Lysozyme
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac102438w

Silica colloidal crystals formed from 330 nm nonporous silica spheres inside of 75 μm i.d. fused silica capillaries were evaluated for the efficiency of capillary electrochromatography of proteins. Three proteins, ribonuclease A, cytochrome C, and lysozyme, each covalently labeled with fluorophor, were well separated over a distance of 1 cm by isocratic electromigration, using 40:60 acetonitrile/water with 0.1% formic acid. A van Deemter plot showed that the plate height for lysozyme, which was the purest of the three proteins, was diffusion-limited for electric fields ranging from 400 to 1400 V/cm. The plate height for lysozyme was below 50 nm at almost all of the migration velocities, and it approached 10 nm at the highest velocity. Eddy diffusion was negligible. Lysozyme migrated over a 12 mm separation length with more than 106 plates in 1.5 min. These results indicate that silica colloidal crystals are well suited for electrically driven separations of large, highly charged analytes such as proteins. The 106 plates observed for a separation length of barely more than a centimeter means they are potentially valuable for miniaturized separations in microchip and lab-on-a-chip devices.