Adaptation of Capillary Isoelectric Focusing to Microchannels on a Glass Chip

• Published in: Analytical chemistry (Washington) Vol. 71; no. 3; pp. 678 - 686
• Main Authors: Hofmann, Oliver, Che, Diping, Cruickshank, Kenneth A, Müller, Uwe R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.02.1999
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Glass; Hydrogen-Ion Concentration; Isoelectric Focusing - instrumentation; Isoelectric Focusing - methods; Oligopeptides - isolation & purification; Other chromatographic methods; Fluorescence detector; Chemical analysis; Multicomponent analysis; Ultraviolet detector; Zone electrophoresis; Isoelectrical focusing; Capillary electrophoresis; Biological macromolecule; Optimization; Quantitative analysis; Operating conditions
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9806660

As a first step toward adaptation of capillary isoelectric focusing (cIEF) to microchannels on a glass chip, we have compared the three most common mobilization methods:  chemical, hydrodynamic, and electroosmotic flow (EOF)-driven mobilization. Using a commercial cIEF apparatus with coated or uncoated fused-silica capillaries, both chemical and hydrodynamic mobilization gave superior separation efficiency and reproducibility. However, EOF-driven mobilization, which occurs simultaneously with focusing, proved most suitable for miniaturization because of high speed, EOF compatibility and low instrumentation requirements. When this method was tested in a 200-μm-wide, 10-μm-deep, and 7-cm-long channel etched into planar glass, a mixture of Cy5-labeled peptides could be focused in less than 30 s, with plate heights of 0.4 μm (410 plates/s) upon optimization. For a total analysis time of less than 5 min, we estimate a maximum peak capacity of approximately 30−40. Interestingly, the order of migration was found to be reversed compared to capillary-based focusing.