Adaptation of capillary isoelectric focusing to microchannels on a glass chip.
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-micron-wide, 10-micron-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 micron (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.
Hofmann
O
O
Vysis, Inc., Downers Grove, Illinois 60515, USA. o.hofmann@ic.ac.uk
Che
D
D
Cruickshank
K A
KA
Müller
U R
UR
D005898:Glass; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D009842:Oligopeptides / Q000302:isolation & purification*