Diode Laser-Based Concentration Gradient Detector for Detection of Capillary Isoelectric Focusing

Abstract

A diode laser-based concentration gradient detector was used in a compact capillary isoelectric focusing (CIEF) instrument which also includes a short piece of 20- μm-i.d. capillary. The diode laser was mounted with a focusing lens and driving circuit in an 8-cm-long, 2-cm-diameter aluminium barrel, and was powered by a 9-V battery. The size of the whole instrument is about 25 cm × 15 cm—much smaller than that of any other capillary electrophoresis (CE) instrument. In order to focus the laser beam into the narrow capillary, optical geometry of the detector was optimized. This diode laser-based detector is the first one which has been applied to CE with capillaries narrower than 50 μm. The detection limit in concentration for ovalbumin reaches 2.0 × 10−7 M, which corresponds to a 7.0 fmol on-column detection limit for the protein. This on-column detection limit of the universal detector is within the same order of magnitude as that of a diode laser-based fluorescence CE detector. The resolution of the instrument is about 0.02 pH units, and small amounts of protein samples in a wide isoelectric point range can be separated and detected by the compact instrument without derivatization in less than 12 minutes.