Axially illuminated fluorescence imaging detection for capillary isoelectric focusing on Teflon capillary

Abstract

An axially illuminated laser induced fluorescence (LIF) whole column imaging detection (WCID) method was developed for capillary isoelectric focusing (CIEF). In this method, an argon ion laser was used as the excitation source and the isoelectric focusing process was monitored dynamically by a thermoelectrically (TE) cooled (charge coupled device) CCD camera. Low refractive index PTFE capillary was used in the experiment, and electroosmotic flow (EOF) and protein adsorption were minimized by dynamic conditioning of the capillary with methyl cellulose (MC). With the addition of a small amount of high refractive index organic solvent such as glycerol in the sample mixture, the laser beam was axially propagated with total internal reflection. Stray light and scattering light originating from the wall of the capillary were minimized. The instrumentation is simple and the method is sensitive. Two naturally fluorescent proteins (R-phycoerythrin and green fluorescent protein) were separated, and a concentration detection limit (LOD) of 10⁻¹³ M or mass LOD of 10⁻¹⁹ mol for R-phycoerythrin was obtained.