On-Line Optical Fiber Detection in a Preparative Free-Flow Electrofocusing Apparatus

Abstract

Many analytical isoelectric focusing (IEF) instruments are equipped with on-line detection, e.g., conductivity or UV-vis absorbance. Most of their preparative counterparts have not integrated such detection systems and thus require labor-intensive off-line analysis to quantify separation results. This paper describes the incorporation of an optical fiber based, on-line detection system that allows one to follow the evolution of the protein bands in a preparative IEF apparatus. An array of four optical fibers was designed to deliver light to the annulus of a free-flow electrophoresis apparatus, to detect the transmitted light passing through the separation media and to determine the protein concentration at vertical positions along the annulus of a vortex-stabilized focusing chamber using a 1024 bit CCD line camera. The final concentration of the major myoglobin band was 21.0 mg/mL at electric field strengths as high as 333 V/cm. Spectrophotometric analysis indicated a final concentration of 18.9 mg/mL, 10% less than that reported by the optical fibers.