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.

Free‐flow electrophoresis in the proteomic era: A technique in flux

“…Furthermore, a progress in automation will certainly facilitate a continuous operation of instruments and help to rapidly process the fractionated sample for its further analysis. Bottenus and Ivory 108 developed an online detection system based on optical fibers coupled to a prototype of a vortex‐stabilized, also membrane‐less FFE system. While currently limited to a wavelength between 400 and 700 nm and therefore not suitable for the detection of the most protein species, this innovation promises that an online‐detection system in the UV range will be available in the near future.…”