Theoretical concepts of on-line liquid chromatographic-biochemical detection systems. II. Detection systems based on...

A theoretical concept for on-line liquid chromatography-biochemical detection (LC-BCD) using labelled affinity proteins as reporter molecules is presented. The BCD system is based on the post-column addition of labelled affinity proteins such as fluorescein-labelled streptavidin to the LC effluent. After a short reaction time, free and analyte-bound label are separated during passage through a column packed with an immobilised-ligand support. The bound fraction passes the column unretained and is measured downstream by means of a conventional HPLC detector. The theoretical model presented here relates the detector response to the most important instrumental and biochemical parameters such as dispersion, reaction time, concentration and affinity of the affinity protein and the number of binding sites. The theoretical concept is validated using fluorescein-labelled streptavidin and biotin as model system.