High-performance liquid chromatographic postcolumn reaction detection based on a competitive binding system.
Postcolumn reactions are typically employed to improve detection in high-performance liquid chromatography (HPLC) separation techniques. This study proposes the use of competitive binding principles in designing novel postcolumn reaction schemes. The feasibility of this approach was tested by using the HPLC determination of biotin and biocytin as a model system. The effluent from the HPLC column was merged with a reagent stream containing avidin, whose binding sites were occupied by the dye HABA (2-[4'-hydroxyphenylazo]benzoic acid). HABA was displaced by the analytes from the avidin-HABA complex and the free dye was determined with a UV-vis detector at 345 nm. The procedure was optimized with respect to reactor design, reagent concentrations, and the flow rate of reagent solution. Analytical characteristics of the developed procedure were determined and compared with the direct detection of biotin and biocytin at 220 nm. The postcolumn reaction scheme improved the selectivity and sensitivity of the detection of biotin and biocytin while maintaining similar detection limits.