The use of chitosan microparticles as chromatographic support has received much attention. In this study, the effects of process parameters, namely, chitosan molecular weight, chitosan concentration, molar ratio of amino group to aldehyde group, volume ratio of water to oil phase and stirring speed on the size and size distribution of chitosan microparticles and their application for immunoaffinity extraction were extensively investigated. Size distribution analysis indicated that the average diameter of the microparticles was 124μm with Span value of 1.1. The obtained microparticles exhibited low non-specific adsorption and kept stable in the pH range 4.0-10.0. Immunoaffinity chromatography (IAC) column was prepared by coupling antibody against enrofloxacin (ENR) with chitosan microparticles. Further characterization indicated that the binding capacity of the column was 4392ng ENR/mL gel and the variation of ENR extraction efficiency among columns was less than 5.2%. When challenged with ENR-fortified bovine milk samples, recoveries of ENR by immunoaffinity extraction were found to be in the range of 85.9% to 101.9%, demonstrated the feasibility of the prepared IAC columns for sample clean-up in ENR residue determination.
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