Preparation and evaluation of molecularly imprinted polymeric microspheres by aqueous suspension polymerization for use as a high-performance liquid chromatography stationary phase

Molecularly imprinted polymeric microspheres (MIPMs) were prepared using 4aminopyridine as imprinting molecule by aqueous suspension polymerization. The MIPMs were packed into stainless steel column (250 mm×4.6 mm i.d.) for selective separation and quantitative determination of 4-aminopyridine and 2-aminopyridine. The influence of mobile phases adjusted with different pH buffers (acetate solution) on capacity factors were investigated. The results showed that the capacity factor (kʹ) for 4aminopyridine increased with increasing pH of mobile phase while that of 2aminopyridine was subtly changed. The studies showed that the capacity factors for substrates were affected by both of MIPM stationary phase and mobile phase. In low pH (pH<5.5) mobile phase, the 4-aminopyridine was eluted prior to 2-aminopyridine and its capacity factor (kʹ) was lower. In high pH (pH>5.5) mobile phase, however, the capacity factor (kʹ) for template molecule exhibited far higher than that of non-imprinting molecule. Therefore, the imprinted microspheres against 4-aminopyridine were used to separate and simultaneous determinate 4-aminopyridine and 2-aminopyridine. The responses of peak areas versus concentrations of substrates present good linearity with R>0.999. This method may be used to separate and simultaneous determinate multicomponental homologous compounds or isomers, which are very expensive or unavailable or whose physicochemical properties are very similar, by changing the pH of mobile phases.