Molecularly imprinted microspheres (MIMs) were prepared using 4-aminopyridine (4-AP) as template molecule by aqueous microsuspension polymerization. The MIMs were packed into stainless steel column (250×4.6 mm I.D.) for selective separation of 4-aminopyridine (4-AP) and 2-aminopyridine (2-AP). The influences of pH, kinds and concentration (c) of buffer on capacity factors were investigated in detail. The relationships of capacity factor (k′) with pH and concentration of buffer are quantitatively described firstly. The effects of pH of phosphate and acetate buffer on capacity factors are very different. The relationship between k′ and pH can be described by the following equation: k′=-8.23 + 9.23 pH-0.99 pH 2 (in phosphate buffer) with R 2=0.9775 and k′=6.79-3.76 pH + 0.68 pH 2 (in acetate buffer) with R 2=0.9866. Furthermore, the capacity factors were also greatly affected by the concentration of acetate buffer in mobile phase while non-imprinted molecule is poorly changed. It increases with decreasing the concentration of buffer-especially in low concentration buffer (c acetate<0.02 mol/L, final concentration in mobile phase). The fit curve of log k′ to log c is described by equation: log k′=-0.571-1.256×log c-0.186×(log c) 2 with R 2=0.9979. The ratio of acetate buffer to methanol was investigated and the optimal ratio for separation of 4-AP and 2-AP is below 1∶7.5 (V/V). Molecularly imprinted microspheres (MIMs) were prepared using 4-aminopyridine (4-AP) as template molecule by aqueous microsuspension polymerization. The MIMs were packed into a stainless steel column (250 × 4.6 mm ID) The influences of pH, kinds and concentration (c) of buffer on capacity factors were investigated in detail. The relationships of capacity factor (k ’) with pH and concentration of buffers are quantitatively The relationship between k ’and pH can be described by the following equation: k’ = - 8.23 ​​+ 9.23 pH-0.99 pH 2 (in phosphate buffer ) with R2 = 0.9775 and k ’= 6.79-3.76 pH + 0.68 pH 2 in acetate buffer with R2 = 0.9866. Furthermore, the capacity factors were also greatly affected by the concentration of acetate buffer in mobile phase while- imprinted molecule is poorly changed . It increases with decreasing the concentration of buffer-especially in low concentration buffer (c acetate <0.02 mol / L, final concentration in mobile phase). The fit curve of log k ’to log c is described by equation: log k’ = -0.571-1.256 × log c-0.186 × (log c) 2 with R 2 = 0.9979 The ratio of acetate buffer to methanol was investigated and the optimal ratio for separation of 4-AP and 2-AP is below 1: 7.5 V / V).