Abstract In this paper we report a method for the synthesis of molecular imprinted polymers for use in sample preparation with aqueous biological materials. Highly cross-linked bulk polymers were synthesized in the presence of the template molecule, 2,6-pyridinedicarboxylic acid (DPA) using acrylamide (ACD) and 4-vinylpyridine (VP) as functional monomers. Conditions are described for the optimization of the template complex with temperature, copolymer mixture and crosslinker type. Selective binding of the template molecule is demonstrated in comparison to structural isomers and analogs for molecular imprinted polymers (MIPs) synthesized with three different crosslinkers, ethyleneglycol dimethacrylate (EGDMA), bisacrylamide and N, N′-1,3-phenylene bismethacrylamide (PBMA). The chromatographic capacity factors and selectivities for a series of structural analogs were compared. Molecular imprinted polymers prepared with equimolar ratios of ACD and VP and either PBMA or bisacrylamide resulted in highly selective binding for the template versus analogs with similar structure and chemistry. Multiple molecular dissociation constants were measured with the maximum binding capacities for EGDMA, PBMA and bisacrylamide measuring 17, 27 and 90 μmol/g, respectively.