Macroporous molecularly imprinted monolithic polymer columns for protein recognition by liquid chromatography

Macroporous cytochrome c (cyc)-imprinted monolithic polyarylamide columns were prepared, and applied for the template protein recognition by HPLC. With cyc (18.8 mg) as template, the imprinted monolithic materials were in situ polymerized in an HPLC column tube, with methacrylamide (450 mg), methacrylic acid (15.8 mg), piperazine diacrylamide (720 mg) and ammonium sulfate (390 mg) dissolved in 5 mL of phosphate buffer (pH 7.4), initiated by ammonium persulfate and TEMED. After the reaction, cyc was removed with acetic acid (10%, v/v) containing 10% w/v SDS. The non-imprinted monolithic column was prepared under the same procedure except without cyc. Retention of cyc and its competitive protein, lysozyme (lys), on molecular-imprinting polymer (MIP) and non-imprinted polymer columns was studied. When the pH value of mobile phase was 4.0, on MIP column, the retention factors of cyc and lys were 2.0 and 1.3, respectively. However, those on non-imprinted polymer column were very similar, both as 1.1. Even in competitive environment, a mixture of cyc and lys could be separated on MIP column under gradient elution, with resolution as 1.2. These results indicate that protein-imprinted monolithic polymer columns could offer obvious affinity and specific recognition to the template protein.