The analytical and preparative separation of proteins and peptides will be explored using metalloporphyrins covalently immobilized onto silica supports. Incorporation of appropriate metal ions (e.g.,Zn++,Ni++,Fe+++,etc.) within the immobilized porphyrin structure is expected to yield stationary phases superior to existing immobilized metal ion affinity (IMA) phases based on immobilized iminodiacetates. The extremely high stability constants for metal ion-porphyrin complexes should enable polypeptide separations to occur without loss of the metal ion, and this would represent a significant advantage over currently available IMA phases thereby achieving reproducible high performance analytical separations of peptides/proteins. Further, the aromatic macrocycle of the immobilized porphyrin will provide a stationary phase that exhibits a unique mixed mode retention mechanism, with both metal ion affinity and pi-pi interactions occurring simultaneously, possibly increasing solute selectivity. With the proper choice of eluent components, the relative influence of each retention mechanism can be varied to optimize a given protein/peptide separation. The proposed research will build upon recent efforts by Selective Technologies Inc., that have already led to the successful commercialization of the FulleSep (TM) column, a column packed with a tetraphenylporphyrin- silica phase that exhibits extraordinarily high selectivity for the separation of fullerenes.
