Investigation of immobilized biopolymers for metal binding

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Malachowski, Lisa Lyn

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This research focuses on the utility of immobilized poly amino acids for metal remediation and preconcentration. The biohomopolymer poly-L-histidine (PLHis) was immobilized onto controlled pore glass (CPG) and its metal binding capabilities evaluated through the use of a flow injection analysis - flame atomic absorption system (FIA-FAAS). The metal binding capability of PLHis-CPG was determined through the analysis of the generated breakthrough curves. The polymer likely coordinates cationic metals through the imidazole side chain (pKa ≈ 6) present on each histidine residue with both strong and weak binding sites for Cu2+, Cd2+, Co2+, and Ni2+. It has also been shown that the protonated imidazole side chain present in acidic conditions is capable of binding metal oxyanions such as chromates, arsenates, and selenites; although oxyanion binding currently exhibits interferences from competing anions in solution, such as sulfate and nitrate. Poly-L-Aspartic Acid (PLAsp) and Poly-L-Glutamic Acid (PLGlu) were also individually immobilized onto controlled pore glass (CPG) and compared using their metal binding capabilities. Elemental combustion analysis was used to yield polymer coverage approximations. Formation constants and site capacities of both polymers for Cd2+ were determined through equilibrium and breakthrough studies. Additionally, the metal selectivity of PLAsp and PLGlu was evaluated when breakthrough curves were run with several metals present in solution at one time. Both polymers exhibited similar binding trends and binding strengths for all of the metals studied. This likely reflects the absence of a predetermined tertiary structure of the polymers on the surface and the relatively high residue-per-metal ratio (~20:1), which places less stringent requirements on the steric hindrance between the side chains and the resultant ìwrappingî of the peptide around the metal. Initial attempts at determining formation constants of PLAsp and PLGlu through competitive binding experiments with either EDTA or oxalate present were unsuccessful due to complications caused by the current immobilization procedure. Therefore, alternate immobilization procedures were investigated utilizing an epoxide linker. These methods eliminate the formation of an amine functionality on the surface. Additionally, a combinatorial approach was used in an attempt to elucidate an optimal copolymer primary structure for successful binding of a target metal. This approach included screening the library for successful binding with micro x-ray fluorescence (MXRF) and obtaining the sequence of the successful copolymer through Edman Degradation. A considerable amount of the metal binding experiments conducted in this research used the analysis of breakthrough curves generated through flow injection-flame atomic absorption spectrometry. Solution flow rate is a critical parameter in breakthrough analysis. Due to the absence of an inexpensive, on-line flow meter for flow injection analysis systems, an electronic flow meter was constructed to measure the flow rate during the FIAAS measurements. Thus, flow rates can be measured while collecting breakthrough data, and continuous monitoring of flow rates is possible.