Date of Award
Doctor of Philosophy (PhD)
Mechanical Engineering with a specialization in Chemical Engineering
Affinity chromatography., Separation (Technology), Biomolecules--Analysis.
Affinity chromatography is used to separate biomolecules based on highly specific interactions, such as receptor and ligand, enzyme and substrate or antigen and antibody. Traditional chromatographic separation systems have the disadvantages of low flowrates, high pressure drop across the column, low capacity and poor reusability. Ceramic monoliths have been previously used as active support for affinity chromatographic columns due to their compact geometry and outstanding mechanical properties. In addition, mathematical models have been developed in order to determine the adsorption and elution times depending on the monolith geometry. The reversible interaction between L-asparagine and L-asparaginase has been exploited, using a ceramic monolith coated with agarose as support, cyanate esters as spacer arm and asparagine as ligand. As an alternative, this work uses a ceramic monolith with a polymer coating, poly (L-lysine) as spacer arm and asparagine as ligand. This method provides a more environmentally-friendly and safe approach since cyanide chemistries are avoided in the preparation of the spacer arm. The polymer coating based on poly (acrylic acid) was tested to characterize its mechanical and adhesion properties, and infrared spectroscopy was used to characterize its molecular properties. Poly (L-lysine) of different chain lengths was synthetized using the ring-opening polymerization of amino acid N-carboxy anhydrides and characterized by 1H NMR spectroscopy. Coupling reactions between the polymer coating, poly (L-lysine) and L-asparagine were performed using the EDC/NHS method, and proved successful. Finally, the adsorption/elution experiments demonstrated reversible binding between L-asparagine and L-asparaginase and served as a proof of concept for its application in protein separation and purification. The discoveries in this work will make an important contribution to a better understanding of the use of ceramic monoliths as support in affinity chromatography.
Sanchez Santiago, Javier, "Robust support media for high throughput chromatographic separations" (2016). Dissertations. 114.